DE2533701A1 - IMMUNOLOGICAL DIAGNOSTIC PRODUCTS - Google Patents

Description

The invention relates to a diagnostic product for immunochemical determinations based on the agglutination phenomenon, processes for its preparation and its Use.

The invention is in the field of diagnostic agents and involves the application of well known immunological principles a. The invention relates in particular to an improved immunological diagnostic product with improved Sensitivity and stability which can be used for agglutination tests and which is a proteinaceous substance with immunological properties, which is crosslinked by reaction with a functional reagent and based on polystyrene and latex particles are adsorbed which have a diameter of about 0.05 to about 1.0 μm and which are serologically are inert and chemically inert towards the functional reagent, since they contain no reactive groups.

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To describe the phenomena addressed, the terms crosslink, polymerization and / or conjugation can be used (Protein conjugates) can be used. For the sake of simplicity however, the term networking is used.

When animals, including humans, are exposed to "foreign" proteins (such as antigens), their blood forms and their tissue fluids soluble substances (antibodies). Any protein that is normally found in a given animal absent, if introduced into such an animal under the appropriate conditions, formation can occur of antibodies. All immunological test methods are based on this so-called antigen-antibody reaction, whereby in the known diagnostic materials either an antigen, an antibody or a similar protein is used for this purpose the presence or absence of the corresponding antibody or antigen in the test Determine animal.

When an antigen and its corresponding antibody come into contact under suitable conditions, they combine to form a complex that is less soluble than the original ingredients not combined. This "Insoluble" complex is more or less visible to the human eye. Combine certain antigens and antibodies in a relatively short period of time with the formation of large particles that are macroscopically visible. However forms In many antigen-antibody systems, the complex changes very slowly and / or the particle size is so small that certain "Carriers" can be used to make the reaction macroscopically more visible.

In immunochemical processes based on the so-called antigen-antibody reaction build, one often uses inert particles as a carrier for the antigen, the antibody or other immunologically active proteins that have the immunological properties of an antigen or antibody to drive the reaction

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between the antigen and the antibody or the like, if such a process takes place by agglutination of the particles, which is also known as agglutination do. In this application, the antigen, antibody, or other immunologically active protein is either chemically bound to the carrier particles, or physically adsorbed thereon. In immunochemical processes, involving either adsorption or chemical bonding have become a wide variety of diverse Carrier materials used for a wide variety of immunologically active proteins, including polystyrene latex particles different size.

The prior art patents and prior publications differ from the invention in that they merely adsorb uncrosslinked antigen or antibody to the latex carrier or use a carrier particle having a reactive group that is non-crosslinked Antigen or the non-crosslinked antibody with a covalent bond through a coupling agent to the Binding surface of the carrier particle. In contrast to this, according to the invention, in relation to the coupling, as it is for the The above method is given, inert carrier particles are used, and the antigen, the antibody or a other similar immunologically active protein crosslinked, after which the crosslinked antigen, the crosslinked antibody or physically crosslink the other similar protein to form an improved diagnostic product Surface of the carrier particle is adsorbed. Investigations have shown that the diagnostic Product is more stable and sensitive than a product that can be obtained by simply absorbing uncrosslinked antigen, Antibodies or other similar protein attach to the support member receives, which is explained by the following explanations.

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In certain cases the method according to the invention can be more favorable than chemical coupling, as there are fewer physical and chemical changes (denaturation) of the protein can lead. An attempt was made to obtain tetanus toxoid via a carbodiimide reaction on a carboxylated particle to couple, but it has been found that the product formed in this way is not as sensitive as that after obtained by the method according to the invention. It should be noted that the desired sensitivity can only be achieved through the application of the method according to the invention, which is shown in the following examples will be further explained, and could not be achieved by simple adsorption or chemical coupling. For example, it has been shown that on polystyrene latex particles with an average diameter of 0.25 μπι adsorbed, non-crosslinked tetanus toxoid is suitable for the detection of 1 to 2 units of tetanus antibodies per ml of serum, while cross-linked tetanus toxoid adsorbed on the same polystyrene latex particles, 0.01-0.02 units of antibody able to detect per ml.

