NO774240L - PROCEDURE FOR DETECTION AND DETERMINATION OF ANTIGENES - Google Patents

Description

The present invention relates to enzyme-linked immunoassay and provides a new method for the detection and measurement of antigens, and reagents for use therein. The term "antigen" as used here means not only substances which in themselves can induce the production of antibodies in animals, i.e. immunogens, but also substances, sometimes called haptens, which after conjugation with a carrier molecule become capable of producing the production of and reaction with specific antibodies.

There is considerable theoretical and practical interest in being able to detect and measure the presence of antigens. As such substances often have a complex and frequently unknown chemical structure and appear in very low concentrations in biological and other materials,

it is not possible to apply conventional analysis techniques. Consequently, in the last few years a number of techniques have been developed based on the antigen-antibody reaction, in which a reaction component is marked with a radio-active "tracer" or e.g. with an enzyme, so that the reaction products or in some. cases the unused reagent can be detected and/or measured. Although methods based on the use of radioactive markers

have certain theoretical advantages, notably the fact that certain types of marking, especially those using tritium or 14

C, the radioactive tracer itself does not interfere with the antigen-antibody reaction, there are certain disadvantages associated with the use of this technique, in particular the need to provide the necessary equipment to measure the radioactive tracer, to protect the operator from possible harmful effects from the radioactivity and to place the radioactive waste after use. Many radioactive markers must be thrown away at regular intervals due to the radiochemical decomposition of the radioactive isotope. Furthermore, it can be difficult to radiolabel low molecular weight antigens and retain their important antigenic properties. Considerable interest has therefore been shown in methods in which the antigen-antibody reaction is followed using a reagent in which an enzyme acts as a tracer. Such methods are easy to perform and require relatively simple equipment, and involve no health risks. Nevertheless, many of the systems proposed so far have suffered from the disadvantage that it is necessary for good functioning to produce a conjugate between the enzyme used as a tracer and the antigen to be detected. The preparation of these conjugates is not. always easy because the need to preserve the enzyme activity of the enzyme part of the conjugate, and it is particularly cumbersome to have to prepare a separate conjugate for each individual antigen that is intended to be detected. In the method according to the present invention, on the other hand, a single enzyme-containing conjugate can be used on a large number of antigens. In principle, a single such conjugate can actually be used on all antigens.

In an earlier proposal (Engvall et. a_l, J. Immunology 109

(1972) page 129) antibodies are determined by reaction with antigens on an insoluble substrate followed by reaction of the antigen-antibody conjugate on the substrate with an enzyme-labelled anti-immunoglobulin which was known to be able to react with the antibody on the conjugate. With this method, the amount of enzyme that is finally attached to the substrate depends on the amount of antibody in the sample that is being treated, as the enzyme is bound to the substrate by a chain, whereby the antibody to be determined forms a defined and essential link.

Method of the present invention for enzyme-linked immunoassay consists of bringing into contact, simultaneously

or stepwise and in a liquid medium, (1) an antigen (as previously defined) on an insoluble substrate or in the form of an insoluble aggregate, (2) a first antibody (as defined below) and (3) a enzyme-labeled other antibody (eg

hereinafter defined), separate the insoluble and soluble materials and measure the enzyme activity of one or more of them.

The invention is characterized in that said first antibody (2) is also brought into contact with a sample containing the antigen to be detected or measured, which may be the same or different from the antigen (1), the second antibody (3) is thereby linked to the insoluble material in inversely proportional to the quantity of the antigen in the sample to be detected or measured to determine the presence or quantity of the antigen in the sample. The term "first antibody" as used herein means an antibody that can bind both the antigen on the insoluble substrate and the antigen to be detected. The term "other antibody" means antibody specific for all antibodies of the immunoglobulin class to which the first antibody belongs, e.g. all sheep IgG, rabbit IgG or rabbit IgM. If e.g. the. the first antibody is an immunoglobulin that is formed in a special animal, e.g. a sheep, the second antibody is an antibody against immunoglobulins of this species, regardless of their antibody specificities, which are formed in a different animal, e.g. a donkey.

The new method can be easily adapted for detection and measurement

of a variety of antigens. Besides antigens, serum with appropriate content and tissue proteins such as α^-fetoprotein, c^-haptoglobulin and carcinoembryonic antigen, haptens such as hormones, e.g. triiodothyronine norepinephrine, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, insulin and thyroxine, steroids, e.g. cortisol, pro-gesterone, estrone, estradiol and testosterone, drugs, e.g. morphine, amphetamine, barbiturates, diphenylhydantoin, methotrexate and gentamycin, vitamins and nutritional supplements, e.g. pyridoxal-5-phosphate and aflotoxin, herbicides, insecticides and nitrosourinous substances are also used. In each case, the antigen must first be bound either chemically or, less often, physically to a suitable substrate, e.g. in the manner described in more detail below.

