Assay for bone alkaline phosphatase

Monoclonal antibodies highly specific for human bone alkaline phosphatase, especially in the presence of human liver alkaline phosphatase, and their use in assays for human bone alkaline phosphatase are disclosed. A kit using the antibodies in an assay for human bone alkaline phosphatase is also disclosed.

BACKGROUND OF THE INVENTION 
The present invention is method for detecting the presence and 
concentration of bone alkaline phosphatase (BAP) in human body fluids. 
Elevated BAP levels in serum is symptomatic for serious disorders such as 
bone metastasis from carcinomas, such as those of the breast and prostate, 
and Paget's disease (osteitis deformous). 
Paget's disease is a serious disease affecting primarily those over the age 
of 40. The disease is slowly progressive, first involving bone resorption 
followed by a distorted pattern of new growth in bones such as the pelvis, 
femur, skull, tibia, vertebrae, clavicle and humerus. The disease exhibits 
gross symptoms such as bowing of the tibia or femur, enlargement of the 
skull, shortened stature, and severe aching in the affected bones. Neural 
effects may include deafness or spinal cord compression accompanied by 
paresis or paraplegia. 
Until now the only reliable diagnosis for Paget's disease has been by x-ray 
examination. Routine assays for serum BAP are confined to measuring total 
alkaline phosphatase concentration. Several related forms of alkaline 
phosphatase exist in the serum, specifically those of the intestinal, 
placental and the hepatic/renal/skeletal groups. These three broad groups 
C an usually be separated using the time-consuming procedure of 
electro-phoretic assay, but until now assays for distinguishing between 
the clinically important markers of bone and liver alkaline phosphatases 
(LAP) were at best ambiguous and time-consuming. The present invention is 
directed to a convenient highly specific sandwich immunoassay method for 
determining the presence or concentration of BAP and especially so in the 
presence of LAP. 
SUMMARY OF THE INVENTION

DETAILED DESCRIPTION 
The present invention is directed to a sandwich assay highly specific for 
BAP in the presence of LAP and other human alkaline phosphatase 
isoenzymes. (The concept of a sandwich assay in general is described in 
David et al., U.S. Pat. Nos. 4,376,110 and 4,486,530, issued Mar. 8, 1983 
and Dec. 4, 1984, respectively, herein incorporated by reference.) The 
invention also encompasses monoclonal antibodies highly specific for BAP, 
especially so in the presence of LAP. 
Specifically, one embodiment of the invention is a "forward" assay that 
entails a process for the determination of the presence of or 
concentration of BAP in a fluid comprising the steps of: 
(a) contacting a sample of the fluid with a first monoclonal antibody for 
BAP, wherein the first monoclonal antibody is bound to a solid carrier 
insoluble in the fluid in order to form an insoluble complex between the 
first monoclonal antibody and the BAP; 
(b) separating the fluid sample containing unreacted BAP from the insoluble 
complex of the first monoclonal antibody and BAP; 
(c) reacting a measured amount of a second, monoclonal antibody to BAP 
which is labelled and which antibody is soluble in the fluid with the 
insoluble complex of the first monoclonal antibody and BAP, in order to 
form an insoluble complex of the first monoclonal antibody, BAP, and 
second, labeled antibody; 
(d) separating the solid carrier from unreacted second, antibody; 
(e) measuring either the amount of second, antibody associated with the 
solid carrier or the amount of unreacted second, antibody; 
(f) relating the amount of second antibody measured with the amount of 
labelled antibody measured for a control sample prepared in accordance 
with steps (a) through (e), said control sample known to be free of BAP, 
to determine the presence of BAP in said fluid sample, of relating the 
amount of labelled antibody measured in said fluid sample with the amount 
of labelled antibody measured for samples containing known amounts of BAP 
prepared in accordance with steps (a) through (e) to determine the 
concentration of BAP in the fluid sample; 
wherein both monoclonal antibodies used are highly specific for BAP, 
especially in the presence of LAP. 
Another embodiment of the invention is a "reverse" sandwich assay for BAP 
that entails a process for the determination of the presence of or 
concentration of BAP in a fluid comprising the steps: 
(a) contacting a sample of the fluid with a measured amount of the first 
monoclonal antibody for BAP, wherein the first monoclonal antibody is 
labelled, in order to form a soluble complex between the first monoclonal 
antibody and BAP: 
(b) contacting the sample (which now contains the soluble complex) with a 
second monoclonal antibody to BAP, which second antibody is bound to a 
solid carrier insoluble in the fluid, with the soluble complex of the 
first monoclonal antibody and BAP, in order to form an insoluble complex 
of the first monoclonal antibody, BAP, and second, antibody; 
(c) separating the solid carrier from unreacted first, antibody; 
(d) measuring either the amount of first, antibody associated with the 
solid carrier or the amount of unreacted first, labelled antibody; 
(e) relating the amount of first antibody measured with the amount of 
labelled antibody measured for a control sample prepared in accordance 
with steps (a) through (d), said control sample being known to be free of 
BAP, to determine the presence of BAP in said fluid sample, or relating 
the amount of labelled antibody measured for the fluid sample with the 
amount of labelled antibody measured for samples containing known amounts 
of BAP prepared in accordance with steps (a) through (d) to determine the 
concentration of BAP in the fluid sample; 
wherein both monoclonal antibodies used are highly specific for BAP, 
especially in the presence of LAP. 
Yet another aspect of the present invention involves a "simultaneous" assay 
for BAP that entails a process for the determination of the presence of 
concentration of BAP enzyme in a fluid comprising the steps: 
(a) simultaneously contacting a sample of the fluid with a first and second 
monoclonal antibodies for BAP, wherein the first monoclonal antibody is 
bound to a solid carrier insoluble in the fluid and the second monoclonal 
antibody is labelled and provided in a measured amount, in order to form 
an insoluble complex between the first monoclonal antibody and BAP; 
(b) separating the solid carrier from the fluid sample unreacted second, 
antibody; 
(c) measuring either the amount of second, antibody associated with the 
solid carrier or the amount of unreacted second, antibodies; 
(d) relating the amount of labelled antibody measured with the amount of 
labelled antibody measured for a control sample prepared in steps (a) 
through (c), wherein the control sample is known to be free of BAP, to 
determine the presence of BAP in said sample fluid, or relating the amount 
of labelled antibody measured for the fluid sample with the amount of 
labelled antibody measured for samples containing known amounts of BAP 
prepared in accordance with steps (a) through (c),to determine the 
concentration of BAP in the fluid sample; 
wherein the monoclonal antibodies are both highly specific for BAP, 
especially in the presence of LAP. 
