DK153590B - IMMUNOLOGICAL DIAGNOSTIC REAGENT FOR DETERMINATION OF PREGNANCY, PROCEDURE FOR PREPARING THIS REAGENT AND ITS USE FOR DETERMINATION OF PREGNANCY - Google Patents

Abstract

1. An immunological diagnostic reagent for the determination of pregnancy by means of HCG determination and latex agglutination containing discrete particles of an immunologically inert latex polymer having a particle size of 0.01 to 0.9 mu m and a specific weight of 0.01 to 1.1, to which human choriongonadotropin (HCG) has been covalently bound via an amide bridge with a carbodiimide coupling agent at a pH of 5-8, characterized in that the HCG is used in the form of its beta-chain, whereby this beta-chain is substantially free from the alpha-chain and whereby this beta-chain has been separated from the alpha-chain by treating partially purified HCG containing 2000-5000 IU/mg of HCG for 1/2 to 2 hours at a pH of 4-5.5 and a temperature of 20-50 degrees C with a chaotropic agent, namely with a 7-10 molar aqueous solution of urea, a 7-10 molar aqueous solution of lithium bromide, a 4.5-6 molar aqueous solution of guanidine hydrochloride or a 2-3 molar aqueous solution of ammonium thiocyanate, subsequently adjusting the pH of the reaction mixture to 7.5 and separating the mixture of HCG/chaotropic agent by ion exchange and/or further conventional methods in accordance with which molecules can be separated on the basis of differences in the elektrostatic charge, and whereby neither the partially purified HCG nor the beta-chain thereof have been purified further.

Description

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in

The present invention relates to an immunologic diagnostic reagent for the detection of pregnancy by HCG determination and latex agglutination, a method for producing this reagent, and a method for detecting pregnancy.

The use of human chorionic gonadotropin (hereafter referred to as HCG) in the preparation of latex reagents for the detection of pregnancy has been known for a long time. Thus, e.g. U.S. Patent No. 3,857,931, which became publicly available on December 31, 1974, discloses the preparation and use of such reagents. Furthermore, there are commercially available a variety of reagents for the detection of pregnancy which are similar to the latex HCG type. A similar reagent is described in French Patent No. 2,234,789.

The commercially available latex HCG pregnancy tests generally use HCG in a partially purified form which is bound to a serologically inert latex polymer carrier. In performing the test, a drop of the woman's urine is placed on a cleaned slide, mixed with a drop of anti-HCG serum and then mixed with a drop of an aqueous suspension of a latex carrier to which HCG is attached. After a few minutes, the slide is examined to see if an agglutination 20 has occurred, indicating a negative result. This is only a very general indication of the method, as variants exist, both in technique and in materials used.

As several commercial pregnancy tests are based on HCG latex, there are constant efforts in this area of competition to improve these tests, namely in terms of sensitivity, material cost, manufacturing and convenience of handling and use. In the investigation of more specific tests, it has been found that by digestion of HCG in its α and β chain and subsequent purification of the β chain, a highly specific reagent can be prepared [Morgan & Canfield, Endocrinology, Volume 88, page 1045 (1971)].

Methods for purifying the β-chain from HCG are known, but the known methods are extremely expensive, why - although a reagent with improved specificity with a pure β-chain from

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2 The HCG commercialization as a latex-based test in practice is excluded. The only latex-based product on the market in which the β-chain material is used is a product in which β-chain antiserum and non-purified β-chain from HCG antigen are used, which is probably due to the expensive purification of β chain from HCG.

Within the scope of the present invention, there is now found a method according to which crude HCG, which is commercially available, can be treated without further costly purification so as to obtain from the HCG the β chain, which - surprisingly, is immunologically highly reactive .

The HCG β chain of the present invention can be readily bound to immunologically inert latex particles to obtain a reagent which is 2 to 4 times more sensitive than similar known reagents in which commercially available HCG is used. The high sensitivity exhibited by the reagent of the present invention is very advantageous considering that it can be prepared from relatively impure HCG without costly purification techniques that have hitherto been deemed necessary by the use of the β-chain from HCG.