It must be noted that the nature of the enzyme-labeled second antibody in no way depends on the antigen to be detected. It is only necessary to elicit the first antibody in an animal of the same species as that which elicited the immunoglobulin used to stimulate the production in other species of the second

the antibody. There is therefore no need to produce

a large spectrum of enzyme-marked antibodies, and the difficulties involved in linking the enzymes to the antibodies therefore only have to be solved once.

Any enzyme could be used as a label on the second antibody. Oxidases such as horseradish peroxidase, hydrolytic enzymes such as alkaline phosphatase or dehydrogenases such as glucose-6-phosphate dehydrogenase are particularly suitable. Ideally, an enzyme used as a marker should have the following properties: (1) it is relatively cheaply available in high purity, (2) it has a high activity per unit weight, (3) it is readily water-soluble and stable under normal storage conditions, (4) it enables the use of a measurement method that is simple, rapid, sensitive and inexpensive, (5) it should be free from biological fluids and (6) biological fluids should not contain any substrate inhibitors or activators for it, (7) it should contain suitable chemical groups for conjugation to another antibody that enable conjugation to have only a minimal effect on both enzyme and antibody activity. 3-galactosidase satisfies these requirements. Other possible but less preferred enzymes are glucose oxidase, acetylcholinesterase-glucoamylase, lysozyme, glucose-6-phosphate dehydrogenase and malate dehydrogenase.

The enzyme used is preferably linked to the second antibody using the linking method described below,

which can be applied to any combination of antibody and enzyme provided that the latter contains a mercapto group or can be modified so that it will contain such a group. However, if necessary, other connection methods can be used. These include

a) One-step glutaraldehyde binding in which the glutaraldehyde is used to link proteins in a complicated reaction that gives

high molecular weight heterogeneous conjugates, (see Avrameas, S., Immunochemistry 6^43 (1969)).

b) Two-step glutaraldehyde binding in which the enzyme horseradish peroxidase reacts with only one molecule of glutaraldehyde. This limits enzyme-enzyme conjugation and is an improvement over the one-step method. However, this method probably cannot be applied to many enzymes besides horseradish peroxidase, as these will react with more than one molecule of glutaraldehyde.

(see Avrameas. S and Ternynck, Immunochemistry £5 1175 (1971)). c) Oxidation of saccharide residues in the enzyme followed by Schiff base formation. Horseradish peroxidase has several oligosaccharide groups, and their oxidation to aldehyde groups (using periodate) which can react with amino groups is based on this binding method. Other enzymes can also contain oligo-saccharide groups and can in principle also be used. However, high molecular weight conjugates are formed by this method. (Nakane P.K. + Kawaoki, A.J. Histochem. Cytochem. 2_2 108.4 (1974)). d) Dimaleimide binding using N,N<1->o-phenylenedimaleimide after introduction of -SH groups in the antibody using 2-mercaptoethylamine which reduces previously existing -S-S- bridges to two -SH groups. (Kato et al European J. Biochem. 6_2 285

(1976)). e) Other bifunctional reagents that can be used include toluene-2,4-diisocyanate, p_',p-difluoro-m,m'-dinitrofenyl sulfone, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, but these give generally worse results than those described above. f) Binding can also be achieved with hetero-bifunctional reagents, i.e. reagents containing two different functional groups in each molecule, e.g. m-maleimidobenzyl N-hydroxy-succinimide ester, which has been used in a two-step reaction to couple insulin to 3-galactosidase. (T. Kitagawa + T. Aikawa J. Biochem. 1_9 233-236 (1976)). g) Non-covalent cross-linking by e.g. to link antibodies to horseradish peroxidase to form a conjugate that is non-covalent but relatively stable in nature. This method has the disadvantages that (i) During storage, the enzyme-antibody conjugate can dissociate slowly.

(ii) The antibodies can inhibit the enzymes they are raised against.

(ii) It takes several months to produce the antibodies and antibodies produced by different manufacturers have different properties.

Due to the greater speed and ease associated with

work with relatively large amounts of antibodies, it is often easier if the first and second antibodies are produced in relatively large animals such as horses, donkeys or sheep rather than small animals such as rats, mice, guinea pigs or hamsters, although the use of such is not excluded. As already indicated, the first and second antibodies must be produced in different types of animals. It has been found easy to raise the first antibody in a sheep and the second antibody in a donkey, but any other pair of different types can be used if desired.

The possibility of interference by the second substance in the antigen-first-antibody reaction is reduced if the second antibody is not produced against all of the first antibody, but only against the Fc fragment of the immunoglobulin that constitutes the first antibody.

The other antibodies can mainly be produced in a known manner

way, but the preferred method is to 'prime' the animals after

followed by production of antibodies and giving "booster dose" rings when serum antibody levels have passed the peak. The blood normally contains peak antibody levels shortly after "boost" things. When a suitably high level (or a maximum level)

of anti-immunoglobulin has accumulated in the bloodstream of the animal, the latter is bled and the serum containing the anti-immunoglobulin is separated.

