GB1591660A - 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 silicone 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 enzyme-linked-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-tetoprotein 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, a- and (3- 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 (3cells 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. On the other hand, ornithine 5-aminotransferase (referred to hereinafter as OATase) from rat liver catalyzes the metabolic conversion between ornithine and a-ketoglutaric acid in animal tissue, and plays also an important role in the main function of liver.

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 hereinafter 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, time-consuming 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 EIA is one of a group of binding assays in which the molecular 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 the incubation followed by washing, the bound antigen 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 antigen-antibody 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.

This technique has the disadvantage of involving an extremely complicated procedure of solid phase washing for removal of unreacted substances and of removal of the supernatant from the centrifugation procedure, and also of having its accuracy and reproducibility lowered by markedly deteriorated quantification at low concentrations of a substance being assayed, which may result from an abnormal rise in blank values due to the enzymelabelled-antibody or antigen being nonspecifically bound to the carrier or the inner wall of the assay tube while taking part in the antigen-antibody reaction with the substance being assayed.

In another method, sometimes, antibody and antigen are adsorbed onto a polystyrene tube as the carrier and further the polystyrene tube with the antibody or the antigen adsorbed thereto is used as a receptacle of the assay. In this case, the material cost of polystyrene is generally high and therefore such material is unsuitable for throwing away after using it, and further in the case of polystyrene tube, it is very difficult practically to standardise the space for the reaction. Non-specific binding of the enzyme-labelledantibody or antigen to the carrier or the inner wall of the receptacle mentioned above, is in proportion to the volume for the reaction. Reaction medium, particularly, for the antigen-antibody reaction is incubated generally with stirring. This technique has disadvantages as, for example, the volume of enzyme-labelled antibody or antigen non-specifically bound to the carrier or the inner wall of the receptacle mentioned above in each receptacle for assay is very difficult to keep constant owing to e.g. differences in the conditions for stirring, and therefore, its accuracy and reproducibility is much lowered.

Sensitivity for assay in the case of polystyrene tubes is at most about 3 x 10-1 to 3 x mole. 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 a new, simple, satisfactorily accurate, sensitive and reproducible enzyme-linked-immunological technique for the assay of antigen or antibody has been discovered using silicone (including other materials treated with silicone) as the carrier in place of those so far known. More precisely, the inventor replaced the above-mentioned known carriers with ordinary silicone support material or a piece of silicone support material treated on the surface with a silane derivative, to which carrier antibody or antigen was bound in the solid phase (by adsorption) without-the aid of a binder. This led to pronounced inhibition of the phenomenon of non-specific adsorption by the above-mentioned enzyme-labelled complex, while enabling antibody or antigen to be fixed in the solid phase on a piece of silicone both in a constant quantity and in an extremely stable state, with a consequent marked improvement in the sensitivity, accuracy and reproducibility of assay. Furthermore, the entire process of assay was greatly simplified since it was now completely unnecessary to centrifuge each specimen several times during solid phase washing for the removal of the unreacted antigen, antibody or enzyme-labelled complex from the product of antigen-antibody reaction, as had usually been the case before.

The idea of fixing antibody or antigen in the solid phase to silicone carrier in EIA is unprecedented and originates with the invention. Simple, convenient, easy immobilization of antibody or antigen made feasible by the use of highly plastic silicone material is of great significance in minimizing the loss of the activity of antibody or antigen during the process of immobilization.

Silicone used in the invention may be, for example as is ordinarily used in the laboratory, or alternatively any suitable carrier, e.g. certain kinds of plastics, quartz, glass, ceramics, paper, metal or other materials with a similar surface treated with a commercial silicone coating agent or silicone oil may be used.