The present invention relates to an immunologic diagnostic reagent 20 for detection of pregnancy by HCG determination and latex agglutination, which reagent contains separate particles of an immunologically inert latex polymer having a particle size of 0.01-0.9 µm and a density of 1.01-1.1, to which human chorionic gonadotropin (CHCG) is covalently linked to a carbodiimide coupling agent 25 at a pH of 5-8 via an amide bridge, which reagent is characterized in that HCG is used in its form. β-chain, which is essentially free of the α-chain and where this β-chain is separated from the α-chain by 1 / 2-2 hours treatment of partially purified HCG containing 2000-5000 ΙΕ / mg HCG at a pH of 4-5.5 and a temperature of 20-50 ° C with a chaotropic agent, namely with a 7-10 molar aqueous solution of urea, a 7-10 molar aqueous solution of lithium bromide, a 4.5-6 molar aqueous solution of guanidine hydrochloride or a 2-3 molar aqueous solution of ammonium thiocyanate, e setting the pH of the reaction mixture to 7.5 and separating the mixture of HCG and chaotropic agent by ion exchange and / or 3

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other conventional methods by which molecules can be separated due to differences in the electrostatic charge and in which neither the partially purified HCG nor the β chain thereof are further purified. The β-chain thus obtained from HCG exhibits a high immunological sensitivity, although it still contains a large amount of proteins, for example urine proteins, which are derived from crude starting material.

The invention also includes a process for preparing this reagent which is characterized by using HCG in the form of its β-chain, which is obtained by treating partially purified HCG 10 containing 2000-5000 ΙΕ / mg HCG in 1 / 2-2 hours at a pH of 4-5.5 and at a temperature of 20-50 ° C with a chaotropic agent, namely with a 7-10 molar aqueous solution of urea, a 7-10 molar aqueous solution of lithium bromide, a 4.5-6 molar aqueous solution of guanidine hydrochloride or a 2-3 molar aqueous solution of ammonium thiocyanate, thereby obtaining separation of the α chain and the β chain, the β chain being isolated free of the α the chain by adjusting the pH of the reaction mixture to 7.5 and separating the mixture of HCG and chaotropic agent by ion exchange and / or other conventional methods by which molecules are separated due to differences in the electrostatic charge without the β chain are subjected to further purification. The invention also relates to a method of detecting pregnancy by determining the presence of chorionic gonadotropin in body fluids, which method is characterized by mixing a sample of this fluid with chorionic gonadotropin antiserum and then contacting this mixture with an aqueous suspension of the immunological diagnostic reagents according to the invention and the results obtained are observed.

The β-chain from HCG obtained by the process of the present invention can be readily bonded to immunologically inert latex carrier particles in a manner known per se. It has been found that the reagent obtained has a 2 to 4 times as much immunological sensitivity as commercialized preparations which have commercially available HCG bound to a suitable latex.

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4

In the process of the present invention, a relatively unclean HCG is used as the starting material in the form in which it usually occurs in the market. This material contains HCG in the presence of protein-containing material. The content of HCG in such commercially available preparations usually varies between about 10%. 2000 and approx. 5000 international units / mg. The proteinaceous material present in such preparations does not immunologically contaminate HCG but merely "dilutes" it. In this specification, such HCG compositions are referred to as "partially purified" HCG.

In contrast to the "partially purified" HCG mentioned above, those skilled in the art of purified HCG understand such material which contains approx.

10,000 - approx. 20,000 ΙΕ / mg. Within the scope of the present invention, it has now been found that "partially purified" HCG, i. thus HCG containing approx. 2,000 - approx. 5,000 µg / mg can be treated with a chaotropic agent and the β-chain isolated therefrom - although still containing a substantial proportion of proteinaceous material of "partially purified" HCG - can be used to prepare a highly sensitive latex gestational reagent.

As used herein, the term "chaotropic agent" means an agent capable of cleaving hydrogen bridges in a polypeptide molecule. Chaotropic agents characteristically do not cleave covalent bonds in such molecules. Within the scope of the present invention, it is understood that the chaotropic agent acts more or less on the surface of the HCG molecule and breaks the hydrogen bridges and cleaves the molecule in the α and β chains.

Suitable chaotropic agents which can be used in the present invention include, for example, an aqueous solution of urea, ca. 7 -ca. 10 molar, an aqueous solution of lithium bromide at similar concentration, an aqueous solution of guanidine hydrochloride at a concentration of about 30 4.5 - approx. 6 mol, and an aqueous solution of ammonium thiocyanate, ca. 2 - 3 molar. The urea solution is preferred.