The second antibody is preferably purified by incubating donkey antiserum with a suitable immunoadsorbent, e.g. sepharose or other insoluble carrier that has been activated first by reaction \

i with a suitable bifunctional reagent, for example 1,4-bis(2,3-epoxypropoxy)butane and then with a solution of the immunoglobulin. (or Fc fragment thereof) used to raise the second antibody. This provides an immunoadsorbent that is specific for the second antibody, and the latter can be purified by adsorption onto the immunoadsorbent followed by elution, before or after labeling with an enzyme.

To prepare the labeled second antibody, it is preferred to treat the immunoadsorbent with the second antibody adsorbed thereon with a reagent such as methyl mercaptobutyrimidate hydrochloride to modify the amino groups in the antibody and introduce from 3-5' thiol groups into each antibody molecule. The second antibody with embedded mercapto groups is then eluted from the immunoabsorbent and. is treated so that the enzyme binds to it. Preferably, it is treated with N,N'-o-phenylene-bis-maleimide and then reacted with purified (3-galactosidase which normally originates from Escherichia coil. The enzyme-secondary-antibody conjugate is purified

then finally by chromatography on a diethylaminoethyl agarose gel or other suitable adsorbent to separate the conjugate from unreacted enzyme and unreacted other antibody.

The first antibody is raised in a suitable animal using known techniques. Where, as is often the case, the antigen to be detected or measured is a hapten with a relatively simple chemical structure, e.g. triiodothyronine, estradiol, gentamycin, phenytoin or morphine, it is not capable by itself of producing the formation of the first antibody. It is therefore necessary to conjugate the antigen with a carrier molecule in order to

provide a conjugated molecule capable of eliciting the production of the antibody. For this purpose, it is appropriate to conjugate the antigen with an easily accessible protein which is foreign to the animal in which the first antibody is to be elicited, e.g. bovine serum albumin (BSA). The conjugation can be carried out by reacting the albumin or other protein with e.g. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, followed by reaction of the product with the antigen which for this reaction must contain an amino or carboxyl group. Other known conjugation methods can be used if desired, and ideally

set should approx. 3-30 antigen molecules are built into each albumin molecule or other proteins with a high molecular weight.

The first antibody can be produced by injecting the antigen or antigen-conjugate into a suitable animal, e.g. a sheep, repeated

over a period of weeks or months until a sufficiently high antibody content with the desired binding properties has accumulated in the animal's blood stream. The animal is then bled and the antiserum separated.

The first antibody is purified 'So preferably by contact with the antigen against which the antibody was raised or a related compound on an insoluble support. After absorption, the antibodies can be eluted, e.g. with guanidine hydrochloride solution at high or low pH with solutions of high salt concentration or with non-aqueous solvents and then dialyzed against an appropriate buffer to remove the eluent.

The insoluble carrier used to hold the antigen or related compound both in the above-mentioned purification process and in the method of the invention may be any suitable water-insoluble material to which the antigen or related compound can be readily bound sufficiently to ensure that it remains linked to this during the reactions included in the cleaning or testing method in question. Although it is possible to use a carrier that simply adsorbs the antigen as is the case in the system described by Engvall et al in the already mentioned publication, it is preferred to bind the antigen chemically to the carrier, and this means that it is necessary that the carrier contains functional groups with which the necessary reactions can take place. Carriers consisting of glass, nylon, polyacrylamide, cellulose or dextran can be used. Certain types of immunoadsorbers containing hydroxyl groups can be reacted with 1,4-bis(2,3-epoxypropoxy)butane and the product is reacted with the antigen. This method has been found to be simple, safe, effective and provide a stable covalent bond provided that the antigen or related compound contains hydroxyl, amino or thiol groups. The requirements for an immunoadsorbent for the purification of antibodies are not necessarily the same as for such.

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the immunoadsorbent in the real sample.

The method according to the present invention can be carried out in

many different ways. The amount of first antibody and therefore the marker associated with the insoluble antigen is inversely related to the amount of antigen in the sample to be determined. The enzyme activity, either free or bound to the insoluble or supported antigen is measured. Some of the specific ways to carry out the new procedure are as follows.

In a first method, the first antibody is incubated with a sample containing the antigen to be determined. After the reaction is complete, the supported antigen is added and the mixture is incubated again. The solid phase is then separated from the liquid phase, washed and mixed with an excess of the enzyme-labelled second antibody. After incubation, the solid phase is separated again and washed. The amount of activity found in the insoluble fraction (solid phase) is inversely proportional to the amount of antigen present in the sample, i.e. the supernatant is directly proportional to it. Both fractions can be measured.

