WO1986002736A1

WO1986002736A1 - Immunoassay method for small molecules

Info

Publication number: WO1986002736A1
Application number: PCT/US1984/001737
Authority: WO
Inventors: Paul H. Ehrlich; Alexander Krichevsky
Original assignee: Eks, Inc.
Priority date: 1984-10-29
Filing date: 1984-10-29
Publication date: 1986-05-09
Also published as: EP0198826A4; EP0198826A1; JPS63501096A

Abstract

Method for performing two-site immunoassays for small molecules, which were previously considered too small for binding two antibody molecules and hence which were widely believed not to be detectable using this type of assay. The discovery is believed to be applicable to virtually all immunoassay procedures, including traditional immunoassay procedures as well as those most recently developed. This discovery provides a method for detecting accurately very minute amounts of such agriculturally and medically important low molecular weight molecules as steroids, mycotoxins, antibiotics, small hormones and small peptides.

Description

IMMUNOASSAY METHOD FOR SMALL MOLECULES

This invention relates to a method for performing two-site immunoassays for small molecules. As used herein, small molecules are those im unogens or haptens which were previously considered too small for binding two antibody molecules and hence were widely believed not to be detectable using this type of assay. The invention is believed to be applicable to virtually all immunoassay procedures, including traditional immunoassay procedures as well as those most recently developed.

A preferred assay method is one in which one antibody is present in solid phase and the other antibody is labelled and in liquid phase.

The present invention provides a method for detecting accurately very minute amounts of such agriculturally and medically important low molecular weight molecules as steroids, mycotoxins, antibiotics, small hormones and small peptides.

BACKGROUND OF THE INVENTION

Two-site immunoassays have many advantages over single-site methods, including increased sensitivity, specificity, speed and stability of the assay [A. White, "Monoclonal Antibodies for Steroid Immunoassay," IN: .H. Hunter and J.E.T. Corrie (Eds.), Immunoassays for Clinical Chemistry, Edinburgh, London, Melbourne and New York, Churchill Livingstone (1983) p. 500] . For example, immunoradiometric assay (IRMA) , especially when combined

with monoclonal antibody technology, is simpler to perform, has a lower detection limit, requires a shorter incubation period and counting time, and gives a wider working range than radioimmunoassay (RIA) [W.M. Hunter, et al., "Monoclonal Antibodies for Use in an

Iramunoradiometric Assay for Alpha-Foetoprotein," Journal of Immunological Methods, Vol. 50 (1982) pp. 133-144; W.M. Hunter and P.S. Buάd, "Immunoradiomet ic vs. Radioimmunoassay: A Comparison Using Alpha-Foetoprotein as the Model Analyte," Journal of Immunological Methods, Vol. 45 (1981) pp. 255-273].

Recently, it has been shown that the combination of two monoclonal antibodies can be employed to construct a cooperative immunoassay, that is, one in which a mixture of monoclonal antibodies raised to a particular antigen has a greater affinity than either antibody individually has for that antigen- [P.^* H. Ehrlich and W.R Mojrle, "Cooperative Immunoassay: Ultrasensitive Assays with Mixed Monoclonal Antibodies," Science, Vol. 221 (15 July 1983) pp. 279-281 (hereinafter referred to as Ehrlich and Moyle Science article); P. H. Ehrlich et al., "Mixing Two Monoclonal Antibodies Yields Enhanced Affinity for Antigen", Journal of Immunology, Vol. 128 (1982) pp. 2709-2713 (hereinafter referred to as Ehrlich et al. Journal of Immunology article)] .

However, as implied in the name, a two-site immunoassay requires that two antibodies be able to bind simultaneously to the molecule to be assayed. It has generally been believed that in order to bind two antibodies simultaneously, the molecule of interest roust be of a certain minimum size, since, when one antibody binds to one epitope on a small imraunogen or hapten, this antibody can sterically block any other antibody binding a

J

second epitope IA. Nisonoff, et al.. The Antibody Molecule, New York, Academic Press (1975) p. 65]. Due to the requirement for simultaneous binding of two antibodies, two-site immunoassays have thus far been employed only in the assays of large molecules. For example, alpha-foetoprotein and human chor onic gonado- tropin were assayed respectively using the previously mentioned immunoradiometric and cooperative assays techniques.

