GB1591204A - Preparation of immobilised antigens and antibodies - Google Patents

Description

(54) PREPARATION OF IMMOBILISED ANTIGENS AND ANTIBODIES (71) We, GIST-BROCADES N.V., a Dutch Body Corporate, of 1, Wateringseweg, Delft, Holland, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to a process for the preparation of an antigen or antibody on a glass carrier, to the antigen or antibody on the carrier and to its use in antibody or antigen assay by the enzyme-linked-immunological technique.

The invention concerns a new method of assay of antigen or antibody by the enzymelinked-immunological technique, which is designed to assay antigens (including substances that can be haptens) and antibodies of both low and high molecular weight; more specifically, polypeptide hormones of the endocrine glands such as the pituitary, pancreas, parathyroid, or mucosa of the digestive tract; non-proteinic steroid hormones; other thyroid hormones such as tyrosine derivatives; serum immunoglobulins and albumin; other proteins such as a-fetoprotein and urinary albumin; or HBs antigen, complement, carcinoembryonic antigen, and various types of enzymes in organs and blood; and typically, the pituitary hormones, insulin, immunoglobulins and liver enzymes.

The pituitary gland, in particular, which is connected to the so-called funnel at the bottom of the third ventricle in the hypothalamus, serves as the centre for the endocrine system.

Secreted from the anterior lobe of the gland are thyroid-stimulating hormone (TSH), growth hormone (GH), adrenocorticotroPic hormone (ACTH), concerned in metabolic control, follicle-stimulating hormone (FSH), luteinizing hormone (LH), luteotropic hormone (LTH) and lactogenic hormone (prolactin) involved in sex function; from the intermediate lobe, aand p-melanocyte-stimulating hormones which expand melanophores to darken the colour of the skin; and from the posterior lobe, oxytocin and vasopressin of the antidiuretic hormone (ADH). These, like other hormones in the body, play a major role in the mutual regulation of metabolism of various organs. A study of the variation in the blood level of pituitary hormones or on their behaviours following administration of different drugs or hormones therefore, is of great clinical importance both for accurate diagnosis of various endocrine diseases and precise appraisal of their therapeutic responses and provides a new pituitary function test by estimating the variation in the blood level of pituitary hormones. Insulin, which is secreted from p cells in islets of Langerhans of the pancreas, acts on the cell membrane to increase its permeability for sugar and reduce the concentration of sugar in blood, and operates competitively against the actions of glucagon secreted by a cells.

Consequently, an assay of insulin is also of obvious clinical importance, being useful in investigating the aetiology and pathologic physiology of diabetes mellitus as well as for the treatment of the illness and diagnosis of insulinoma. Immunoglobulin, which exists in blood serum, is a generic term for globulin proteins possessing antibody activity, and is the substance that protects animals from infections. There are five different types of immunoglobulin as classified according to chemical structure, physico-chemical properties and biological activity: IgG, IgA, IgM, IgD and IgE. An assay of these immunoglobulins is also of major significance in assessing the defensive ability of the organism.

Two different techniques are widely known at present for the assay of the hormones and other substances referred to above: biological and immunochemical. The former is rarely used clinically because of its general low sensitivity and the unsatisfactory objectivity and reproducibility of data obtained, in addition to troublesome procedures required by the use of animals or their organs. The latter technique is further classified into so-called radioimmunoassay (referred to hereeinafter as RIA) which utilizes an antiserum (antibody) prepared with a substance to be assayed (e.g. hormone) as antigen and an antigen labelled with a radioisotope; and an assay by the enzyme-linked-immunological technique (referred to hereinafter as EIA), which makes use of an enzyme for labelling antigens or antibodies in place of a radioisotope. Both these techniques are highly useful with a combination of two different advantages: the specificity of antigen-antibody reaction occurring between the antigen or antibody to be assayed and a corresponding antibody or antigen, and the feasibility of microdetermination of radioactivity or the reaction product of the enzyme.

The RIA technique, which is now predominantly used in the micro-determination of polypeptides, peptidic hormones, serum proteins etc., has nonetheless been put to practical use only to a limited extent because of a variety of serious disadvantages resulting from the use of radioisotopes, e.g., the risk of the human body being injured by radioactivity, the necessity for special equipment as countermeasures against public injury by radioactivity and for an expensive apparatus for counting radioactivity, the complicated, troublesome, timeconsuming procedure involved, and the half-life of certain radioisotopes which is too short to permit long-term preservation of an antigen labelled therewith.