The silicone support material or silicone-coated carrier may be in any convenient shape, e.g. cylindrical, globular, flattened or granular in shape, but is most efficient when used in pieces (small fragments). A porous or tubular carrier serving as the receptacle as well is inappropriate because of a tendency to augment the above-mentioned non-specific adsorption and the variability of the amount of antibody or antigen adsorbing. Silicone of such different shapes can usually ensure easy immobilization of antibody or antigen, even when used without special treatment.

It is generally advantageous, however, if its surface is treated as required with aminoalkylsilane or other silane derivatives, either alone or in combination with other chemical binders such as glutaraldehyde.

In addition to antiserum prepared by routine methods, antibody isolated from antiserum, F(ab')2 fragment obtained by pepsin digestion of antibody or Fab' fragment resulting from treatment 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 utilized in EIA by the competitive coupling or the sandwich technique described in the abovementioned Reference (1) written by the inventor and his associate or even in an assay by the converse technique, i.e., that of antibody with antigen used in the solid phase, for the purpose of assaying antigens or antibodies of high molecular weight, such as enzymes or proteins, or those of middle or low molecular weight, such as hormones, present in organs or serum.

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, -D-galactosidase produced by the same bacterium, glucose oxidase produced by fungi, and lysozyme from egg white. Of these -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 routine 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 immobilised on a solid support which comprises contacting the antigen or antibody in aqueous solution with a silicone surface, preferably with a piece or particles of a silicone support material or with a piece or particles of a carrier having a silicone surface, to effect a physical adsorption of the antigen or antibody to the silicone surface.

Preferably the silicone surface is treated with a silane before contact with the antigen or antibody. The silane derivative is preferably an aminoalkylsilane, for example of the general formula: H2N-Alk-Si(OR)3 wherein Alk represents an alkylene group containing at most six carbon atoms and R represents an alkyl group containing at most six, preferably at most three, carbon atoms.

3-Aminopropyltriethoxysilane is most preferred. It is to be understood that alkyl and alkylene groups in the accompanying specification and claims may be straight-or branched-chain.

After treatment with the silane derivative the silicone surface may, if desired, be treated with another chemical binder such as glutaraldehyde.

The immobilised antigens or antibodies in which the antigen or antibody is physically bound to a silicone surface, preferably to a piece or particles of a silicone support material or to a piece or particles of a carrier having a silicone surface 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 ornithine o-aminotransferase (OATase) from rat liver and 2,4-dinitrophenyl-human immunoglobulin G(DNP-h Ig G) (A) Specimens and reagents and preparation of reagents in solution.

(1) Preparation of specimens of OATase from rat liver and of rabbit antibody against it: Rats fed for 1 week on a high-protein diet containing 70% casein were subjected to isolation of the liver, which was then homogenized to isolate OATase from the mitochondria fraction according to the method of Matsuzawa et al. [Matsuzawa, T., Katsunuma, T and Katsunuma N.: Biochem.Biophys., Res, Commun. 32, 161-166 (1968)] and to purify it into crystals. An electrophoretic analysis following ultracentrifugation indicated that the crystals of the enzyme were homogeneous with a mean molecular weight of 170,000. Then, in accordance with the method of the authors cited above, 10 mg of the crystalline enzyme thus obtained was injected subcutaneously with Freund's complete adjuvant into a normal rabbit to prepare antiserum. Ten millilitres of the antiserum were then subjected to salting-out with ammonium sulfate [Arnon, T. and Shapira, E.: Biochemistry 6, 3942-3950 (1967) and DEAE cellulose column chromatography [Levy, H.B. and Sober, H.A.: Proc.

Soc. Exp. Biol. Med., 103, 250-252 (1960)] to obtain 77 mg of 1gG fraction, i.e. rabbit (anti-OATase) IgG.

(2) Preparation of DNP-h IgG and antibody against it: DNP-h IgG was prepared by treating commercial hIgG (Miles Laboratories, Inc., U.S.A.) with sodium 2,4-dinitrobenzene sulfonate in accordance with the method of Eisen et al.