As another example, H.A. Katus and others used a "double sandwich" radioimmunoassay to measure two different epitopes against human myosin light chain in order to differentiate between cardiac-and skeletal-myosin light chains. ["Increased Specificity in Human

Cardiac-Myosin Radioimmunoassay Utilizing Two Monoclonal Antibodies in a Double Sandwich Assay", Molecular Immunology, Vol. 19 (1982) pp..451-455.1

David and Green (U.S. Patent No. 4,376,110 issued

March 8, 1983) describe a number of two-site immunoassays employing monoclonal antibodies. They refer to testing large immunogenic protein molecules which may have dozens of antigenic sites. They further describe their invention as useful for determining the presence or concentration of a wide variety of polyvalent antigenic substances.

As mentioned previously, a two-site immunoassay requires the presence of at least two epitopes on the same molecule with enough distance between these epitopes to allow two antibodies to bind simultaneously without steric hindrance. The length required to avoid steric interference and permit the combination of a hapten or immunogen with two antibody molecules is thought to be at least 20 to 26 angstroms. [A. Nisonoff et al.. The Antibody Molecule, New York, Academic Press (1975) , p.65]. However, it should be noted that these experiments were conducted to determine the distance between binding sites needed to avoid steric hindrance on bifunctional dinitrophenol (Dnp) derivatives, Dnp-n-Dnp, where n represents a spacer which can be varied in length. These synthetic haptens are designed for experimental use only, and do not necessarily mimic naturally occurring small haptens, especially with respect to the limited number of conformations these bifunctional dinitrophenol derivatives can assume. It is generally believed that a much larger molecule than a 26 angstrom, bifunctional dinitrophenol derivative would be the practical limit for a two-site immunoassay.

For instance, during a discussion following the description of immunoassays employing monoclonal antibodies against steroids, Ekins remarked that he thought that monoclonal antibodies are likely to be important solely to assay large molecules using two-site sandwich assay. [R.P. Ekins, quoted in "Discussion (Chapter 11.5)", tt_.^{: W}-^M» Hunter and J.E.T. Corrie, (Eds.) Immunoassays for Clinical Chemistry, Edinburgh, London, Melbourne, New York, Churchill Livingston (1983) p. 500]. The smallest, clinically important molecule that has been tested using two-site immunoassays is insulin, molecular weight approximately 6000. The smallest molecule that can be tested using two-site immunoassays is generally considered to be of a molecular weight above 3200.

Yet, there is a need for the extra sensitivity and specificity provided by imraunoradiometric, cooperative, and other two-site immunoassays to detect the presence or concentration of agriculturally or medically significant small molecules like mycotoxins and steroids.

- -

These compounds are present at extremely low concentrations in collected samples; lack of sensitivity and specificity are the limiting factors in detecting these substances. Increased sensitivity and specificity also improve these immunoassays, because some expensive, cumbersome extraction steps can be eliminated. The avoidance of tagging the antigen with a fluorescent-, enzyme- or radiolabel by utilizing a two-site assay in which one of the antibodies is labelled removes a step where scarce antigens such as mycotoxins and steroids may be destroyed. The cross-reactivity of antibodies to steroids is another reason why assays for these compounds, more sensitive and specific than are currently available, are needed. Often, it is clinically informative to determine the relative abundance of each steroid, but. it may presently be difficult to make this determination given the inadequate specificity of assays currently available.

SUMMARY OF THE INVENTION

This invention is based on the discovery that two-site immunoassays can be performed to detect or measure the concentration of small antigens and small haptens of a size previously considered too small to be assayed using two-site immunological assay methods.

The small molecules that are within the scope of this invention range in size from molecular weights of about 250 to 2500-3000, preferably between about 275 and 1000. The most preferred range is between about 275 and 500.

A major reason for these variations is that molecular weight is not the only parameter in determining

the dimensions of a molecule in space, since a compact, globular molecule may be heavier than an elongated molecule, even though both molecules can be considered to be of the same length in their longest dimension. Furthermore, when two epitopes suitable for binding two antibodies are not located at the two points in a molecule farthest from each other, then a molecule would need be larger before simultaneous binding could occur than if these two binding sites were, in fact, at opposite ends of the small molecule to be studied. Hence the invention cannot be concisely bounded in terms of molecular weights or any other simple parameter. However, even though molecules may assume many different conformations, molecular weight is at least illustrative of the small molecules that are the subject of the present invention; and furthermore, we believe that the molecules in the above-mentioned molecular weight ranges are within the bounds that are unexpected by those skilled in the art.

