Preparation of samples for vitamin B12 and/or folate assay and assay

Vitamin B.sub.12 (cobalamin) and/or folate (folic acid) assay techniques can be simplified, by eliminating the step of heating or boiling the to-be-tested sample prior to its assay. This is accomplished by utilizing compositions and procedures: PA1 (1) which substantially completely liberate all vitamin B.sub.12 and/or folate from endogenous binding protein without heating or boiling; PA1 (2) which substantially completely destroy all endogenous binding protein which is present in the to-be-assayed sample, and which may bind natural and radioactive vitamin B.sub.12 or folate both due to its liberation from vitamin B.sub.12 or folate in the original sample or due to its unbound presence in the original sample; PA1 (3) which substantially completely inhibit or block any undestroyed endogenous binding protein; and PA1 (4) which substantially completely inhibit or destroy any intrinsic factor-blocking antibodies which may be present in the to-be-assayed sample. In preferred embodiments the protein destroying compositions utilized include both a strong base and a sulfhydral compound which is a strong disulfide protein destroying material while the material of choice to bind with undestroyed endogenous binding protein is a vitamin B.sub.12 analogue. The sample is then assayed using known, for example, radioisotrope dilution assay techniques.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to methods and materials for preparing 
mammalian blood and tissue for assay, and to the resulting assay. More 
specifically it relates to methods and materials for preparing human serum 
for assay of vitamin B.sub.12 (cobalamin) and/or folate (folic acid). 
2. Prior Art 
For many years it has been recognized that the assay of the vitamin 
B.sub.12 in humans is a valuable technique for diagnosing and subsequently 
treating certain diseases which cause a deficiency of vitamin B.sub.12, 
such as for example, pernicious anemia, post gastrectomy states, 
nutritional deficiencies, intestinal disorders, and/or other conditions 
where the blood level of vitamin B.sub.12 is frequently depressed. The 
assay of vitamin B.sub.12 in humans has also been useful for diagnosing 
certain myeloproliferative diseases, such as chronic myelogenous leukemia 
where the blood level of vitamin B.sub.12 is frequently elevated. 
Initially vitamin B.sub.12 was assayed for example, using either Euglena 
gracilis or Lactobacillus leichmannii in microbiologic assays. More 
recently, radioisotope dilution (RID) assays for vitamin B.sub.12 have 
been utilized. Such radioisotope dilution assay techniques for vitamin 
B.sub.12 are well documented in the literature, see for example Lau, et 
al. (1965) "Measurement of Serum B.sub.12 Levels Using Radioisotope 
Dilution and Coated Charcoal," BLOOD, 26, 202, as modified by Raven et. 
al. (1968) "The Effect of Cyanide Serum and Other Factors on the Assay of 
Vitamin B.sub.12 by Radio-Isotope Method Using .sup.57 Co-B.sub.12, 
Intrinsic Factor and Coated Charcoal," GUYS HOSPITAL REPORTS, 117, 89; and 
(1969) "Improved Method for Measuring Vitamin B.sub.12 in Serum Using 
Intrinsic Factor, .sup.57 Co-B.sub.12 and Coated Charcoal," JOURNAL OF 
CLINICAL PATHOLOGY, 22, 205. More recently the presence of vitamin 
B.sub.12 analogues in samples has been found, which analogues falsely 
contribute to the apparent vitamin B.sub.12 level found by radioisotope 
dilution assay, unless the binding protein utilized in the assay is 
specific to vitamin B.sub.12, as for example, either pure intrinsic 
factor, or binding protein which has been treated to eliminate or 
inactivate binding proteins which are non-specific to vitamin B.sub.12, 
see Allen U.S. Pat. No. 4,188,189, Allen U.S. application Ser. No. 
069,257, filed Aug. 24, 1979, and Kolhouse, et. al. (1978) "Cobalamin 
analogues are present in human serum and can mask Cobalamin deficiency . . 
. " N. ENG. J. MED. 229, 784. 
In view of these prior art developments, current radioisotope dilution 
assay of vitamin B.sub.12 generally includes the steps of freeing the 
endogenous vitamin B.sub.12 from its endogenous natural binding protein 
by, for example, heating or boiling to-be-tested serum samples according 
to the teaching of Kolhouse et. al., adding an amount of intrinsic factor 
binding protein from which non-specific binding protein has been 
eliminated or inactivated, and radioactive vitamin B.sub.12, for example 
.sup.57 Co-B.sub.12, in a known amount which is adequate by itself to bind 
all of the intrinsic factor. Thus, all of the added intrinsic factor 
binding protein should be bound, either by the vitamin B.sub.12 present 
from in the sample undergoing test, or by the radioactive vitamin B.sub.12 
added to the composition. As both the natural vitamin B.sub.12 and the 
radioactive vitamin B.sub.12 compete dynamically in the reaction to bind 
with the limited amount of intrinsic factor binding protein, the degree to 
which the radioactive count of the intrinsic factor protein bound-vitamin 
B.sub.12 is inhibited or reduced is indicative of the amount of natural 
vitamin B.sub.12 which is present in the sample undergoing testing. The 
inhibition of the binding of the radioactive vitamin B.sub.12 with 
intrinsic factor is determined, both natural and radioactive, for example, 
by separating all unbound vitamin B.sub.12 from the intrinsic factor 
protein bound vitamin B.sub.12 by, for example, using protein-coated 
charcoal which adsorbs the unbound radioactive vitamin B.sub.12. Then the 
radioactivity of the supernatant liquid containing the mixture of 
intrinsic factor bound radioactive vitamin B.sub.12 and intrinsic factor 
bound non-radioactive vitamin B.sub.12 is counted for radioactivity. The 
vitamin B.sub.12 concentration in the sample undergoing testing is then 
determined from the count, often by comparison with a standard chart using 
techniques well known in the art. 
Folate RID analysis is quite similar, although it uses radioactive folate 
and a folate binder for the same purpose. 
As already noted, it has recently been recognized by Kolhouse et. al. that 
vitamin B.sub.12 measurements previously provided were often inconsistent 
with the results obtained by other measuring techniques for vitamin 
B.sub.12, such as the microbiological assays. Most often, the vitamin 
B.sub.12 assay obtained by radioisotope dilution assay techniques had been 
found to be high. The determination by Kolhouse et. al. that there are 
vitamin B.sub.12 analogue substances in mammalian blood and tissue which 
react with the non-specific protein binders which were then in use in 
prior art RID assay techniques explained why many prior art analyses of 
vitamin B.sub.12 tested higher than the amount of vitamin B.sub.12 
actually in the sample. After the determination by Kolhouse et. al. that 
portions of most prior art binding protein included substantial amounts of 
binding protein which was not specific to vitamin B.sub.12 and which was 
capable of binding with vitamin B.sub.12 analogues; the corrective 
techniques were developed by Allen. As a result of Allen's work, current 
RID assays now use only pure or purified intrinsic factor binding protein, 
from which non-specific binding protein has been removed or inactivated, 
so that it is specific in its binding reaction only with vitamin B.sub.12, 
and can not bind with vitamin B.sub.12 analogues. 
Folate RID analysis is far less demanding as there are no similar problems 
with folate analogues and no similar problems with non-specific folate 
binder, however, even improved specific vitamin B.sub.12 RID assay 
techniques, using only intrinsic factor as the binding protein and the 
folate assay, still require the heating (or boiling) of samples prior to 
testing, in order to liberate vitamin B.sub.12 and/or folate in the 
to-be-tested sample from endogenous binding protein. The current heating 
step is difficult to control, tedious and time consuming in terms of 
handling, capping and uncapping test tubes, and does not allow a process 
in which samples can be tested without heating. 
Recently, means of denaturing samples chemically, to liberate vitamin 
B.sub.12 from endogenous binding protein without boiling, have been 
reported. Ithakissios et. al. (1980) "Room Temperature Radioassay for 
B.sub.12 with Oyster Toadfish (Opsanus tau) serum as binder", CLINICAL 
CHEMISTRY, 26, 323 has reported that the use of NaOH at pH 13.6 in the 
presence of KCN provided more than 98% release of vitamin B.sub.12, with 
the release increasing to 100% when Ficol was used to replace human serum 
albumin. It also discussed vitamin B.sub.12 release results for vitamin 
B.sub.12 from protein at pH environments in the range of 2-12. Ithakissios 
et. al. did not discuss the effect of released endogenous binding protein 
in the to-be-assayed sample, or recognize any potential problem which 
might be caused by the presence of abnormally high amounts of endogenous 
binding protein or intrinsic factor-blocking antibodies which may be 
naturally present in some samples. A label for a commercial kit, dated 
October 1980, from RIA Products, Inc., Waltham, Mass "No-Boil Combostat 
T.M. Kit for simultaneous radioassay of FOLATE and VITAMIN B.sub.12 " has 
recently been noted as disclosing the use of NaOH as a denaturing agent in 
the presence of 0.01% KCN and dithiothreitol in a no-boil technique for 
analyzing both folate and vitamin B.sub.12. It also does not discuss the 
effects of undestroyed endogenous binding protein on the vitamin B.sub.12 
assay or recognize any potential problem caused by the presence of high 
amounts of endogenous binding protein or intrinsic factor-blocking 
antibodies in some samples. 
In a similar manner an undated label for a commercial kit, from Diagnostic 
Products Corporation, Los Angeles, Calif., "No-Boil Dualcount", radioassay 
for vitamin B.sub.12 and folic acid, has also been noted as disclosing the 
use of NaOH, KCN and dithiothreitol in another no-boil technique for 
analyzing both folate and vitamin B.sub.12. Again, this technique does not 
discuss or recognize the potential for any problem in the vitamin B.sub.12 
assay due to undestroyed endogenous binding protein or the presence of 
intrinsic factor-blocking antibodies or high amounts of endogenous binding 
protein. 
In each of these prior art techniques KCN is normally added for the primary 
purpose of converting the vitamin B.sub.12 in the sample to its most 
stable form, cyanocobalamin. Similarly, dithiotreitol is normally used in 
folate assays, and therefore in dual vitamin B.sub.12 -folate assays, as a 
means of stabilizing folate during the folate assay. 
Currently, the best documented RID vitamin B.sub.12 -folate dual assay 
technique became available in December 1980 from Corning, Medfield, Mass. 
"Vitamin B.sub.12 [.sup.57 Co]/Folate [ 125I] Radioassay" 
Immophase,.sub.TM (For the simultaneous determination of vitamin B.sub.12, 
and folate in serum, plasma and red blood cells.) The documentation and 
accuracy of this kit and test are greater than for any known prior-art 
kit. 
BRIEF DESCRIPTION OF THE INVENTION 
As has already been indicated, in most standard prior art radioisotope 
dilution assays for vitamin B.sub.12 and/or folate, the to-be-assayed 
sample is heated or boiled, to separate the sample vitamin B.sub.12 and/or 
folate from endogenous binding protein. In more recent techniques the 
separation has been obtained without boiling by using a strong base with 
or without other chemicals. Then a known amount of radioactive vitamin 
B.sub.12 and/or radioactive folate is mixed with a prepared to-be-tested 
sample and a known amount of specific binding protein which is capable of 
binding with both the natural and radioactive vitamin B.sub.12, (but 
incapable of reacting with vitamin B.sub.12 analogues) and/or folate 
binder is added to the mixture. Subsequently, utilizing well-known 
techniques, the radioactivity of the bound sample is compared, for 
example, with a standard curve to determine the amount of natural vitamin 
B.sub.12 or folate present in the sample undergoing assay. 
It has now been discovered that, in accordance with the teaching of the 
present invention, the RID assay techniques can be simplified, improved 
and in many instances, rendered more accurate by eliminating the step of 
heating or boiling the to-be-tested sample prior to its assay. This is 
accomplished by utilizing compositions and procedures: 
(1) which substantially completely liberate all vitamin B.sub.12 and/or 
folate from endogenous binding protein without heating or boiling: 
(2) which substantially completely destroy all endogenous binding protein 
which is present in the to-be-assayed sample, and which may bind natural 
and radioactive vitamin B.sub.12 or folate both due to its liberation from 
vitamin B.sub.12 or folate in the original sample or due to its unbound 
presence in the original sample; 
(3) which substantially completely inhibit or block any undestroyed 
endogenous binding protein; and 
(4) which substantially completely inhibit or destroy any intrinsic 
factor-blocking antibodies which may be present in the to-be-assayed 
sample. 
Failure to consider and carry out these procedures in a no-boil test may 
result in erroneous vitamin B.sub.12 and/or folate RID assay, as 
documented and detailed below. 
This is accomplished by treating vitamin B.sub.12 and/or folate samples 
which are to-be-assayed without heating or boiling, with compositions and 
procedures which substantially completely liberate vitamin B.sub.12 and/or 
folate from endogenous binding protein, without heating or boiling; which 
substantially completely destroy substantially all endogenous binding 
protein present in the sample, whether liberated from vitamin B.sub.12 
and/or folate or otherwise present in the sample; which substantially 
inhibit or block undestroyed endogenous binding protein which has not been 
destroyed; and which inhibits or destroys substantially all intrinsic 
factor-blocking antibodies which are present in the to-be-assayed sample. 