[Eisen, H.N., Carsten, M.E. and Belman, S.: J. Immunol. 73, 296-308 (1954)].

Measurement of the absorbancy by DNP-hIgG thus obtained at 280 nm and 360 nm showed that 8.2 DNP residues were contained in each molecule of DNP-hIgG. Here, commercial rabbit (anti-hIgG) IgG (Miles Laboratories, Inc., U.S.A.) was used as the antibody for the assay of DNP-hIgG.

(3) Buffer Solution A: 0.01 M sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl, lmM MgCl2, 0.1% NaN3 and 0.1% bovine serum albumin (BSA).

(4) Buffer Solution B: 0.25 M sodium phosphate buffer (pH 7.5).

5 Preparation of a silicone carrier with rabbit (anti-OATase) IgG or rabbit (anti-hIgG) IgG fixed in the solid phase: A commercial silicone tube (2.5 mm in inner diameter, 4 mm in outer diameter, Silicone No. 3 SH type, Fuji Macromolecular Industry, Inc.) was cut into pieces, each 3 mm long, and then into halves longitudinally. Each piece was cleaned adequately with a commercial cleanser (Scatt 20-Xo, Nakarai Chemical Co.

Ltd.), then washed several times in water, soaked in Buffer Solution B containing 20-2,000 Fg/ml of rabbit (anti-hIgG) IgG fraction at room temperature for 30 minutes, and then kept at 4"C overnight. Afterwards each silicone piece was washed first in Buffer Solution B and then in Buffer Solution A, then soaked in Solution A and stored at 40C until the time of use.

(6) Preparation of Sepharose 4B with rabbit (anti-OATase) IgG fixed in the solid phase: Rabbit (anti-OATase) IgG was fixed in the solid phase to commercial CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals AB, Sweden) according to the procedure described in Reference (1).

(7) Preparation of -D-galactosidase-labelled rabbit (anti-OATase) Fab' complex or rabbit (anti-hIgG) Fab' complex: Preparation of antibody Fab' from rabbit antibody IgG fraction, and preparation and isolation of a complex of Fab' conjugated with -D-galactosidase produced by E. coli (Boehringer Mannheim GmbH, West Germany) were carried out in accordance with the methods given in Reference (1) and in J. Immunol. 116 (No. 6), 1554-1560 (Kato, K., Fukui, H., Hamaguchi, Y. and Ishikawa, E.) (1976) (hereinafter referred to as Reference 2).

(8) Preparation of standard solution of crystalline OATase or standard DNP-hIgG solution for standard curves: The OATase crystal prepared was dissolved in Buffer Solution A and diluted so as to obtain 10 different standard solutions in a concentration range of 2 x 10-l7 - 6 x 10-13 mol per 50 u DNP-hIgG was dissolved and diluted in the same mannertorepare 11 different standard solutions in a concentration range of 6 x 10-17 - 1 X 1x101 mol per 50 'Ll.

(9) Reagent and method used for the determination of the activity of the bound enzyme: A determination was made with a fluorescence spectrophotometer (MPF-4) (Hitachi) of the intensity of fluorescence of 4-methylumbelliferone released from 4-methylumbelliferyl -D-galactoside (Sigma Chemical Co. U.S.A.) as the substrate by the procedures given in References (1) and (2).

(B) Assay procedure and results obtained: A piece of silicone with rabbit (anti-OATase) IgG fixed in the solid phase was placed in a small test tube containing 100 iil of Buffer Solution A and 50 Ill of standard solution of OATase crystal, incubated at 37"C for 4 hours with shaking, and then kept at 4"C overnight.