We have discovered that two-site immunoassays are particularly useful to determine the presence and concentration of small molecules which are important in agriculture and medicine. Specifically, clinically relevant small molecules which can be tested using two-site immunoassays include steroids, such as aldosterone, cortisone, estradiol, progesterone, and testosterone, small hormones such as triiodothyronine and lutenizing hormone-releasing factor (LH-RH) , as well as antibiotics such as gentiroicins and penicillins. Similarly, mycotoxins, like aflatoxins and trichothecenes, can be detected at very minute concentrations using two-site methods, and these assays are useful in testing grain and other agricultural products. Small peptides such as aspartame and the previously mentioned hormones are also suitable to be assayed by two-site methods.

DETAILED DESCRIPTION OF THE INVENTION

To assay for molecules previously considered too small to oe assayed using two-site immunoassay methods, one must first raise antibodies to these molecules, if the small molecule is an immunogen, a purified sample of the small molecule may be injected into two or more animals to raise two separate and distinct antisera. Likewise, one or more animals could be immunized with the desired immunogenic small molecule and monoclonal antibodies can thus be obtained using the well-known procedures for constructing monoclonal antibody-producing hybridomas as first reported by Milstein and Kohler [Nature, Vol. 256 (1975) pp. 495-497; see also: H. Zola and D. Brooks, "Techniques for the Production and

Characteriziation of Monoclonal Hybridoma Antibodies" IN: J. G. R. Hurrell, (Ed.) Monoclonal Hybridoma Antibodies: Techniques in Applications, Boca Raton, Florida, CRC Press, Inc. (1982) p. 1 (hererinafter referred to methods "H. Zola and D. Brooks")].

Most small molecules which can be assayed using the methods described herein may be haptens and therefore not immunogenic unless coupled to a protein or other suitable group. Because of the small size of the molecules which are the subject of the present discovery, in some cases it may be necessary to construct two conjugated small molecules, that is, the small molecule to be assayed is conjugated by attachment of a protein or other suitable group to two different sites on the small molecule. Then, these two sets of conjugated small molecules are used to immunize at least two mice, or other suitable animals, to produce either polyclonal antisera or monoclonal antibodies as described above. One mouse or other suitable animal can be immunized with these two sets of conjugated small molecules to raise two monoclonal

OMPI _ antibodies suitable to practice the present invention. Methods of constructing conjugated haptens are well known in the art and are often varied to suit the particular hapten to be studied. A procedure for making two conjugates, at different positions, to the steroid, aldosterone, will be given in the experimental examples which follow.

Once at least one pair of antibodies, whether a pair of monoclonal antibodies, polyclonal antisera, or a monoclonal antibody and a polyclonal antiserum has been raised to the small molecule to be assayed, these antibodies must next be tested for simultaneous binding to the small molecules to be assayed, in keeping with the teaching of the present invention. Many different two-site immunoassays, well-known to those skilled in the art, could be used for this purpose. For instance, an immunoradiometric assay could be employed. More specifically, assume, for example, that X is a small molecule, A is one monoclonal antibody raised against X and B is another monoclonal antibody raised against X, but not identical to A. In outline, an immunoradiometric assay would proceed as follows: antibody A attached to a solid support is brought into contact with radiolabelled antioody B mixed with a known quantity of small molecule X; the mixture of antibodies A and B molecule as well as small hapten X is incubated; subsequently, the liquid phase is removed and washed from the solid phase; and the solid support is checked for radioactivity. Radioactivity will be found in association with the solid support only it antibodies A and B are both capable of binding simultaneously to small molecule X.

The same two-site immunometric assay used to test for simultaneous binding can then be used to assay a

OMPI ^™°« ^* -g_

collected sample to determine the presence or the concentration of the small molecule in that sample, if any. Many different types of immunometric assays exist; the use of and methods to conduct a number of these two-site immunometric assays are discussed by David and Green in U.S. Pat. No. 4,376,110.