More specifically, combinations of ingredients and procedures are taught 
which provide the foregoing results. 
As has already been noted, a number of techniques are now publicly known 
and available which indicate that they release, without heating or 
boiling, vitamin B.sub.12 and folate from a to-be-assayed sample. However, 
the present invention is based on the recognition of facts, apparently not 
known, or not understood, or ignored by other RID no-heat methods: 
(1) that optimum release of vitamin B.sub.12 and/or folate from a 
to-be-assayed sample must be achieved to obtain accurate vitamin B.sub.12 
measurement; 
(2) that while boiling apparently substantially destroys endogenous binding 
protein, whether released from vitamin B.sub.12 or otherwise present in 
the to-be-assayed sample, that currently published no-heat methods allow 
substantial amounts of endogenous protein to survive in some blood samples 
which may result in inaccurate assays of vitamin B.sub.12 in these 
samples, as detailed below; and 
(3) that there are antibodies naturally present in some samples which 
antibodies react with or block intrinsic factor protein used for current 
state-of-the-art vitamin B.sub.12 RID assays, in accordance with the 
teaching of Kolhouse et al and Allen, which antibodies may also result in 
inaccurate assays of vitamin B.sub.12, as also detailed below. 
In the absence of the recognition of these problems and the use of the 
procedures and compositions of the present invention to deal with them, 
seriously inaccurate vitamin B.sub.12 assays will be obtained for a 
clinically unacceptable number of vitamin B.sub.12 abnormal patients using 
current no-boil techniques and certain of the Kolhouse et. al and Allen 
RID assay procedures, even though the Kolhouse et. al. and Allen 
procedures provide the greatest accuracy for boiling techniques. For 
example, undestroyed or uninhibited endogenous binding protein present in 
a sample after no-boil release is available during the assay incubation 
period to react with both sample and radioactive vitamin B.sub.12. This 
may result in a false, either high or low vitamin B.sub.12 reading, 
depending on the technique which is used to separate bound and unbound 
vitamin B.sub.12. 
In another instance, where antibodies capable of binding with intrinsic 
factor are present in the sample, but have not been destroyed by boiling, 
in the Kolhouse et. al. and Allen RID assay procedures using intrinsic 
factor as the binding protein, the intrinsic factor which is added and 
expected to only react with and bind with sample and radioactive vitamin 
B.sub.12 may react with intrinsic factor blocking antibodies, therefore 
making some intrinsic factor unavailable to react with radioactive and 
sample vitamin B.sub.12. This reaction of intrinsic factor thus results in 
a lower amount of both types of vitamin B.sub.12 being bound with 
intrinsic factor, due to the added competition for the intrinsic factor 
from the antibody, which in turn will be seen as a lower bound radioactive 
vitamin B.sub.12 -intrinsic factor concentration, and be analyzed, in the 
absence of the teaching of the present invention, as a higher amount of 
vitamin B.sub.12 in the assayed sample. 
It is also clearly apparent, without the need for detailed explanation, 
that failure to release substantially all vitamin B.sub.12 or folate from 
endogenous binding protein in a to-be-assayed sample will result in an 
erroneous low vitamin B.sub.12 or folate reading. 
Finally, it has recently been noted that some subjects with abnormally high 
amounts of both vitamin B.sub.12 and endogenous binding protein in their 
blood serum, such as subjects with chronic myelogenous leukemia, may be 
assayed at lower vitamin B.sub.12 levels than actually exist, due to the 
competition of the excess endogenous binding protein for radioactive 
vitamin B.sub.12, as explained above in the discussion of uninhibited 
endogenous binding protein in a sample after no-boil release. 
Based on these discoveries, it is now proposed that samples being prepared 
for vitamin B.sub.12 and/or folate assay be treated with compositions 
which liberate vitamin B.sub.12 from endogenous protein, substantially 
destroy endogenous binding protein and intrinsic factor (IF) blocking 
antibodies, and which react with or inhibit any undestroyed endogenous 
binding protein, rendering it unavailable to bind with any form of vitamin 
B.sub.12. In preferred embodiments the protein destroying compositions 
utilized include both a strong base and a sulfhydral compound which is a 
strong disulfide protein destroying material while the material of choice 
to bind with undestroyed endogenous binding protein is a vitamin B.sub.12 
analogue. Based on these discoveries, it is also now proposed that large 
numbers of samples from low vitamin B.sub.12 patients with and without 
IF-blocking antibodies and high vitamin B.sub.12 CML patients be used to 
assure that the above objectives are met. 
Based on these teachings, other types of compositions and related 
techniques can be readily determined and applied to the practice of the 
present invention. 
The foregoing and other objects, features and advantages of the invention 
will be apparent from the following more particular description of 
preferred embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION 
Based on the problems which are not adequately recognized or dealt with in 
currently published no-boil vitamin B.sub.12 and/or folate assays, and a 
careful analysis of these problems using a large number of test samples, 
procedures and compositions have been developed in the present invention 
which compositions and procedures avoid the problems even though they are 
not recognized or dealt with in currently published no-boil vitamin 
B.sub.12 RID assays. 
The preferred compositions of the present invention generally include a 
vitamin B.sub.12 analogue to be added to samples undergoing no-boil 
vitamin B.sub.12 or RID assay. The vitamin B.sub.12 analogue combines with 
endogenous binding protein in the sample whether released from the sample 
vitamin B.sub.12 or otherwise present in the to-be-assayed sample. By 
binding, and thus inhibiting this endogenous binding protein with added 
vitamin B.sub.12 analogue, the endogenous binding protein is unavailable 
to combine with radioactive vitamin B.sub.12 and/or to recombine with 
vitamin B.sub.12. Therefore, the errors which would be caused by such a 
binding combination or recombination are avoided. The added vitamin 
B.sub.12 analogue should be one that is not substantially bound by 
intrinsic factor and thereby does not substantially interefere with the 
ability of intrinsic factor to bind radioactive vitamin B.sub.12 and 
vitamin B.sub.12 in the to-be-assayed sample. Vitamin B.sub.12 analogues 
useful in the practice of the present invention include, but are not 
limited to, cobinamide (Cbi), CN-Cbl(bde-OH) and (3,5,6,Me3BZA)(CN,OH) 
Cba, or other vitamin B.sub.12 analogues. Vitamin B.sub.12 analogue used 
should be used at least in an amount great enough to bind substantially 
all endogenous binding protein which could be expected to be in a sample 
being prepared for assay. Vitamin B.sub.12 analogue in the range of about 
1 ng to about 10,000 ng per 200 ul of serum sample is useful. Preferred 
amounts of vitamin B.sub.12 analogue are in the range of about 20 ng to 
about 1000 ng per 200 ul of serum sample. The vitamin B.sub.12 analogue 
should be added to the to-be-assayed no-boil sample either prior to 
vitamin B.sub.12 release, during vitamin B.sub.12 release or immediately 
after vitamin B.sub.12 release, but always prior to or at the same time as 
the added radioactive vitamin B.sub.12. By its addition at any of these 
times the vitamin B.sub.12 analogue is available to immediately bind 
endogenous binding protein before it can combine with any substantial 
amount of released vitamin B.sub.12 or added radioactive vitamin B.sub.12. 
Surprisingly, vitamin B.sub.12 analogue not only binds endogenous binding 
protein, but, as subsequently shown in Table I, it also assists in the 
release of vitamin B.sub.12 from endogenous binding protein. 
A strong base is used in preferred compositions of the no-boil assay of the 
present invention. It is believed that a strong base, in combination with 
other ingredients of the present invention, serves to both assist in the 
release of vitamin B.sub.12 from endogenous binding protein and also 
serves to assist in substantially destroying and/or inactivating both the 
endogenous binding protein, and any intrinsic factor-blocking antibodies 
which may be present in the sample. The amount of base used should be 
enough, in combination with the other ingredients, to release 
substantially all of the vitamin B.sub.12 and/or folate from endogenous 
binding protein, to destroy and/or inactivate substantially all of the 
endogenous binding protein, and to inhibit or destroy substantially all of 
the intrinsic factor-blocking antibodies. Preferred strong bases include, 
but are not limited to, NaOH, KOH, LiOH and other strong bases. Useful 
concentrations of base are in the range of about 0.02 N to about 4.0 N, 
during the preparation of samples for assay, that is prior to the steps 
involving partial neutralization and the addition of intrinsic factor (IF) 
or binding protein that is substantially specific for binding vitamin 
B.sub.12 and/or folate. Preferred concentrations of base are in the range 
of about 0.3 N to 1.0 N during the preparation of samples for assay. The 
base is preferrably added to the sample at the time the action to prepare 
the sample for assay is initiated. 
At least one additional compound to assist in releasing vitamin B.sub.12 
from endogenous binding protein and to assist in substantially destroying 
endogenous binding protein and IF-blocking antibodies is included in 
preferred compositions of the present no-boil assay preparation. The 
additional compound is preferably selected for its protein destroying 
character. One family of disulfide protein destroying materials are the 
sulfhydral compounds. Included within the family of useful sulfhydral 
compounds are betamercaptoethanol (BME), thioglycolate, thioglycerol and 
dithiothreitol (DTT), although other sulfhydral compounds or other protein 
destroying materials may be used in the practice of the present invention. 
The amount of protein destroying material used should be enough, in 
combination with the other ingredients, to release substantially all of 
the vitamin B.sub.12 from endogenous binding protein, to destroy and/or 
inactivate substantially all of the endogenous protein, and to inhibit or 
destroy substantially all of the intrinsic factor-blocking antibodies. 
Useful concentrations of sulfhydral compounds are in the range of about 
0.004 N to 0.4 N during the preparation of samples for assay. Preferred 
concentrations of sulfhydral compounds are in the range of about 0.04 N to 
about 0.1 N during the preparation of samples for assay. The protein 
destroying material is added to the sample at the time the sample is 
prepared for assay, and preferably prior to, or at the same time as, the 
radioactive vitamin B.sub.12 is added to the sample to thus eliminate or 
minimize the combination of endogenous binding protein with the added 
radioactive vitamin B.sub.12. 
One preferred vitamin B.sub.12 assay using the no-boil sample preparation 
and procedure of the present invention is now described. Generally, the 
assay procedure is substantially the same as that described in U.S. Pat. 
No. 4,188,189, with the exception that: (1) the sample and added reagents 
are not boiled or heated; and (2) additional or substitute compounds, in 
accordance with the teaching of the present invention are utilized. During 
the assay the reagents and test specimens are all at ambient room 
temperature, in the range of about 15.degree. C. to about 35.degree. C. 
Any reagents or specimens which have been frozen or refrigerated are 
allowed to come to room temperature. This is not a heating step as the 
term heating is used herein. To initiate the assay, 200 ul of to-be-tested 
serum or standard is pipetted into each sample tube, each tube being 
labelled to indicate its identity. Then, an aqueous 50 ul solution 
containing: 
60 pg .sup.57 Co-B.sub.12 
100 ng cobinamide 
10.0 ug KCN 
2000.0 ug DTT 
is added. Now 50 ul of 2.0 N NaOH is pipetted into each tube and after 
thorough mixing the tubes are allowed to incubate at ambient conditions, 
without heating, for about 20 minutes. Subsequently the assay is completed 
when, an aqueous 1000 ul solution having a pH of 6.4 and containing: 
0.01 M NaPO4, pH7 
0.127 M NaCl 
0.162 M boric acid 
intrinsic factor in an amount capable of binding about 50 pg of vitamin 
B.sub.12 
is added to substantially neutralize the solution and allow binding of both 
sample and radioactive vitamin B.sub.12 with intrinsic factor (IF). This 
mixture is then allowed to incubate for about one hour without heating. 
The simultaneous or subsequent addition of charcoal then results in the 
unbound vitamin B.sub.12, in the solution, whether sample or radioactive 
tracer vitamin B.sub.12, to be bound to the charcoal, which is 
subsequently removed from the supernatant liquid. The radioactive count of 
either the supernatant, which now contains sample vitamin B.sub.12 and 
tracer radioactive vitamin B.sub.12 bound to IF binder, or the charcoal 
which contains remaining unbound sample vitamin B.sub.12 and unbound 
radioactive vitamin B.sub.12, can be utilized to indicate the amount of 
vitamin B.sub.12 in the sample undergoing test, using one or more well 
known and documented techniques. The resulting preferred assay preparation 
and assay of the present invention gave highly accurate vitamin B.sub.12 
assay results, as verified by comparison with the Corning radioassay 
mentioned previously as currently being the most highly tested and 
accurate RID boiling assay. 
The foregoing preferred procedures, can be modified in many ways and still 
remain within the teaching of the present invention which provides 
techniques and compositions for preparing vitamin B.sub.12 for assay 
without recourse to heating or boiling during any step in the procedure. 