The EIA technique is the one developed a few years ago, and its characteristics, together with the mechanism of reactions involved and some examples of application were previously expounded by the inventor and his associate. ["Enzyme-linked Immunoassay" by K. Kato and E. Ishikawa, Procedures of Immunologic Experiments IV, edited by the Japanese Immunologic Society, p. 1137-1152, 1975 (referred to hereinafter as Reference (1))].

EIA is one of a group of binding assays in which the molecule recognition properties of antibodies are used. An enzyme is used as label and in the immunoassay the labelled component of an antibody/antigen reaction binds to its complementary binding site. The amount bound depends on the concentrations of the other components of the system and if one of these is varied this causes a change in the distribution of the labelled component between the bound and unbound fractions. The properties of the label are used to determine its distribution, and thus a calibration curve can be constructed relating concentration of the label to the concentration of the component varied.

In the competitive coupling technique, labelled and unlabelled antibody compete for solid-phase antigen. The sandwich technique requires the antigen to have at least two binding sites. Antigen reacts with excess solid-phase antibody, and after incubation followed by washing, the bound nantigen is treated with excess labelled antibody. After further washing the bound label is assayed, and this provides a direct measure of the amount of antigen present.

In principle, the EIA technique resembles RIA, being based on the specificity of antigenantibody reaction. However, it is completely free from the above-mentioned faults of the RIA technique since it utilizes an enzyme instead of a radioisotope as the label. Polysaccharides such as cellulose, Sephadex or Sepharose 4B are known as the carrier so far used for this technique. "Sephadex" and "Sepharose" are registered Trade Marks. Such a carrier is often employed with an antibody combined therewith by chemical or covalent bonding through the action of a bifunctional binding agent. Sometimes, a polystyrene tube with an antibody adsorbed on its surface is used. In general, this technique has the disadvantage of involving an extremely complicated procedure of solid phase washing for removal of substances unreacted and also of having its accuracy and reproducibility lowered by markedly deteriorated quantification at low concentrations of a substance being assayed. This may result from an abnormal rise in blank values due to the enzyme-labelled antibody or antigen being non-specifically bound to the carrier or the inner wall of the receptacle for assay during the antigen-antibody reaction with the substance being assayed.

Accordingly, the EIA technique, despite its advantages over RIA, has rarely been put to practical use in the field of clinical testing, which requires a prompt, convenient and accurate assay of a number of specimens under ordinary laboratory conditions.

As a result of research and experimentation there has been found a new method of assay of antigens and antibodies by the enzyme-linked-immunological technique, which differs from previously available techniques, in employing a glass carrier treated with an organic silane derivative as a solid phase and hence is simple, convenient and highly accurate and reproducible. In place of the above-mentioned carriers previously used, the inventor used glass treated with an organic silane derivative on its surface, to which an antibody or antigen (or any other substance that can be a hapten in aqueous solution was fixed by physical adsorption on the solid phase without using a special binder. This led to substantial inhibition of the abovementioned non-specific binding to the carrier or the inner wall of the assay tube by an enzyme-labelled antibody or antigen, with resultant marked improvement in sensitivity, accuracy and reproducibility. Furthermore, the entire process was significantly simplified, because the step of centrifugation during solid phase washing became completely unneces sary. The washing step, with previous techniques, was usually repeated several times for each specimen in order to remove unreactive substances in the reaction mixture. This invention is the first to embody the concept of physical adsorptive immobilization of antibody or antigen on the surface of silane derivative-treated glass. It is highly useful, as no special binder is required for the immobilization, in minimizing the loss of the activity of bound antibody or antigen as well as sparing reagents. The glass used for this purpose may be, for example of such a composition was is ordinarily used in the laboratory. It may be of any convenient shape, for example cylindrical, globular, flattened or granular. A porous glass is disadvantageous, because it tends to enhance the above-mentioned non-specific binding. The glass is treated with an organic silane derivative which can react with and be bound to the surface of glass. Desirable for this purpose are silane derivatives with a silicon-alkoxy or -halogen bond, for example, in the molecule, when viewed in terms of the reaction with the surface of glass.