The silicone piece was then washed twice in 1 ml of Buffer Solution A and, after addition of 150'ill of Buffer Solution A containing 1,650 units of rabbit (anti-OATase) antibody (Fab')- -D-galactosidase complex, incubated at 370C for 6 hours with shaking. Then the silicone piece was washed twice again in 1 ml of Buffer Solution A and transferred to another small test tube, where it was pre-incubated at 300C for 5 minutes with 100 Rl of Buffer Solution A added in preparation for the subsequent determination of the activity of the bound enzyme. In an assay with rabbit (OATase) IgG fixed in the solid phase to Sepharose 4B, 100 Ill of a 10-fold dilution with Buffer Solution A of Sepharose 4B was used in accordance with the procedures given in References (1) and (2).

In assaying DNP-hIgG, reagents appropriate for this purpose were subjected to the same procedures except for the use of 2,000 units of rabbit (anti-hIgG) antibody (Fab')- -D- galactosidase complex.

To the silicone piece referred to above or Sepharose 4B thus incubated, 50'ill of 3 x 10-4 mol 4-methylumbelliferyl-B-D-galactoside was added before incubation with shaking at 30"C for 5-20 minutes. From the intensity of fluoresence of the resultant 4methylumbelliferone the standard curves shown in Figure 1 were obtained. In Figure 1 the ordinate gives (3-D-galactosidase activity bound (units/assay tube) and the abscissa gives rat ornithine 6-aminotransferase or DNP-human IgG (femto mol/assay tube). Standard curve (a) [marked with open circles] represents the calibration curve for OATase obtained with Sepharose 4B used as the carrier as before, and standard curve (b) [marked with closed circles] that with a silicone piece in place of Sepharose. As is apparent from Figure 1, the former procedure yields extremely high blank values due to nonspecific binding of the enzyme-labelled complex to the inner wall of the test tube, so that it is completely impossible to determine a quantity of OATase less than approximately 0.1 femto mol. In contrast, the latter is much more sensitive with blank values lowered to about one-seventh of those with the former, so that it is well capable of determining even a quantity lower than 0.03 femto mol. Such lowered blank values with the consequent marked elevation of sensitivity were also observed in an assay of DNP-hIgG [standard curve (c), marked with open squares].

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

(1) Buffer Solutions A and B: prepared as described in Example 1 (A)(3) and (4).

(2) Preparation of a silicone carrier with guinea pig (anti-p Ins.) antibody IgG fixed in the solid phase: In accordance with the technique described in Reference (1), commercial guinea pig (anti-p Ins.) serum (Miles Laboratories, Inc. U.S.A.) was subjected to Na2SO4 fractionation Kekwick, R.A.: Biochem. J., 34, 1248-1257 (1940) and DEAE cellulose column chromatography [Levy, H.B. and Sober, H.A.: Proc. Soc. Exp. Biol. Med. 103, 250-252 (1960)]. Ten millilitres of guinea-pig antiserum were processed to obtain approximately 75 mg of IgG fraction. Then the guinea-pig (anti-p Ins.) IgG was added to Buffer Solution B so as to give a concentration of 2.5 mg/ml before fixing it in the solid phase to a piece of silicone by the technique described in Example 1 (A)(5).

(3) Preparation of -D-galactosidase-labelled p Ins. complex: This was done as described in Reference (1). Used as the p Ins.-enzyme complex was a 10-fold dilution with Buffer Solution A of an eluate fraction from a Sepharose 6B column (1.5 x 40 cm) with an enzyme activity close to its peak.

(4) Preparation of standard p Ins. solutions: Commercial p Ins. ("Actrapid" 40 U/ml, Novo Industri A/S, Denmark) was diluted with Buffer Solution A to prepare 12 different standard solutions in a concentration range from 0.1 - 400 'LU per 20 coil.

(5) Reagent and method used to determine the activity of bound (3-D-galactosidase: as in Example 1 (A)(9).