For example, the size of the serum sample or standard can be increased, 
say doubled, or reduced substantially, without any need to modify the 
amount of the other sample preparation ingredients. For efficiency, the 
tracer, endogenous protein and antibody destroying chemicals, vitamin 
B.sub.12 analogue, and strong base, or any combination thereof, may all be 
combined in a single pipetted liquid aliquot and added to the sample 
simultaneously, within the teaching of the present invention. In a similar 
manner, and of special interest and use to laboratories which make many 
vitamin B.sub.12 assays, various combinations of the tracer, endogenous 
binding protein and antibody destroying chemicals, vitamin B.sub.12 
analogue, and strong base may be prepared in liquid form, and/or in dry 
form, for reconstitution with liquid when needed, for use in the teaching 
of the present invention. Such materials could also be packaged together, 
for example, as a kit, thus allowing any laboratory to obtain the test 
materials from an outside source as a package, without the need to 
maintain individual chemicals and compositions in stock or the need to go 
through extensive test preparations. In any of the suggested combinations, 
these materials, other than the radioactive tracer, are those materials 
which are required to prepare a sample for no-boil vitamin B.sub.12 assay, 
in accordance with the teaching of the preferred embodiments of the 
present invention. 
As folate is often tested simultaneously with vitamin B.sub.12, the 
foregoing example may be readily modified for the simultaneous testing of 
folate. For example, the addition of radioactive folate such as 
125I-labelled folate at the time the radioactive vitamin B.sub.12 is 
added, and folate binder to bind the sample and radioactive folate at the 
time of the addition of the IF binder. These modifications make the 
process suitable for the simultaneous assay of vitamin B.sub.12 and 
folate, using well known dual assay techniques. 
In a similar manner, the process of the present invention may be modified 
by using its no-boil processes and compositions for the assay of folate 
alone. 
BASIS OF THE PRESENT INVENTION 
The compositions and procedures of the present invention, as already noted, 
have been conceived, and developed and evolved utilizing information and 
recognizing problems which were either not previously known, and which 
were apparently not known or considered of importance in currently 
published no-boil vitamin B.sub.12 RID assays. These problems, as 
previously detailed include: 
(1) the need for the release of substantially all of the vitamin B.sub.12 
from endogenous binding protein in a to-be-assayed sample in order to 
obtain accurate vitamin B.sub.12 measurement; 
(2) the need to destroy or inactivate substantially all of the endogenous 
binding protein whether released from liberated vitamin B.sub.12 or 
otherwise present in the to-be-assayed sample. (Previously published 
no-heat methods allow substantial amounts of endogenous binding protein to 
survive in some blood samples, which may result in inaccurate assays of 
vitamin B.sub.12 in those samples); and 
(3) the need to destroy or inactivate antibodies naturally present in some 
samples which antibodies are of the character which could otherwise react 
with or block intrinsic factor protein used in RID assays. (Such 
IF-blocking antibodies result in inaccurate assays of vitamin B.sub.12 in 
previously published no-heat methods.) 
Clearly the non-heating separation of vitamin B.sub.12 from endogenous 
binding protein requires compositions which break the binding linkage 
between the vitamin B.sub.12 and endogenous binding protein. The use of 
bases, which are known protein destroying materials, suggests itself. 
However, a strong base by itself does not release substantially all 
vitamin B.sub.12 from endogenous binding protein. Furthermore, a strong 
base does not, by itself destroy or inactivate substantially all 
endogenous binding protein. Finally, a strong base does not destroy 
substantially all IF-blocking antibodies which may be present in a 
non-heating sample. 
With this recognition of the problem, various materials alone and in 
combination were tested as to their ability to release vitamin B.sub.12 
from endogenous binding protein and destroy or inactivate endogenous 
binding protein. These materials were tested by applying them alone and in 
various combinations, to known amount of radioactive .sup.57 Co-B.sub.12 
which has been bound to various forms of endogenous binding protein by 
preincubation. More specifically, .sup.57 Co-B.sub.12 was bound to 
endogenous binding protein in human plasma, which contains two such 
binding proteins known as R protein (R) and transcobalamin II (TCII), and 
also bound with R individually and TCII individually. For use as a control 
sample, .sup.57 Co-B.sub.12 was also preincubated with buffer solution 
substantially free of any binding protein. 
As set forth in Table I, following the preincubation with radioactive 
vitamin B.sub.12 and the subsequent treatment with the individual or 
combined materials, each sample was substantially neutralized, incubated 
for 60 minutes, without IF or any added binder, and then treated with 
charcoal, which adsorbs substantially all of the bound radioactive vitamin 
B.sub.12 that is released and remains released from endogenous binding 
protein. Therefore, using this procedure, the lesser the amount of .sup.57 
Co-B.sub.12 found in the supernatant liquid, as reported for example in 
Table I, the greater the amount of .sup.57 Co-B.sub.12 which was released 
by the material or materials, and the better the materials are in an assay 
preparation composition. 
TABLE 1A 
______________________________________ 
Release of [.sup.57 Co]B.sub.12 from plasma and various binders 
% [.sup.57 Co]B.sub.12 
Sample Cbi BME NaOH in supernatant 
______________________________________ 
plasma - - - 94.4 
R - - - 100.0 
TC II - - - 74.3 
buffer - - - 0.4 
plasma + - - 95.9 
R + - - 99.8 
TC II + - - 76.1 
buffer + - - 0.1 
plasma - + - 44.9 
R - + - 100.0 
TC II - + - 83.7 
buffer - + - 0.3 
plasma - - + 40.8 
R - - + 31.3 
TC II - - + 6.0 
buffer - - + 0.3 
plasma + + - 71.1 
R + + - 100.0 
TC II + + - 84.8 
buffer + + - 0.2 
plasma + - + 26.9 
R + - + 4.4 
TC II + - + 5.0 
buffer + - + 0.0 
plasma - + + 2.5 
R - + + 10.8 
TC II - + + 1.5 
buffer - + + 0.5 
plasma + + + 1.4 
R + + + 0.9 
TC II + + + 1.0 
buffer + + + 0.1 
______________________________________ 
As set forth by Table I, the various individual and combined materials had 
varying effects on the release of .sup.57 Co-B.sub.12. 
Now again, referring to Table IA, when a combination of cobinamide (Cbi), 
betamencaptoethanol (BME) and NaOH (strong base) were used, for each of 
Samples 29-32, the lowest percent of .sup.57 Co-B.sub.12 was found to 
remain in the supernatant, as compared to all other combinations. 
Therefore, the greatest release of vitamin B.sub.12 from binding protein 
was achieved using these three materials in combination. 
Special note is also called to Samples 13-16 in Table I in which a strong 
base, NaOH was used at a concentration of 0.5 N during the release 
analysis. It will be noted that in Samples 13-15 substantial amounts of 
.sup.57 Co-B.sub.12 remained in the supernatant liquid, and therefore was 
not released substantially completely by denaturing with a strong base in 
the absence of other materials. This raises questions as to the 
effectiveness of a strong base, by itself, as a denaturing agent. It is 
noted that all solutions had KCN present in them. 
Now, taken as a whole, the data in Table I makes it abundantly clear that 
in a controlled experiment with a known amount of radioactive vitamin 
B.sub.12, and no excessive amounts of endogenous binding protein or 
IF-blocking antibodies, that the combination of Cbi (a vitamin B.sub.12 
analogue), BME (a disulfide protein destroying composition), and NaOH (a 
strong base) provides the most substantially complete vitamin B.sub.12 
release composition. In a similar manner, the data in Table IB also shows 
that this same composition also provides substantially complete folate 
release from endogenous folate binding protein. 
Table IIA set forth comparative data as to vitamin B.sub.12 analysis of a 
large number of normal subjects using the standard "boiling" techniques of 
Kolhouse et. al. and Allen, and a prototype non-boiling technique of the 
present invention, still using BME as the disulfide protein destroying 
composition. Table IIB contains comparative data for folate analysis for 
some normal and patient samples. Table IIC contains comparative data for 
vitamin B.sub.12 and simultaneous folate analysis on a few normals and two 
vitamin B.sub.12 deficient patients. Note the total agreement of results 
as to the amount of vitamin B.sub.12, folate, and vitamin B.sub.12 and 
folate, using both the boil and the no-boil techniques, which is well 
within both scientific and clinical tolerances. 
TABLE 1B 
______________________________________ 
Release of .sup.125 I--folate from Serum 
% .sup.125 I--folate in supernatant 
Serum* Treated** Untreated*** 
______________________________________ 
A 2.6 82.9 
B 1.7 45.4 
C 3.2 97.0 
D 1.8 57.6 
E 2.2 74.9 
F 1.9 74.6 
G 1.5 52.4 
______________________________________ 
*These sera had high endogenous folate binding protein levels. Most norma 
sera have no free endogenous folate binding ability, i.e. untreated is 
&lt;2%. 
**Same as in Table 1A complete conditions. 
***Neutralizing solution, i.e. TrisHCl, was added before Cbi, BME & NaOH. 
Assay Conditions for Table I 
A. These assays had sample volumes of 200 ul and were incubated at room 
temperature for 20 minutes with 45.4 pg CO.sup.57 B.sub.12 in 5 ul of 
H.sub.2 O. The samples were: (a) 40 ul Buffer A (.05 M KPO.sub.4 pH 7.5, 
.5 M NaCl, 150 mg HSA/ml) plus 160 ul .15 M NaCl); (b) 200 ul plasma; (c) 
40 ul salivary R protein (1.2 ng B.sub.12 binding ability) plus 40 ul 
Buffer A plus 120 ul saline; (d) 160 ul TC II (0.59 ng B.sub. 12 binding 
ability) plus 40 ul Buffer A. Following this incubation 25 ug of KCN was 
added in 5 ul of H.sub.2 O to every test tube and eight different 
conditions were introduced. (See Table 1) They were all possible 
combinations of the following: (a) 1 ug cobinamide in 5 ul of H.sub.2 O; 
(b) 40 ul of 1 M BME; and (c) 100 ul of 2 N NaOH. H.sub.2 O was added whe 
an ingredient(s) was missing. The reaction, in a volume of 355 ul, was 
allowed 20 minutes at room temperature before neutralizing those tubes 
containing NaOH with 275 ul 1 M TrisHCl pH 7.5. All the test tubes had 
their volume brought up to 1.5 ml with 0.1 M NaCl and the assay went 
through a final 60 minutes at room temperature. The reaction was ended 
with 0.5 ml BSA coated charcoal. The assay tubes were spun 10,000 .times. 
g for 20 minutes and 1.0 ml supernatant was removed for counting. 
B. The complete assay was also used to investigate .sup.125 I--folate 
release. 
TABLE IIA 
______________________________________ 
Serum B.sub.12 values for normals using 
boiling and non-boiling methods. 
Serum Serum B.sub.12 (pg/ml) 
Normal: Boiled Non-boiled 
______________________________________ 
1. 390 350 
2. 260 319 
3. 230 258 
4. 325 419 
5. 475 510 
6. 365 465 
7. 205 242 
8. 540 590 
9. 410 527 
10. 390 496 
11. 150 211 
12. 636 490 
13. 300 357 
14. 460 589 
15. 390 490 
16. 305 316 
17. 265 326 
18. 370 420 
19. 200 292 
20. 325 235 
21. 230 290 
22. 295 382 
23. 425 546 
24. 700 785 
25. 280 310 
26. 165 171 
27. 200 254 
28. 185 220 
29. 150 223 
30. 325 413 
31. 345 440 
32. 170 208 
33. 410 468 
34. 300 378 
35. 480 518 
36. 230 298 
37. 230 245 
38. 345 363 
39. 250 319 
40. 300 335 
41. 250 304 
42. 500 580 
43. 240 261 
______________________________________ 
TABLE IIB 
______________________________________ 
Serum folate values in patients on whom 
folate values were requested 
in Colorado General Hospital boiling versus non-boiling assays 
serum folate (ng/ml) 
Serum boiled* non-boiled 
______________________________________ 
1 6.1 6.8 
2 3.3 2.6 
3 22.0 16.3 
4 7.6 7.2 
5 2.8 2.5 
6 20.0 35.7 
7 2.5 1.6 
8 12.0 10.8 
9 4.5 4.7 
10 11.0 10.3 
11 3.4 4.0 
12 3.4 4.0 
13 5.9 9.3 
14 3.0 3.5 
15 10.0 6.9 
16 23.0 20.5 
17 11.5 8.8 
18 2.1 2.5 
19 3.4 3.0 
20 3.1 3.2 
21 8.6 8.2 
22 4.5 4.6 
23 3.3 4.0 
24 13.5 15.6 
25 6.1 6.2 
26 8.5 8.0 
27 3.2 3.0 
28 &gt;25.0 28.0 
29 23.0 18.8 
30 11.5 8.4 
31 3.3 2.6 
32 15.0 17.2 
______________________________________ 
*Becton-Dickinson kit in our hospital lab. (normal range = 3-5 ng/ml) 
TABLE IIC 
______________________________________ 
Separate versus combined assays for Vitamin B.sub.12 and folate using 
the non-boiling technique. 