Those derivatives with a silicon-alkylamino, carboxyalkyl, mercaptoalkyl; hydroxyalkyl or carboalkoxyalkyl bond, for example, in the molecule are desirable, when viewed in terms of the relation to the antibody or antigen in question: it will be appreciated that, as the antigen or antibody is physically adsorbed on the silane derivative-treated glass, suitable groups are those which do not promote reaction with the antigen or antibody. In treating the surface of glass with such an organic silane derivative, all generally known methods of treatment for coupling may be used. It has been found to be possible to use an antigen or antibody in the solid phase adsorbed to a carrier easy to treat and handle, namely, silane derivative-treated glass. The invention enables the use in the solid phase of the antigen or antibody in question without any substantial loss of its reactivity by utilizing as the carrier glass which is treated on the surface with for example, an amino-, carboxy-, hydroxy-, or mercaptosilane derivative, as required.

In addition to antiserum prepared by the routine procedure, antibody isolated from antiserum, fragment F(ab')2 obtained by digesting antibody with pepsin, or fragment F(ab') resulting from treatment of F(ab')2 with a reducing agent such as 2-mercaptoethylamine may be used as the antibody to be fixed in the solid phase. Antibody in the solid phase thus obtained can be used in EIA by the competitive coupling or sandwich technique described in the above-mentioned Reference (a) elaborated by the inventor and even in an assay of antibody with antigen fixed in the solid phase, which is not mentioned in the Reference (1), in order to estimate antigens and antibodies of high molecular weight such as enzymes or proteins in organs or blood serum or those of middle or low molecular weight, notably hormones.

The enzymes that may be used for the purpose of the invention to label antigens or antibodies include peroxidase (HRP) in routine use in this field, alkaline phosphatase from the intestinal mucosa of animals or produced by E. coli, p-D-galactosidase produced by the same bacterium, glucose oxidase produced by fungi, and lysozyme from egg white. Of these, p-D-galactosidase produced by E. coli is the most satisfactory in view of the stability of the enzyme itself and easy availability of standard products of high specific activity and purity in addition to the high sensitivity with which its reaction products can be assayed.

The assay method of the invention is of great clinical significance, since, free from difficulties involved in handling of radioactive substances as in the RIA technique, it enables clinical laboratory tests to be performed conveniently with satisfactory accuracy and sensitivity in a short period of time even in laboratories equipped with no more than routing appliances for assay, e.g., an apparatus for the assay of reaction products of enzymes.

The present invention also provides a process for the preparation of a composite comprising an antigen or antibody on a glass support which comprises treating the glass surface with an organic silane derivative and then contacting the antigen or antibody in aqueous solution, with the treated glass surface, preferably with a piece or particles of glass to provide plysical adsorption of the antigen or antibody to the treated surface of the glass.

Preferred organic silane derivatives are of the general formula: (R2-Alk)4-flSi(R i)n I wherein R1 represents a halogen atom or an alkoxy group with at most six carbon atoms, R2 represents an amino, carboxy, mercapto, hydroxy or carboalkoxy group, Alk represents an alkylene group with at most six carbon atoms and n is 1, 2 or 3, or are aminoalkyl silanes of the general formula: H2N-Alk-Si(OR)3 II wherein R represents an alkyl group with at most six carbon atoms and Alk is as hereinbefore defined. It is to be understood that alkyl groups or moieties and alkylene groups in the accompanying specification and claims may be straight- or branched-chain.

The symbol R in the aminoalkylsilanes of formula II preferably represents an alkyl group with at most three carbon atoms. 3-Aminopropyltriethoxysilane is most preferred.

The immobilised antigens or antibodies in which the antigen or antibody is physically adsorbed on an organic silane derivative-treated glass surface, preferably to a piece or particles of glass, which may be prepared by the processes hereinbefore described constitute a further feature of the present invention.

It is to be understood that the term antigen as used in this specification and the accompanying claims includes any substance that can be a hapten.

The following Examples illustrate the invention.

EXAMPLE 1 Assay of human immunoglobulin G (hIgG) (I) (A Reagents and preparation of their solutions: 1) Buffer Solution A: 0.01M phosphate buffer, pH 7.0, containing 0.1%bovine serum albumin (BSA), 0.1 %NaN3, lmM MgCl2 and 0. 1M NaCl.