In summary, they state that such assays employ a quantity of unlabelled antibody bound to a solid support that is insoluble in the fluid being tested and a quantity of soluble antibody bearing a label such as a radioactive isotope that permits detection and/or a quantitative estimate of the amount of the ternary complex formed between solid phase antibody, antigen, and labelled antibody.

In immunometric assays known to the prior art, typically a "forward" assay, in which the antibody^' bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody:antigen complex, is employed. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen if any, and then contacted with a solution containing a known quantity of labelled antibody.

After a second incubation period to permit the labelled antibody to complex with the antibody bound to the solid support through the unlabelled antigen, the solid support is washed a second time to remove the unreacted labelled antibody. In a simple "yes/no" assay to determine whether the antigen is present in the sample being tested, the washed solid support is tested to detect the presence of labelled antibody, for example, by

-^JR£4

O PI -lO-

measuring emitted radiation if the label is a radioactive element. The amount of labelled antibody detected is compared to that for a negative control sample known to be free of the antigen. Detection of labelled antibody in amounts substantially above the background levels indicated by the negative control is interpreted to indicate the presence of the suspect antigen. It is best that several dilutions of the antigen-containing fluid be tested, since a great excess of antigen will give a background reading. Dilution of the sample would effectively eliminate this possibility. Quantitative determinations can be made by comparing the amount of labelled antibody with that obtained for standard samples containing known quantities of the antigen.

This kind of assay is frequently referred to as a

"two-site" or "sandwich" assay, since the antigen has two antibodies bonded to its surface at different locations. This and related techniques are described by Wide [Kirkham and Hunter (Eds.), Radioimmunoassay Methods, Edinburgh, E. & S. Livingston (1970). pp. 199-206]. A sandwich assay designed to detect human chorionic gonadotropin is descriDed in the Ehrlich et al. Journal of Immunology article previously mentioned.

The particular system of one solid phase/one liquid phase, two-site immunometric assay used is not limited and workers skilled in the art will consider many variations. For instance, many substances are suitable as the immobilizing support for the solid phase in this type of assay such as polyvinyl chloride microtiter plates, filter paper, plastic beads, or test tubes made from polyethylene, polystyrene, polypropylene or other suitable material. Antibodies can also be linked to particular materials such as agarose, cross-linked dextran and other

O PI ^*•__-

polysaccharides using well-known techniques such as the method described to bind antibodies to polysaccharide polymers in U.S. Pat. No. 3,645,852. Likewise, a number of well-known methods exist for labelling a liquid phase

5 antibody. A liquid phase antibody can be radiolabelled with 125I using the procedures of D. M. Klinman and J.

C. Howard [IN: "Protein Iodination for Labelling

Hybridoma Antibodies", R. H. Kennett et al. (Eds.),

Monoclonal Antibodies - Hybridomas: A New Dimension In

10 Biological Analyses, New York, Plenum Press (1980) p. 401-402 hereinafter referred to as "the method of D.M. Klinman and J.C. Howard")]. Similarly, this antibody could be marked using a fluorogenic label as in U.S. Pat. No. 3,940,475 or enzyme markers as in U.S. Pat. No.

15 3,645,090.

In some cases, a small molecule, which is the subject matter of the present application', may have enough symmetry in its conformation or chemical structure to

20 possess two binding sites for the identical antibody, and therefore, any two-site immunoassays described above can be performed using one antibody in place of two different antibodies. An example of a small molecule which would be suitaole for this particular embodiment of the present

25 invention is merphyrin. In most other cases, two nonidentical antibodies, that is, two antibodies which do not bind to the same epitope, would be required.

A detailed description of an immunoradiometric 30 assay as contemplated for use in connection with the present discovery is given below as Example 3.

Two-site immunoassays, other than the immunoradiometric or sandwich assays described above, are 35 also suitaole to carry out the teaching of the present

assay. A mixture of two monoclonal antibodies can be tested in a cooperative immunoassay, as described in the Ehrlich and Moyle Science article and the Ehrlich et al. Journal of Immunology article previously discussed. If a synergistic effect using this type of assay is observed, this result can be taken as a demonstration that the two nonidentical monoclonal antibodies are capable of binding to the same small molecule simultaneously; and therefore, these particular antibodies and the same assay procedures can be used to test a collected sample. Antibodies that display a synergistic effect in regard to a specific antigen are often capable of forming an extremely stable complex with that antigen.