Separate Assays 
Combined Assays 
folate B.sub.12 folate B.sub.12 
Set A ng/ml pg/ml ng/ml pg/ml 
______________________________________ 
1 12.8 245 11.5 190 
2 8.5 360 9.2 305 
3 17.2 300 14.1 260 
4 15.2 200 16.4 185 
5 16.2 400 16.0 370 
6 5.8 320 5.2 360 
7 15.5 340 11.5 270 
8 7.6 &lt;10 7.4 &lt;10 
9 23.0 80 23.0 40 
______________________________________ 
This supports the accuracy and utility of the compositions of the present 
invention for testing vitamin B.sub.12 and/or folate in normal subjects 
and in some vitamin B.sub.12 deficient patients. 
Table III is of interest as showing the application of a "boiling" and one 
prototype non-boiling technique of the present invention to samples from a 
larger number of known vitamin B.sub.12 deficient patients. But for serum 
Sample #17, the agreement achieved by both techniques is statistically 
excellent, again showing the scientific accuracy and utility of the 
present invention. However, the large discrepency in serum Sample #17 is 
clinically unacceptable. Using the assay data from this prototype 
"non-boiled" assay of the present invention would have resulted in the 
patient who was the source of Sample #17 being classified "normal". 
Therefore, the prototype "no-boil" assay of Sample #17 represents a 
clinically unacceptable lapse in this prototype composition and technique. 
The failure to diagnose this single patient correctly could have led to a 
failure to treat this patient with vitamin B.sub.12. This could have had 
disastrous consequences for this patient since untreated vitamin B.sub.12 
deficiency can lead to potentially fatal blood and/or neurologic 
abnormalities, with the latter being especially serious since frequently 
they cannot be reversed by later treatment with vitamin B.sub.12, possibly 
resulting in commitment to a mental institution for life. 
Subsequent analysis indicated that many of the "deficient" samples set 
forth in Table III, including Sample #17, had IF-blocking antibodies 
(which have been labelled "anti-IF blocking antibody" in Table III). In 
Table III "+" indicates the presence and "-" indicates the absence of such 
antibodies. Further analysis indicated that Sample #17 had only a 
moderately high amount of IF blocking antibodies, but more importantly, 
that its antibodies were particularly resistant to destruction by the 
prototype assay composition used. (IF-blocking antibodies have many 
different structures and thus differ in their resistance to destruction.) 
It was also determined that these IF-blocking antibodies compete with 
vitamin B.sub.12 for IF in the test. 
TABLE III 
______________________________________ 
Serum B.sub.12 values for patients with clinical evidence 
of B.sub.12 deficiency using boiling and non-boiling methods. 
Serum B.sub.12 (pg/ml) 
anti IF 
Serum boiled non-boiled 
blocking antibody 
______________________________________ 
1. 41 &lt;10 + 
2. 48 16 - 
3. 53 55 - 
4. 48 &lt;10 + 
5. 31 34 + 
6. 54 39 - 
7. 50 &lt;10 + 
8. &lt;10 &lt;10 - 
9. 25 &lt;10 - 
10. 75 33 + 
11. &lt;10 &lt;10 + 
12. 16 31 + 
13. 51 60 - 
14. 39 59 - 
15. 24 38 - 
16. &lt;10 13 + 
**17. 52 290 + 
18. 18 61 - 
19. 22 59 - 
20. &lt;10 &lt;10 - 
21. 70 35 + 
22. 10 30 - 
23. 95 113 - 
24. &lt;10 &lt;10 - 
25. 60 75 - 
26. 85 104 - 
27. &lt;10 &lt;10 - 
28. 43 60 - 
29. 32 45 - 
30. 39 68 - 
31. 44 15 - 
32. 38 49 - 
33. &lt;10 24 - 
34. &lt;10 &lt;10 - 
35. &lt;10 34 - 
36. 28 &lt;10 - 
37. &lt;10 &lt;10 + 
38. 70 55 - 
39. 42 68 - 
40. 58 56 - 
41. 38 &lt;10 + 
42. &lt;10 &lt;10 - 
43. &lt;10 19 - 
______________________________________ 
**high value by nonboiling is due to incomplete destruction of antiIF 
antibody.? 
Thus, when not destroyed or inhibited, the antibodies make amounts of IF 
unavailable to bind with sample and radioactive vitamin B.sub.12, which 
unavailability in turn causes an inaccurate, smaller amount of vitamin 
B.sub.12 and radioactive B.sub.12 to be bound by IF which in turn results 
in a lower radioactive count in the IF-containing supernatant. This lower 
count is then interpreted as the presence of additional vitamin B.sub.12 
in the sample, while what was really present was IF-blocking antibodies. 
Therefore, a different composition, capable of destroying substantially 
all IF-blocking antibodies, when such antibodies are present in any 
sample, was devised. 
Reconstitution of the prototype composition of the present invention to 
include a stronger disulfide protein destroying material, DTT, instead of 
BME, results in a composition which is more effective in destroying IF 
blocking antibodies. Using such a DTT modified composition, and retesting 
some of the samples in Table III, resulted in substantially the same 
vitamin B.sub.12 analysis of the samples, with the exception that Sample 
#17 now showed a low vitamin B.sub.12 content of 55 pg/ml. In retrospect, 
it appears that classic boiling techniques apparently destroy both 
endogenous binding protein and IF-blocking antibodies when they are 
present in a sample, but that "no-boil" chemical techniques may release 
vitamin B.sub.12 from endogenous binding protein without destroying or 
inactivating either substantially all endogenous binding protein or 
substantially all IF-blocking antibodies. These problems are now overcome 
by the preferred compositions and no-boil procedures of the present 
invention which release substantially all vitamin B.sub.12 for assay, 
which destroy substantially all endogenous binding protein, which bind or 
inhibit substantially all undestroyed endogenous binding protein, and 
which destroy or inhibit substantially all IF-blocking antibodies. 
SUPPORTING DATA 
With the initial recognition of the problems of no-boil analysis of vitamin 
B.sub.12, including the need for: full release of substantially all 
vitamin B.sub.12 ; and for the first time the destruction or inactivation 
of substantially all endogenous binding protein; then the conception of a 
prototype solution; its reduction to practice followed by the recognition 
of another problem and the identification of that problem as being related 
to the presence of IF-blocking antibodies in some of the samples, and 
therefore the need for the destruction or inactivation of substantially 
all such IF-blocking antibodies which may be in any sample; then the 
compositions and procedures of the preferred embodiments of the present 
invention were reformulated and again reduced to practice, with the result 
being an apparently highly accurate vitamin B.sub.12 and/or folate no-boil 
RID assay. 
Steps and procedures were then taken to check the accuracy and validity of 
the preferred embodiments of the present invention. Referring to Table IV, 
which is similar to Table I, a "non-specific binding" comparison was made 
between the highly accurate reference Corning boil assay and the no-boil 
assay of the present invention. "Non-specific binding" is a test of the 
ability of the samples to bind .sup.57 Co-B.sub.12 following the release 
process of either the Corning reference assay or followng the release 
process of the present invention. Effectively a Corning reference boil or 
the no-boil procedure of the present invention were carried out, except 
that in each instance a blank reagent was substituted for the IF binders. 
The subsequent use of charcoal to separate unbound vitamin B.sub.12 and 
unbound radioactive vitamin B.sub.12 from vitamin B.sub.12 and radioactive 
vitamin B.sub.12 bound to endogenous binding protein gave "non-specific 
binding" defined as: 
##EQU1## 
Now referring to Table IV, it is seen that the no-boil assay of the 
present invention not only agrees with the Corning reference assay for: 
Normal vitamin B.sub.12 subjects; and deficient (Low) vitamin B.sub.12 
patients, without antibodies (Antibody Negative); but also for subjects 
known to be suffering from chronic myelogenous leukemia (CML), a disease 
which usually produces subjects with both extremely high vitamin B.sub.12 
levels and extremely high levels of endogenous binding protein. More 
specifically, the non-specific binding of the present invention actually 
appears to be superior to the Corning reference analysis, as indicated by 
a comparison of the mean for each group. 
The "Unsaturated B.sub.12 -Binding Ability" in Table IV, indicates the 
amount of endogenous binding protein in the initial samples which is not 
bound to endogenous vitamin B.sub.12. Such unbound endogenous binding 
protein may interfere with the accuracy of an assay which does not destroy 
or inhibit substantially all endogenous binding protein. 
TABLE IV 
______________________________________ 
Comparison of Non-Specific Binding Using the Reference Boil 
Assay and the No-Boil Assay of the Present Invention 
Non-Specific Binding 
Subject Unsaturated No-Boil Assay 
or B.sub.12 -Binding 
Corning Reference 
of the Present 
Patient Ability Boil Assay Invention 
(#) (pg/ml) (%) (%) 
______________________________________ 
Normal 
1 470 2.3 1.3 
4 360 2.4 1.2 
6 170 2.6 1.4 
12 100 2.4 1.0 
20 710 3.0 1.1 
21 520 2.2 1.1 
30 340 1.7 1.0 
36 230 1.8 1.0 
39 580 1.3 1.2 
40 920 2.5 1.4 
41 2,400 3.2 1.3 
43 560 2.8 1.2 
45 920 3.1 1.3 
49 160 1.7 0.9 
Mean 603 2.4 1.2 
Low B.sub.12, 
Antibody 
Negative 
1 870 2.2 0.6 
2 2,600 2.1 1.4 
3 1,300 2.2 1.2 
4 2,300 1.9 0.8 
5 1,400 1.7 0.8 
6 850 2.6 1.2 
7 1,800 1.7 0.9 
8 1,400 1.7 1.2 
9 1,100 1.9 1.2 
10 1,200 1.5 0.9 
11 900 1.5 1.0 
12 1,100 3.3 1.4 
Mean 1,402 2.0 1.0 
Chronic 
Myelo- 
genous 
Leukemia 
1 2,400 3.9 1.7 
2 3,200 4.5 1.7 
3 4,500 6.3 2.1 
4 1,200 3.9 1.5 
5 3,900 6.0 2.3 
6 17,000 12.4 7.4 
7 2,000 9.1 6.5 
8 1,800 5.3 2.1 
9 14,000 3.1 2.7 
10 3,500 6.7 2.7 
11 15,000 4.0 2.5 
12 860 2.2 1.2 
13 15,000 4.2 2.4 
14 7,900 -0.3 0.4 
15 15,000 4.4 3.4 
16 7,100 0.0 0.8 
17 7,300 0.6 2.2 
18 7,400 -2.1 0.4 
Mean 7,170 4.1 2.4 
______________________________________ 
Table V is a relatively straight forward comparison between a large number 
of normal subjects (50) who should have normal vitamin B.sub.12 levels, 
using both the Corning Reference assay and the no boil assay of the 
preferred embodiment of the present invention. It is similar to Table II, 
but the samples used in Table II and in Table V were not the same. Both 
the scientific and clinical agreement using both assays is excellent for 
each and every sample assayed and for the mean of all the samples. It 
should be noted that Table V also defines a"Normal" vitamin B.sub.12 range 
using the preferred processes and compositions of the present invention. 
This Normal range is defined as "140-704 pg/ml", as compared to "148-696 
pg/ml" for the Reference assay, mathematically and scientifically an 
insignificant difference. 
Table VI sets forth a comparison between known vitamin B.sub.12 deficient 
samples without antibodies, using the Reference assay and the no-boil 
assay of the present invention. Again, both assays produce results which 
are scientifically and clinically in complete agreement. All samples were 
found to be "below the normal range" using both techniques, and the mean 
from both assays was exactly the same. This is a strong confirmation of 
the accuracy of the present invention in its ability to identify vitamin 
B.sub.12 in vitamin B.sub.12 deficient subjects, in the absence of 
IF-blocking antibodies. 
Table VII is of extremely great interest as supporting the present 
invention's accuracy when used to identify subjects with low vitamin 
B.sub.12 who have IF-blocking antibodies in their blood serum. In this 
table, the "Capacity to Block vitamin B.sub.12 binding to IF" is an 
indicator of the amount of IF which could and would be bound by 
IF-blocking antibodies present initially in the serum. This ranges from a 
low of 600 pg/ml, for sample 76, to a high of 360,000 pg/ml for sample 96, 
with a mean of 29,000 pg/ml. Now despite the varied, and often extremely 
high levels of IF-blocking antibodies in these samples, there is complete 
agreement between the reference assay and the assay of the present 
invention in identifying each and every sample, 100% of the samples, as 
being vitamin B.sub.12 deficient. More specifically, the vitamin B.sub.12 
value obtained with the present invention actually appears to be superior 
to the reference assay as indicated by a comparison of the mean for each 
group. 