In another method, the first antibody is reacted with the enzyme-labelled second antibody to form a complex which is then added to the excess of the sample containing the antigen to be determined and incubated. The insoluble carrier carrying the antigen is then added, and after incubation the solid and liquid phases are separated. The determination of the enzyme activity of the liquid phase gives a result that is directly proportional to the amount of antigen in the sample.

In a third method, the complex of first and second antibodies, the sample containing the antigen to be determined, and the carrier carrying the antigen are incubated simultaneously, and after separation of the solid phase, the enzyme activity in the liquid phase is determined. Again, the result is directly proportional to the concentration of antigen in the sample.

As already indicated, for a quantitative determination by it;

first and second methods above, the amount of first antibody used be in excess of what is required to react all the antigen in the sample, and the carrier carrying the antigen must be added in a sufficient excess to ensure complete adsorption of all the first antibody that has not reacted with the antigen in the sample. In practice, the easiest way to ensure this is to perform a series of determinations with different concentrations of first antibody in geometric or arithmetic series. If this is done, it is a simple matter to find a suitable concentration of the first antibody which enables the performance of an accurate determination.

The following examples illustrate the invention.

i i Example 1

1. Production and marking of other antibody

(a) Preparation of Fc fragment of ovine immunoglobulin

(IgG)

The method used for the production of sheep Fc fragment is based on Porter's (R.R. Porter, 1959, Biochem J. 7_3, 119).

320 ml of serum was collected from 28 sheep. 150 ml of serum was incubated with stirring in an ice bath with 30 g of moist diethylaminoethyl (DEAE) - A 50 Sephadex previously equilibrated with 10 mM phosphate buffer at pH 6.5. After 1 hour, the gel was filtered on a coarse sintered glass funnel and washed with 50 ml of 10 mM phosphate buffer pH 6.5. The filtrate plus wash was incubated once more with 30 g of moist DEAE-Sephadex, filtered and washed with 100 ml of phosphate buffer. Filtrate and wash (300 mL) were treated with 150 mL of saturated (NH^^SO^ solution which was slowly added to the continuously stirred solution. The precipitate was collected and washed with 100 mL of 20% (NH^)^SO^-l. The final precipitate was dissolved in 50 ml of 50 mM phosphate buffer at pH 8.0

and dialyzed overnight at 4°C against 5 1 of distilled water. The solution was then freeze-dried.

1.50 g of freeze-dried sheep IgG obtained in this way was dissolved in

50 ml of 0.1 M tris-HCl buffer at pH 7.2 containing 10 mM cysteine and 2 mM EDTA. The buffer was produced immediately

before use. 0.6 ml of a crystalline suspension of Papain (Sigma, twice crystallized) in 50 mM acetate buffer at pH 4.5 was added. The solution was incubated at 37°C for 4 hours with occasional shaking. 0.222 g of iodoacetamide was then added and the solution immediately applied to a column (80 x 5 cm) of G.100 Sephadex equilibrated with 10 mM acetate buffer at pH 5.6 at 4°C.

The second peak eluted from the column with 10 mM acetate buffer f is at pH 5.6 contains a mixture of Fc" and F thaw fragments. The first peak contains unused IgG. The second peak was applied to a column (40 x 2.5 cm) of carboxymethyl cellulose (Whatman CM 32) that was equilibrated with 10 mM acetate buffer at pH 5.6 at 4° C. A peak was eluted from the column by washing with 10 mM acetate buffer pH 5.6 and several small peaks and a major peak were eluted when a gradient from 10 mM to 500 mM acetate pH 5.6 was applied (1000 mL in each compartment of a gradient mixer).The major peak was collected and rechromatographed on carboxymethyl cellulose column, this time a gradient from 10 mM to 300 mM acetate buffer at pH 5.6

with 1000 ml in a mixing vessel applied. A single peak was eluted. This was collected, completely dialyzed against distilled water and freeze-dried.

b) Production of other antibody (donkey-anti-sheep-immunoglobulin)

A stable emulsion was prepared by mixing 1 part saline solution containing sheep Fc fragment (0.75 mg/ml) and Bacillus Calmette-Buerin (1.5 mg/ml) with 2 parts Marcol 52 excipient. 6 intramuscular injections (1 ml) of the emulsion were given in the lower leg. The production of donkey-anti-sheep antibodies was regulated and the animal was ready for boosting after 2 months. An emulsion consisting of 2 parts excipient to 1 part saline solution (sheep Fc 0.75 mg/ml) was injected intramuscularly at 6 sites (0.5 ml) in the lower legs. The blood samples 10 days later and in the following month contained large amounts of antibodies.

c) Production of immunoadsorbent for purification of donkey anti-sheep immunoglobulin

Sepharose 4B was activated with 1,4-bis-(2,3-epoxypropoxy)-butane by incubating equal volumes of the gel, the bisepoxide and 0.6M NaOH containing 2 mg per ml of sodium borohydride with continuous stirring at 25°Ci for 8 hours and then washed. Sheep IgG was prepared from a pool of normal serum by precipitation with 33% ammonium sulfate solution. A solution of sheep IgG in 0.1 M NaHCO 3 buffer at pH 10 was added using 10 mg of sheep

IgG per ml of gel. The activated gel was incubated with IgG for 48 h and the immunoadsorbent finally washed and processed

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with 0.1M ethanolamine at pH 10 for 24 hours.