The present invention will be illustrated in. more detail with reference to the following examples, which should not be construed as limiting the scope of the invention.

* EXAMPLES

Example 1

Two monoclonal antibodies to a small steroid for use in a sandwich immunoassay can be raised by immunizing one mouse or other suitable animal using the method of Zimmering and co-workers [P.E. Zimmering et al. , Biochemistry, Vol 6 (1967) pp. 154-164] . Once antisera are thus raised, monoclonal antibodies can be produced according to the methods described by H. Zola and D.

Brooks, as previously cited. After monoclonal antibodies are isolated, these antibodies can be tested for simultaneous binding by the assays described in Examples 3 or 4.

OMPI _ -13

Example 2

It is not always possible to raise two different antibodies by the procedures of Example 1; in many cases, two different hapten conjugates must be used as immunogens, since the method of coupling a small hapten may not allow the production of two distinct antioodies that can bind simultaneously. The immunization of one animal with two conjugates would result in the production of two types of antibodies. However, in the case of antisera, the two populations of antibodies may not be separable (or at least considerable effort may be necessary to separate them) . Therefore, the use of two different animals would provide an easy method of keeping the two antibody populations separate. For monoclonal hybridoma antibodies, the two conjugates could be injected into one animal because separation of different types of antibodies would be'effected by cloning of cell lines.

An example of two conjugates of one hapten is aldosterone. Aldosterone -18,21-dihemisuccinyl- bovine serum albumin can be prepared according to the method of Erlanger et al. [Journal of Biological Chemistry, Vol. 228 (1957) pp. 713-727] . Immunization protocols are given in the same article. The production of 3-oxime aldosterone conjugates is described by J.T. McKenzie and J.A. Clements [Journal of Clinical Endocrinology and Metabolism, Vol. 38 (1974) p. 622] . Use of this conjugate as an immunogen is described by T. Ogihara et al. [Journal of Clinical Endocrinology and Metabolism, Vol. 45 (1977) p. 726]. The conjugation of aldosterone by reaction at the 3 carbon and at the 18 and 21 carbons provide two conjugates attached to a carrier molecule at opposite ends of the aldosterone molecule. Thus, the antibodies produced by these conjugates are most likely to be able to

bind simultaneously to aldosterone. Two antisera, one antiserum and one monoclonal antibody, or two monoclonal antibodies raised using these conjugates can be tested for their ability to both bind to aldosterone using the procedures outlined in Examples 3 or 4.

Example 3

An immunoradiometric assay with one solid phase^' antibody and one radiolabelled liquid phase antibody can be carried out as follows: Monoclonal antibodies A and B raised to a small hapten X (as in Examples 1 and 2) are purified from ascites fluid by well-known procedures such as the methods of H. Zola and D. Brooks, cited previously. To test whether antibodies A and B can bind simultaneously to small hapten X, fifty microliters of 0.1 mg/ml antibody A in 0.01 M potassium phosphate, 0.15 M NaCl, 0.02% sodium azide, pH 7.5 is incubated overnight at

4°C in polyvinyl chloride microtiter plate wells. Antibody B is radiolabelled with 125I, by the method

D.M. Klinman and J.C. Howard, as referred to earlier.

Antibody A solution is removed from the microtiter plate, then the wells in the plate are rinsed with distilled water three times and the wells are dried by banging the plates on a paper towel. A sample containing a known quantity of small hapten X is mixed with radiolabelled antibody B, and fifty microliters of the mixture containing approximately 20,000 to 50,000 cpm of raαiolabelled antibody B is added to each microtiter plate well. After an incubation of about 17-24 hours at 20°C, the radioactive solution is aspirated and the wells washed as before. Each well is cut from the plate with a scissor and counted in a gamma counter. Radioactivity attached to a well greater than background counts (background counts would be the amount of radioactivity bound even if small

hapten X was not mixed with antibody B before antibody B was added to the microtiter wells) , depends on the ability of antibodies A and B to bind simultaneously to small hapten X. It is best to use several concentrations of hapten X, because an excess amount of hapten will give background radiation bound to the plastic. If any of the concentrations give higher than background radioactivity, then antibodies A and B can bind hapten X simultaneously.