It is well known that antigens, antibodies or other proteins form insoluble products with bifunctional Reagents can be crosslinked. The crosslinked and insoluble antigen can, for example, be in columns or batches guided procedures can be used to determine the specific Separate antibodies from raw sera. The antibody can be recovered in a semi-pure state or pure by elution using various eluents. However, the subject of the invention is not this method, apart from the fact that the crosslinked antigen is in the form of a Coating is applied to the latex particle and is therefore in fact an insoluble antigen. When the latex is not introduced into the reaction mixture, no insoluble products are obtained. The coating with the crosslinked Antigen, or the adsorption of the crosslinked antigen to form a useful insoluble product, requires the presence the latex particles.

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The invention therefore relates to (1) a method for producing the immunological diagnostic product,

(2) the immunological diagnostic product as such,

(3) the use of the immunological diagnostic product in immunological test procedures; and (4) diagnostic equipment to carry out immunological test methods, the immunological prepared in the manner according to the invention contains diagnostic product.

The inventive method for producing an immunological diagnostic product that is based on the particles of a chemically and serologically inert polystyrene latex carrier material absorbed crosslinked protein which has immunological properties and with its immunological Counterpart to enter into the antigen-antibody agglutination reaction is capable of being

a) a protein that has immunological properties and is able to enter into an antigen-antibody reaction, mixed in a buffered aqueous medium with a bifunctional reagent,

b) the mixture is heated or at for a time sufficient to carry out the crosslinking of the protein Let stand room temperature,

c) separating the unreacted bifunctional reagent from the mixture and the crosslinked protein and

d) the crosslinked protein with inert polystyrene latex carrier particles with a particle size in the range of about 0.05 to about 1.0 ρ mixed to the adsorption effecting the crosslinked protein on the particles; or you can alternatively

e) a protein which has immunological properties and is able to enter into an antigen-antibody reaction with a bifunctional reagent and inert polystyrene latex carrier particles with a particle size in the range from about 0.05 to about 1.0 μτα. mix in a buffered aqueous medium,

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f) the mixture for a time sufficient to allow crosslinking of the protein and adsorption of the crosslinked To effect protein on the particles, heat or let stand at room temperature and

g) the unreacted bifunctional reagent from the medium split off.

The term "immunological counterpart" as used herein is either an antigen, antibody, or similar protein that possesses such properties and which undergoes a specific reaction with the corresponding or opposite antigen or antibody.

The immunological diagnostic product per se comprises chemically and serologically inert polystyrene latex carrier particles with a Particle size in the range from about 0.05 to about 1.0 μm, which is none have reactive groups and which carry a cross-linked protein that absorbs the immunological properties of antigens or has antibodies and is able to initiate the antigen-antibody agglutination reaction with its immunological counterpart enter into.

The application of the immunological diagnostic according to the invention Product in immunological testing involves performing an immunological test based on the gut known antigen-antibody reaction, which consists of that one has a crosslinked antigen, antibody, or similar crosslinked protein reactant (Any protein that has the immunological properties of antigens and antibodies can be used for this and is able to enter into the antigen-antibody reaction with its immunological counterpart) the chemically and serologically inert polystyrene latex carrier particles which have no reactive groups and are of a particle size in the range from about 0.05 to about 1.0 μΐη have adsorbed is, with the corresponding body fluid from which one is

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presumes that they represent the immunological counterpart Contains reactants, brings them into intimate contact and observes the mixture thus formed to determine whether ^ a macroscopic insoluble complex forms (agglutination) .