TABLE V 
______________________________________ 
Comparison of Serum Vitamin B.sub.12 Values 
in 50 Normal Subjects Using the Reference 
Boil Assay and the No-Boil Assay of the Present Invention 
Serum Vitamin B.sub.12 Values 
Normal Corning Reference 
No-Boil Assay of 
Subjects Boil Assay the Present Invention 
(#) (pg/ml) (pg/ml) 
______________________________________ 
1 243 180 
2 322 365 
3 343 267 
4 273 265 
5 350 450 
6 430 410 
7 225 172 
8 270 225 
9 429 320 
10 303 203 
11 730 690 
12 397 375 
13 182 192 
14 234 250 
15 330 330 
16 397 420 
17 455 435 
18 181 186 
19 363 340 
20 360 320 
21 175 130 
22 220 203 
23 540 510 
24 345 343 
25 338 373 
26 303 297 
27 385 345 
28 400 445 
29 360 340 
30 580 550 
31 197 180 
32 795 790 
33 380 380 
34 442 427 
35 465 450 
36 232 250 
37 308 350 
38 250 250 
39 193 245 
40 322 285 
41 385 365 
42 162 200 
43 215 180 
44 352 500 
45 700 740 
46 218 163 
47 377 323 
48 233 250 
49 450 520 
50 150 161 
Mean 346 339 
Normal Range.sup.a 
148-696 140-704 
______________________________________ 
.sup.a Mean .+-. 2 standard deviations after log normalization to correct 
for skewness of the data to higher values.? 
TABLE VI 
______________________________________ 
Comparison of Serum Vitamin B.sub.12 Values in 12 Patients Without 
Anti-IF Blocking Anitbodies Using the Reference Boil Assay 
and the No-Boil Assay of the Present Invention 
Serum Vitamin B.sub.12 Values 
Low B.sub.12, No-Boil Assay 
Antibody Negative 
Corning Reference 
of the Present 
Patients Boil Assay Invention 
(#) (pg/ml) (pg/ml) 
______________________________________ 
1 18 65 
2 128 120 
3 108 25 
4 120 100 
5 5 38 
6 92 99 
7 77 89 
8 78 103 
9 107 103 
10 117 117 
11 80 104 
12 88 98 
Mean 85 85 
Number of patients below 
(12) (12) 
the normal range 
% of patients below 
(100%) (100%) 
the normal range 
______________________________________ 
TABLE VII 
__________________________________________________________________________ 
Comparison of Serum Vitamin B.sub.12 Values in 111 Patients with 
Anti-IF Blocking Antibodies Using the Reference Boil Assay 
and the No-Boil Assay of the Present Invention 
Serum Vitamin B.sub.12 Values 
Low B.sub.12, No Boil Assay 
Antibody Positive 
Capacity to Block 
Corning Reference 
of the Present 
Patients B.sub.12 Binding to IF 
Boil Assay 
Invention 
(#) (pg/ml) (pg/ml) (pg/ml) 
__________________________________________________________________________ 
1 200,000 0 0 
2 17,000 0 24 
3 21,000 110 120 
4 18,000 0 20 
5 24,000 0 0 
6 1,300 0 5 
7 2,300 70 120 
8 21,000 0 16 
9 1,000 39 75 
10 4,900 0 19 
11 6,400 0 26 
12 6,400 0 60 
13 15,000 30 60 
14 23,000 0 0 
15 25,000 15 25 
16 3,600 83 90 
17 4,200 47 40 
18 15,000 0 19 
19 1,200 100 28 
20 2,400 13 0 
21 1,200 86 24 
22 1,200 15 0 
23 22,000 106 84 
24 1,400 80 24 
25 1,500 117 31 
26 1,100 5 5 
27 8,700 99 78 
28 700 69 5 
29 6,400 5 0 
30 25,000 33 0 
31 5,100 25 34 
32 26,000 91 62 
33 25,000 38 18 
34 5,800 72 60 
35 2,500 29 19 
36 6,400 5 0 
37 4,700 76 56 
38 3,600 44 42 
39 2,300 28 18 
40 7,700 0 0 
41 23,000 5 5 
42 9,000 41 18 
43 18,000 24 19 
44 27,000 29 0 
45 3,900 122 68 
46 1,800 142 135 
47 4,300 5 0 
48 2,400 125 110 
49 18,000 35 22 
50 4,200 38 0 
51 42,000 22 10 
52 4,000 18 0 
53 50,000 36 0 
54 25,000 33 0 
55 4,400 29 20 
56 21,000 28 11 
57 5,500 100 118 
58 33,000 23 0 
59 4,200 31 61 
60 3,700 54 39 
61 140,000 25 7 
62 4,100 31 26 
63 210,000 70 86 
64 5,500 90 33 
65 20,000 26 5 
66 3,600 47 48 
67 67,000 33 18 
68 4,400 51 37 
69 4,100 74 47 
70 170,000 33 40 
71 10,000 0 5 
72 12,000 25 20 
73 12,000 40 28 
74 200,000 55 36 
75 1,400 10 13 
76 600 30 20 
77 10,000 79 40 
78 19,000 123 94 
79 1,500 10 5 
80 2,300 27 27 
81 8,700 41 44 
82 2,900 46 40 
83 260,000 39 10 
84 800 78 48 
85 7,700 110 15 
86 2,900 96 32 
87 800 45 29 
88 180,000 125 102 
89 2,700 10 15 
90 6,400 16 14 
91 21,000 0 5 
92 4,400 115 57 
93 2,200 94 51 
94 8,700 92 48 
95 320,000 5 0 
96 360,000 23 13 
97 1,000 68 0 
98 17,000 24 5 
99 1,400 106 31 
100 3,000 36 0 
101 22,000 5 0 
102 2,900 36 42 
103 2,900 39 21 
104 2,400 35 0 
105 900 130 93 
106 20,000 33 19 
107 900 25 13 
108 2,700 46 0 
109 2,900 57 19 
110 54,000 70 40 
111 140,000 41 37 
Mean 29,000 45 31 
Number of patients (111) (111) 
below normal range 
% of patients below (100%) (100%) 
normal range 
__________________________________________________________________________ 
It is therefore clear that using the preferred embodiments of the present 
invention, the no-boil assay of the present invention is not rendered 
ineffective or inoperative as to any sample by the presence of IF blocking 
antibodies. 
Now referring to Table VIII, a substantial number of samples from CML 
patients were tested using both the reference assay and the assay of the 
present invention. As there is some indication, which will be detailed 
below, that some no-boil assays may be sample concentration sensitive, 
both "Standard" sample concentrations and "Diluted" sample concentrations 
were run for both assays. Reviewing the information set forth in Table 
VIII, it is again seen that the assay of the present invention is 
completely in agreement with the reference assay. In the single instance, 
Patient #12, in which a CML patient was determined to be in a normal 
vitamin B.sub.12 range by the reference assay, the present no-boil assay 
also identified that, and only that, subject as being normal. 
As noted in the prior art discussion, there are currently two known 
"no-boil" assays now being marketed, one from each of RIA and Diagnostic 
Products. Table IX sets forth comparisons of the Non-Specific Binding 
Character of each of these commercial no-boil assays, with the "Corning 
Reference Boil Assay" for a number of "Normal" vitamin B.sub.12 subjects, 
a number of known vitamin B.sub.12 deficient subjects without antibodies 
(Low B.sub.12, antibody negative) and a number of CML subjects. The 
results set forth in Table IX indicate that the no-boil RIA and Diagnostic 
Products tests are completely accurate in destroying endogenous binding 
protein, as measured by non-specific binding in samples from normal and 
deficient subjects without IF-blocking antibodies. However, both of those 
commercial kits failed to destroy, or inhibit or block endogenous binding 
protein in samples from CML patients, as measured by non-specific binding, 
and as compared to both the procedure of the Reference assay and the 
procedure of the present invention as set forth in Table IV for the same 
CML subjects. Therefore, the no-boil procedure of the present invention is 
more accurate, at least as to CML patients, than existing commercial 
no-boil kits, when all are compared to the Corning boil assay as a 
reference. 
TABLE VIII 
______________________________________ 
Comparison of Serum Vitamin B.sub.12 Values in 29 Patients with 
Chronic Myelogenous Leukemia Using the Reference Boil Assay 
and the No-Boil Assay of the Present Invention 
Serum Vitamin B.sub.12 Values 
No-Boil Assay 
Corning Reference 
of the Present 
Boil Assay Invention 
Chronic Myelogenous 
Volume Assayed 
Volume Assayed 
Leukemia Patients 
Standard Diluted Standard 
Diluted 
(#) (pg/ml) (pg/ml) (pg/ml) 
(pg/ml) 
______________________________________ 
1 &gt;2,000 4,100 &gt;2,000 4,200 
2 &gt;2,000 3,500 &gt;2,000 2,700 
3 &gt;2,000 2,300 1,900 1,700 
4 &gt;2,000 3,300 &gt;2,000 2,600 
5 &gt;2,000 4,400 &gt;2,000 4,600 
6 &gt;2,000 7,500 &gt;2,000 8,600 
7 &gt;2,000 14,000 &gt;2,000 17,000 
8 &gt;2,000 4,700 &gt;2,000 4,800 
9 &gt;2,000 6,300 &gt;2,000 7,000 
10 &gt;2,000 5,000 &gt;2,000 6,100 
11 &gt;2,000 6,300 &gt;2,000 6,600 
12 460 500 410 450 
13 &gt;2,000 6,800 &gt;2,000 7,600 
14 &gt;2,000 6,300 &gt;2,000 7,400 
15 &gt;2,000 5,800 &gt;2,000 5,700 
16 &gt;2,000 6,600 &gt;2,000 7,500 
17 &gt; 2,000 8,500 &gt;2,000 10,000 
18 &gt;2,000 4,900 &gt;2,000 5,900 
19 &gt;2,000 13,000 &gt;2,000 15,000 
20 &gt;2,000 4,700 &gt;2,000 4,900 
21 &gt;2,000 16,000 &gt;2,000 18,000 
22 &gt;2,000 7,000 &gt;2,000 8,000 
23 &gt;2,000 15,000 &gt;2,000 17,000 
24 &gt;2,000 13,000 &gt;2,000 16,000 
25 &gt;2,000 7,000 &gt;2,000 8,400 
26 1,700 1,300 1,800 1,600 
27 1,900 1,600 1,900 1,600 
28 &gt;2,000 4,200 &gt;2,000 4,700 
29 &gt;2,000 3,700 &gt;2,000 4,400 
Number of patients 
28 28 28 28 
above the 
normal range 
% of patients above 
(97%) (97%) (97%) (97%) 
the normal range 
______________________________________ 
TABLE IX 
__________________________________________________________________________ 
Comparison of Non-Specific Binding Using the Reference Boil Assay and 
the 
No-Boil Assays of RIA Products, Inc. and Diagnostic Products, Inc. in 
both their Standard Forms and as Modified to Make them Approach 
the No-Boil Assay of the Present Invention 
Non-Specific Binding 
Corning 
RIA Products 
Diagnostic Products 
Subject Unsaturated 
Reference 
No-Boil No-Boil 
or B.sub.12 -Binding 
Boil ComboStat Kit 
Dualcount Kit 
Patient Ability 
Assay Standard 
Modified 
Standard 
Modified 
(#) (pg/ml) 
(%) (%) (%) (%) (%) 
__________________________________________________________________________ 
Normal 
1 470 2.3 1.5 - 0.8 
4 360 2.4 0.8 - 0.8 
6 170 2.6 1.0 -1.1 
12 100 2.4 1.6 -1.2 
20 710 3.0 1.9 -0.5 
21 520 2.2 0.8 -1.1 
30 340 1.7 0.8 -1.4 
36 230 1.8 0.5 -0.9 
39 580 1.3 0.5 -1.0 
40 920 2.5 1.6 -0.8 
41 2,400 3.2 4.2 0.8 
43 560 2.8 1.2 -0.2 
45 920 3.1 2.2 -0.5 
49 160 1.7 0.3 -1.6 
Mean 603 2.4 1.3 -0.8 
Low B.sub.12, Antibody Negative 
1 870 2.2 0.6 1.5 
2 2,600 2.1 0.7 0.1 
3 1,300 2.2 0.6 0.9 
4 2,300 1.9 0.5 -0.7 -5 
1,400 1.7 0.4 -0.5 
6 850 2.6 1.0 0.7 
7 1,800 1.7 0.4 -0.8 
8 1,400 1.7 0.4 -0.4 
9 1,100 1.9 0.6 -0.6 
10 1,200 1.5 0.0 0.4 
11 900 1.5 0.3 -0.6 
12 1,100 3.3 1.5 0.1 
Mean 1,402 2.0 0.6 0.0 
Chronic Myelogenous Leukemia 
1 2,400 3.9 9.7 1.1 0.6 1.6 
2 3,200 4.5 9.3 0.9 3.6 2.0 
3 4,500 6.3 13.3 1.7 3.5 2.0 
4 1,200 3.9 5.9 0.7 1.0 1.4 
5 3,900 6.0 18.5 2.2 4.4 2.3 
6 17,000 12.4 37.0 7.8 16.3 7.2 
7 2,000 9.1 26.2 5.6 6.4 5.3 
8 1,800 5.3 11.1 1.4 0.6 0.9 
9 14,000 3.1 16.3 6.2 18.1 6.1 
10 3,500 6.7 18.0 2.2 0.9 1.2 
11 15,000 4.0 14.8 5.9 17.4 5.6 
12 860 2.2 1.3 -0.1 -0.7 0.0 
13 15,000 4.2 46.6 6.4 18.6 6.6 
14 7,900 -0.3 37.7 4.6 12.2 3.6 
16 7,100 0.0 11.3 4.7 11.2 3.6 
17 7,300 0.6 14.0 4.5 11.1 4.6 
18 7,400 -2.1 32.7 3.0 8.9 1.9 
Mean 7,170 4.1 19.0 3.5 7.4 3.1 
__________________________________________________________________________ 
Table IX also indicates that when the assay material of the kits was 
"Modified" in the manner indicated in Table XV, to bring the commercial 
kits into closer agreement with the compositions and procedures of the 
present invention, then the results for these modified kits came into 
closer agreement with the Reference assay. However, neither the need for 
the modifications nor the modifications themselves would have been 
appreciated or made without the recognition of the problems and the 
solutions of the present invention. 