2 Buffer Solution B: 0.25M sodium phosphate buffer, pH 7.5.

3 Preparation of glass rods with rabbit (anti-hIgG) antibody IgG fixed in the solid phase: A commercial Pyrex rod (3mizen in diameter, 5mm in length) heated at 5000C for 5 hours was soaked in a 2% solution of 3-aminopropyltriethoxy silane (Nakarai Chemical Co., Ltd.) in acetone at 450C for 24 hours, and then taken out of the acetone solution, washed in purified acetone, and then dried. "Pyrex" is a registered Trade Mark. The glass rod with its surface so coated with aminoalkylsilyl radical was washed again in Buffer Solution B, then soaked for 30 minutes in the same buffer solution containing 2.0 mg/ml of commercially available rabbit (anti-hIgG) antibody IgG fraction (Miles Laboratories, Inc. U.S.A.), and then kept overnight at 40C. The resulting glass rod with rabbit (anti-hIgG) IgG fixed in solid phase was washed -and then soaked for 2 hours in Buffer Solution A, and then kept at 4"C until the time of use.

(4) Preparation of Sepharose 4B with rabbit (anti-hIgG) antibody IgG fixed in the solid phase: The rabbit (anti-hIgG) antibody IgG was adsorbed on commercial CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals AB, Sweden) in accordance with the procedure given in Reference (1) by the inventor.

(5) Preparation of rabbit (anti-hIgG) antibody-(Fab')- p-D-galactosidase complex: Preparation of antibody Fab' .against rabbit (anti-hIgG) IgG by pepsin digestion -and reductive treatment, and preparation and isolation of a complex of the Fab' conjugated with p a D-galactosidase from E. coli as the labelling enzyme (Boehringer Mannheim GmbH, West Germany) were carried out according to the procedures given in Reference (1) and in Eiji Ishikawa, J. Immunology, 116 (No. 6), 1554-1560 (1976), (hereinafter referred to as Reference 2).

(6) Preparation of standard hIgG solution for standard curves: Commercial hIgG (Miles Laboratories, Inc. U.S.A.) was diluted with Buffer Solution A to prepare 9 different standard solutions in a concentration range of 4 x 1 0- 17- - 1 X 10- 14 mol/ 5 0 1.

(7) Reagent and method used for the determination of enzyme activity: The activity of p-D-galactosidase bound as described in (5) above was determined by the techniques described in References (1) and (2). A fluorescence spectrophotometer (MPF-4 type) (Hitachi) was used to estimate the intensity of fluorescence of 4-methyl-umbelliferone released from 4-methylumbelliferyl-p-D-galactoside (Sigma Chemical Co. U.S.A.).

(B) Assay procedure and results obtained: A glass rod with rabbit (anti-hIgG) IgG fixed in the solid phase was placed in a small test tube containing 100 CL1 of Buffer Solution A and, after addition of 50 ,a1 of standard hIgG solution, incubated at 370C for 4 hours with shaking and then kept overnight at 40C. The glass rod was washed twice with 1 ml of Buffer Solution A and, after addition of 150 ILl of the same buffer solution containing rabbit (anti-hIgG) antibody (Fab')-p-D-galactosidase complex, incubated at 370C for 6 hours with shaking. Then the glass rod was washed twice again with 1 ml of Buffer solution A and then transferred using tweezers to another small tube, where it was preincubated at 30"C for 5 minutes with 100 ILl of Buffer Solution A added in preparation for the subsequent determination of enzyme activity. The same procedure in accordance with References (1) and (2) was applied to rabbit (anti-hIgG) IgG-coupled Sepharose 4B. For the glass rod and Sepharose 4B so incubated, determination was made of the enzyme activity of p-D-galactosidase bound to the carrier in the solid phase. As a result, the standard curves shown in Figure 1 were obtained by plotting the measurements on a logarithmic scale. In Figure I the ordinate gives p-D-galactosidase activity bound (units/ assay tube) and the abscissa gives human IgG (femto mol/ assay tube). Here, standard curve (a) [marked with open circles] represents the enzyme activity with conventional Sepharose 4B used as the carrier, and standard curve (b) [marked with closed circles] that with a glass rod used as such in place of Sepharose 4B. As is apparent from the curves, there was a marked improvement in the sensitivity of assay with the latter technique, whereby even as low a concentration of hIgG as 0.27 femto mol is well determined in contrast to the former technique which is unable to measure concentrations below 0.6 femto mol because of very high blank values due to the nonspecific binding (adsorption) to the inner wall of the tube of the antibody labelled with the enzyme. Then, an estimate was made of the hIgG content of a 10 8 dilution of human serum with Buffer Solution A by using calibration (standard) curve (b).

As a result, a value of 4.1 x 10-l7 mol per 50 IL1 of diluted specimen was obtained, a value which corresponds well to the hIgG content of normal human serum.