(d) Purification and labeling of donkey anti-sheep other antibody

Donkey anti-sheep immunoglobulin was purified by incubating donkey anti-serum (prepared as described above) with the immunoadsorbent. Thiol groups were introduced into the immunoglobulin while it was adsorbed to the immunoadsorbent using methyl-4-mercapto-butyrimidate/HCl to modify the amino groups of the immunoglobulin. The degree of introduction was limited to give between 3 and 5 thiol groups per globulin molecule.

The immunoadsorbent-antigen complex was washed and suspended in 0.1 M N-ethylmorpholine/HCl at pH 7.5 at a concentration of 1 ml. immunoadsorbent in a total volume of 10 ml. A solution of methyl 4-mercaptobutyrimidate/HCl in 0.2M Na 2 CO 3 (10 mg/ml) was prepared immediately before use. 0.1 ml of this solution was then added for every 10 ml of the suspension. The suspension was stirred for 30 min. at room temperature and then washed with 0.1 M N-ethylmorpholine/HCl at pH 7.5 which contained 1 mg of dithiothreitol per 10 ml puff. The gel was then thoroughly washed with HCl containing 0.1 M NaCl to pH 3.5.

The immunoglobulin containing embedded thiol groups thus prepared was eluted with HCl containing 0.1 M NaCl at pH 2.5. The simplest method to achieve this was to pack the immunoadsorbent in a small column and collect the effluent in tubes containing puffs. As the next preparation step is carried out in 0.1M acetate buffer at pH 5.0, the effluent was collected in sufficient 0.1M acetate at pH 6.0 to give a final pH of 5.0.

2 ml of 0.1 M acetate buffer at pH 5.0 containing 2.5 mg of the immunoglobulin modified with thiol groups was cooled in an ice bath. This solution was then slowly added dropwise to a 1 ml saturated solution of N,N<1->o-phenylene-bis-maleimide in 0.1M acetate buffer at pH 5.0 on an ice bath. The mixture was then incubated at 30°C for 20 min. The immunoglobulin was then separated from the excess N,N-o-phenylene-bis-

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maleimide by passing the solution through a column (20 x 1.5 cm) of G. 25 Sephadex equilibrated at room temperature with 0.01 M phosphate buffer containing 0.01 MgCl 2 at pH 6.0.

5.0 mg of ammonium sulfate field 3-galactosidase from Escherichia coli was dissolved in purified activated immunoglobulin solution and incubated at 30°C for 4 hours. Sufficient. N NaOH to adjust the pH of the reaction mixture to 7.5 and sufficient 2M 2-mercaptoethanol to give a final concentration of 10 mM was added.

The immunoglobulin-enzyme conjugate solution was applied to a column (60 x 0.9 cm) of DEAE-Agarose (Biogel) equilibrated at 4°C with 10 mM Tris-HCl buffer at pH 7.5 containing 10 mM 2-mercapto-ethanepl , 10 mM MgCl2 and 50 mM sodium chloride. The solution was washed on the column with the equilibrating buffer. A gradient from 50 mM to 200 mM sodium chloride in the same buffer was applied with 250 ml in the mixing vessels. The immunoglobulin that is not conjugated with the enzyme does not adsorb on the column and is eluted before the gradient is applied. When the gradient is applied, the immunoglobulin that is labeled with enzyme is eluted before free enzyme. The eluted fraction containing the labeled immunoglobulin is freeze-dried and provides the enzyme-labeled second antibody for use according to the invention.

2. Preparation of first antibody

(a) Preparation of tri iodothyronine ( T^ )- cone jugate

250 mg of bovine serum albumin (BSA) was dissolved in 25 ml of distilled water and 1000 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was dissolved in 50 ml of distilled water. Both solutions were cooled to 4°C and 2/3 of the EDC solution slowly mixed with the BSA solution. 150 mg of purified T3 was dissolved in 25 ml of ethanol:2M-NH4OH (25:1, vol./vol.) and the solution cooled to 4°C was added dropwise to the mixture with continuous stirring while maintaining the pH at 7.0 by dropwise addition of 0/25M-HC1. The reaction mixture was stirred for a further 10 min. and the other EDC solution added dropwise while the pH was kept at 7.0.

The mixture was stirred overnight at 4°C. In the morning, the solution was centrifuged and the supernatant dialyzed against 3 batches of 0.1M NaCO3/NaHCO3 at pH 11.0 and then distilled water. The product was finally freeze-dried. The introduction of T3i BSA was found to be 4.3 mol T3 per mol BSA.