The invention containing the immunological diagnostic product Diagnostic equipment for performing the immunological test procedure essentially comprises a container for that on particles made from a chemically and serologically inert polystyrene latex carrier which does not contain any reactive groups and has a particle size in the range of about 0.05 to about 1.0 µm (the "Latex Reagent") of adsorbed crosslinked protein, which has immunological properties and is able to initiate the antigen-antibody reaction with its immunological counterpart enter into; a human positive control container; and / or a container for the antibody or the antiserum. The kit may contain additional components that are used in the test, such as additional ones Controls, diluents, slides, micropipettes, pipettes, and mixing sticks.

It has been shown that certain detergents for the maintenance the stability of the latex reagent are important. Without such detergents, the latex reagent tends to balling up while standing and / or giving false positive results with serum or plasma specimens; d. H. cause non-specific agglutination. It has therefore proven advantageous to use certain detergents, such as Triton X-100 (a neutral detergent from Rohm and Haas Inc.) and sodium taurocholate (an anionic detergent from Nutritional Biochemicals) in the latex reagent.

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In the present invention, as the immunological reagent, there can be used any protein that can be used for immunological diagnostic methods suitable is. Such a protein possesses immunological properties and is able to with its corresponding one immunological counterpart to enter into the antigen-antibody reaction. Examples of such proteins are: Tetanus toxoid, human chorionic gonadotropin, gammaglobulin, human albumin, Rheumatoid factor, generally viral and bacterial antigens, hepatitis antigens, rabies, gonnorrhea, and shyphilis Measles. In general, the invention relates to crosslinking any suitable proteinaceous substance followed by the absorption of the material obtained on chemically and serologically inert polystyrene latex particles that are not reactive Have groups. The proteinaceous substances can be converted into (1) antibodies; (2) antigens, a) virus and bacterial extracts, b) hormones (HCS, HCG, insulin etc.) and c) allergic and tumor extracts; (3) enzymes (catalase, peroxidase, urease etc.) and (4) haptens.

The concentration of the protein used depends in part on its purity and can, as in the case of pure HCG (Human chorionic gonadotropin) are only 0.06 mg / ml. In the latter case, however, the pure protein is using a serologically inert protein such as bovine serum albumin "spread" onto the latex particles (see Example 5). this takes place on the one hand to save the expensive protein, such as HCG, and on the other hand to prevent steric Obstruction that could occur if the molecules of the antigen (such as HCG) are too close together to allow a allow maximum reactivity with the antibody.

The bifunctional reagents to be used according to the invention can be classified with regard to their reactivity with different groups of a protein molecule. Examples of such reactive groups are: -SH (sulfides, disulfides), -NH ₀

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- y -

(et-amino groups, ^ -amino groups (on lysine residues) and amide groups), -COOH (glutamic acid and aspartic acid residues,
C-terminal residues), guanido groups, -OH (serine, threonine), phenolic groups (tyrosine) and imidazole groups (histidine). The following are examples of suitable bifunctional
Reagents and the appropriate reactive groups indicated:

(1) N-substituted maleimide derivatives (-SH);

(2) alkylating agents (ie, alkyl halides) (-SH, imidazole groups, -NH ₂ ); (3) bifunctional aryl halides (-NH ₂ /
Phenol groups, -SH, imidazole groups); (4) bifunctional
Isocyanates (-NH-); (5) bifunctional acylating reagents (OC -amino groups, ε -amino groups); (6) bifunctional
Imidoester (-NH ₉ ); (7) glutaraldehyde (-SH, -NH ₀ ); (8) bis-diazotized compounds (e.g. benzidine); (9) Carbodiimides (-NH ₂ , -COOH). The term "bifunctional reagent" means a reagent having two reactive groups capable of interacting with the side chains of the amino acids of the protein
react and build bridges in between.

The concentration of the bifunctional reagent depends on the reagent used. In most cases the amount to be used is roughly known from published data relating to the use of the reagent. In the case of the carbodiimide reaction, the amount used in Example 5 is 8 mg / ml, while the range of maximum effectiveness extends from about 5 to about 20 mg / ml. The amount of the necessary
Carbodiimide may be slightly different in the case of a different protein.