As shown in Table X, the RIA Products and Diagnostic Products kits were 
also compared with the Corning Reference assay for the same 50 normal 
subjects tested by the no-boil assay of the present invention in Table V. 
It will be seen that both of these commercial kits were in acceptable 
agreement with the Corning Reference assay in the values obtained for all 
of the normal subjects and in the normal ranges indicated by these values. 
However, when the RIA Products and Diagnostic Products Kits were used to 
test a number of patients who were vitamin B.sub.12 deficient, without 
IF-blocking antibodies, as set forth in Table XI, the RIA Products 
procedure failed to identify two-thirds of the subjects as being vitamin 
B.sub.12 deficient, while the Diagnostics Products kit identified 100% of 
the vitamin B.sub.12 deficient subjects Table XI should be compared with 
Table VI, in which the same tests were reported for the no-boil assay of 
the present invention, the no-boil assay of the present invention giving 
100% accuracy in assay results. As discussed below, the failure of the RIA 
Products kit to correctly identify many vitamin B.sub.12 deficient 
patients without IF-blocking antibodies is believed to be due to the high 
amount of DTT used in its procedure which subsequently interferes with the 
ability of IF to bind vitamin B.sub.12 in the assay itself, such 
interference being greater with prepared serum samples than in prepared 
non-serum samples containing known amounts of vitamin B.sub.12 which are 
used to construct a standard curve. 
Now at this time, as set forth in Table XII, samples which were known to be 
vitamin B.sub.12 deficient, and to have IF-blocking antibodies were 
assayed using both the RIA Products and Diagnostic Products kits and 
compared with the Corning Reference assay. Table XII should be compared 
with Table VII which set forth similar tests using the assay of the 
present invention. A smaller number of samples are used in Table XII than 
in Table VII due to the fact that many of the samples used in Table VII 
were depleted and therefore unavailable for further testing. 
TABLE X 
______________________________________ 
Comparison of Serum Vitamin B.sub.12 Values in 50 Normal Subjects 
Using the Reference Boil Assay and No-Boil Assays of 
RIA Products, Inc. and Diagnostic Products, Inc. 
Serum Vitamin B.sub.12 Values 
Corning RIA Products 
Diagnostic Products 
Normal Reference No-Boil No-Boil 
Subjects Boil Assay 
ComboStat Dualcount Kit 
(#) (pg/ml) Kit (pg/ml) 
(pg/ml) 
______________________________________ 
1 243 203 347 
2 322 357 406 
3 343 603 750 
4 273 243 275 
5 350 445 412 
6 430 420 493 
7 225 181 175 
8 270 340 283 
9 429 463 420 
10 303 235 283 
11 730 750 725 
12 397 333 315 
13 182 247 220 
14 234 310 310 
15 330 400 426 
16 397 450 470 
17 455 455 520 
18 181 204 177 
19 363 410 426 
20 360 400 467 
21 175 220 234 
22 220 303 285 
23 540 645 587 
24 345 377 446 
25 338 480 460 
26 303 417 340 
27 385 240 460 
28 400 525 480 
29 360 545 450 
30 580 660 690 
31 197 290 287 
32 795 860 860 
33 380 527 555 
34 442 395 575 
35 465 480 570 
36 232 350 283 
37 308 410 450 
38 250 397 420 
39 193 350 357 
40 322 366 553 
41 385 565 565 
42 162 194 347 
43 215 360 365 
44 352 645 585 
45 700 800 1,060 
46 218 240 375 
47 377 345 545 
48 233 312 310 
49 450 780 535 
50 150 200 177 
Mean 346 415 442 
Normal Range.sup.a 
148-696 172-853 187-901 
______________________________________ 
.sup.a Mean .+-. 2 standard deviations after log normalization to correct 
for skewness of the data to higher values.? 
TABLE XI 
______________________________________ 
Comparison of Serum Vitamin B.sub.12 Values in 12 Patients 
Without Anti-IF Blocking Antibodies Using 
the Reference Boil Assay and the No-Boil Assays of RIA 
Products, Inc. and Diagnostic Products, Inc. 
Serum Vitamin B.sub.12 Values 
Low B.sub.12, RIA Products 
Antibody Corning No Boil Diagnostic Products 
Negative Reference ComboStat No-Boil 
Patients Boil Assay 
Kit Dualcount Kit 
(#) (pg/ml) (pg/ml) (pg/ml) 
______________________________________ 
1 18 110 36 
2 128 224 145 
3 108 175 54 
4 120 235 140 
5 5 117 29 
6 92 224 107 
7 77 177 61 
8 78 170 119 
9 107 183 128 
10 117 240 175 
11 80 155 138 
12 88 185 146 
Mean 85 183 106 
Number of (12) (4) (12) 
patients below 
the normal 
range 
% of patients 
(100%) (33%) (100%) 
below the 
normal range 
______________________________________ 
TABLE XII 
______________________________________ 
Comparison of Serum Vitamin B.sub.12 Values in 12 Patients 
with Anti-IF Blocking Antibodies Using 
the Reference Boil Assay and 
the No-Boil Assays of RIA 
Products, Inc. and Diagnostic Products, Inc. 
Serum Vitamin B.sub.12 Values 
Low B.sub.12, RIA Products 
No-Boil Assay 
Antibody Corning No-Boil Diagnostic Products 
Positive Reference ComboStat No-Boil 
Patients Boil Assay 
Kit Dualcount Kit 
(#) (pg/ml) (pg/ml) (pg/ml) 
______________________________________ 
3 110 245 195 
13 30 140 96 
15 15 180 38 
16 83 470 168 
18 0 150 31 
32 91 250 136 
34 72 125 113 
45 122 185 155 
46 142 205 191 
52 18 107 43 
59 31 110 58 
64 90 295 57 
83 39 200 17 
Mean 67 222 107 
Number of (12) (5) (10) 
patients below 
the normal 
range 
% of patients 
(100%) (42%) (83%) 
below the 
normal range 
______________________________________ 
However, even in this abbreviated comparison of 12 vitamin B.sub.12 
deficient subjects with IF-blocking antibodies, the RIA Products kit 
identified less than 50% of the deficient subjects as being deficient and 
the Diagnostic Products kit identified only 10 to 12 (83%) as being 
vitamin B.sub.12 deficient. That the RIA Products kit is particularly 
inaccurate with some vitamin B.sub.12 deficient patient samples with 
IF-blocking antibodies is supported by the value of 470 pg/ml as set forth 
for patient #16 in Table XII, since this value is higher than the mean 
value of 415 pg/ml that was obtained for the 50 normal subjects using the 
RIA Products kit as set forth in Table X. Both of these commercial kits 
provide results which are not only inaccurate, but also totally clinically 
unacceptable as to patient diagnosis and treatment. By comparison, 
referring to Table VII, the no-boil assay of the present invention is 
clinically acceptable as to patient diagnosis and treatment, in that it 
identified 100% of the vitamin B.sub.12 deficient subjects. The fact that 
Table VII tested 111, instead of only 12 IF-blocking antibody positive 
subjects, makes the proven accuracy and precision of the no-boil assay of 
the present invention even more evident. 
As in Table VIII using the present invention, subjects with CML are 
reported in Tables XIII and XIV, using the RIA Products and Diagnostic 
Products kits. Again, Tables XIII and XIV are somewhat abbreviated due to 
the depletion and unavailability of some samples. The central columns in 
Tables XIII and XIV, marked above and below with an * set forth the 
results which would be obtained using the commercial kits as they are 
intended to be used. It is noted that when so used the RIA Products kit 
identified only 6 of 17 samples as being high in vitamin B.sub.12, and 
that the Diagnostic Products kit identified only 10 of 17 samples as being 
high in vitamin B.sub.12. Therefore, these commercial tests would be 
unreliable in identifying subjects with high vitamin B.sub.12, as a tool 
in diagnosing CML. By comparison, as set forth in Table VIII, the no-boil 
assay of the present invention identifies 100% of the CML patients with 
high vitamin B.sub.12 levels as being high. 
TABLE XIII 
__________________________________________________________________________ 
Comparison of Serum Vitamin B.sub.12 Values in 18 Patients with 
Chronic Myelogenous Leukemia Using the Reference Boil Assay, and 
the No-Boil Assay of RIA Products, Inc. in both Standard Form and as 
Modified to Make it Approach the No-Boil Assay of the Present Invention 
Serum Vitamin B.sub.12 Values 
RIA Products 
Chronic 
Corning Reference 
* No-Boil ComboStat Kit 
Myelogenous 
Boil Assay 
Standard Assay 
Modified Assay 
Leukemia 
Volume Assayed 
Volume Assayed 
Volume Assayed 
Patients 
Standard 
Diluted 
Standard 
Diluted 
Standard 
Diluted 
(#) (pg/ml) 
(pg/ml) 
(pg/ml) 
(pg/ml) 
(pg/ml) 
(pg/ml) 
__________________________________________________________________________ 
1 &gt;2,000 
4,100 
1,000 
4,300 
&gt;2,000 
4,500 
2 &gt;2,000 
3,500 
1,000 
2,700 
&gt;2,000 
3,200 
3 &gt;2,000 
2,300 
720 2,100 
1,700 
1,700 
4 &gt;2,000 
3,300 
1,400 
3,100 
&gt;2,000 
3,500 
5 &gt;2,000 
4,400 
890 4,200 
&gt;2,000 
5,300 
6 &gt;2,000 
7,500 
300 7,000 
1,700 
8,600 
7 &gt;2,000 
14,000 
580 15,000 
&gt;2,000 
18,000 
8 &gt;2,000 
4,700 
1,400 
4,300 
&gt;2,000 
4,400 
9 &gt;2,000 
6,300 
290 6,000 
&gt;2,000 
7,000 
10 &gt;2,000 
5,000 
990 5,800 
&gt;2,000 
6,700 
11 &gt;2,000 
6,300 
310 6,600 
&gt;2,000 
7,300 
12 460 500 550 580 500 570 
13 &gt;2,000 
6,800 
320 6,000 
2,000 
7,300 
14 &gt;2,000 
6,300 
460 6,600 
&gt;2,000 
7,500 
15 &gt;2,000 
5,800 
310 5,000 
&gt;2,000 
6,200 
16 &gt;2,000 
6,600 
500 7,200 
&gt;2,000 
7,500 
17 &gt;2,000 
8,500 
510 8,600 
&gt;2,000 
9,800 
18 &gt;2,000 
4,900 
540 5,100 
&gt;2,000 
5,800 
Number of 
(17) (17) (6) (17) (17) (17) 
patients above 
the normal 
range 
% of patients 
(94%) 
(94%) 
(33%) 
(94%) 
(94%) 
(94%) 
above the 
* 
normal range 
__________________________________________________________________________ 
TABLE XIV 
__________________________________________________________________________ 
Comparison of Serum Vitamin B.sub.12 Values in 18 Patients with 
Chronic Myelogenous Leukemia Using the Reference Boil Assay, and 
the No-Boil Assay of Diagnostic Products in both Standard Form and as 
Modified to make it Approach the No-Boil Assay of the Present Invention 
Serum Vitamin B.sub.12 Values 
Diagnostic Products 
Chronic 
Corning Reference 
* No-Boil Dualcount Kit 
Myelogenous 
Boil Assay 
Standard Assay 
Modified Assay 
Leukemia 
Volume Assayed 
Volume Assayed 
Volume Assayed 
Patients 
Standard 
Diluted 
Standard 
Diluted 
Standard 
Diluted 
(#) (pg/ml) 
(pg/ml) 
(pg/ml) 
(pg/ml) 
(pg/ml) 
(pg/ml) 
__________________________________________________________________________ 
1 &gt;2,000 
4,100 
1,700 
4,700 
&gt;2,000 
4,400 
2 &gt;2,000 
3,500 
1,400 
3,000 
&gt;2,000 
2,400 
3 &gt;2,000 
2,300 
890 1,900 
1,800 
1,700 
4 &gt;2,000 
3,300 
1,700 
2,800 
&gt;2,000 
2,100 
5 &gt;2,000 
4,400 
1,200 
4,400 
&gt;2,000 
4,700 
6 &gt;2,000 
7,500 
500 6,500 
&gt;2,000 
8,200 
7 &gt;2,000 
14,000 
1,400 
17,000 
&gt;2,000 
21,000 
8 &gt;2,000 
4,700 
1,800 
5,200 
&gt;2,000 
4,100 
9 &gt;2,000 
6,300 
680 6,600 
&gt;2,000 
7,300 
10 &gt;2,000 
5,000 
1,700 
5,900 
&gt;2,000 
6,700 
11 &gt;2,000 
6,300 
640 5,900 
&gt;2,000 
7,500 
12 460 500 480 510 520 400 
13 &gt;2,000 
6,800 
600 6,600 
&gt;2,000 
7,400 
14 &gt;2,000 
6,300 
990 6,900 
&gt;2,000 
8,600 
15 &gt;2,000 
5,800 
640 5,500 
&gt;2,000 
6,100 
16 &gt;2,000 
6,600 
900 7,000 
&gt;2,000 
8,400 
17 &gt;2,000 
8,500 
990 9,900 
&gt;2,000 
11,000 
18 &gt;2,000 
4,900 
1,100 
5,400 
&gt;2,000 
5,400 
Number of 
(17) (17) (10) (17) (17) (17) 
patients above 
the normal 
range 
% of patients 
(94%) 
(94%) 
(56%) 
(94%) 
(94%) 
(94%) 
above the 
normal range 
__________________________________________________________________________ 
Again, referring to Tables XIII and XIV, when the RIA Products kit and 
Diagnostic Products Kit were each varied, first by dilution, and then by 
modifications as set forth in Table XV, and then by both modification and 
dilution, the kits both gave completely accurate results in identifying 
the CML patients with high vitamin B.sub.12. However, in the absence of 
the teaching of the present invention, one would not know to modify the 
RIA Products or Diagnostic Products kits in this manner. 