(B) Assay procedure and results obtained: A silicone piece with guinea-pig (anti-p Ins.) IgG fixed in the solid phase was placed in a small test tube containing 110 Fl of Buffer Solution A and, after addition first of 20 'Ll of p Ins. solution (standard solution as used here) and then of 20 Rl of p Ins.-enzyme complex, incubated with shaking at 300C for 2 hours. Then the silicone piece was washed several times with 1 ml of Buffer Solution A and then transferred to another small test tube of the same shape, where it was preincubated at 30"C for 5 minutes with 100 Ill of Buffer Solution A. A determination of the enzyme activity of -D-galactosidase thus bound in accordance with the method employed in Example 1 gave the results shown in Figure 2. In Figure 2 the ordinate gives -D-galactosidase activity bound (fluorescence intensity) and the abscissa gives porcine insulin (1l units/assay tube). (In which fluorescence intensity of 100 is equivalent to 1 x 10-7 mol of 4-methylumbelliferone). Thus, no less satisfactory standard curves than those with previously available techniques were obtained both quite simply and conveniently and in a short time by applying the invention, using a piece of silicone as the carrier, in EIA by the competitive coupling technique.

Example 3 Assay of rat liver OATase (II) (A) Specimens and reagents and preparation of reagent solutions: The same procedures as described in Example 1 were employed in preparing OATase specimens from rat liver mitochondrial fraction and in preparing other reagent solutions.

However, rabbit (anti-OATase) IgG was fixed in the solid phase to a piece of silicone by, in addition to the method used in Example 1 (A)(5), soaking any one of the following silicone carriers in 2 mg/ml of the former substance: a silicone piece cleansed well with the cleanser Scatt 20--X as in Example 1, soaked in a 2% solution of 3-aminopropyltriethoxysilane (Nakarai Chemical Co. Ltd.) in acetone at 45"C for 24 hours and then cleansed with purified acetone and dried; a silicone piece treated in the same way with aminoalkylsilane, soaked for 1 hour in a 1% aqueous solution of glutaraldehyde (Nakarai Chemical Co. Ltd.) and then well washed in Buffer Solution B; or a silicone piece washed as in Example 1 and treated with glutaraldehyde as described above.

(B) Assay procedure and results obtained: An enzyme immunoassay was made by the sandwich technique as in Example 1 both in the presence and in the absence of 6.8 femto mol of antigen (rat liver OATase) to be assayed. The results obtained are shown in the Table below, where the figures indicate mean values from 2 repeated assays.

Silicone piece Activity of enzyme bound treated with (units/piece) 3-Aminopropyl- Glutaralde- In the absence In the presence triethoxysilane hyde of antigen of antigen + + 1.8 317 + - 2.0 305 + + 1.5 285 1.8 270 For all the intervals there was an extremely low blank value obtained in the absence of antigen to be assayed. The silicone piece may be used as it is, as in Example 1, but can provide a more sensitive assay if treated before use with aminoalkylsilane either alone or in appropriate combination with glutaraldehyde.

Example 4 Assay of 2,4-dinitrophenyl-human immunoglobulin G (DNP-hIgG) (II) The basic procedure pertinent to the assay of DNP-hIgG in Example 1 was used again here, except that in place of a silicone piece used for immobilizing the rabbit (anti-hIgG) IgG in Step (A)(5), a glass rod carrier coated with siliconiser was employed here. The carrier was obtained by cleansing a commercial Pyrex rod adequately with a chemical cleanser and water, drying and then soaking it in a 10% solution of the commercial silicone coating agent Dry Film (DSC-87 (Pierce Co. U.S.A.) at room temperature for 5 hours and then allowing it to dry in a current of air at approximately 50-60"C. Besides, DNP-hIgG was used in this Example in an amount of 50 'Ll of a 6.0 femto mol solution, with the result that a p-D-galactosidase activity equivalent to approximately 92% of that in Example 1 was exhibited both in the blank samples (no DNP--hIgG present) and in the DNP-hIgG samples. "Pyrex" is a registered Trade Mark.