The chaotropic agents for the treatment of "partially purified" HCG of the present invention are used at a pH range of from ca. 4 to 5

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5.5, preferably approx. 5. An inorganic acid, preferably hydrochloric acid, is used to adjust the preferred pH range of the chaotropic agent. For the individual chaotropic agents, the preferred pH range may vary slightly. Thus, e.g. the preferred pH range using urea as a chaotropic agent of approx. 4.5.

The chaotropic agent is allowed to act for approx. 1/2 to approx. 2 hours, preferably for approx. 1 hour, in aqueous solution on the "partially purified" HCG. Then the pH of the reaction mixture is adjusted to 7.5.

A temperature of approx. 20 to 50 ° C, preferably approx. 37 ° C.

To adjust the pH in the HCG solution after the cleavage, a suitable inorganic base, e.g. an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide.

To prevent reunification of the α- and (5-chain from HCG after treatment with the chaotropic agent, the mixture of HCG and chaotropic agent is separated by ion exchange (layer or column), electrophoresis and / or other conventional methods, according to which molecules can be separated because of differences in the electrostatic charge, ion exchange chromatography is preferred. Any conventional ion exchange material can be used to decompose the α and β chain from HCG. A preferred material is a hydrophilic, water-insoluble, crosslinked dextran polymer gel. This material and the method of preparation thereof are described in British Patent Specification No. 854,715. The preferred gel material commercially available from Company Pharmacia Fine Chemicals, Uppsala, Sweden, under the trademark "Sephadex" consists of a three-dimensional, macroscopic network of dextran compounds which are interconnected or interconnected.This material is capable of absorbing water during swelling. e for water uptake is inversely proportional to the degree of crosslinking of dextran compounds. The gel material can be obtained in a variety of degrees in terms of porosity. Similar known substances in the art may also be used. Using this separation technique, the β chain is obtained from HCG which is essentially free of the contaminating α chain. It has been found that this technique allows the recovery of substantially more than 80% of the HCG antigenic properties. The "partially purified" 35 β-chain from HCG is then bound to a suitable immunologically inert latex carrier.

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The term "immunologically inert latex polymers" and "immunologically inert latex polymer carrier particles" encompasses water-insoluble latex polymers having a particle size of approx. 0.01 - 0.9 and a density roughly equivalent to water. This means that the density of the particles 5 after the binding of the β-chain from HCG is approximately equal to water or is slightly above it, ie. of 1.01 - approx. 1.1, whereby the particles remain in aqueous suspension. The latex particles must have a sufficient charge density on the surface for their repulsive forces to be large enough to prevent an agglutination 10 after binding to the β-chain from HCG. The latex particles must be inert to the immunological diagnostic tests and preferably have reactive groups capable of forming a covalent bond with the HCG β chain. Such groups are e.g. carboxy groups, amino groups and groups which can be converted therefrom. Typical, particularly suitable groups of latex particles for the purpose of the invention are those containing an active hydrogen atom, e.g. -COOH, -CONH2 and primary amino groups.

Particularly suitable latex carriers are those which are commercially available in the form of an aqueous latex suspension having a solids concentration of 20 usually 50-60%. For the purposes of the present invention, many species of latex polymers are suitable as long as they meet the above criteria. The present invention encompasses the use of all suitable latex polymers.

According to the present invention, the β-chain from HCG is covalently bonded to a water soluble carbodiimide coupling agent to the latex polymer particles via an amide bridge.

Examples of suitable latex polymers include carboxyl-containing polymers and copolymers such as polystyrene, polyvinylpyridine, styrene butadiene copolymers, vinyl acetate / acrylate copolymers and vinyl chloride-acrylate copolymers. The usual carboxyl groups can be incorporated into the latex by conventional emulsion polymerization techniques or they can be incorporated later by reaction of the already prepared latex polymer or copolymer in known manner. Thus, e.g. a monomer such as acrylic acid, methacrylic acid, itaconic acid, maleic acid or the like is added 7

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to the monomer mixture. On the other hand, the carboxyl groups can be formed in the usual manner on the surface of the latex, e.g. by oxidation of hydroxyl groups or by hydrolysis of acrylate or methacrylate groups on the surface. The latexes can be prepared by many conventional emulsion polymerization techniques, e.g. batchwise emulsion polymerization, graft emulsion polymerization or semi-continuous or continuous emulsion polymerization. For introduction of a carboxyl group, graft emulsion polymerization is preferred, in which a graft latex - before starting the reaction - is added to the reaction vessel to control the number of particles. In this case, a carboxy group-containing monomer is polymerized on the surface of the podela-tex particles.