As already mentioned, those used according to the invention are
Polystyrene latex particles chemically and serologically inert,
contain no reactive groups and have a particle size which corresponds to a diameter in the range from about 0.05 to about 1.0 μm. An important feature is that the polystyrene latex particles are chemically inert to the bifunctional reagent. Particle with an average

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Diameters of about 0.25 to about 0.81 μm are most effective, with those particles with an average diameter of 0.25 and 0.81 μm being preferred. The polystyrene latex particles of the appropriate particle size can conventionally be obtained in the form of emulsions or suspensions under various trade names from a wide variety of manufacturers, for example Lytron W® latices from Monsanto Company, St. Louis, Missouri; Dylex l- * latices from Sinclair-Koppers Company, Pittsburgh, Pennsylvania; and Dow Latex Particles from Dow Chemical Company of Indianapolis, Indiana; and also acrylic polymers such as Acryl vx particles from Colab Laboratories, Inc., Chicago Heights, Illinois (polymethacrylic acid esters); and Ubatol U-7001 from Stanley Chemical, Cambridge, Massachusetts. Specific examples of suitable polystyrene latex preparations include Lytron 615 (Monsanto Company), Bacto-latex (Difco Laboratories), and Dylex K-31 (Sinclair-Koppers Company).

Any buffering system can be used with respect to the constituents and the conditions of the reaction mixture are chemically inert with a molarity in the range of 0.01 to 1.0 mol / l use. The choice of the buffer system depends on the optimal one pH of a given reaction mixture. For example, phosphate buffers are for the carbodiimide reaction with a pH range of 6.0 to 8.0 preferred, while for reactions which proceed at a pH of 8 or more, for example when using bis-diazotized benzidine, borate buffer or bicarbonate carbonate buffers are preferred. In general the bifunctional reagents are in an aqueous buffer system with a pH range of 6 to 10 is used. The adsorption of the crosslinked protein can also take place within the same pH range, but this depends on the type of protein used Protein and the type of latex used. However, the crosslinking of a protein can also be used in a specific one

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-M-

pH and adsorption of the crosslinked protein to the Latex particles take place at a different pH. The latter conditions make the method described below A necessary.

The essence of the invention resides in the cross-linking of the immunological active protein before or during adsorption on polystyrene particles, which in contrast to US Pat. No. 3,088,875, which the adsorption of non-crosslinked proteins {antigens and antibodies) on polystyrene latex particles, and NL-PS 7 201 308 teaches that the chemical bonding of non-crosslinked Describes protein with a coupling agent to the polystyrene latex particles. A new feature of the invention is that the crosslinked protein is firmly fixed (adsorbed) to the latex particles, and the obtained one Product is more stable and sensitive than products that have been adsorbed without crosslinking.

The diagnostic products according to the invention are extremely sensitive and stable in storage and can be used in direct slide agglutination tests can be used, for example, to determine the state of immunity by determining the Antibody content. These tests can be done on body fluids, such as whole blood, serum, plasma, or urine will. In practice, a drop of the latex reagent is added to an appropriate amount of the test sample (body fluid) applied to a slide. After mixing, the presence of the specific antibody can be confirmed by agglutination or agglomeration of the latex particles into large, easily visible clumps can be observed. Occurs under these conditions if no agglutination occurs, it is due to the absence of the specific antibody and thus a lack of immunity close. This principle can also be used for indirect tests to determine the presence of other proteins, for example, the determination of human chorionic gonadotropin in urine to determine pregnancy. In this case

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will inhibit agglutination as a positive reaction considered. Furthermore, antigen levels can be determined when the antibodies are adsorbed on the particles.

According to the invention there are two effective methods of manufacture of the diagnostic product according to the invention provided.