A preferred embodiment of the above forward, reverse or simultaneous assays 
has the first antibody the product of a cell line different than that of 
second monoclonal antibody. Another preferred embodiment of the above 
forward, reverse or simultaneous assays occurs when the first and second 
monoclonal antibodies are a product of the same cell line. Further 
preferred embodiments within the above two preferred embodiments described 
immediately above include assays wherein the labelled antibody is labelled 
with a radioisotope, an enzyme, biotin, avidin, a chromogenic substance, 
or a fluorogenic substance. A preferred embodiment of the particular 
labelling preferred embodiment entails having the monoclonal antibodies 
chosen from the group consisting of BA1F 419, BA1G 017, BA1G 121, BA1G 
151, and BA1G 339. Especially preferred embodiments occur when the BA1F 
419, BA1G 017, BA1G 121, BA1G 151, or BA1G 339 monoclonal antibody is 
labelled with the radioactive isotope .sup.125 I, or when the antibody is 
labelled with an enzyme other than alkaline phosphatase (e.g., 
beta-galactosidase, horse radish peroxidase, etc.), or when the antibody 
is labelled with biotin, and wherein the amount of labelled antibody is 
measured by adding a measured amount of streptavidinconjugated enzyme 
label where the enzyme is other than an alkaline phosphatase. 
Other preferred embodiments include the above forward, reverse or 
simultaneous assays, wherein the unlabelled antibody is bound directly or 
indirectly to a plastic bead to a porous membrane, and especially so when 
the unlabelled antibody is bound to microparticles, which microparticles 
are in turn bound to a porous membrane. Similarly, preferred embodiments 
of the above forward, reverse or simultaneous assays and wherein the 
labelled antibody is labelled with a radioactive isotope, an enzyme, 
biotin, avidin, chromogenic substance, or a fluorogenic substance includes 
embodiments wherein the unlabelled monoclonal antibody is bound directly 
or indirectly to a porous membrane, and especially so when the unlabeled 
monoclonal antibody is bound to microparticles, which microparticles are 
in turn bound to a porous membrane. 
When discussing the present invention, various terms have specific 
connotations. Thus, the term "fluid" means human serum, plasma, whole 
blood, urine or tumor ascites. The term "solid carrier" means common 
supports used in immunometric assays made from natural and synthetic 
material. The support required is insoluble in water and can be rigid or 
non-rigid. Among such supports are filter paper, filtering devices (e.g., 
glass membranes), plastic beads (such as polystyrene beads), test tubes or 
(multiple) test wells made from polyethylene, polystyrene, polypropylene, 
nylon, nitrocellulose, and glass microfibres. Also useful are particulate 
materials such as agarose, cross-linked dextran and other polysaccharides. 
It will be understood to one skilled in the art that for the present assay 
the capture antibody can first bind to the BAP antigen then bind to the 
carrier through, for instance, an anti-mouse IgG antibody, an 
avidin-biotin system, or the like. 
Preferred embodiments of the invention have the capture antibody being 
bound to a porous membrane. By porous membrane we mean flexible or rigid 
matrix made from any of a variety of filtration or chromatographic 
materials including glass fibres and micro-fibres and natural or synthetic 
materials. Fluids should be able to flow into and pass easily through the 
membrane. The membrane should also preferably have pores of at least 0.1 
.mu. and preferably at least 1.0 .mu.. The porous membrane can be used by 
itself or as part of a more elaborate device. Such devices includes the 
ICON.RTM. and like devices described in Valkirs et el., U.S. Pat. Nos. 
4,632,901 and 4,727,019, issued Dec. 20, 1986 and Feb. 23, 1988, 
respectively, herein incorporated by reference. Another such device is the 
TEST-PAK.RTM. device of Abbott Laboratories (North Chicago, Ill.), 
described in European Patent Application No. 217,403, published Apr. 8, 
1987. Still other devices containing the present porous membrane include 
the device of Bauer et al., U.S. Pat. No. 3,811,840, issued May 21, 1974, 
Cole et al., U.S. Pat. No. 4,407,943, issued Oct. 4, 1983, Cole et al., 
U.S. Pat. No. 4,246,339, issued Jan. 20, 1981, Geigel et al., U.S. Pat. 
No. 4,517,288, issued May 14, 1985, F. S. Intengan, U.S. Pat. No. 
4,440,301. issued Apr. 3, 1984, M. E. Jolly, U.S. Pat. No. 4,704,255, 
issued Nov. 3, 1987, Tom et al., U.S. Pat. No. 4,366.241, issued Dec. 28, 
1982, or Weng et al., U.S. Pat. No. 4,740,468, issued Apr. 26, 1988, all 
of which are herein incorporated by reference. 
The capture monoclonal antibody can be directly or indirectly bound to the 
membrane. The direct binding can be a covalent or non-covalent one by 
methods well known in the art (for example, the use of glutaraldehyde and 
aminosilanes). See, for example, "Immobilized Enzymes", Ichiro Chibata, 
Halstead Press, New York (1978), Cuatrecasas, J. Bio. Chem. 245: 3059 
(1970), and March et al., Anal. Biochem. 60, p149 et seq. (1974). The 
non-covalent binding takes advantage of the natural adhesion to the 
non-synthetic and especially the synthetic fibers by antibodies. Thus, 
appropriately buffered solutions can be mixed with the membrane then 
evaporated leaving a coating of the desired antibody on the membrane. 
The non-direct method for applying the antibody to the membrane employs 
microparticles which are bound to the membrane, or both matrix of 
membrane, on the surface of the membrane, or to other particles which are 
in turn bound to the membrane. The particles can be any shape but 
preferably spherical. The size of the particles should be such that they 
do not migrate through the membrane to any significant degree. The size of 
the particles may vary, but in general they should be slightly larger than 
the minnimum pore size of the membrane and smaller than the maximum pore 
size, or in the alternative, should be larger than the maximum pore size. 
(Thus, the particles can be bound with the matrix of membrane, on the 
surface of the membrane, or to other particles which are in turn bound to 
the membrane) The particles can be made up of a variety of naturally 
occurring or synthetic materials. Exemplary of such particles are those 
made from polyethylene, polyacrylates, polyacrytamide, or naturally 
occurring materials such as cross-linked polysaccharides like agarose, 
dextran, cellulose, starch and the like. The primary requirement is that 
materials do not contribute a signal, usually light absorption, that would 
cause the zone in which the particles were located to have a different 
signal than the rest of the membrane. 
The antibody can be covalently or non-covalently bound to the particle. The 
binding to the particle uses methods similar to those discussed above for 
binding the antibody directly to the membrane. 