EXAMPLE 2 Assay of pig insulin (p Ins.) (A) Reagents used.

(1) Buffer Solutions A and B: prepared as described in Example 1 (A) (1) and (2).

(2) Preparation of a glass rod with guinea-pig (anti-p Ins.) antibody IgG in the solid phase: The technique for the fractionation of IgG was similar to that given in Reference (1).

Commercial guinea-pig (anti-p Ins.) serum (Miles Laboratories, Inc. U.S.A.) was subjected to the fractionation of y-globulin with Na2SO4 (Kekwick, R.A.: Biochem. J. 34, 1248-1257 (1940)] and then to DEAE-cellulose column chromatography [Levy, H.B. and Sober, H.A.: Proc. Soc. Exp. Biol. Med. 103, 250-252 (1960)] to obtain approximately 75 mg of IgG fraction by processing 10 ml of guinea-pig antiserum. Then the guinea-pig (anti-p Ins.) IgG was added to Buffer Solution B in a concentration of 2.5 mg/ml to fix it on an aminosilanetreated glass rod in accordance with the technique described in Example 1 (A) (3).

(3) Preparation of p-D-galactosidase-p Ins. complex: The technique given in Reference (1) was used. An eluate from a Sepharose 6B column (1.5 x 40 cm) with a maximal enzyme activity in the neighbourhood of its peak was diluted 10-fold with Buffer Solution A before using it as spins. - enzyme complex.

(45 Preparation of standard p Ins. solution: Commercial p Ins. ("Antrapid", 40 U/ml, Novo Industri A/S, Denmark) was diluted with Buffer Solution A to prepare 8 different standard solutions in a concentration range of 0.1-300 U/ 20 ul.

(5) Reagent and method used for the determination of the activity of p-D-galactosidase so bound: as in Example 1(A) (7).

(B) Assay procedure and results obtained: A glass rod with guinea-pig (anti-p Ins.) IgG fixed in the solid phase was placed in a small test tube containing 110 CL1 of Buffer Solution A and, after addition first of 20 IL1 of p Ins.

solution (standard solution as used here) and then of 20 CL1 of p Ins. - enzyme complex, incubated at 30"C for 2 hours with shaking. Then the glass rod was washed several times with 1 ml of Buffer Solution A and transferred to another small tube of the same shape, where it was preincubated at 30"C for 5 minutes with 100 it1 of Buffer Solution A. A determination was then made by the method employed in Example 1 of the enzyme activity of pa D-galactosidase thus bound. The results obtained are shown in Figure 2 (in which a fluorescence intensity of 100 is equivalent to 1 x 10-7 mol of 4-methylumbelliferone). In Figure 2 the ordinate gives p-D-galactosidase activity bound (fluorescence intensity) and the abscissa gives porcine insulin (,u-units/assay tube). It was shown that the invention which utilizes a glass rod as the carrier even when applied in connection with EIA by the competitive coupling technique, yield no less satisfactory standard curves both quite simply and within a short period of time when compared with previously available methods.

EXAMPLE 3 Assay of human immunoglobulin G(hIgG) (II) The basic procedure described in Example 1 was used here, except that in step (A) (3) of the treatment the glass rod was not treated with 3-aminopropyltriethoxysilane. A commercial Pyrex glass rod heated at 500"C for 5 hours and then allowed to cool was used as the carrier which was soaked in a solution of rabbit (anti-hIgG) IgG (2mg/ml) to fix the latter substance onto the solid phase. An enzyme-linked-immunoassay was then made by the sandwich technique, as in Example 1, using a standard solution containing 4.9 femto mol per 50,ul of hlgG along with 2,050 units of rabbit (anti hIgG) antibody (Fab')-p-D-galactosidase com plex. The activity of the enzyme so bound was found to be as follows: Blank interval (hIgG not coexistent): 6.7 (units/tube) hIgG interval : 49.4 (units/tube) The results obtained are less satisfactory than in Example 1, the values cited above being approximately 68 per cent of those in Example 1. The method used here, however, proved to have a much higher sensitivity than methods utilizing Sepharose 4B as the carrier.

WHAT WE CLAIM IS: 1. Process for the preparation of a composite comprising an antigen or antibody on a glass support which comprises treating the glass surface with an organic silane derivative and then contacting the antigen or antibody in aqueous solution with the treated glass surface to provide physical adsorption of the antigen or antibody to be treated surface of the glass.