(b) Preparation of first antibody (T3~antibodies)

A stable emulsion was prepared by mixing 1 part saline solution containing Tween 80 (50%), T3 bovine serum albumin (2 mg/ml)

and Bacillus Calmette-Guerin (BCG 1 mg/ml) with 2 parts mareol S2 adjuvant. 6 doses (1 ml each) of the emulsion were injected subcutaneously in the back and 4 intramuscular injections (0.75 ml) were given in the calves. Antibody titers were regulated and after 36 weeks the animals were boosted. For reinforcement, 6 intramuscular injections (0.5 ml) were given in the calves. The emulsion consisted of parts excipient to 1 part salt solution containing T3~BSA conjugate (2 mg/ml). The blood samples 10 days after reinforcement and the following 2 months contained large amounts of T3~antibodies.

Ce) Preparation of immunoadsorbent for purification of first

antibody

Sepharose 4B was activated with 1,4-bis-(2,3-epoxypropoxy)-butane as previously described. The activated Sepharose (4 ml)

was added to phosphate buffer (pH 12.0, 0.025M, 20 ml) containing T3 (50 mg). The buffer was stirred for 45 hours at room temperature. The gel was then washed with phosphate buffer (pH 12.0, 0.025M, 250 ml), sodium chloride solution (150 ml, 0.1M) and distilled water (100 ml). 9 mg of T3 was added per ml of swollen gel. All remaining oxirane groups were blocked by suspending the gel in 4 times the volume of aqueous ethanolamine (IM,

pH 11) and shake for 4 hours. The gel was then exhaustively washed

with distilled water to remove excess ethanolamine.

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(d) Purification of first antibody

Anti-T₂ serum (prepared as described above, 20 ml) was mixed for 1 hour at room temperature with T₂-substituted Sepharose (1 ml). Non-specifically adsorbed proteins were removed from the immunoadsorbent by thorough washing with phosphate-buffered saline (0.05M, pH 7.0, 0.2H NaCl). The gel was packed into a syringe as previously described and washed with hydrochloric acid (pH 3) to remove the phosphate buffer. T₂ antibodies were eluted by washing with guanidine hydrochloride (4 ml, 6M, pH 2) and collected in barbitone buffer (1 ml, 0.051, pH 8.6, 0.1% bovine serum albumin). The T^ antibodies were exhaustively dialyzed against this buffer for

to remove the guanidine hydrochloride.

3. Preparation of applied antigen (T-,) for use in the measurement

The procedure is as described in the preparation of immuno-adsorbent for the purification of the first antibody (2 c above) with the exception that the concentration of T., is less. This lowers the concentration of substituted in the gel.

Measurement of T-. which use reagents whose preparation is described above is carried out in the manner already described.

"Universal Reagent" can be used in several different labels to determine antigens. These include:

Label la

Incubation of first antibody and free antigen followed by addition of immunoadsorbent, and after washing addition of the marker.

Label lb

A simultaneous incubation of first antibody, free antigen

and immunoadsorbent. The label is added to the washed immunoadsorbent.

in

Label 2a

An incubation of the first antibody, free antigen and labeling. This is followed after a suitable time by the addition of the immunoadsorbent.

Label 2a can be modified by incubating the first antibody with the enzyme label before adding free antigen (Label 2b).

Label 3

Label 3 includes simultaneous addition of first antibody, labelling, free antigen and immunoadsorbent. This label can also be modified somewhat by pre-forming the label-first antibody complex.

The following examples describe the results of the tests performed using the "Universal Reagent" followed by the labels described above.

Enzyme immunomeasurement of triiodothyronine (T3) using Universal Reagents

The measurement of T3" was carried out using several different methods.

Example 2

Label la

T3 antiserum (sheep, 1:2000 dilution) in barbitone buffer

(0.1 ml, 0.05M, pH 8.6, 0.1% w/v) was mixed with T3 standards prepared in barbitone buffer (0.1 ml) and kept at 4°C for 24 hours. Barbitone buffer (0.1 ml, 0.3% w/v Tween 80) containing T3 immunoadsorbent (50 µg) was added, mixed, kept at 4°C for 1 h, washed twice with barbitone-buffered saline (5% w/v volume of NaCl) and once with barbitone buffer. Enzyme labeling (100 µl, donkey anti-sheep IgG-galactosidase) was mixed with the washed immunoadsor-

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bent, kept at 4°C for 24 hours, washed as previously described and the enzyme activity associated with the immunoadsorbent was measured by standard methods.

Results

Non-specific binding of marker to immunoadsorbent = 0.05<x>Binding calculated after subtracting NSB.