The particles are usually applied to the membrane in an area smaller than 
the surface area of the part of the membrane that it is applied to. 
Several methods known in the art can be employed. One such method employs 
various mechanical means (or directly) to apply a suspension, frequently 
aqueous ("latex") to the membrane. 
The methods and use of microparticle for the instant invention are further 
discussed in Weng et el., U.S. Pat. No. 4,740,468, issued Apr. 26, 1988 
(see especially columns 13, 14 and 15), Brown et al., European Patent 
Application No. 217,403, published Apr. 8, 1987, and A. S. Rubenstein, 
European Patent Application No. 200,381, published Nov. 5, 1986. 
The separation steps for the various assay formats (e.g. forward, 
simultaneous, and reverse) can be performed by methods known in the art. 
Where indicated, a simple washing with buffer followed by filtration or 
aspiration is sufficient. After washing, it is sometimes appropriate, as 
with particulate supports to centrifuge the support, to aspirate the 
washing liquid, add wash liquid again and aspirate. For membrane and 
filters, additional washing with buffer may often be sufficient, 
preferably drawing the liquid through the membrane or filter by applying 
vacuum to the opposite side of the membrane or filter or contacting the 
opposite side of the filter or membrane with a liquid absorbing member 
that draws the liquid through, for instance, by a capillary action. 
Moderate temperatures are normally employed for carrying out the assay. 
Constant temperatures during the period of the measurement are generally 
required only if the assay is performed without comparison with a control 
sample. The temperatures for the determination will generally range from 
about 10.degree. C.-50.degree. C., more usually from abut 
15.degree.-45.degree. C. 
The concentration of BAP which may be asseyed will generally vary from 
about 10.sup.-4 to 10.sup.-10 M, more usually from about 10.sup.-5 to 10 
.sup.-8 M. Considerations such as whether the assay is qualitative, 
semi-quantitative or quantitative, the particular detection device and the 
concentration of BAP will normally determine the concentration of other 
reagents. 
The term "labelled antibody" indicates the unique anti-BAP, highly 
non-cross-reactive monoclonal antibodies of the present invention that are 
labelled by conventional methods to form all or part of a signal 
generating system. Thus, the present monoclonal antibodies can be 
covalently bound to radioisotopes such as tritium, carbon 14, phosphorous 
32, iodine 125 and iodine 131 by methods well known in the art. For 
example, .sup.125 I can be introduced by procedures such as the 
chloramine-T procedure, enzymatically by lactoperoxidase procedure or the 
by the are labelled Bolton-Hunter technique. These techniques plus others 
are discussed in H. Uan Vunakis and J. J. Langone, Editors, Methods in 
Enzymolgy, Vol. 70, Part A, 1980. See also U.S. Pat. No. 3,646,346, issued 
Feb. 29, 1972, and Edwards et al., U.S. Pat. No. 4,062,733, issued Dec. 
13, 1977, respectively, both of which are herein incorporated by 
reference, for further examples of radioactive labels. 
Chromogenic labels are those compounds that absorb light in the visible 
ultraviolet wavelengths. Such compounds are usually dyestuffs and include 
quinoline dyes, triarylmethane dyes, phthaleins, insect dyes, azo dyes, 
anthraquimoid dyes, cyanine dyes, and phenazoxonium dyes. 
Fluorogenic compounds include those which emit light in the ultraviolet or 
visible wavelength subsequent to irradiation by light. The fluorogens can 
be employed by themselves or with quencher molecules. The primary 
fluorogens are those of the rhodamine, fluorescein and umbelliferone 
families. The method of conjugation and use for these and other fluorogens 
can be found in the art. See, for example, J. J. Langone, H. Van Vunakis 
et al., Methods in Enxymology, Vol. 74, Part C, 1981, especially at page 3 
through 105. For a representative listing of other suitable fluorogens, 
see Tom et al., U.S. Pat. No. 4,366,241, issued Dec. 28, 1982, especially 
at column 28 and 29. For further examples, see also U.S. Pat. No. 
3,996,345, herein incorporated by reference. 
These non-enzymatic signal systems are adequate for the present invention. 
However, those skilled in the art will recognize that enzyme-catalyzed 
signal system is in general more sensitive than a non-enzymatic system. 
Thus, for the instant invention, catalytic labels are the more sensitive 
non-radioactive labels. 
Catalytic labels include known in the art and include single and dual 
("channelled") enzymes such as alkaline phosphatase, horseradish 
peroxidase, luciferase, .beta.-galactosidase, glucose oxidase, (lysozyme, 
malate dehydrogenase, glucose-6-phosphate dehydrogenase,) and the like. 
Examples of dual ("channeled") catalytic systems include alkaline 
phosphatase and glucose oxidase using glucose-6-phosphate as the initial 
substrate. A second example of such a dual cataivtic system is illustrated 
by the oxidation of glucose to hydrogen peroxide by glucose oxidase, which 
hydrogen peroxide would react with a leuco dye to produce a signal 
generator. (A further discussion of catalytic systems can be found in Tom 
et al., U.S. Pat. No. 4,366,241, issued Dec. 28, 1982, herein incorporated 
by reference. (See especially columns 27 through 40.) Also, see Weng et 
al., U.S. Pat. No. 4,740,468, issued Apr. 26, 1988, herein incorporated by 
reference, especially at columns 2 and columns 6, 7, and 8. 
The procedures for coupling enzymes to the antibodies are well known in the 
art. Reagents used for this procedure include glutaraldelyde, p-toluene 
diisocyanate, various carbodiimide reagents, p-benzoquinone m-periodate, 
N, N.sup.1 -o-Phenylenedimaleimide and the like (see, for example, J. H. 
Kennedy et al., Clin. Chim Acta 70, 1 (1976)).As another aspect of the 
invention, any of the above devices and formats may be provided in a kit 
in packaged combination with predetermined amounts of reagents for use in 
assaying BAP. Where an enzyme is the label, the reagents will include an 
antibody that is highly specific for BAP as described above and will also 
be conjugated to the appropriate enzyme, substrate for the enzyme or 
precursors therefor including any additional substrates, enzymes, and 
cofactors and any reaction product to provide the detectable chromophore 
or fluorophore. The relative amount of the various reagents may be varied 
widely, to provide for concentrations in solution of the reagents which 
substantially optimize the sensitivity and specificty of the assay. The 
reagents can be provided as dry powders, usually lyophilized, including 
excipients, which on dissolution will provide for a reagent solution 
having the appropriate concentrations for performing the assay. 
Chemiluminescent labels are also applicable. See, for example, the labels 
listed in C. L. Maier, U.S. Pat. No. 4,104,029, issued Aug. 1, 1978, 
herein incorporated by reference. 