Method a

The selected protein and the bifunctional reagent are mixed in a buffered aqueous medium. The networking the protein molecules can be removed either by heating the solution for a short time or by letting the solution stand take place at room temperature for a longer period of time. The unreacted bifunctional reagent is then dialysed removed. The crosslinked protein solution is then added to the polystyrene latex particles and is at room temperature or adsorbed by them by heating the solution for a certain period of time. The latex reagent formed will centrifuged, after which the centrifuged material again is suspended. The centrifugation and the resuspension is repeated several times to remove any adsorbed Remove protein.

Method B.

The polystyrene particles, the protein and the bifunctional reagent are mixed in a buffered aqueous medium, heated or allowed to stand and then removes the bifunctional reagent and excess protein by repeated washing (by centrifuging and resuspending the centrifuged material), which is then attached to the polystyrene particles adsorbed protein remains.

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In the two methods A and B, the temperature "onetwa 5 may extend to about 60 ⁰ C, and it can be applied to 24 hours reaction time of 0.1. Of the above methods, method B is preferred.

A particularly preferred embodiment of the invention is the so-called tetanus latex reagent used for the tetanus latex agglutination test, which consists of a tetanus toxoid latex reagent which comprises crosslinked tetanus toxoid attached to chemically and serologically inert polystyrene latex carrier particles which contain no reactive groups, is adsorbed and is produced in the manner described. The tetanus latex test is a direct slide agglutination test to determine the status of tetanus immunity by determining the tetanus antibody content. This test can be done on whole blood, serum, or plasma. It is done in the way that one drop of the specific crosslinked tetanus toxoid latex reagent together with one drop of the Applying sample to a slide. After mixing, the presence of the antibody can be confirmed by a rapid agglutination of the latex particles into large, easily visible clumps can be observed. There is no agglomeration no tetanus antibodies are present.

In this way, amounts as small as 0.01 to 0.02 units / ml of the tetanus antibody can be determined. An example the use of the test system is illustrated in Table I below. It has been proven that a salary of 0.1 units / ml or more confers protective immunity. To perform quick overview examinations, the whole blood obtained by puncturing fingertips on a microscope slide with a sufficient amount of one Diluent mixed to make a 1:10 dilution of the To reach sample. A drop of the latex reagent is then added, followed by a sufficient amount of the antibody if necessary is present, agglutination of the latex reagent occurs. In this way, as little as 0.1 units / ml of tetanus

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antibodies can be detected in whole blood. Thus, one has obtained one with the whole blood diluted 1:10 positive agglutination for a protective antibody content (i.e., which is greater than 0.1 units / ml), while the failure to agglutinate indicates that the tetanus antibody level is low or zero.

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Table I.

Results of the tetanus latex test

Serum dilution

no dilution

1: 5

Tetanus antibody titer (units / ml)

interpretation

cn o co

1. 2. 3.

0.02 - 0.1 <0.02

protective immunity protective immunity questionable no immunity

1. The positive agglutination at both concentrations indicates a protective one Immunity so that no further immunization is required.

2. If this patient is at risk, the doctor can take one Give toxoid enhancers but not tetanus immunoglobulin.

3. The negative agglunitation at both concentrations indicates that none protective immunity exists and that toxoid immunization is required. If the patient is at risk, the doctor can use both tetanus immunoglobulin administer as well as perform a tetanus toxoid immunization.

