Detection of HCG with solid phase support having avidin coating

Human chorionic gonadotropin (HCG) is detected by enzyme immunoassay by bonding biotinylated antibody to an avidin coupled paper disc, reacting the antibody on the disc with the solution suspected of containing HCG, and determining the amount of HCG on the disc by enzymatic assay detection techniques.

FIELD OF THE INVENTION 
This invention relates generally to enzyme immunoassay (EIA) techniques, 
and to the determination of human chorionic gonadotropin (HCG) in 
particular. 
BACKGROUND 
Enzyme immunoassay (EIA) methods have been described, Guesdon J., Ternynck, 
T., Avrameas, S., "The Use of Avidin-Biotin Interaction in Immunoenzymatic 
Techniques," J. Histochem Cytochem 27:1131-1139, 1979. Guesdon et al 
tested the effects of labeling antibodies, antigens and enzymes with 
biotin in EIA procedures. The biotin-avidin interaction is well-known and 
has been studied rather exhaustively, and has been used in purification of 
reagents by coupling avidin to a Sepharose chromatographic column, 
conjugating biotin to a protein to be purified, binding the biotinylated 
protein to the column, washing the column, and then eluting the purified 
protein-biotin conjugate protein from the column. Liu, F., Zinnecker, M. 
Hamaoka, T. and Katz, D. H., J. Biochemistry 18:690, 1979. 
The measurement of HCG in serum or urine is of interest in the diagnosis of 
early pregnancy, detection of ectopic pregnancies, and in monitoring 
various tumors. Walsh, P. R., Wang M., Gittermann, M. L., CLINICAL 
IMMUNOCHEMISTRY, American Association for Clinical Chemistry, 1978, 
Natelson, S. et al, Eds., pp. 306-328. 
The use of filter papers, usually in the form of small discs, in various 
assay procedures is well-known. However, efforts to develop an assay 
method for HCG using filter paper discs has not been successful. After 
considerable work, it was discovered that the methods based upon binding 
HCG antibody to a disc and reacting the disc with a solution suspected of 
containing HCG, e.g., urine, were of poor reliability and sensitivity. 
Moreover, discs and filter paper devices cannot be manufactured by known 
techniques in a satisfactory manner. The present invention overcomes these 
disadvantages and makes possible a rapid, reliable and simple enzyme 
immunoassay for HCG in urine, or other solution. 
SUMMARY OF THE INVENTION 
A method has been developed for determining chorionic gonadotropin (HCG) 
which is highly sensitive, extremely simple to carry out, and can be 
adopted to home use kits. The invention comprises the method and a kit or 
system for carrying out this detection. The concept of this invention has 
broader application to specific antigen determinations, and can also be 
adapted for determination of antibodies using the same concept by simply 
reversing the role of the antigen and antibody. 
The method of this invention, exemplified by the determination of human 
chorionic gonadotropin (HCG) comprises, in its most preferred form: 
Coating all or part of a solid phase support with avidin, by chemical 
bonding, physical absorption or any other suitable technique, attaching 
biotinylated anti-HCG antibody to the support through the avidin-biotin 
coupling, exposing the support thus prepared to a sample, e.g., a 
solution, suspected of containing HCG and to labelled anti-HCG antibody. 
The result is a solid phase support having bound thereon HCG and a 
detectable label which is present in proportion to the amount of HCG 
substrate or developer for the label, or counted if the label is 
radioactive, as a measure of the concentration of HCG in the sample. 
Alternatively, the labeled species may be removed from the support and 
detected in any conventional manner. Enzyme labelling, e.g., with 
.beta.-galactosidase, is a preferred approach, but other enzymes and 
radiolabels may be used. 
The invention can also be utilized in the technique for determining the 
presence or absence, and measuring the quantity of antibodies. In this 
instance, an antigen to the specific antibody is biotinylated and bound 
through avidin to a support, a like antigen is labelled and the reaction 
is carried out in the same manner, except that the antibody is the species 
to be determined.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
The solid phase supports in the assay are, in this example, paper discs 
approximately 0.7 cm in diameter, Whatman (Trademark) grade 541. The paper 
discs are first activated with CNBr according to the procedure described 
by R. Axen et al. (Nature, 214:1302, 1967). The activated discs were 
allowed to react with avidin at 4.degree. C. overnight. After reaction, 
the solution was removed and the discs were blocked with 50 mM 
ethanolamine, pH=8.0, at room temperature for 2 hours. The discs were then 
washed extensively with 0.1M NaOAC, 0.5M NaCl buffer, pH=4.0, and 0.1M 
NaHCO.sub.3, 0.5M NaCl buffer, pH=8.3, alternately, 3 times. 