The substrates for the catalytic systems include simple chromogens and 
fluorogens such as para-nitrophenyl phosphate (PNPP), .beta.-D-glucose 
(plus possibly a suitable redox dye), homovanillic acid, o-dianisidine, 
bromocresol purple powder, 4-alkyl-umbelliferone, luminol, 
para-dimethylaminoiophine, paramethoxylophine, AMPPD, and the like. 
Depending on the nature of the label and catalytic signal producing system, 
one would observe the signal, by irradiating with light and observing the 
level of fluorescence: providing for a catalyst system to produce a dye, 
fluorescence, or chemiluminescence, where the dye could be observed 
visually or in a spectrophotometer and the fluorescence could be observed 
visually or in a fluorometer; or in the case of chemiluminescence or a 
radioactive label, by employing a radiation counter. Where the appropriate 
equipment is not available, it will normally be desirable to have a 
chromophore produced which results in a visible color. Where sophisticated 
equipment is involved, any of the techniques is applicable. 
The monoclonal antibodies useful in the present invention are obtained by 
the process discussed by Milsrein and Kohler and reported in Nature 256, 
495-497, 1975. The details of this process are well known and will not be 
repeated here. However, basically it involves injecting a mouse with an 
immunogen. (In the current case, the immunogen used is a source rich in 
BAP, such as a human osteosarcoma cell.) The mouse is subsequently 
sacrificed and cells taken from its spleen are fused with myeloma cells. 
The result is a hybrid cell, referred to as a "hybridoma," that reproduces 
in vitro. The population of hybridomas is screened and manipulated so as 
to isolate individual clones each of which secretes a single antibody 
species to the antigen. Each individual antibody species obtained in this 
way is the product of a single B cell from the immune animal generated in 
response to a specific antigenic site recognized on the immunogenic 
substance. 
When an immunogenic substance is introduced into a living host, the host's 
immune system responds by producing antibodies to all the recognizable 
sites on the substance. This "shotgun" approach to producing antibodies to 
combat the invader results in the production of antibodies of differing 
affinities and specificities for the immunogenic substance. Accordingly, 
after the different hybridoma cell lines are screened to identify those 
that produce antibody to BAP, the antibodies produced by the individual 
hybridoma cell lines are preferably screened to identify those having the 
highest affinity for BAP stimulating their original production before 
selection for use in the present invention. Selection based on this 
criterion is believed to help provide the increased sensitivity in the 
immunometric assays of the present invention using monoclonal antibody 
compared to the polyclonal antibody used in the prior art which, at best, 
has an affinity for the antigen which is roughly the average of the 
affinities of all antibodies produced by the immune system. Preferably, 
the monoclonal antibody selected will have an affinity compatible with the 
desired sensitivity and range for the test system under consideration. 
Preferably the antibody will have an affinity of at least about 10.sup.8 
liters/mole and, more preferably, an affinity of at Least about 10.sup.9 
liters/mole. 
Specifically, as an initial screen, the hybridomas from the initial fusions 
were screened using a "one-site" assay. In the assay, microtiter plates 
were coated with goat anti-mouse IgG. Culture supernatant from the initial 
hybridomas are added to the microtiter plates, incubated, and then washed 
to bind the anti-BAP monoclonal antibody indirectly to a solid support. 
Cross reactivity of the antibodies was checked by adding crude detergent 
extract of BAP (from SAOS-2 cells) or a crude butanol extract of LAP (from 
human liver samples), washing the solid support, then measuring the 
activity of the bound BAP or LAP by measuring its effect on PNPP substrate 
(para-nitrophenyl phosphate). Those antibodies that bound at least 2.0 
times more BAP than LAP (as measured by either enzymes activity on PNPP) 
were grown and expanded. The hybridomas were subsequently given a 
definitive RIA screen for BAP vs LAP cross-reactivity. The RIA used 
.sup.125 I labelled BAP or LAP in a competitive assay format. A sample of 
hybridoma supernatant and measured amounts of labelled and unlabelled BAP 
or LAP were combined then incubated with a solid support coupled to sheep 
anti-mouse IgG. The solid support was washed and was placed in a gamma 
counter. Varying proportions of labelled or cold BAP or LAP were used to 
perform a saturation analysis for each antibody. Antibodies that were less 
than 20% crossreactive for LAP in the presence of BAP (as determined by 
the concentration of unlabeled antigen required to inhibit 50% of the 
binding of the labelled antigen) were selected for use in the two-site (or 
"sandwich") immunometric assay. It is monoclonal antibodies that are less 
than 20% crossreactive with LAP in the presence of BAP that are referred 
to in this disclosure as "highly specific for BAP, especially in the 
presence of LAP". 
As foreshadowed above, yet another aspect of the present invention are 
monoclonal antibodies that are highly specific for BAP, especially in the 
presence of LAP. Preferred embodiments of this aspect of the invention 
include antibodies BA1F 419, BA1G 017, BA1G 121, BA1G 151, and BA1G 339. 
The hybridomas that produce antibodies BA1B 067, BA1F 419, BA1G 017, BA1G 
121, BA1G 151, and BA1G 339, which are referred to by the designation of 
the antibody they produce, were deposited with the American Type Culture 
Collection (Rockville, Md.) as follows: 
______________________________________ 
Hybridoma Deposit Date Accession Number 
______________________________________ 
BA1B 067 January 26, 1989 
HB 10004 
BA1F 419 January 26, 1989 
HB 10005 
BA1G 017 January 26, 1989 
HB 10002 
BA1G 121 January 26, 1989 
HB 10007 
BA1G 151 January 26, 1989 
HB 10003 
BA1G 339 January 26, 1989 
HB 10006 
______________________________________ 
As stated earlier in this discussion, for the two site assay of the instant 
invention to selectively detect BAP in the presence of LAP, the capture 
antibody and the labelled antibody both must be highly non crossreactive 
with LAP in the presence of BAP. This requirement is graphically 
demonstrated by FIGS. 1 through 9. FIGS. 1 and 2 show the specificity of 
antibody BA1G 121 for BAP over LAP. The sequential saturation analyses of 
these figures employed labelled BAP antigen followed by addition of cold 
(crude) BAP antigen (for FIG. 1) or LAP antigen (FIG. 2). The analyses 
demonstrated that the 50% inhibition concentration of cold antigen to 
differ greatly for BAP and LAP for BA1G 121, thus the antibody was 
specific for BAP epitopes and highly non-crossreactive with LAP epitopes. 