The presence or unknown concentration of small hapten X in a sample is determined in a similar manner. This sample is mixed with radiolabelled antibody B while antibody A is adsorbed to a solid support, and the subsequent steps listed above are conducted. The amount of radioactivity attached to a well is proportional to the amount of small hapten X in the sample, and its concentration can be judged by comparing this amount of radioactivity bound to the solid support with the amount bound when known concentrations of small hapten X are assayed in this system.

Example 4

A cooperative immunoassay with two liquid phase antioodies starts with fifty microliters of radiolabelled small hapten X and fifty microliters of a sample containing the unlabelled small hapten to be measured (both in 1% horse serum, 99% 0.01M potassium phosphate, 0.15 M NaCl, 0.02% sodium azide, pH 7) are mixed with 100 microliters 0.3M potassium phosphate (pH 7.5) .

Subsequently, 100 microliters of a mixture of two antioodies that can bind simultaneously are added (the mixture of antibodies is also diluted in 1% horse serum, 99% phosphate buffered saline) . The ability of the two antibodies in the mixture to bind simultaneously can be

O H established by showing the higher affinity in a radioimmunoassay of the mixture for a known quantity of small hapten X than either antibod separately, or alternatively, by the ability to use these antibodies in a sandwich assay, in accordance with the technique described in the Ehrlich et al. Journal of Immunology article. The antibody mixture is composed as follows: the amount of each antibody that had previously been determined to bind 50% of radiolabelled small hapten X in the absence of unlabelled small hapten X by the procedure described here (wherein each individual antibody replaces the mixture of antibodies) are mixed in a 1:1 ratio. The dilution of this mixture that had previously been determined to bind 30% to 50% of radiolabelled small hapten X by the procedure described here in the absence of any unlabelled small hapten X is then used for the assays with unlabelled small hapten X.

The antibodies, buffers and radiolabelled and unlabelled small hapten X are incubated one hour at 37°C followed by 18 hours at 5°C. Ten microliters of 50% normal mouse serum in phosphate buffered saline is then added to each assay tube. An appropriate amount of rabbit anti-mouse IgG antiserum is then added. (The appropriate amount is determined by performing the described assay with no unlabelled small hapten X and varying amounts of rabbit anti-mouse IgG antiserum. The amount of antiserum that precipitates the highest amount of radioactivity is the appropriate amount.) The tubes are incubated 10 minutes at 37°C and then one hour at room temperature. The precipitate is sediraented by centrifugation, the supernatant is aspirated, and the radioactivity in the precipitate is counted and is proportional to the amount of small hapten X present in the sample, that is, the lower the count, the higher the concentration of small hapten X present in the sample.

OMPI The invention described and claimed herein is not to be limited by the examples described above or by the particular small molecules, both small i munogens and small haptens, listed in the present application, since these examples and choices are merely intended as illustrative of aspects of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the invention which is intended to be limited only in accordance with the appended claims.

Claims

WE CLAIM:

1. A method for performing a two-site immunoassay to determine the presence or concentration of a small molecule in a sample, comprising:

(a) contacting such sample with an effective assaying amount of a first antibody to such small molecule;

(b) contacting such sample with an effective assaying amount of a second antibody to such small molecule, such effective assaying amount of the first and second antibody being sufficient to form a first antibody:small molecule:second antibody complex; and (c) detecting the presence of or measuring the quantity of first antibody:small molecule:second antibody complexes formed.

2. A method according' to claim 1, wherein the first and second antibodies are nonidentical.

3. A method according to claim 1, wherein the small molecule is a small hapten.

4. A method according to claim 1, wherein the molecular weight of the small molecule is greater than 250 and less than 2500-3000.

5. A method according to claim 1, wherein the molecular weight of the small molecule is greater than 275 and less than 1000.

6. A method according to claim 1, wherein the molecular weight of the small molecule is greater than 275 and less than 500.