A similar measurement was performed using donkey anti-sheep Fc-galactosidase as a marker. The procedure was as described above with the exception that (1) the antiserum and standards were kept together for 8 hours, (2) the antiserum dilution was 1:1600.

Example 3

Label 2a

Purified T3 antiserum (sheep, 1:1500 dilution, 0.1 ml) and T3 standards (0.1 ml) in barbitone buffer (0.05 M, pH 8.6, 0.1% w/v BSA) were mixed with marking (25 µl, donkey anti-sheep IgG enzyme) and kept at 4°C for 24 hours. Barbiton pouf f is I

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(0.1 mL, 0.3% w/v Tween 80) containing T3 immunoadsorbent (50 µg) was added with mixing and kept at 4°C for 1 hour. The immunoadsorbent was washed and the bound enzyme activity was measured as described in Example 2.

Results

Example 4 -

Label 2b

Purified T3 antibodies (1:1500 dilution) in barbitone buffer (0.05M, pH 8.6, 0.1% w/v BSA, 8.5 ml) were mixed with the enzyme label (donkey anti-sheep IgG-galactosidase conjugate, 5.1 ml) and stored at 4°C until use.

T3 standards in barbitone buffer (50 µl) were mixed with the label-first antibody complex (200 µl) and additional barbitone buffer (0.5 µl), then kept at 4°C for 4 hours. T3 immunoadsorbent (50 µg) in barbitone buffer containing Tween 80 (0.4%) was added to the tubes and incubated at 4°C for 1 hour. The immunoadsorbent was washed again with barbitone buffer containing 5%, then 1% sodium chloride. The enzyme activity linked to the immunoadsorbent bee measured by the usual methods.

In i Results

This experiment was repeated with a donkey anti-sheep F - enzyme label. The results were similar to those shown above.

Example 5

Label 3

T3 standards in barbitone buffer (100 µl) were mixed with the label-first antibody complex (200 µl) and T3 immuno-adsorbent (25 µg) in barbitone buffer (100 µl) containing Tween 80 (0.4% w/v) , and incubated at 4°C for 2 hours. The immunoadsorbent was washed and the bound enzyme was measured as described for Example 4.

Results

Example 6

Label 2b

The procedure was as previously described for example 4 except that the standards were prepared in stripped plasma. Thyroid-binding globulin was destroyed by heating the plasma samples in a water bath at 70°C for 1 hour. The results obtained were similar to those previously described in example 4.

Example 7

Enzyme immunomeasurement of human growth hormone (HGH) using Universal reagent

Label la

HGH antiserum (rabbit, 1:20000 dilution) in barbitone buffer (0.1 ml, 0.05M, pH 8.6, 0.65M NaCl, 0.5% w/v BSA) was mixed with HGH standards dissolved in barbitone buffer (0.1 ml) and kept at 4°C for 24 hours. HGH immunoadsorbent in barbitone puff f is (0.1 ml, 10 ug) was. then added, mixed, kept at 4°C for 1 hour and washed once with barbitone buffer (2 ml, 0.05M, pH 8.6) containing 5% NaCl and once with buffer containing 1% NaCl. Enzyme label (50 µl, donkey anti-rabbit F c-galactosidase) was added to the washed immunoadsorbent, mixed, kept at room temperature for 24 hours, washed as described above, and the enzyme activity associated with the immunoadsorbent was measured by the usual methods.

Non-specific marker binding to immunoadsorbent =

0.08

<x>Binding calculated after subtracting NSB.

Example 8

Enzyme immunoassay for insulin using Universal reagent Label la

Insulin antiserum (guinea pig, 1:10000 or 1:40000 dilution) in phosphate buffer (0.1 ml, 0.0411, pH 7.4, 0.5% w/v BSA) was mixed with bovine insulin standards prepared in phosphate buffer (0.1 ml) and kept at 4°C for 24 hours. Insulin immunoadsorbent in phosphate buffer (0.1 ml, 25 µg) was added, mixed and the tubes kept at 4°C for 30 min. The immunoadsorbent was then washed with phosphate buffer (2 ml) containing 5% and then 1% sodium chloride. Enzyme labeling (50 µl, donkey anti-guinea pig IgG-galactosidase conjugate) was added to the tubes, mixed and kept at room temperature for 24 hours. The immunoadsorbent was washed as previously described and the galactosidase activity associated with the immunoadsorbent was measured by the usual methods.

in

IN

IN

in Results

Antiserum dilution 1:10000.

Non-specific binding =0.05 of enzyme labeling in immuno-adsorbent.