A similar set of experiments with BA1B 067 (FIGS. 3 and 4) shows the 50% 
inhibition level in the competitive RIA saturation analyses for BAP and 
LAP to be approximately the same. Thus, BA1B 067 does not have the 
required high specificty for BAP over LAP. Those skilled in the art would 
have predicted that a sandwich or two site assay using one monoclonal 
antibody highly specific for BAP and non-crossreactive with LAP and 
another monoclonal antibody specific for alkaline phosphatases in general 
but cross-reactive for BAP and LAP would give a highly specific assay for 
BAP over LAP. For instance, the highly BAP-specific monoclonal antibody 
could be the capture antibody bound to the solid support. Thus, the sample 
containing the BAP analyte (and LAP also) could be incubated and the 
unbound BAP, LAP and other antigens in the test solution be removed in a 
wash step. The (labelled) antibody that was non specific for BAP could 
then be incubated with the complex-containing solid support, followed by 
washing and the addition of chromogen, fluorogen or chemuluminescent agent 
if necessary. The capture antibody highly specific for BAP should, under 
normal circumstances, enable the assay to distinguish BAP over LAP. A 
sandwich assay such as this was performed, using the highly specific BA1F 
419 antibody as the capture antibody and the cross-reactive BA1B 067 
antibody as the labelled antibody (the saturation analysis data for BA1F 
419 is given in Example 1). The results of the assays with crude BAP and 
crude LAP are shown in FIGS. 5 and 6. The dose response curves, set forth 
in FIG. 5, show no significant difference in the detection of LAP and BAP 
in in vitro conditions. If the same highly specific capture antibody is 
used but substituting a highly specific (labelled) antibody BA1G 017 the 
assay responds in a markedly different manner, as graphically set forth in 
FIG. 6. The assay essentially does not respond to a difference in the 
concentration of LAP, while there is a positive dose response curve for a 
change in BAP concentration. (The slopes of other highly specific 
antibodies used in conjunction with the capture antibody BA1F 419 are also 
set forth below in conjunction with Example 4.) 
The requirement for both the capture and the labelled antibody to be highly 
specific for BAP especially in the presence of LAP, exists not only in 
sandwich assays for partially purified human samples but with human 
patient samples obtained in the normal ways for diagnostic purposes. 
FIG. 7 charts the results of a sandwich assay again employing highly 
specific monoclonal antibody BA1F 419 as the capture antibody and the 
crossreactive monoclonal antibody BA1B 067 as the labelled antibody. 
Little difference is seen in dose response curves in human sera taken from 
patients whose sera will have elevated levels of LAP (i.e., patients with 
various forms of liver disease) and those patients whose sera will have 
elevated levels of BAP (i.e., patients with Paget's Disease or healthy 
juvenile ("JUV-1") patients). In contrast, FIGS. 8 and 9 set forth the 
results of the sandwich assay using the same highly non-crossreactive 
capture antibody (BA1F 419) but this time using two highly BAP-specific 
antibodies (BA1G 339 and BA1G 121, whose RIA data is presented below in 
Example 2) as labelled antibodies. The difference in dose response curves 
for these two assays (as depicted in FIGS. 8 and 9) over the assay 
depicted in FIG. 7 is dramatic. The dose response curves in FIGS. 8 and 9 
show that the assays described in the graphs have a positive response to 
increasing concentrations of BAP-containing sere (Paget's disease patients 
and normal juvenile (i.e., "JUV-1", "JUV-2", and "JUV-3") patients) versus 
the flat response to increasing concentrations of sera containing elevated 
levels of LAP and normal levels BAP (i.e. patients with liver disease). 
Yet another aspect of the present invention is a kit for detecting the 
presence or concentration of BAP, comprising a monoclonal antibody for BAP 
which is bound or can be bound to a solid carrier, a labelled monoclonal 
antibody, and a signal generating substance if required, wherein both 
monoclonal antibodies are highly specific for BAP, especially in the 
presence of LAP. Thus, the antibodies and assay formats discussed above 
and exemplified below can be supplied as a kit. Once again, the solid 
carrier can either be bound to the capture antibody at the time the 
reaction between the capture antibody and the BAP takes place, or the 
solid carrier can be coated with a substance that binds the capture 
antibody (e.g. sheep antimouse antibody) such that the solid carrier binds 
to the capture antibody after the antibody binds to the BAP antigen. 
The kit can additionally contain substrate for the enzyme or the requisite 
precursors for the substrate, including any additional substrates, 
enzymes, and cofacters and any reaction partner of the enzymic product 
required to provide the detectable chromophore or fluorophore. In 
addition, other additives such as ancillary reagents may be included, for 
example, stabilizers, buffers, and the like. The relative amounts of the 
various reagents may vary widely, to provide for concentrations in 
solution of the reagents which substantially optimize the sensitivity and 
specificty of the assay. The reagents can be provided as dry powders, 
usually lyophillized, including excepients, which on dissolution will 
provide for a reagent solution having the appropriate concentrations for 
performing the assay. 
Preferred embodiments of the kit use combinations of the antibodies BA1F 
419, BA1G 017, BA1G 121, BA1G 151, or BA1G 339 as either the capture or 
the labelled antibody, or alternatively where the kit uses one of these 
antibodies as both the capture and the labelled antibody. 
Experimental 
The abbreviations used in the following Procedures and Examples have the 
same meaning as is commonly used in the art. Thus, for example. "MEM" 
stands for modified Eagle's medium, "NP-40" stands for Nonidet P-40, "PBS" 
stands for phosphate-buffered saline, "PNPP" stands for 
para-nitrophenyiphosphate, "BSA" stands for bovine serum albumin, and the 
like. The following Procedures and Examples are set forth of specific 
instances of the general teaching set forth above on how to make and how 
to use the present invention. 
Procedure 1 
Purification of Human Bone Alkaline Phosphatase 
BAP was extracted from the SAOS-2 human esteosarcoma cell line (ATCC#HTB 
85) which were grown in complete MEM supplemented with 8% horse serum and 
2% fetal calf serum. The cells were scraped from the culture flask and 
washed twice in PBS by centrifugation followed by incubation in an 
extraction buffer containing 1% NP-40 in a 0.1 M Tris-HCl buffer, pH 8.0. 
The cells were extracted for one hour at 25.degree. C. with gentle 
stirring followed by centrifugation at 10,000 g for 15 min to remove the 
cellular debris. 