Reference is now made to Table XV. This table sets forth the assay 
preparation conditions for preferred embodiments of the present invention 
as well as the standard assay preparation conditions for the RIA Products 
no-boil kit and the Diagnostic Products no-boil kit. It also sets forth 
data indicative of the types of modifications which were made in each of 
these kits as discussed in Tables IX, XIII and XIV. Table XV also allows 
ease of comparison between the preferred embodiments of the present 
invention and the standard embodiments of the RIA Products and Diagnostic 
Products kits. It is immediately apparent that each of the no-boil tests 
utilizes the same intial volume of serum sample. The initial volume of 
sample is not critical, and is a matter of choice, but of course, all 
other procedures and ingredients must be adjusted to accomodate the volume 
of sample utilized. It is next noted that the standard RIA Products and 
Diagnostic Products procedures do not utilize a vitamin B.sub.12 analogue 
in the assay preparation, while the preferred embodiments of the present 
invention utilize cobinamide analogue. It is next noted that each of the 
procedures utilizes the strong base NaOH, but that the concentration of 
the base in the preferred embodiments of the present invention is about 
22% to about 65% greater than the concentration of base in the RIA 
Products kit and the Diagnostic Products kit, respectively. It is 
theorized that the concentration of the base is an important factor, in 
combination with the other ingredients and their concentrations, in 
providing the most complete release of endogenous vitamin B.sub.12 from 
endogenous binding protein, and in the initial inactivation or destruction 
of both endogenous binding protein and IF-blocking antibodies. One of the 
several theories of the present invention is that no-boil assay 
preparation does not substantially completely destroy or inhibit 
endogenous binding protein which is released from sample endogenous 
vitamin B.sub.12 or which is otherwise present in the serum sample, thus 
allowing the undestroyed or uninhibited endogenous binding protein to 
recombine with the sample endogenous vitamin B.sub.12 and/or the added 
radioactive vitamin B.sub.12, either of which reactions will result in 
erroneous sample vitamin B.sub.12 readings. 
TABLE XV 
__________________________________________________________________________ 
Conditions of No-Boil Assays in Standard Form and Those Modified in 
Accord with the Present Invention 
Incubation 
Time During 
Conditions during Extraction Extraction 
Serum (Min) 
Sample 
100 ng Dilution 
.mu.l after 
After 
After 
Volume, .mu.l 
Cobinamide 
NaOH 
DTT of Serum 
Dilution 
DTT NaOH 
__________________________________________________________________________ 
No-Boil Assay of the 
200 + 0.33M 
0.043M 
0.67 300 0 20 
Present Invention 
RIA Products No-Boil 
ComboStat Kit 
Standard 200 - 0.20M.sup.b 
.sup. 0.052M.sup.c 
.sup. 0.40.sup.d 
500 15 5 
Modified.sup.a 
200 + 0.25M 
0.065M 
0.50 400 0 20 
Diagnostic Products 
No-Boil Dualcount Kit 
Standard 200 - 0.27.sup.b 
.sup. 0.017M.sup.d 
.sup. 0.27.sup.d 
750 30 15 
Modified.sup.a 
200 + 0.44 
0.029M 
0.44 450 0 15 
__________________________________________________________________________ 
.sup.a Modifications were made with the individual kit reagents to 
approach as closely as possible the method of the present invention, 
subject to the constraints imposed by the manufacturers' 
.sup.b Based on measurements obtained by Titration with 
.sup.c Based on information obtained from RIA Products, 
.sup.d Calculated from information supplied in the package labels. 
044515710440x v 
Therefore, the addition of an analogue which is capable of binding 
substantially all endogenous binding protein, whether released from 
vitamin B.sub.12 or otherwise present in the sample, was theorized and 
provided a serum assay preparation process and composition which would 
provide more accurate assays. Cobinamide is the vitamin B.sub.12 analogue 
of choice due to its ease of availability and low cost. However, any other 
vitamin B.sub.12 analogue which is capable of binding with endogenous 
binding protein, and which will not inhibit or interfere with the 
intrinsic factor utilized as the binder in the assay is of substantial 
importance in the procedures and compositions of the present invention. 
Each of the procedures utilizes potassium cyanide, not shown in Table XV. 
The presence of cyanide in the preparation for assay has long been a 
standard procedure in order to convert the vitamin B.sub.12 being tested 
to its most stable form, cyanocobalamin. As has previously been noted, 
Ithakissios, et. al. has indicated that the combination of a strong base 
with cyanide provides excellent vitamin B.sub.12 release. The present 
invention has verified that the combination of cyanide and a strong base 
by themselves do not provide optimum vitamin B.sub.12 release, as set 
forth in Table I, for which all of the procedures used cyanide, and as set 
forth in Tables IX, XIII and XIV for the RIA Products and Diagnostic 
Products kits, both of which use a strong base and cyanide. It has thereby 
been verified that this combination is apparently not as useful in 
releasing substantially all vitamin B.sub.12 as has been reported. 
Each of the procedures utilize DTT in the extraction procedure. It is first 
noted that both the RIA Products and Diagnostic Products kits are intended 
for the simultaneous determination of both vitamin B.sub.12 and folate, 
and that it is well known in the art to utilize a sulfhydral compound such 
as DTT to stabilize the folate. However, in the present invention, the 
sulfhydral compound is utilized primarily for its known ability as a 
disulfide protein destroying compound, a previously unappreciated or 
unrecognized use. As such, it has been theorized that it has utility in 
both releasing endogeneous vitamin B.sub.12 from endogenous binding 
protein and in substantially destroying both endogenous binding protein 
and IF-blocking antibodies which are, or may be, present in the sample 
undergoing assay. As demonstrated in Table III, and the discussion related 
thereto, the selection of the type and the amount of protein destroying 
compound may be critical in those samples which contain IF-blocking 
antibodies which are resistant to destruction. It is noted that the 
concentration of DTT utilized in the preferred embodiments of the present 
invention lies intermediately between the concentration of DTT utilized in 
the RIA Products kit and the amount utilized the Diagnostic Products kit. 
However, it is further noted that as set forth in the Tables, both the RIA 
Products and the Diagnostic Products kits failed to identify a substantial 
number of low vitamin B.sub.12 samples containing IF-blocking antibodies 
and a substantial number of high vitamin B.sub.12 CML samples. It is 
further noted that the RIA Products kit, which contains the greatest 
amount of DTT, gave the worst results for the analysis of vitamin B.sub.12 
in CML patients. It is also further noted that the RIA Products kit failed 
to identify a substantial number of low vitamin B.sub.12 samples without 
IF-blocking antibodies. It is theorized that the high concentration of DTT 
may have provided the additional margin of error in the CML analyses 
and/or in low vitamin B.sub.12 without IF-blocking antibody analyses. 
Therefore, it is difficult at this time, with scientific certainty, to 
indicate the specific concentration of sulfhydral compound which should be 
utilized in the preparation of samples for vitamin B.sub.12 assay. It 
would appear, that the relationship between the sulfhydral compound and 
the amounts and types of other ingredients is of importance in providing 
an optimum no-boil test procedure. It is, of course, noted that the --SH 
moiety of the sulfhydral compounds is what supplies the disulfide protein 
destroying capability, and that a compound such as DTT has twice an many 
--SH moieties as a compound such as BME. Therefore, the selection of the 
sulfhydral compound for use in combination with the other ingredients of 
the present invention should consider both the --SH concentration and 
structure. 
It is also theorized that the cobinamide aids in the release of endogenous 
vitamin B.sub.12 from endogenous binding protein. This is supported by the 
data in Table I which indicate that the greatest amount of vitamin 
B.sub.12 release occurred in those samples which included cobinamide (Cbi) 
in the assay preparation composition. Therefore, the cobinamide has 
utility not only to bind endogenous binding protein in the sample, but 
also to aid in the release of vitamin B.sub.12 in the sample. In 
hindsight, it may be theorized that the analogue acts to displace vitamin 
B.sub.12 from the endogenous binding protein, thus diluting the 
concentration of endogenous binding protein-vitamin B.sub.12 complex in 
the solution and driving the reaction which releases endogenous binding 
protein from vitamin B.sub.12 in the direction of additional release. 
It is also of interest to note that both the RIA Products and the 
Diagnostic Products kits allow substantial incubation time, at room 
temperature, before the strong base is added to the serum undergoing assay 
preparation, while the preferred embodiments of the present invention add 
the strong base simultaneously or with other assay preparation 
ingredients. It is theorized that since it is now recognized that there is 
a need to destroy or inhibit substantially all endogenous binding protein 
in the sample and prevent it from recombining with vitamin B.sub.12 or 
combining with radioactive vitamin B.sub.12, and as a strong base is a 
good protein destroying material, that the simultaneous or early addition 
of the strong base inhibits the recombination of endogenous binding 
protein with vitamin B.sub.12 and inhibits the combining of endogenous 
binding protein with radioactive vitamin B.sub.12 to maintain the amount 
of released vitamin B.sub.12 of both kinds at optimum levels. 
Based on the experimental results achieved by the preferred embodiments of 
the present invention and the comparative results achieved by out original 
prototype procedure and by the RIA Products and Diagnostic Products kits, 
it appears, and is theorized, that the concentration of the solutions (or 
volume) at the time of extraction, the nature and amounts of the 
ingredients, and the various incubation times, may all be of some 
importance in situations where samples containing IF-blocking antibodies 
or CML patient samples are being analyzed. Analysis of the data obtained 
with the RIA Products kit for the vitamin B.sub.12 deficient patient 
samples without IF-blocking antibodies indicates that this may also be 
true at the time of the actual assay. These finding verify the importance 
of using samples from vitamin B.sub.12 deficient patients, both with and 
without IF-blocking antibodies, and from CML patients, in selecting 
individual ingredients, and their combinations and amounts and 
concentrations and various incubation times, in both the preparation and 
assay procedures used in vitamin B.sub.12 determinations. The importance 
of using more than one, and more than just a few patient samples from each 
of the individual patient groups to verify the assay has also been shown 
since the use of only some of the patient samples as set forth in Tables 
II, IX, XI, XII, XIII and XIV would have indicated that our original 
prototype procedure and the current procedures of the RIA Products and 
Diagnostic Products kits were acceptable both scientifically and 
clinically, whereas the use of additional and other patient samples as set 
forth in the same tables clearly establishes that none of these procedures 
is acceptable, either scientifically or clinically. 