Commercially available latexes suitable for the purposes of the present invention include, e.g. carboxylated styrene butadiene co-polymers available under the "Dow" 816 and "Dow" 421 marks from the Dow Chemical Company.

As the coupling agent, a water-soluble carbodiimide of the general formula is used

R-N = C = N-R

Wherein R represents a 5- or 6-membered cycloalkyl group; a straight or branched chain alkyl group having 2 to 12 carbon atoms, e.g. ethyl, n-propyl, isopropyl, n-butyl, sec. butyl, tert. butyl, amyl, hexyl, octyl, nonyl, undecyl or dodecyl; a monoaryl-substituted lower alkyl group, e.g. benzyl or α- or β-phenylethyl; a monoaryl group such as phenyl, morpholinyl or piperidyl; a morpholinyl-substituted lower alkyl group, e.g. morpholinoethyl; a di (lower alkyl) amino-lower alkyl group or a pyridyl-substituted lower alkyl group such as o-, β- or ϊ-methyl- or -ethylpyridyl; as well as acid addition salts and quaternary amines thereof.

Preferred coupling agents of the present invention are 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate and

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The reaction between the "partially purified" β-chain from HCG and the suitable latex support is preferably carried out in the presence of the carbodiimide coupling agent in aqueous medium, preferably at room temperature (8). However, the temperature may also be between 5 and 40 ° C. In order to ensure that the β-chain from HCG is effectively bound to the latex carrier, such an amount of water-soluble carbodiimide, which is approx. 0.05 - about 2% by weight, based on the weight of the carrier particles.

It is particularly preferred to use approx. 1% by weight of coupling agent 10 (based on the weight of the carrier particles). The proportion of carbodiimide coupling agent may vary within wide limits, but is dependent on e.g. the number of carboxyl groups and the presence of additional negatively charged groups (e.g. sulfonate groups) on the surface of the latex particles.

The pH value of the coupling reaction is important and should be between approx.

5 and 8, preferably between ca. 6 and 7. It is preferred to bring the pH of the latex suspension to the desired range, i.e. that is, between 6 and 7 before being added to the reaction mixture.

The reaction ends at between approx. 5 minutes and approx. 24 hours.

Generally, 1.5 - 2.5 hours are sufficient. The product obtained is a water-insoluble material which is suspended in an aqueous medium and exhibits a pH of approx. 7 - approx. 8.5, preferably 8. The density of the material corresponds to approx. water, which means that the suspension of the product is stable. The product can e.g. is isolated by centrifugation and is a white, slightly thixotropic, viscous, and clayey material. Chemically, the product consists of a monomolecular layer of "partially purified" β-chain from HCG, which is physically and / or chemically linked via an amide bridge to separated particles of an immunologically inert carrier. Any contaminating proteins present, as discussed above, have no serological effect on the sensitivity of the β-chain from HCG.

For commercial purposes, the reagent obtained according to the present invention is introduced in the form of an aqueous solution which contains approx.

0.5 - approx. 3.5% by weight of β-chain from HCG bound to it 9

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immunologically inert carrier. In particular, it is preferred that such suspensions contain between ca. 1 and approx. 2.5% by weight of such particles. In a preferred aspect, the (5-chain from HCG is attached via an amide bridge to a carboxylated butadiene-styrene copolymer, which has a monomer ratio of about 45% butadiene and 55% styrene, and contains about 1 to 5% by weight, usually 3% by weight, carboxyl groups.

The aqueous suspension of the "partially purified" (5-chain from HCG, which is bound to the immunologically inert carrier), according to the present invention, is preferably provided in the form of a test set containing in another container the appropriate antiserum, i.e. The above-mentioned antiserum can be prepared in a manner known per se, so rabbits or goats can be immunized with highly purified HCG or purified β-chain from HCG, after which the antisera are collected and their titers determined, the storage bin.