The BAP preparations were purified by running the supernatants through an 
anti-alkaline phosphatase immunoaffinity column consisting of purified 
anti-alkaline phosohatase monoclonal antibodies (BA1B 067, an antibody 
that is specific for both BAP and LAP, and is obtained according to 
Procedure 3 below) coupled to activated agarose beads Affigel-10 Bio-Rad 
Laboratories, Richmond, Calif., according to the packate insert. The 
column was washed with extraction buffer followed by elution with buffer 
containing 125 mM KCl and 10 mM lysine at pH 11. Fractions were collected 
and assayed for alkaline phosphatase activity. Each fraction was assayed 
for alkaline phosphatase activity by combining 50 .mu.l of appropriately 
diluted sample (i.e., anywhere between 2 through 200 times, depending on 
the concentration of the sample) with 100 .mu.l of PNPP solution (1 PNPP 
tablet (Sigma) in 3 ml of water) in microtiter plates and measuring the 
rate of PNPP (substrate) turnover an 405 nm in a Vmax microtiter plane 
reader (Molecular Devices, Palo Alto, Calif.) in the kinetic reading mode 
at 25.degree. C. One unit (U) of activity was defined as the quantity of 
enzyme that catalyzes the hydrolysis of 1 .mu.mole of substrate per minute 
under these conditions. One unit of activity was considered to equal 
approximately one microgram of pure BAP (or LAP). Protein-containing 
fractions were also analyzed by SDS polyacrylamide gel elecnrophoresis 
(10-15% gradient reducing gels, Pharmacia Phast gel system, Pharmacia, 
Uppsala, Sweden). The electrophoretic analysis indicated a purity for BAP 
of greater than 95%. After electrophoretic analysis, the 
protein-containing fractions were incubated with sheep anti-mouse IgG 
(North Valley Farms, San Diego, Calif.) coupled to cyanogen 
bromide-activated Sepharose 4B beads (Pharmacia, Upsala, Sweden) to remove 
mouse IgG. The yields of BAP from the crude detergent extract were 
typically approximately 50%. 
Procedure 2 
Purification of Human Liver Alkaline Phosphatase 
LAP was extracted from human liver samples by suspending the diced, washed 
samples in an extraction buffer consisting of 30% butanol in a buffer at 
pH 7.5 containing 2 mM MgCl.sub.2, 0.025 mM ZnCl.sub.2, 10 mM Tris-HCl, 
and homogenizing the mixture with a Polytron homogenizer (Brinkman 
Instruments, Westbury, N.Y.). The homogenate was incubated at 25.degree. 
C. for 16 hr followed by 8 hr at 4.degree. C. with gentle stirring. 
Centrifugation was performed at 9000 g for 30 min (Beckman Instruments, 
Model J2-21, Palo Alto, Calif.) and the aqueous phase was separated from 
the butanol phase and the pellet. The aqueous phase was clarified by 
centrifuging at 20,000 g for 20 min. The LAP preparations were purified 
and analyzed as in Procedure 1. Electrophoretic analysis as in Procedure 1 
of the protein-containing fractions showed that the LAP thus obtained was 
greater than 90% pure. After electrophoretic analysis, the 
protein-containing fractions were incubated with sheep anti-mouse IgG as 
in Procedure 1. The yields of LAP from crude butanol extract were 
typically approximately 33%. 
EXAMPLE 1 
Production and Isolation of Monoclonal Antibodies BA1F 419, BA1G 017, BA1G 
121, BA1G 151, and BA1G 339. 
Balb/c and A/J mice were immunized with SAOS-2 cells which had been 
extracted with a Triton-X containing buffer. Mice were immunized using 
complete Freunds adjuvant for the initial injection followed by incomplete 
Freunds adjuvant at day 14. Boosts (e.g., BA1B - 1 boost, BA1F 419- 2 
boosts, BA1G's - 1 boost) were repeated at 14 day intervals with PBS. 
Fusion of P3.653 myeloma cells and spleen cells from immunized animals 
were performed according to Kohler and Milsrein (1975), as modified and 
described in Oi and Herzenberg, Selective Methods in Cellular Immunology, 
Mishell, B. B., and Shigii, S. M., Eds., W. H. Freeman and Company, San 
Francisco, Chapter 17, (1980). Serum titers and initial screening of 
clones were done by the RIA procedure described below. Hybridomas which 
were identified to be secreting alkaline phosphatase reactive antibodies 
were grown in mouse ascites. 
Alkaline phosphatase-reactive antibodies were partially purified for 
immunoenzymetric assays by sodium sulfate fractionation ("salt cut") of 
mouse ascites. The salt cut was performed by determining the amount of 
ascites fluid collected, and adding dropwise sufficient 25% (w/v) sodium 
sulfate to the ascites with mixing to give a solution wherein the 
concentration of salt was 18%. The salt solution is then rotated for two 
hours at room temperature. The solution is then spun in a JA-20 centrifuge 
(Beckman Instruments, Palo Alta, Calif.) at 10,000 rpm for twenty minutes. 
The supernatant is removed and the pellet is resuspended in the 18% salt 
ascites solution. The solution is stirred or shaken again at room 
temperature for 10 to 15 minutes, spun again at 10,000 rpm, the 
supernatant is removed and the pellet is resuspended in a minimum volume 
of IX PBS. The buffered solution is dialyzed in PBS at 4.degree. C. 
overnight. A portion (10 .mu.l) of the sample is diluted with 1X PBS (1 
ml) and the concentration determined at 280 nm. (Formula: Absorbance at 
280 nm divided by 1.4 (constant for mouse IgG).times.dilution 
factor=concentration of salt cut ascites in mg/ml). No further 
purification is required to use the antibodies in the assay set forth 
below. 
EXAMPLE 2 
Competitive Radioimmunoassay for BAP-Specific, non-LAP Cross-reactive 
Monoclonal Antibodies 
A. Radiolabelling LAP and BAP 
The purified BAP and IAP enzymes from Procedures 1 and 2 were labelled with 
.sup.125 I using Chloramine-T to give a specific activity of approximately 
18 .mu.Ci/.mu.g of protein. 
B. Radioimmunoassy (RIA) 
i) Determining Antibody Titer 
The general procedure for the assay entailed adding hybridoma supernatant 
sample (25 .mu.l) to microtiter plate wells followed by the addition of 50 
.mu.l of 1-125 labeled, purified BAP. Sepharose 4B beads (Pharmacia, 
Uppsala, Sweden) coupled with cyanogen bromide (Cuatrecacas, Methods in 
Enzyology, J. Bio. Chem. 245:3059 (1970) to sheep anti-mouse IgG were 
added and the plates were incubated overnight at 25.degree. C. with gentle 
shaking. The Sepharose beads from each plate were washed with 0.1% Tween 
and PBS and collected with a cell harvester on paper discs and the discs 
were counted in a gamma counter (Iso-Data, Rolling Meadows, Ill.). This 
assay was used to measure the ascites titer for anti-BAP antibodies. (The 
titers for clones BA1F 419, BA1G 017, BA1G 121, BA1G 151, BA1G 339 and 
BA1B 067 are set forth in Table 1, below). 
ii) Determining Specificity for BAP in the presence of LAP 
This RIA procedure was also used for (simultaneous) saturation analysis of 
BAP for each of the antibodies. The results of this analysis using 
radiolabelled BAP displaced by either unlabelled BAP or unlabelled LAP are 
set forth in Table 1 below and FIGS. 1 through 4 above. (In general, 
varying amounts of crude (unlabelled) LAP or BAP (from Procedures 1 and 2) 
along with fixed amount of radiolabelled BAP were used to get the results 
set forth above.) Saturation analysis was performed by first determining 
the 50% titer point of the antibody sample by the above RIA procedure. 