Biotinylation of affinity purified rabbit anti-HCG antibody was 
accomplished by incubation of the antibody with 
N-hydroxysuccinimidyl-biotin in borate buffered saline (BBS) at pH=8.3 at 
room temperature for 2 hours. Excess N-hydroxysuccinimidyl-biotin was then 
removed by dialysis or gel filtration. 
.beta.-Galactosidase-linked antibody was prepared by reacting affinity 
purified mouse anti-HCG monoclonal antibody with .beta.-galactosidase 
through a cross-linking reagent M-Maleimidobenzol-N-Hydroxysuccinimide 
(MBS) according to procedure published by F. T. Liu et al., J. 
Biochemistry 18:690 (1979). 
The immunoassay is accomplished by the interactions of the assay components 
as described below: 
In a typical Disc-Avidin Biotin-Ab-HCG-Ab-.beta.-Galactosidase (HCG ElA) 
example, the disc resides in a chamber with a lyophilized pellet of 
Anti-HGC-.beta.-Galactosidase, 250 .mu.l of urine sample is used to 
dissolve the pellet and rehydrate the disc. The disc-avidin-biotin-Anti 
HCG (BAD), the anti-HCG-.beta.-Galactosidase, and the urine sample 
incubate simultaneously at room temperature for 10 minutes. The disc is 
then removed and washed extensively with a buffer solution or under a 
stream of tepid water. The disc is finally incubated with 
O-nitrophenyl-galactopyranoside (ONPG), a synthetic substrate for 
.beta.-Galactosidase. The enzyme activity is related to the amount of HCG 
present in the urine sample. 
A dose-response curve has been demonstrated using the above protocol. For 
qualitative assays, urine sample with 400 mIU/ml HCG can be easily 
detected with the naked eye. This HCG level corresponds to approximately 3 
days after missed period. 
One feature of the invention is the application of the method and protocol 
to a kit which can be used by an untrained individual in the privacy of 
her own home to detect the presence or absence of HCG as an indicator of 
possible pregnancy. As the above protocol indicates, this detection can be 
made as early as three days after a menstrual period has been missed. A 
missed period may indicate pregnancy, and it may also indicate a possible 
complication, in some individuals an early indication of pregnancy is 
important. There has been a long standing need for a kit which can be used 
without need for a laboratory or skilled technicians. Kits may also be 
used in or out of the clinic or laboratory to monitor various tumors. 
Kits include all reagents necessary to complete a qualitative or 
semi-guantitative determination of HCG in urine. The basic constituents of 
a kit are: (1) Solid phase support, typically filter paper discs, treated 
with avidin, the avidin being bound to the paper to provide sites which 
are reactor with biotinylated anti-HCG antibody; (2) Labeled anti-HCG 
antibody; (3) Wash reagents; and (4) Developing reagent. In the case given 
as a preferred embodiment, which is exemplary and non-limiting, the 
developing reagent is O-nitrophenyl-galactopyranoside (ONPG). Development 
of a .beta.-galactoside labeled HCG-antibody species provide a color 
change which is visible to the naked eye. Other color, or visible effect 
producing label and developer combinations may be applied; however, the 
system described is presently preferred. Buffers, saline reagents, etc. 
may also be included but it is preferable to incorporate these into the 
various reagents described above. Kits, according to this invention, would 
typically include a vessel having a predetermined volume indication, 
instructions for use, and such accessories as may be convenient for the 
user. 
FIG. 1 depicts an example of an article of manufacture, such as component 
of a kit, which embodies a principle of this invention. The device 
depicted in FIG. 1 comprises the solid phase support, a layer of avidin, 
and a layer of biotinylated antibody (or antigen if an antibody is to be 
detected). Of course, these many layers need not be perfectly congruent 
with one on the other and, if the substrate were porous, would not be 
physically discrete layers but in effect would have the described 
structure. The substrate may be porous or non-porous. For example, filter 
paper and polystyrene may both serve very well. 
Any solid phase material which is inert in the reactions, and to which 
avidin can be bound, either by chemical reaction or physical attachment, 
may be used. Filter paper discs or sheets are a common solid phase support 
material as are discs or plates of polystyrene. 
The next component is biotinylated antibody (or antigen) which is not 
coated over the entire surface, as was the case in the earlier described 
examples. Rather the biotinylated antibody (or antigen) is "printed" on 
only selected areas of the avidin coated surface in any desired pattern. 