Finally, reference to Table XV indicates what modifications were made to 
the standard RIA Products kit and the standard Diagnostic Products kit, in 
terms of the addition of a vitamin B.sub.12 analogue, cobinamide, the 
adjustment of the concentrations of the strong base, NaOH and sulfhydral 
compound (DTT) and the volume and therefore the concentration of the test 
solutions and procedures. Reference to Tables IX, XIII and XIV indicates 
that utilizing these modifications, the commercially available kits now 
provided improved conditions, of the type achieved by the preferred 
embodiments of the present invention. The kits could not be modified with 
complete accuracy or certainty to match the preferred embodiments of the 
present invention, since their initial ingredients presented some 
constraints and since further, their actual contents were not known with 
complete certainty. Nevertheless, the modifications made did improve the 
kits and bring them to clinically acceptable levels. 
The term "clinically acceptable" as utilized herein, is intended to mean 
substantially 100% accuracy. For example, referring again to Table III in 
which Sample #17 was missed, apparently due to the presence of difficultly 
destroyed IF-blocking antibodies, this inaccuracy could have resulted in 
failure to diagnose that patient's vitamin B.sub.12 deficiency. Failure to 
identify and treat that deficiency could have resulted in the development 
of potentially fatal blood and/or neurologic abnormalities. Neurological 
abnormalities, once developed, may be quite serious, as they are not 
always subject to reversal by subsequent treatment with vitamin B.sub.12. 
Referring to Table XVI, a comparison between the standard curves utilized 
for the Corning reference boil assay, the RIA Products no-boil assay, the 
Diagnostic Products no-boil assay, with the no-boil assay of the present 
invention is made. These standard curves are indicative of the number of 
radioactive counts bound to intrinsic factor which in turn correspond with 
the amount of non-radioactive vitamin B.sub.12, in the known samples, in 
pg/ml. Initially, it is noted that the standard curves for the Corning 
reference assay, and the no-boil assay of the present invention are 
substantially indistinguishable from one another. While the standard 
curves for the RIA Products kit and the Diagnostic Products kit do vary 
from the Corning assay, as long as they are reproducible for those kits, 
this variation represents no problem. However, in any of the four assays 
set forth in Table XVI, a false variation, i.e. one not related to the 
amount of vitamin B.sub.12 in the sample to-be-tested, of as little as 10% 
can result in a major variation in the amount of non-radioactive vitamin 
B.sub.12 which is determined to be present in a to-be-tested sample. Now, 
based upon the teaching and analysis of the present invention, it is seen 
that false variations of as much as 46% of bound radioactive vitamin 
B.sub.12 are possible utilizing the RIA Products and Diagnostic Products 
kits, due to their apparent failure to completely release all bound 
vitamin B.sub.12, and/or completely substantially inhibit or destroy all 
endogenous binding protein, and/or completely inhibit or destroy all 
IF-blocking antibodies and/or bind undestroyed or uninhibited endogenous 
binding protein. 
In preferred embodiments of the present invention, after the samples were 
prepared for assay, they were neutralized and allowed to incubate with 
soluble IF, followed by the addition of albumin coated charcoal to adsorb 
the unbound sample vitamin B.sub.12 and radioactive vitamin B.sub.12. In a 
modification of the present invention, after the sample is prepared for 
assay, and neutralized, rather than adding the IF binder to the solution 
as a soluble ingredient, another method of adding IF to the system can be 
utilized, and is actually preferred as giving more reliable assay results. 
The form of IF which was used, was an article consisting of small glass 
beads to which IF is covalently attached. Such IF-glass beads were made 
available on an experimental basis from Corning. When utilizing such 
IF-glass beads, the final steps of the assay procedure vary from the 
procedures previously described. Previously, albumin coated charcoal was 
added to the liquid to adsorb the unbound vitamin B.sub.12, both sample 
and radioactive, and the supernatant liquid, containing sample and 
radioactive vitamin B.sub.12 bound to IF, was subjected to analysis for 
radioactive counts. Now, utilizing IF-glass beads, the sample and 
radioactive B.sub.12 becomes bound to the IF on the glass beads. Because 
the glass beads have a much greater density than the liquid composition to 
which they are added, they are easily centrifuged to the bottom of the 
tube. Then, subsequent removal of the supernatant liquid leaves one with 
the natural and radioactive vitamin B.sub.12 bound to the IF on the glass 
beads. Then, the easily separated and handled glass beads are subjected to 
radioactive count in order to determine the amount of natural vitamin 
B.sub.12 in the sample. For the sake of accuracy, it is noted that all 
data concerning the present invention which are set forth in Tables IV-XVI 
for the preferred embodiments of the present invention were gathered 
utilizing IF-glass beads rather than by the addition of soluble IF to the 
solution followed by the removal of unbound vitamin B.sub.12 utilizing 
charcoal. For at least two reasons relevant to the present invention, it 
is believed that the use of IF-glass beads in the completion of the assay 
provides more accurate assay results than the free IF and charcoal 
technique. 
One reason is that IF-blocking antibodies do not bind as well to IF-glass 
beads as they do to soluble IF. A second reason is that non-specific 
binding to endogenous binding protein is present in the supernatant with 
unbound vitamin B.sub.12 in the IF-glass assay and thus can not lead to a 
falsely low reading of sample vitamin B.sub.12 as can occur with the 
soluble IF and charcoal assay where non-specific binding to endogenous 
binding protein is present in the supernatant fraction together with 
vitamin B.sub.12 bound to IF and thereby can lead to a falsely low reading 
of sample vitamin B.sub.12. 
TABLE XVI 
______________________________________ 
Standard Curves Obtained with the Reference Boil Assay, 
the No-Boil Assay of the Present Invention, and the No-Boil 
Assays of RIA Products, Inc. and Diagnostic Products, Inc. 
% Binding of [.sup.57 Co]Vitamin B.sub.12 Observed 
No-Boil 
Assay Diagnostic 
Non- Corning of-the RIA Products 
Products 
radioactive 
Reference Present No-Boil No-Boil 
Vitamin B.sub.12 
Boil Assay 
Invention 
ComboStat 
Dualcount 
Added (pg) 
(%) (%) Kit (%) (%) 
______________________________________ 
0 (100) (100) (100) (100) 
50 -- -- 94 88 
100 86 83 -- 79 
150 -- -- 81 -- 
250 78 76 -- -- 
300 -- -- -- 57 
400 -- -- 52 -- 
500 61 61 -- -- 
600 -- -- -- 37 
1,000 45 45 26 -- 
1,200 -- -- -- 22 
2,000 29 30 14 -- 
2,400 -- -- -- 11 
______________________________________ 
Of course, IF bound to solid non-interferring material other than glass for 
use in completion of the assay would have similar improved properties in 
carrying out the assay. 
As shown with regard to a modification of the preferred embodiments and in 
the discussion of Table I and Table II, much of the initial work for the 
present invention was carried out in a manner which allowed for the 
simultaneous analysis of folate along with the analysis for vitamin 
B.sub.12. It is more specifically noted that the no-boil release procedure 
and composition of the present invention are also useful for the no-boil 
release of folate for testing in serum. As noted in Table XVII, the 
simultaneously RID assay of both vitamin B.sub.12 and folate is capable of 
utilizing the teaching of the present invention. It would appear that when 
folate alone is being assayed, that the vitamin B.sub.12 analogue may not 
be needed in the procedure and composition, although it can be present if 
this makes the preparation of common reagents for vitamin B.sub.12 and 
folate assays more convenient. 
The data utilized in analyzing and perfecting the present invention is of 
great value in showing the need for proper scientific and clinical 
procedures in determining the accuracy of an assay. If only normals had 
been tested for vitamin B.sub.12, or if only low vitamin B.sub.12 samples 
without IF-blocking antibodies had been tested, or if only a small number 
of samples low in vitamin B.sub.12 and including IF-blocking antibodies 
had been tested, or if no CML patients had been tested or insufficient 
numbers of any of these groups to determine the utility and limits of the 
prototype procedure and composition had been tested, the results could 
have resulted in assay procedures and compositions which were ineffective 
in detecting low vitamin B.sub.12 samples with and/or without difficult to 
destroy IF-blocking antibodies and/or abnormally high vitamin B.sub.12 
samples containing high amounts of endogenous binding protein. This could 
have resulted in faulty assays of the type which were found to exist for 
the RIA Products and Diagnostic Products commercial kits. It is believed, 
that for the first time, the present invention has recognized the fact 
that each sample's IF-blocking antibodies may be unique, and in some 
instances that the IF-blocking antibodies may be sufficiently difficult to 
destroy that they may lead to inaccurate assays in some samples. Only the 
large number of samples ultimately studied in the present invention, as 
set forth in Table V, could have led to the perfection of an analysis 
which is accurate regardless of the presence or absence of IF-blocking 
antibodies, and regardless of the difficulty with which the IF-blocking 
antibodies are destroyed. 
TABLE XVII 
______________________________________ 
Simultaneous Assay of Vitamin B.sub.12 and Folate 
using the Corning Reference Boil Assay and the No-Boil 
Assay of the Present Invention. 
Serum Vitamin B.sub.12 
Serum Folate 
Corning No-Boil Assay 
Corning No-Boil Assay 
Reference of the Present 
Reference 
of the Present 
Serum Boil Assay 
Invention Boil Assay 
Invention 
Sample 
(pg/ml) (pg/ml) (ng/ml) (ng/ml) 
______________________________________ 
A 350 360 4.2 4.0 
B 350 330 6.2 6.4 
C 155 165 6.4 6.6 
D 340 340 8.3 8.9 
E 450 470 15.0 18.5 
F 86 5 10.0 8.5 
G 5 5 6.8 5.5 
H 96 65 37.5 33.5 
I 86 82 6.2 5.1 
J 0 0 10.3 8.0 
______________________________________ 
In conclusion it is seen that the objectives of the present invention have 
been achieved. The present invention discloses and teaches methods for the 
preparation of mammalian blood, tissue and serum for assay of vitamin 
B.sub.12 and of folate, and of both vitamin B.sub.12 and folate without 
the need to heat or boil the samples as a step in their preparation for 
assay. In achieving these objects, optimum procedures and compositions for 
the release of vitamin B.sub.12 and folate from a to-be-assayed sample 
were determined. Additionally, the recognition of problems in vitamin 
B.sub.12 RID no-heat methods caused by the presence of endogenous binding 
protein, and procedures and compositions for substantially destroying or 
inactivating such endogenous binding protein were developed. Also, 
procedures and compositions for binding any undestroyed or uninhibited 
endogenous binding protein, utilizing endogenous binding protein binding 
vitamin B.sub.12 analogues were developed. Finally, the recognition of the 
problem that there are antibodies naturally present in some samples which 
react with or block intrinsic factor protein was made, and procedures and 
compositions for substantially inhibiting or destroying such IF-blocking 
antibodies were made. The procedures and compositions are convenient to 
use, require less steps than the boiling assay techniques and are 
time-saving. Additionally, as detailed throughout the present application 
the preferred embodiments of the present invention are highly accurate, 
and in some instances actually superior to the Reference boil assay. 
Certain preferred compositions have been detailed in the present 
application, and other combinations and concentrations have been taught. 
It is noted that the basic combination of ingredients, that is a 
combination of at least two protein destroying compositions, with or 
without a vitamin B.sub.12 analogue to bind the endogenous protein, 
utilized in the various procedures of the present invention are useful in 
obtaining the results desired. In preferred embodiments, the amount of 
endogenous binding protein binding vitamin B.sub.12 analogue should be 
sufficient to completely bind the greatest amount of endogenous binding 
protein capability found in any sample, as for example, CML sample #6 in 
Tables IV and IX. Of course, to allow for margins of error, ten to one 
hundred times or more vitamin B.sub.12 analogue could be utilized with the 
teaching of the present invention. In a similar manner, the greatest 
amount of, and the most resistant, IF-blocking antibodies determined 
should be provided for in the compositions and the procedures of the 
present invention. 
As used herein, the term "heating" is intended to mean the action of 
actively adding energy to a substance to increase its temperature, but 
does not include changes in temperature due to contact with an ambient 
environment. "Boiling" and "boil" are used herein in their ordinary sense, 
so long as they are caused by the active addition of energy. "Without 
heating", is the absence of the active addition of energy to a substance. 
"No-boil" and "without boiling" mean that a substance has not been 
actively caused to boil. While the compositions and procedures of the 
present invention are specifically novel in that they require no heating 
or boiling in the preparation of samples for vitamin B.sub.12 and/or 
folate assay, they may be used for heating or boiling and still remain 
within the teaching of the present invention. 
While it is noted that certain preferred embodiments have been set forth, 
the non-preferred embodiments also have utility in those situations in 
which there is neither IF-blocking antibodies or substantial amounts of 
endogenous binding protein. It is also noted that the compositions of the 
present invention are readily amenable to being placed into convenient kit 
form and provided to users for use in accordance with the procedures of 
the present invention. 
While the invention has been particularly shown and described with 
reference to preferred embodiments thereof, it will be understood by those 
skilled in the art, that the foregoing and other modifications or changes 
in form and details may be made therein without departing from the spirit 
and scope of the invention.