Varying amounts of crude BAP or LAP were then added to the appropriate 
dilution of antibody sample along with a fixed amount of purified .sup.125 
I BAP tracer antigen. The rest of the procedure was as described above for 
the RIA. The results of the competitive assays are set forth below in 
Table 1 below. 
TABLE 1 
______________________________________ 
Concentration of Cold Antigen for 50% Inhibition 
Ascites 
Titer ng ng 
Antibody 
Reactivity (RIA) Isotype 
BAP LAP 
______________________________________ 
BA1F 419 
Non-crossreactive 
1/6,400 IgG1 16.8 &gt;200 
BA1G 017 
Non-crossreactive 
1/124,000 
IgG2a 5.0 &gt;100 
BA1G 121 
Non-crossreactive 
1/129,000 
IgG2a 6.0 &gt;200 
BA1G 151 
Non-crossreactive 
1/256,000 
IgG2a 8.8 &gt;200 
BA1G 339 
Non-crossreactive 
1/2,000 IgG2a 15.8 &gt;200 
BA1B 067 
Crossreactive 
1/256,040 
IgG2a 4.0 7 
______________________________________ 
EXAMPLE 3 
"One-site" Immuno-assited Enzyme Assay 
A one-site immunoenzymetric assay for the detection of BAP or LAP was 
performed by drying down sheep anti-mouse IgG (North Valley Farms, San 
Diego, Calif.) in 10 mM sodium phosphate buffer (pH 7.0) on microtiter 
plates overnight at 37.degree. C. The plates were rinsed with distilled 
water followed by a 2 minute wash in a 0.1% Tween-20/PBS solution and then 
rinsed again with distilled water. Plates were blocked with a PBS solution 
containing 1% BSA and 0.1% Tween-20. for 30 min an 37.degree. C. and 
washed as before. The plates were incubated with 100 .mu.l of culture 
supernatant from Example 1 for 2 hr at 37.degree. C., washed (as above), 
and then incubated with crude detergent or butanol extract of either BAP 
or LAP (from Procedures 1 or 2) for 1 hr at 37.degree. C. followed by 
washing with distilled water. PNPP substrate (1 PNPP tablet (Sigma) in 3 
ml rarer was added for 30 min and the absorbance was read at 405 nm in a 
Vmax microtiter plate reader. 
EXAMPLE 4 
Sandwich Assays Specific for Human BAP 
Procedure A 
Biotinylation of Anti-BAP Monoclonal Antibodies 
Monoclonal antibodies BA1G 017, BA1G 121, BA1G 151, BA1G 339, and BA1B 067 
(the latter for comparative purposes) were each biotinylated in the 
following manner. The antibody (1 mg) was dissolved in 0.2M sodium 
bicarbonate buffer at pH 8.2 (1 ml). To this solution was added biotin 
N-hydroxy-succinimide ester (120 mg). The resultant solution was incubated 
on a rotator at 25.degree. C. for 1.5 hours then dialyzed against PBS for 
16 hours with two changes of PBS. 
Procedure B 
The Sandwich Assay--Microtiter Plate 
The sandwich assay was performed by drying a PBS solution of the capture 
antibody (BA1F 419) for this assay in a microtiter plate and washing and 
blocking as described above in Example 3. A solution (5 .mu.g/ml) of 
biotin-labeled antibody (either BA1G 017, BA1G 121, BA1G 151, BA1G 339, or 
for comparison, BA1B 067) was added together with 50 .mu.l of the various 
dilutions of antigen sample (from either Procedures 1 or 2 above) and the 
plates were incubated for 4 hours at 25.degree. C. The antibody and 
antigen dilutions were made in a solution consisting of 5% non-fat dry 
milk (Alba), 0.01% anti-foam A emulsion (Sigma Chemical Co., St. Louis, 
Mo.) and 0.001% Thimerosol in PBS. The plates were washed and 50 .mu.l of 
streptavidin-conjugated horseradish peroxidase (Jackson Laboratories Inc., 
Avondale, Pa.) (0.1 .mu.g/ml in 0.1% Tween 20% PBS) was added for 1 hr at 
25.degree. C. The plates were washed with distilled water then incubated 
for 15 min with a solution of 0-Phenylenediamine substrace (Sigma). The 
plates were read at 490 run in a microtiter plate spectrophotometer 
(Bio-Tek ELISA reader, Bio-Tek instrument Co.. Burlington, Vt.) The 
results of assays using BA1F 419 as the capture antibody and either BA1G 
017, BA1G 151, BA1G 339, or (for comparison) BA1B 067 as the labelled 
antibody in an in vitro assay set forth below in Table 2 and in FIGS. 5 
and 6. 
TABLE 2 
______________________________________ 
BAP Tandem Assay: Slope of the Dose-Response Curves.sup.1 
Ratio 
BAP/LAP 
Antibody 
Reactivity BAP LAP Response 
______________________________________ 
BA1G 017 
Non-cross reactive 
1.09 -0.00633 
.sup.2 
BA1G 151 
Non-cross reactive 
2.36 0.01430 
165.0 
BA1G 339 
Non-cross reactive 
1.79 0.00587 
305.0 
BA1B 067 
cross reactive 
36.50 33.10000 
1.1 
______________________________________ 
.sup.1 O. D. (Abs. 490)/.mu.g/ml total ALP 
.sup.2 Cannot be calculated, implies absolute specificity. 
FIGS. 7, 8, and 9 above show the results of assays analyzing human sera 
expected to be rich in either BAP and LAP. BA1F 419 was again used as the 
capture antibody and antibodies BA1G 339 (FIG. 9) and BA1G 121 (FIG. 8) 
plus BA1B 067 (for comparison, FIG. 7) were used as the biotin-labelled 
antibodies. The procedure for these assays using human sera followed that 
of the above in vitro assays substituting the sera for the extracted 
antigen. 