^*^JR£Λ»

O PI l^ WIPO A_*]

7. A method according to claim 1, wherein the small molecule is a steroid.

8. A method according to claim 7, wherein the steroid is aldosterone.

9. A method according to claim 7, wherein the steroid is cortisone.

10. A method according to claim 7, wherein the steroid is estradiol.

11. A method according to claim 7, wherein the steroid is progesterone.

12. A method according to claim 7, wherein the steroid is testosterone.

13. A method according to claim 1, wherein the small molecule is a mycotoxin.

14. A method according to claim 13, wherein the mycotoxin is an aflatoxin.

15. A method according to claim 13, wherein the mycotoxin is a trichothecene.

16. A method according to claim 1, wherein the small molecule is a small peptide.

17. A method according to claim 16, wherein the small peptide is aspartame.

18. A method according to claim 1, wherein the small molecule is an antibiotic.

19. A method according to claim 18, wherein the antibiotic is a gentimicin.

20. A method according to claim 18, wherein the antibiotic is penicillin.

21. A method according to claim 1, wherein the small molecule is a small hormone.

22. A method according to claim 21, wherein the small hormone is triiodothyronine.

23. A method according to claim 21, wherein the small hormone is lutenizing hormone-releasing factor (LH-RH) .

24. A method according to claim 1, wherein the small molecule is merphyrin.

25. A method according to claim 1, wherein at least one of such first or second antibodies has a label providing a detectable signal, such signal is a member of the group consisting of a radioactive isotope, enzyme, fluorescent compound, chemiluminiescent compound, ferromagnetic atom or particle.

26. A method according to claim 1, wherein

(a) an aliquot of a small molecule having a label providing a detectable signal, such signal is a member of the group consisting of a radioactive isotope, enzyme, fluorescent compound, chemiluminescent compound, ferromagnetic atom or particle, is contacted with effective assaying amounts of the first and second antibodies, and after

first antibody:small molecule:second antibody complexes are formed, these complexes are separated from those antibodies and small molecules which are not a part of these complexes;

(b) an aliquot of a small molecule, having a label as in step (a) , is mixed with an assay sample and is contacted with effective assaying amounts of the first and second antibodies, and after first antibody:small molecule:second antibody complexes are formed, these complexes are separated from those antibodies in addition to those labelled and unlabelled small molecules which are not a part of these complexes;^, and

(c) the intensity of signal detectable from the complexes of step (b) is compared with the intensity of signal detectable from the complexes of step (a) , a decrease in the intensity of the signal in step (b) indicating the presence of the small molecule in the assay sample and is inversely proportional to the concentration of the small molecule in the assay sample.

27. A method according to claim 2, wherein the nonidentical first and second antibodies comprise at least two monoclonal antibodies.

28. A method according to claim 2, wherein the nonidentical first and second antibodies comprise one polyclonal antisera and at least one monoclonal antibody.

29. A method according to claim 2, wherein the nonidentical first and second antibodies comprise two nonidentical polyclonal antisera.

30. A first antibody to a small molecule and a second antibody to a small molecule, wherein the first and second antibody can form a first antibody:small molecule: second antibody complex.

31. A first antibody and a second antibody according to claim 30, wherein the first and second antibodies are nonidentical.

32. A first antibody and a second antibody according to claim 31, wherein the nonidentical first and second antibodies comprise two monoclonal antibodies.

33. A first antibody and a second antibody according to claim 31, wherein the nonidentical first and second antioodies comprise a polyclonal antisera and monoclonal antibody. _>

34. A first antibody and a second antibody according to claim 31, wherein the nonidentical first and second antibodies comprise two nonidentical polyclonal antisera.

35. A method for raising at least one first antibody to a small molecule and at least one second antibody to a small molecule, wherein the first and second antibodies are nonidentical and can form a first antibody: small molecule:second antibody complex, by immunizing at least one animal with a first conjugated small molecule and by immunizing at least one other animal with a second conjugated small molecule, wherein the first and second conjugated small molecules are constructed by conjugating the small molecule at different sites on the small molecule.

O PI

36. A method for raising at least one first antibody to a small molecule and at least one second antibody to a small molecule, wherein the first and second antibodies are nonidentical monoclonal antibodies and can form a first antibody:small molecule:second antibody complex, by immunizing one animal with a first and a second conjugated small molecules, wherein the first and second conjugated small molecules are constructed by conjugating the small molecule at different sites on the small molecule.