WHAT WE CLAIM IS: 1. Process for the preparation of a composite comprising an antigen or antibody on a silicone support which comprises contacting the antigen or antibody in aqueous solution with a silicone support material or with a piece or particles of a carrier having a silicone surface, to provide adsorption of the antigen or antibody to the silicone.

2. Process according to claim 1 in which the antigen or antibody in aqueous solution is contacted with a piece or particles of a silicone support material.

3. Process according to claim 1 or 2 in which the silicone surface is first treated with a silane derivative.

4. Process according to claim 3 in which the silane derivative is an aminoalkylsilane.

5. Process according to claim 4 in which the aminoalkylsilane is of the general formula: R2N-Alk-Si(OR)3 wherein Alk represents an- alkylene group with at most six carbon atoms and R represents an alkyl group with at most six carbon atoms.

6. Process according to claim 5 in which R is an alkyl group with at most three carbon atoms.

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

8. Process according to any one of claims 3 to 7 in which, after treatment with the silane derivative, the silicone surface is treated with glutaraldehyde.

9. Process for the preparation of a composite comprising an antigen or antibody on a silicone surface substantially as hereinbefore described.

10. Process for the preparation of a composite comprising an antigen or antibody on a silicone surface substantially as hereinbefore described with especial reference to any one of Examples 1 to 4.

11. Immobilized antigen or antibody prepared by a process claimed in any of claims 1 to 10.

12. Immobilized antigen or antibody, wherein the antigen or antibody is bound to a silicone support material or to a piece or particles of a carrier having a silicone surface.

13. Immobilised antigen or antibody, wherein the antigen or antibody is bound to a piece or particles of a silicone support material.

14. Immobilized antigen or antibody according to claim 12 or 13, wherein the antigen or antibody is linked to the silicone surface via a silane derivative.

15. Immobilized antigen or antibody according to claim 14 wherein the silane derivative

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (32)

1. **WARNING** start of CLMS field may overlap end of DESC **.

   Silicone piece Activity of enzyme bound treated with (units/piece)

   3-Aminopropyl- Glutaralde- In the absence In the presence triethoxysilane hyde of antigen of antigen + + 1.8 317 + - 2.0 305 + + 1.5 285 1.8 270 For all the intervals there was an extremely low blank value obtained in the absence of antigen to be assayed. The silicone piece may be used as it is, as in Example 1, but can provide a more sensitive assay if treated before use with aminoalkylsilane either alone or in appropriate combination with glutaraldehyde.

   Example 4 Assay of 2,4-dinitrophenyl-human immunoglobulin G (DNP-hIgG) (II) The basic procedure pertinent to the assay of DNP-hIgG in Example 1 was used again here, except that in place of a silicone piece used for immobilizing the rabbit (anti-hIgG) IgG in Step (A)(5), a glass rod carrier coated with siliconiser was employed here. The carrier was obtained by cleansing a commercial Pyrex rod adequately with a chemical cleanser and water, drying and then soaking it in a 10% solution of the commercial silicone coating agent Dry Film (DSC-87 (Pierce Co. U.S.A.) at room temperature for 5 hours and then allowing it to dry in a current of air at approximately 50-60"C. Besides, DNP-hIgG was used in this Example in an amount of 50 'Ll of a 6.0 femto mol solution, with the result that a p-D-galactosidase activity equivalent to approximately 92% of that in Example 1 was exhibited both in the blank samples (no DNP--hIgG present) and in the DNP-hIgG samples. "Pyrex" is a registered Trade Mark.