2. Process according to claim 1 in which the glass support is in the form of a piece or particles of glass.

3. Process according to claim 1 or 2 in which the silane derivative is of the general formula: (R2-Alk)4.11Si(R1)fl I wherein R, represents a halogen atom or an alkoxy group with at most six carbon atoms, R2 represents an amino, carboxy, mercapto, hydroxy or carboalkoxy group, Alk represents an alkylene group with at most six carbon atoms and n is 1,2 or 3.

4. Process according to claim 1 or 2 in which the silane derivative is an aminoalkylsilane of the general formula: H2N-Alk-Si(OR)3 II wherein R represents an alkyl group with at most six carbon atoms and Alk is as defined in

Claims (1)

1. claim 3.

   5. Process according to claim 4, in which R is an alkyl group with at most 3 carbon atoms.

   6. Process according to claim 5, in which the aminoalkylsilane is 3-aminopropyltriethoxysilane.

   7. Process for the preparation of a composite comprising an antigen or antibody physically adsorbed on a glass support treated with an organic silane derivative, substantially as hereinbefore described.

   8. Process for the preparation of a composite comprising an antigen or antibody physically adsorbed on a glass support treated with an organic silane derivative, substantially as hereinbefore described with especial reference to Example 1 or 2.

   9. A composite comprising an antigen or antibody prepared by a process claimed in any of claims 1 to 8.

   10. Immobilized antigen or antibody, wherein the antigen or antibody is physically adsorbed on an organic silane derivative-treated glass surface.

   11. Immobilized antigen or antibody according to claim 10, wherein the glass is in the form of a piece or particles of glass.

   12. Immobilized antigen or antibody according to claim 10 or 11, wherein the silane derivative is of the general formula: (R2-Alk)4 nSi(Rl)n wherein R1 represents a halogen atom or an alkoxy group with at most six carbon atoms, R2 represents an amino, carboxy, mercapto. hydroxy or carboalkoxy group, Alk represents an alkylene group with at most six carbon atoms and n is 1,2 or 3.

   13. Immobilized antigen or antibody according to claim 10 or 11, wherein the silane derivative is an aminoalkylsilane of the general formula: H2N-Alk-Si(OR)3 in which R represents an alkyl group with at most six carbon atoms, and Alk is as defined in claim 12.

   14. Immobilized antigen or antibody according to claim 13, wherein R is an alkyl group with at most three carbon atoms.

   15. Immobilized antigen or antibody according to claim 14, wherein the aminoalkylsilane is 3-amino-propyltriethoxysilane.

   16. Immobilized antigen or antibody wherein the antigen or antibody is physically adsorbed on an organic silane derivative-treated glass surface, substantially as hereinbefore described.

   17. Immobilized antigen or antibody wherein the antigen or antibody is physically adsorbed on an organic silane derivative-treated glass surface, substantially as hereinbefore described with especial reference to Example 1 or 2.

   18. An immunoassay method using an immobilised antigen or antibody as claimed in any of of claims 9 to 17.

   19. An enzyme-immunoassay method using an immobilised antigen or antibody as claimed in any one of claims 9 to 17.

   20. A method according to claim 19 in which antibody isolated from antiserum, fragment F(ab')2 obtained by digesting antibody with pepsin, or fragment F(ab') resulting from treatment of F(ab') 2with a reducing agent is used as the antibody.

   21. A method according to claim 20 in which the reducing agent is 2-mercaptoethylamine.

   22. A method according to claim 19, 20 or 21 in which the competitive coupling or the sandwich technique is used.

   23. A method according to any one of claims 19 to 22 in which an assay of antibody with antigen fixed in the solid phase is used to estimate antigens and antibodies of high molecular weight or those of middle or low molecular weight.

   24. A method according to claim 23 in which the antigens and antibodies of high molecular weight are enzymes or proteins in organs or blood serum and those of middle or low molecular weight are hormones.

   25. A method according to any one of claims 19 to 24 in which the enzyme used to label antigen or antibody used in the enzyme-immunoassay method is peroxidase, alkaline phosphatase, p-D-glactosidase, glucose oxidase or lysozyme.

   26. A method according to claim 25 in which the enzyme is p-D-galactosidase.

   27. An immunoassay method according to claim 18 or 19 substantially as hereinbefore described.

   28. An immunoassay method according to claim 18 or 19 substantially as hereinbefore described with especial reference to Example 1 or 2.