The invention is further illustrated by the following examples:

Example 1.

100 mg of commercially available crude HCG containing approx. 2700 IU / mg is cleaved by the method of Morgan & Canfield in Endocrinology, volume 88, page 201045 (1971). According to this method, the crude HCG is dissolved in 15 ml of a 10 M aqueous solution of urea and the pH of the resulting solution is adjusted to 4.5 by hydrochloric acid. The solution is incubated for 1 hour at 37 ° C, then 3 ml of a 0.03M aqueous solution of glycine is added, and the pH is adjusted to 7.5 by sodium hydroxide.

A solution thus obtained containing the separate α and β chain from HCG is subjected to ion exchange chromatography to physically separate the α and β chain and avoid a reunification. The chromatography is carried out on a 2.5 x 40 cm chromatography column which, to a height of 20 cm, is filled with "Sephadex" A-50 (Pharmacia). The product has a particle size (dry) of approx. 40 - 120 microns, a layer volume / ml / g in the dry gel of

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ίο 25 - 33 and a capacity of approx. 3 g hemoglobin / g gel. The column is equilibrated with an aqueous solution of glycine (0.03M) and urea (8M) of pH 7.5. The flow rate is set at 60 ml / hr, after which the solution containing the α and β chain from HCG 5 is applied to the column. The column is then eluted with 500 ml of glycine / urine solution and the eluate containing the α-chain from HCG is collected at once and discarded.

The column is again eluted with 500 ml of 0.2M glycine, 1.0M sodium chloride solution and 8M pH 7.5 urea solution. The eluates Ί0 are collected in one batch, which is diluted with an equal amount of distilled water and then brought to 20 ml by ultrafiltration. The fraction containing the β chain from HCG is then equilibrated with 0.1 M sodium borate buffer of pH 8.0 containing 0.02 wt% sodium azide.

15 By analytical disk electrophoresis on samples from the first and second eluates, it can be shown that the β-chain from HCG is free of α-chain, but still containing urine proteins, whereas the first eluate contains α-chain contaminated with a certain proportion. β-chain HCG and urinary proteins. By latex agglutination, it can be shown that more than 80% HCG activity from the raw starting material can be recovered.

Example 2.

According to Example 1, from "crude" commercially available HCG-produced β-chain from HCG, covalently binds to immunologically inert latex support particles: g 5 ml of a latex of carboxylated styrene-butadiene copolymer ("Dow" No. 816) is washed , ground, adjusted to a concentration of approx. 78-82 mg / ml and then poured into a suitable agitator container.

During stirring, 1 ml of β-chain from HCG prepared according to Example 1 is added and adjusted to a concentration of 3,000 IU 30 HCG / ml.

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When the β-chain from HCG is well dispersed in the latex, 3 ml of a 1% solution of 1-cyclohexyl-3- (2-morpholino-ethyl) carbodiimide-metho-p-toluenesulfonate is added dropwise with stirring. When the addition of the coupling agent is complete, stir for a further 2 hours.

The coupled latex is then centrifuged for 30 minutes at 5-10 ° C and 1500 rpm, separating the latex completely from the suspension medium. The supernatant is analyzed to determine HCG activity and then discarded. The residue is washed with 10 ml of distilled water and centrifuged as described above, then the supernatant is analyzed for HCG activity and discarded. The residue is washed with 10 ml of a pH 8 - 8.2 buffer containing 0.1M tris-HCl and 0.01% Merthiolate. The latex is again centrifuged in the manner described above, after which the supernatant is examined for HCG activity and discarded. The residue is washed again with the above buffer and then resuspended in 10 ml of this buffer. The cycle of centrifugation, supernatant analysis, washing and resuspending is repeated until for two consecutive cycles after washing with buffer, the supernatant is free of HCG activity. The coupled washed latex is then ready to be placed in a diagnostic reagent kit and resuspended in 20 ml of the aforementioned buffer.

A typical reagent kit (reagent garnish) containing the β-chain from the HCG made according to the present invention will contain in a separate container anti-HCG serum. Such anti-sera are formed in the usual way in goats, e.g. by immunizing the animals with highly purified HCG or the purified β-chain from HCG, collection of antisera and control of the titre. To eliminate cross-reaction between anti-HCG and urine proteins, the subtractive affinity chromatin raf is used.