The embodiment of the invention relating to the crosslinked tetanus toxoid latex reagent also relates to a method for preparing a diagnostic product containing crosslinked tetanus toxoid which is used in an agglutination test to determine tetanus immunity and which is based on particles of a polystyrene latex having a particle size of about 0, 05 to about 1.0 μπι, preferably with an average diameter of 0.25 μια, comprises adsorbed crosslinked tetanus toxoid, and which is characterized in that one

a) Tetanus toxoid with a bifunctional reagent such as N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride, in a buffered aqueous system, for example in a 0.05 M sodium phosphate buffer (pH = 8.0) mixed;

b) the mixture is heated for a time sufficient to crosslink the tetanus toxoid or at room temperature lets stand;

c) the unreacted bifunctional reagent (in this case N-ethyl-N- (3-dimethylaminopropyl) -carbodiimide hydrochloride) separating from the mixture and the crosslinked tetanus toxoid; and

d) the crosslinked tetanus toxoid with a polystyrene latex with a particle size of about 0.05 to about 1.0 μΐπ, preferably with a diameter of about 0.25 μm, mixed to effect the adsorption of the material on the particles. Alternatively, the diagnostic product containing the crosslinked tetanus toxoid can be prepared in that man

e | the tetanus toxoid with the bifunctional reagent and the Polystyrene particles mixed in the buffered medium;

f) the mixture for a period of time necessary for the tetanus toxoid to crosslink and for the toxoid to be absorbed by the Particles are sufficient, heated or allowed to stand at room temperature; and

g) separating the unreacted bifunctional reagent from the mixture.

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In the embodiment of the invention, which deals with the networked Tetanus latex reagent includes the immunological diagnostic product as such chemically or serologically inert polystyrene latex carrier particles which do not have reactive groups, a particle size of about 0.05 to about 1.0 jam, preferably have a mean diameter of about 0.25 μm and to which the crosslinked tetanus toxoid is absorbed.

The use of the tetanus latex reagent includes a method of determining the state of tetanus immunity by determination the tetanus antibody content in a body fluid using the antigen-antibody agglutination reaction, and which consists in that a) a body fluid, e.g. B. Serum, with the crosslinked tetanus toxoid, which at Polystyrene latex particles having a particle size of from about 0.05 to about 1.0 µm, preferably an average diameter of about 0.25 μπι is adsorbed, brings into contact, and b) observing the occurrence or non-occurrence of agglutination.

The tetanus toxoid latex diagnostic kit includes test kit, which is predominantly a crosslinked tetanus toxoid latex reagent prepared according to the invention as described above A container containing a positive human tetanus control serum and a container containing the diluent for the tetanus test (phosphate buffered saline PSA) containing container. The equipment can also include a microscope slide, calibrated micropipettes with a pipetting device and mixing sticks.

Another particularly preferred embodiment of the invention is a so-called HCG latex reagent (human chorionic gonadotropin), which is made from an HCG latex reagent prepared in the manner described which is made from crosslinked HCG that on chemically and serologically inert polystyrene latex backing

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particles that do not contain reactive groups, is adsorbed, and that for the detection of human chorionic gonadotropin im Urine or serum used by pregnant women.

The placenta hormone HCG is over during pregnancy excreted urine and detection of its presence generally constitutes a positive test for pregnancy The HCG pregnancy test according to the invention is based on the principle of inhibiting latex agglutination and is designed in such a way that meaningful test results can be obtained even at a very early pregnancy. The HCG pregnancy test can test HCG in concentrations as low as 2 to 4 international units per Detect ml of urine without any protein present in the urine, extreme pH values, the specific gravity, Sediments, red or white blood cells in the urine, or most medicines interfere. If you have the crosslinked HCG-containing Latex reagent mixed with the HCG-specific antibody on a slide or in a test tube, agglutination into large, visible lumps occurs very quickly. However, if the antibody is at an optimal If the concentration is mixed with urine or serum containing HCG, the antibodies are neutralized and form soluble ones Immune complexes. If this mixture is then mixed with the HCG latex reagent, no agglutination is observed, because the antibodies are neutralized and latex agglutination is inhibited. This shows the presence of HCG in the urine or in the serum and thus the pregnancy diagnosis as negative, i.e. H. no agglutination reaction, while the absence of HCG leads to a positive latex agglutination reaction.