Procedure C 
The Sandwich Assay--Bead Format 
Polystyrene beads (5/16inch diameter) were prepared by linking the antibody 
covalently to aminopolysterene beads using dimethyl suberimidate (DMS). 
I) Polystyrene beads are first nitrated in a 100 ml round bottom flask 
equipped with a stirbar to which 25.0 ml of concentrated sulfuric acid 
(H.sub.2 SO.sub.4) followed by 12.5 ml of concentrated nitric acid 
(HNO.sub.3) is added. Chill, using an ice bath, until a temperature of 
5.degree.-10.degree. C. is maintained for a minimum of five minutes. Add 
100 polystyrene beads making sure that all beads are in thorough contact 
with the acid mixture. React for about 20 minutes at 5.degree.-10.degree. 
C. with gentle stirring. The temperature should not exceed 10.degree. C. 
Filter the beads using a Buchner funnel. Retain the acid mixture for 
disposal later. Pour the beads into cold (4.degree.-8.degree. C.) 
deionized water. Use a volume of water sufficient to completely immerse 
the beads. Drain the water and repeat the rinse twice more. 
The beads are then aminated in a 100 ml round bottom flask equipped with a 
stirbar to which add 29.5 ml of concentrated hydrochloric acid (HCl) and 
29.5 g of stannous chloride (SnCl.sub.2) is added. The mixture should be 
maintained at room temperature (20.degree.-25.degree. C.). Add the 
nitrated polystyrene beads making sure that all beads are in thorough 
contact with the acid mixture and react for two hours. After the reduction 
is complete, drain the beads using a Buchner funnel into a separate acid 
container for later disposal. (Note: Under no circumstances should the 
nitration and amination acid mixtures come in contact. Contact results in 
an explosive reaction.) All rinses employ enough buffer/water to 
thoroughly immerse the beads. Rinse the beads for approximately 1 minute 
by immersing in 0.1 M HCl. Drain and repeat the 0.1 M HCl rinse. Rinse the 
beads for approximately 1 minute by immersing in deionized water. Drain 
and repeat the deionized water rinse. Rinse the beads for approximately 1 
minute by immersing in 0.1M NaOH. Drain the beads and rinse twice more 
using deionized water. Store the beads protected from light in 0.20 M 
Sodium Phosphate, pH 7.0, 0.1% Sodium Azide at 2.degree.-8.degree. C. 
II) The functionalized beads were treated in a solution of 0.05M DMS in 
0.25M triethanolamine at pH 9.3 for 20 minutes at room temperature with 
gentle agitation. The beads were then washed in 0.05M sodium phosphate 
buffer at pH 8.0. The beads were then immersed in a solution of the same 
sodium phosphate buffer containing the BA1G 151 (any other antibody could 
be used at this point) antibody (0.05-0.1 mg/ml) am 2.degree.-8.degree. C. 
for 18-24 hours. The beads were washed in 0.1M sodium phosphate buffer 
that is 1.0M in sodium chloride at pH 6.0 for 1 hour followed by 
incubation in the same buffer that additionally contains 0.2% Tween 20 for 
15-20 minutes. The beads were washed in the same buffer but without Tween 
20 for 8-12 minutes followed by rinsing in deionized water with agitation 
for 5 minutes. The beads were blocked with 0.1% BSA (Miles Laboratories, 
Naperville, Ill.) in 0.05M sodium phosphate buffer at pH 7.2 for 3 hours 
at 53.degree. C. with agitation at 20 minute intervals. The beads were 
drained and rinsed three times with 1.0M sodium chloride solution, 0.1M 
sodium phosphate buffer at pH 6.0, and then stored in 0.05M sodium 
phosphate buffer at pH 7.2 that also contains 0.1% sodium azide at 
2.degree.-8.degree. C. for future use. (All antibodies used for the bead 
assay were purified by the HPLC procedure of Procedure 3). 
The antibody (e.g., BA1G 151) was labelled by the Glucose Oxidase 
Lactoperoxidase (GOLP) iodination method using Enzymobeads (Bio-Rad, 
Calif.) according to the directional insert to a specific activity of 
approximately 7-8 .mu.Ci/.mu.g of protein. 
The antigen for calibration and validation was extracted from SAOS-2 cells 
and LAP was extracted from human liver as described above. The extracted 
antigen was diluted into a matrix consisting of 10% BSA, 0.1% sodium 
azide, 0.1% mannitol, 0.001% NP-40, 0.1M sodium phosphate-citrate buffer 
at pH 7.0. The antigen was diluted based on total alkaline phosphate 
activity measured as above. Calibration was typically made at 0, 0.1, 0.2, 
0.4, and 0.6 Units/ml. 
The assay format using the above reagents and calibrators was as follows: 
A) add the sample or calibrator to the assay test tube; 
B) add a solution (100 .mu.l) of the radiolabelled antibody (diluted to 
give 200,000 cpm per tube) to the tubes; 
C) add one bead to the tube; 
D) incubate the mixture on a shaker at room temperature for 2 hours; 
E) wash the bead 3 times with Tandem.RTM.-R wash reagent (Hybritech 
Incorporated, San Diego, Calif., a detergent solution containing 0.3% 
sodium azide as a preservative); and 
F) detect the signal present on the bead with a gamma counter. 
The above bead assay constitutes a preferred embodiment of the invention. 
The most preferred conditions for the bead assay format used BA1G 151 as 
both the capture (i.e. bead) and the labelled antibody. Other 
combinations, such as BA1G 151 as the capture and BA1G 121 as the labelled 
antibody, and vice versa, and BA1G 121 as both capture and labelled 
antibody, work well and are preferred bead assay conditions. Other 
combinations of the BA1F 419, BA1G 017, BA1G 121, BA1G 151, or BA1G 339 
successfully detected BAP in the bead assay format and are included within 
the scope of the present invention. 
Procedure 3 
Production of Cross-Reactive Antibody BA1B 067 
Monoclonal Antibody BA1B 067 was produced along the lines of Examples 1 and 
3. Initially the analysis of Example 2 was not carried out on the antibody 
as purified samples of BAP and LAP were to be obtained only after BA1B 067 
had been produced and purified. In addition to the purification step set 
forth in Example 1, BA1B 067 was further purified by high pressure liquid 
chromatography column on a Bio-Rad HPLC instrument equipped with a TSK 
DEAE anion-exchange column (Bio-Rad Laboratories, Richmond, Calif.) which 
was eluted with a linear gradient of 20 mM Tris-HCl, pH 8.5 to 300 mmol 
sodium chloride, 20 mmol Tris-HCl pH 7.0. This purification was used only 
for affinity column manufacture and the bead assays.