For this procedure, 150 ml of washed "Sepharose" 4 B (Pharmacia) is activated by that of Primus et al. in J. of Immuno., Vol. 188, page 55 (1977). Mix 0.5 g% of urine protein in 200 ml of 0.1 M sodium borate buffer pH 8.0 with the activated Sepharose 4 B for 18 hours at 4 ° C to link the protein thereto. The product is washed

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12 with 1 liter 0.1M sodium borate buffer of pH 8.0 to remove unbound protein and then washed with 500 ml of 1.5M aqueous 8.0-pH ethanol solution 8.0 to reduce the activated groups in Sepharose 4 B The gel, which is then re-equilibrated with 5 sodium umborate buffer, has a pH of 8.0. The urine protein adsorbate obtained is poured into a 4.5 cm diameter chromatography column. To this column is poured in the following order: 10 ml normal goat serum (1: 2 in 0.1M sodium borate buffer is pH 8.0), 300 ml 0.1M sodium borate buffer pH 8.0 and 100 ml 3, OM ammonium thiocyanate in 0.1M pH 7.0 phosphate sodium phosphate. The column is then re-equilibrated with 500 ml of 0.1 M sodium borate buffer.

The anti-HCG serum is diluted with an equal amount of 0.1M sodium borate buffer pH 8.0 and poured onto the column. To control the adsorption process, an automatic affinity chromatograph 15 is recycled. The diluted antisera are automatically passed through the apparatus until everything is chromatographed. A sample of 15 ml is chromatographed at a flow rate of 60 ml / hour. The column eluate is continuously examined by adsorbance and - before protein appears in the eluate - the current is stopped for 1 hour so that the sample can react with the adsorbent. With 100 ml of 3M ammonium thiocyanate, non-adsorbed protein (specific antiserum for HCG) is first eluted and then adsorbed protein containing urine protein antibodies. The non-adsorbed fractions are combined and concentrated after ultrafiltration to the original sample volume.

25 Latex agglutination shows that recovery of antibody activity after adsorption is greater than 90%. The specificity of the antisera is determined by immunodiffusion and confirmed by immunoelectrophoresis.

Example 3

The sensitivity of the β-chain prepared from Example 1 from HCG 30 is shown as follows: 13

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An antiserum against HCG, goat serum, adsorbed with urinary proteins is titrated in a medium containing physiological saline solution. In the presence of constant amounts of latex conjugates, anti-HCG is diluted and the final dilution at which agglutination or flocculation can still be observed after 120 minutes incubation. The final dilution is considered the final product and titre of this antiserum and the corresponding antigen. The results are summarized in Table I. The latex conjugates being compared are β-chain latex according to Example 2, a similarly prepared α-chain latex and a commercial pregnancy test containing native HCG bound to a latex.

Table I

Dilution Latex conjugate

Anti-HCG HCG α β 15 __ 1: 2,000 3-4 3-4 2-3 1: 4,000 3-4 3 3-4 1: 8,000 3 - 4 neg. 3 - 4 1: 16,000 neg. neg. 3-4 20 1: 32,000 neg. neg. 2 1: 64,000 neg. neg. track

A value of 3 - 4 means strong reaction, 3 indicates medium reaction, 2 indicates medium to weak reaction, trace indicates very weak reaction, and "neg." indicates no reaction.

The higher the dilution of antiserum, the less HCG needed for its neutralization. The values for native HCG listed in Table I roughly correspond to a sensitivity of 1.0 - 1.25 IU HCG / ml urine. The sensitivity, ie the last determinable amount of β-chain latex is approx. 0.25 ΙΕ / ml urine.

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14

This correlation can be displayed as follows:

An antiserum test determines the amount of HCG in international units it reacts to. A number of dilutions of this sample in the above buffer are added to the urine samples to be tested. In each case, 2 ml of buffered antiserum is combined with 1 ml of urine in a test tube. 100 microliters of the latex to be tested is added to each sample and mixed by inversion. The lower end of the tube is heated for 2 hours to 37 ° C. Thereafter, agglutination or flocculation should be seen unless there is sufficient HCG in the urine to inhibit the reaction. The comparison is made between a pregnancy test containing native HCG and a β-chain product of Example 2. The results are set forth in Table II below. The comments for this are the same as for Table I.