Example 9

Enzyme immunomeasurement of cortisol using Universal reagent Label la

Cortisol antiserum (sheep, 1:20000 dilution) in phosphate buffer (0.1 ml, 0.1M, pH 7.4, 0.1% w/v gelatin, 0.83% w/v NaCl, 0.1% thiomersal) was mixed with cortisol standards prepared in phosphate buffer (0.1 ml), and kept at 4°C

for 24 hours. Phosphate buffer (0.3% w/v Tween 80) containing cortisol immunoadsorbent (0.1 ml, 25 µg) was

then added, mixed, kept at 4°C for 1 hour, washed once with phosphate buffer saline (3% w/v) and once with phosphate buffer. Enzyme labeling (25 µl, donkey anti-sheep IgG-galactosidase conjugate, 0.5% w/v Tween 80) was added to the washed immunoadsorbent, mixed, kept at 4°C for 24 h, washed as above and measured (as usual ) for enzyme activity bound to the solid phase.

Results

It is good to use sets of materials when carrying out the method according to the present invention on special antigens such as a set comprising an insoluble carrier to which an antigen is bound which may be the same or different.

from what the kit is intended to detect or measure, a suitable first antibody, and a suitable enzyme-labelled second antibody,

and means for detecting the enzyme activities required in the performance of the method, and preferably for calibration purposes included a solution containing a known quantity of the antigen which the kit is intended to detect or measure.

It can be advantageous to have the solid phase attached

a rod that can be introduced into and removed from the other components in the measuring mixture. This would facilitate washing of the solid phase and increase the accuracy of the measurement.

E.g. a set for T^ measurement can consist of the following components: 1. an insoluble carrier, either as a suspension in a buffer (eg barbitone) or in a lyophilized one. form with antigen bound to it.

IN

2. the first antibody (anti-T^ produced in sheep) together with the second antibody (produced in donkey-anti-sheep IgG Fc, the second antibody labeled with 3-galactosidase, this component preferably being in a lyophilized form. 3 a substrate (e.g. nitrophenyl-galactoside) at a final concentration of about 10-3 Mi a buffer (e.g. barbitone) preferably with a pH of 7 - 9 and a molarity of 0.010 - 0.200, this component is preferably in a lyophilized form 4. a solution containing a known amount of antigen T_.

(e.g. 10 nanomoles per litre), this component preferably being in a lyophilized form.

Claims (1)

Procedure for enzyme-linked immunoassay comprising bringing in a liquid contact medium simultaneously or stepwise (1). an antigen (as defined above) on an insoluble support or in the form of an insoluble aggregate, (2) a first antibody (as defined above), and (3) an enzyme-labeled second antibody (as defined above) in contact, insoluble and soluble materials are separated, and the enzyme activity is determined either in the soluble or in the insoluble separated materials, characterized in that said first antibody (2) is also brought into contact with a sample containing the antigen to be detected or measured which may be the same as or different from the antigen (1), the second antibody (3) thereby being linked to the insoluble material in the opposite proportion to the amount of the antigen in the sample to be detected or measured, whereby the presence or amount of the antigen in the sample is determined. j 2. Method according to claim 1, character i- performed by reacting the first antibody (2) with (1) and the sample, the solid phase is separated from the liquid phase, and all the first antibody that is bound to the separated solid phase is then reacted with the second antibody (3) in a other liquid medium free of (2) and the sample, followed by separation of the solid phase from the second liquid medium and detection or measurement of the enzyme activity in the solid phase. 3. Method according to claim 1, characterized in that the second antibody (3) is reacted with the first antibody (2) to form a complex of the two antibodies and the complex is then reacted with the antigen with (1) and the sample. 4. Method according to claim 2 or 3, characterized in that said first antibody or complex is reacted with the sample and then with (1). 5. Method according to claim 2 or 3, characterized in that said first antibody or complex is reacted with (1) and the sample at the same time. 6. Method according to each of claims 1 - 5, characterized in that the amount of said first antibody is more than sufficient to react with all the antigen present in the sample, but not more than sufficient to react with all the antigen in the sample plus all the antigen in 1). 7. Method according to each of claims 1-6, characterized in that the antigen in (1) is the same as the antigen in the sample and is carried by said insoluble carrier. 8. Method according to each of claims 3-7, characterized in that the enzyme activity is determined in the liquid separated phase to determine the presence or quantity of the antigen in the sample. 9. Method according to each of claims 1-8, characterized in that the first antibody (2) is any one of those previously stated herein. 10. Method according to each of claims 1-8, characterized in that the second antibody (3) is any one of those previously specified here. 11. Method according to each of claims 1-10, characterized in that the second antibody (3) is labeled with any of the enzymes specified here before. 12. Material set for use in carrying out the method according to each of claims 1-11, characterized in that it comprises an insoluble carrier (1). to which is bound an antigen which may be the same as or different from the type that the kit is intended to detect or measure or contains said antigen (1) in the form of said insoluble aggregate, a suitable first antibody (2). a suitable enzyme-labeled second antibody (3) and means for detecting the enzyme activity required in carrying out the aforementioned manner of walking. 13. Kit according to claim 12, characterized in that it comprises a solution containing a. known amount of the antigen that the kit is intended to detect or measure.