   WHAT WE CLAIM IS: 1. Process for the preparation of a composite comprising an antigen or antibody on a silicone support which comprises contacting the antigen or antibody in aqueous solution with a silicone support material or with a piece or particles of a carrier having a silicone surface, to provide adsorption of the antigen or antibody to the silicone.
2. 2. Process according to claim 1 in which the antigen or antibody in aqueous solution is contacted with a piece or particles of a silicone support material.
3. 3. Process according to claim 1 or 2 in which the silicone surface is first treated with a silane derivative.
4. 4. Process according to claim 3 in which the silane derivative is an aminoalkylsilane.
5. 5. Process according to claim 4 in which the aminoalkylsilane is of the general formula: R2N-Alk-Si(OR)3 wherein Alk represents an- alkylene group with at most six carbon atoms and R represents an alkyl group with at most six carbon atoms.
6. 6. Process according to claim 5 in which R is an alkyl group with at most three carbon atoms.
7. 7. Process according to claim 6, in which the aminoalkylsilane is 3-aminopropyltriethoxysilane.
8. 8. Process according to any one of claims 3 to 7 in which, after treatment with the silane derivative, the silicone surface is treated with glutaraldehyde.
9. 9. Process for the preparation of a composite comprising an antigen or antibody on a silicone surface substantially as hereinbefore described.
10. 10. Process for the preparation of a composite comprising an antigen or antibody on a silicone surface substantially as hereinbefore described with especial reference to any one of Examples 1 to 4.
11. 11. Immobilized antigen or antibody prepared by a process claimed in any of claims 1 to 10.
12. 12. Immobilized antigen or antibody, wherein the antigen or antibody is bound to a silicone support material or to a piece or particles of a carrier having a silicone surface.
13. 13. Immobilised antigen or antibody, wherein the antigen or antibody is bound to a piece or particles of a silicone support material.
14. 14. Immobilized antigen or antibody according to claim 12 or 13, wherein the antigen or antibody is linked to the silicone surface via a silane derivative.
15. 15. Immobilized antigen or antibody according to claim 14 wherein the silane derivative

    is an aminoalkylsilane.
16. 16. Immobilized antigen or antibody according to claim 15 wherein the aminoalkylsilane is of the general formula: H2N-Alk-Si(OR)3 in which Alk represents an alkylene group with at most six carbon atoms, and R represents an alkyl group with at most six carbon atoms.
17. 17. Immobilized antigen or antibody according to claim 16 wherein R is an alkyl group with at most three carbon atoms.
18. 18. Immobilized antigen or antibody according to claim 17 wherein the aminoalkylsilane is 3-aminopropyltriethoxysilane.
19. 19. Immobilized antigen or antibody according to any one of claims 12 to 18 in which the antigen or antibody is linked to the silicone surface previously treated with a silane derivative and glutaraldehyde.
20. 20. Immobilised antigen or antibody wherein the antigen or antibody is bound to a silicone surface, substantially as hereinbefore described.
21. 21. Immobilised antigen or antibody wherein the antigen or antibody is bound to a silicone surface, substantially as hereinbefore described with especial reference to any one of Examples 1 to 4.
22. 22. An immunoassay method using an immobilised antigen or antibody as claimed in any one of claims 12 to 21.
23. 23. An enzyme-immunoassay method using an immobilised antigen or antibody as claimed in any one of claims 11 to 21.
24. 24. A method according to claim 23 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')2 with a reducing agent is used as the antibody.
25. 25. A method according to claim 24 in which the reducing agent is 2 mercaptoethylamine.
26. 26. A method according to claim 23, 24 or 25 in which the competitive coupling or the sandwich technique is used.
27. 27. A method according to any one of claims 23 to 26 in which an assay of the 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.
28. 28. A method according to claim 27 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.
29. 29. A method according to any one of claims 23 to 27 in which the enzyme used to label antigen or antibody used in the enzyme-immunoassay method is peroxidase, alkaline phosphatase, (3-D-galactosidase, glucose oxidase or lysozyme.
30. 30. A method according to claim 29 in which the enzyme is (3-D-galactosidase.
31. 31. An immunoassay method according to claim 22 or 23 substantially as hereinbefore described.
32. 32. An immunoassay method according to claim 22 or 23 substantially as hereinbefore described with especial reference to any one of Examples 1 to 4.