The term "printed" is used to describe the best known technique for 
manufacturing the multiple indicator substrate of this invention, and the 
ability to "print" the antibody on selected areas is a very important 
feature of this invention. It has been extremely difficult to provide any 
reliable pattern on filter papers or other substrates because the reagent 
migrates through or across the substrate. The present invention obviates 
this entire problem. The biotinylated antibody couples immediately with 
the avidin, thereby fixing the area on the avidin coated support where a 
particular antibody will be found. This coupling prevents migration during 
or after printing, and during reaction, of the antibody (or antigen). 
Thus, upon development of the multiple indicator substrate, the antibody, 
and any antigen to which it is bound, will be found exactly where it was 
printed. Printing may be acccomplished in the same manner that ink is 
printed on paper, i.e., by application on type or other patterned means 
coated with the biotinylated antibody to the avidin coated support. This 
permits very efficient, high volume production of multiple indicator 
support, which may have several different antibodies found thereon. The 
substrate may include indicia formed of ink or other material as well as 
the indicia formed of antibody (or antigen). In FIG. 1, for example, the 
top and center rows indicate, respectively, square or round areas having 
some other indicia therein upon which the biotinylated antibody is 
printed, while the lower row indicates biotinylated antibody printed in 
the form of letters. Obviously, any symbol, shape or alphanumeric indicia 
may be used. 
These multiple indicator substrates may be used in the clinical or research 
laboratory, or may be included in a kit for consumer use. Several 
different antibodies (or antigens), and antibodies of different activity 
may be printed on the substrate, thus permitting, with one such multiple 
indicator substrate, several diagnostic tests to be performed at one time, 
or give semi-quantitative results respecting a particular antigen or 
antibody. 
DISCUSSION 
Efforts to provide a procedure and a kit in which the HCG was bound to a 
solid, fibrous substrate, such as filter paper (which is available in high 
purity and convenient sizes) were not successful. Two problems plagued the 
method to the point that the approach was being considered unfeasible. 
First, low sensitivity was a serious concern. Secondly, and even more 
serious, was the poor reproducibility. After considerable effort, and 
consideration of the problem, the inventors determined that the problems 
were somehow involved in the binding of the reagents to the filter paper. 
It is not known for certain what the problem is, fundamentally, and no 
problem was considered likely since it is relatively easy to bind antigen 
to filter paper, or equivalent porous members. It is postulated, only as a 
possible explanation, which has not been proven by firm data, that some 
type of steric hindrance occurs when antibody is bound to the paper which 
prevents complete, quantitative and reproducible reaction of the treated 
filter paper by the antibody. In any event, regardless of the actual 
molecular phenomenon which occurred, poor sensitivity, and a narrow assay 
range was the result. This immunoassay range did not seem profitable to 
pursue. In addition, the requirements for individual coupling, handling 
and storage of solid phase antibodies for different immunoassays was 
considered prohibitively time consuming and costly. 
After consideration of the problem, and various evaluations of alternative 
approaches, and upon experimentation, the technique set forth above was 
developed by the inventors, and was proved out as a viable, simple, 
reliable and reproducible method of high sensitivity which finds direct 
application to the determination of HCG in urine, and has greater 
applicability for other antibody-antigen type reactions. As pointed out 
above, the same procedure, reversed to bind the antigen to the paper 
through biotin-avidin coupling, rather than the antibody binding described 
above, may be used to detect the presence of an antibody. Thus, the terms 
antigen and antibody may be substantially reversed in the specification 
and claims to give the fully equivalent, converse of the specific terms 
used above and hereinafter. 
The general applicability of the present invention has been established. 
For example, an avidin coated substrate was coated with 
biotinylated-AntiDNP-IgG. This avidin-biotinylated-AntiDNP-IgG coated 
substrate was then reacted with DNP.sub.49 -BSA and finally with 
Anti-DNP-.beta.-galactosidase. The presence of DNP.sub.49 -BSA was 
determined in the final reaction by production of color on the coated area 
of the substrate. The results can be quantitatively determined by reading 
the absorbance of the color at 420 nm in the conventional manner. 
In another example, the avidin coated portion of a substrate was coated 
with biotinylated ragweed allergen, then incubated simultaneously with 
human serum containing Anti-Ragweed allergen-IgE and with goat-Anti-Human 
IgE-.beta.-galactosidase. The presence of Anti-Ragweed IgE is determined 
by the color produced in the area coated as described, and can be measured 
photometrically as described above. 
It will be understood that in giving the preferred embodiment and 
application of the invention the concept and scope of the invention is 
exemplified thereby without being limited to the specific reagents, 
labels, developers, or applications.