Table II

Concentration Latex conjugate of HCG in the sample (IU / ml) HCG β 0.0 3-4 3-4 20 0.1 - 2 0.2 - ± 0.25 3 - 4 neg.

0.3 - neg.

0.4 - neg.

0.5 3 - 4 neg.

0.75 2 - 3 1.0 2 1.25 neg.

These figures show the enhanced sensitivity of the β-chain latex produced according to Example 2 relative to the gestational test containing native HCG.

Claims (7)

An immunologic diagnostic reagent for the detection of pregnancy by HCG determination and latex agglutination, containing reagent separated particles of an immunologically inert latex polymer having a particle size of 0.01-0.9 µm and a density of 1.01-1 1, to which human chorionic gonadotropin (HCG) is covalently linked to a carbodiimide coupling agent at a pH of 5-8 via an amide bridge, characterized in that HCG is used in the form of its β-chain which is in the substantially free of the α-chain, and wherein this β-chain is 10 separated from the α-chain by 1 / 2-2 hours of partially purified HCG containing 2000-5000 ΙΕ / mg HCG at a pH of 4-5, 5 and a temperature of 20-50 ° C with a chaotropic agent, namely with a 7-10 molar aqueous solution of urea, a 7-10 molar aqueous solution of lithium bromide, a 4.5-6 molar aqueous solution of guanidine hydrochloro-15 or a 2-3 molar aqueous solution of ammonium thiocyanate, adjusting the pH of the reaction mixture to 7.5 and separating the mixture of HCG and chaotropic agent by ion exchange and / or other conventional methods by which molecules can be separated due to differences in the electrostatic charge and wherein neither the partially purified HCG nor the β chain thereof is further purified. Reagent according to claim 1, characterized in that the latex is a carboxylated styrene-butadiene copolymer. Reagent according to Claim 1, 25, characterized in that the water-soluble carbodiimide coupling agent is illustrated by the general formula RN = C = NR where R represents cycloalkyl of 5-6 carbon atoms, straight or branched alkyl of 2-12 carbon atoms, monoaryl substituted lower 30 alkyl, monoaryl, morpholino, piperidyl, morpholino-substituted lower alkyl, di (lower alkyl) amino-lower alkyl or pyridyl-substituted lower alkyl, or acid addition salts thereof or quaternary amines thereof. DK 153590B Reagent according to claim 3, characterized in that the coupling agent is 1-cyclohexyl-3- (2-morpholinomethyl) carbodiimide-metho-p-toluenesulfonate or 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride. A method for preparing a highly sensitive immunologic diag nostic reagent for pregnancy detection by HCG assay and latex agglutination, wherein the human chorionic gonadotropin (HCG) at a pH of 5-8 by a carbodiimide cob adjuvant is bonded to separated particles of an immunologically inert 10 latex polymer having a particle size of 0.01-0.9 µm and a density of 1.01-1.1, characterized by the use of HCG in the form of its ( 5-chain obtained by treating partially purified HCG containing 2000-5000 ΙΕ / mg HCG for 1 / 2-2 hours at a pH of 4-5.5 and at a temperature of 20-50 ° C with a chaotropic agent, namely with a 7-10 molar aqueous solution of urea, a 7-10 molar aqueous solution of lithium bromide, a 4.5-6 molar aqueous solution of guanidine hydrochloride, or a 2-3 molar aqueous solution of ammonium thiocy -anate, thereby separating the α-chain and (5-chain, 20-β-chain is isolated from the α-chain by adjusting the pH-v the value in the reaction mixture of 7.5 and separation of the mixture of HCG and chaotropic agent by ion exchange and / or other conventional methods by which molecules are separated due to differences in the electrostatic charge without subjecting the β chain to further purification. Process according to claim 5, characterized in that the latex is a carboxylated styrene-butadiene copolymer and that the coupling agent is 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide-metho-p-toluenesulfonate or 1-ethylene. 3- (3-Dimethylaminopropyl) hydrobiimide hydrochloride. A method for detecting pregnancy by determining the presence of chorionic gonadotropin in body fluids, characterized in that a sample of this fluid is mixed with chorionic gonadotropin antiserum, after which this mixture is contacted with an aqueous suspension of the immunologic diagnostic reagent of claim 1. , and the results obtained are observed.