The embodiment dealing with the crosslinked HCG latex reagent the invention further relates to a method for producing a crosslinked HCG-containing latex reagent as diagnostic product used in agglutination tests for pregnancy

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Shaft detection can be used, and the networked HCG used, which μπι on particles made of a polystyrene latex with a particle size of about 0.05 to about 1.0, preferably a mean diameter of about 0.25 μm, adsorbed is and that consists in that one

a) HCG mixed with a bifunctional reagent, for example N-ethyl-N ¹ - (3-dimethylaminopropyl) -carbodiimide hydrochloride, in a buffered aqueous medium, for example a 0.05 M sodium phosphate buffer (pH = 8.0)?

b) the mixture for a time necessary to crosslink the HCG is sufficient, heated or left to stand at room temperature;

c) the unreacted bifunctional reagent (in this case N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride) separates from the mixture and the crosslinked HCG; and

d) the cross-linked HCG with a polystyrene latex, the particles with a particle size of about 0.05 to about 1.0 μΐη, preferably having a mean diameter of about 0.25 µm, mixed to prevent adsorption on the latex particles to effect. Alternatively, the HCG latex diagnostic product can be prepared by

e) HCG mixed with the bifunctional reagent and the polystyrene particles in the buffered medium;

f) the mixture for a period of time sufficient to crosslink the HCG and adsorb the HCG on the particles, heated or left to stand at room temperature; and

g) separating the unreacted bifunctional reagent from the mixture.

The immunological diagnostic product per se with which the embodiment relating to the crosslinked HCG latex reagent is concerned comprises chemically and serologically inert polystyrene latex carrier particles, which have no reactive groups, a particle size of about 0.05 to about 1.0 μm and preferred

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have an average diameter of about 0.25 and to which the crosslinked HCG is adsorbed.

The use of the HCG latex reagent to prove pregnancy by determining the HCG content in a body fluid using the antigen-antibody agglutination reaction consists in that one

a) a body fluid, such as urine or serum, with the crosslinked HCG, the polystyrene latex particles with a particle size of about 0.05 to about 1.5 μπι, preferably with a mean diameter of about 0.25 μπι, is adsorbed, brings into contact and b) observes the occurrence or non-occurrence of agglutination.

The HCG latex diagnostic kit predominantly comprises a container containing the HCG latex reagent described, a Anti-HCG serum container and a freeze-dried, Containers containing HCG positive control urine. The equipment can also include a microscope slide, micropipettes, Pipetting devices and mixing sticks included.

The following examples serve to further illustrate the invention.

example 1

Production of networked! Tetanus toxoid adsorbed on polystyrene particles and comparison of this material with uncrosslinked tetanus toxoid adsorbed on polystyrene particles'

Four different diagnostic reagents are prepared. In the. Case A mix the polystyrene particles, the tetanus toxoid and the carbodiimide, heat the mixture and wash it repeatedly. In this case, the protein is crosslinked in the presence of the polystyrene. In case B one applies the same Procedure on, with the difference that you do not have a carbodiimide

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used. In case C, the tetanus toxoid and the carbodiimide are mixed and heated, then dialysis is carried out and finally clogs the polystyrene. In case D one applies the measures of method A, with the difference, dialyzing the tetanus toxoid prior to mixing with the polystyrene and the carbodiimide.

The actual reaction conditions used are as follows, with all reagents remaining the same:

Reagents;

0.05 m sodium phosphate buffer (pH = 8.0)

This buffer is obtained by mixing 2.7 l of a 0.2 M NaH ₂ PO 4 solution with 47.3 ml of a 0.2 M Na ₂ HPO ₄ solution

and diluting to 200 ml with distilled water.

Polystyrene latex: polystyrene beads, average diameter = 0.25 μm, available in the form of a 50% emulsion from Monsanto Co.

Tetanus Toxoid Concentrate: The material is with a concentration of about 500 Lf units / ml. The concentrate is for use in a ratio of 1: 5 with the phosphate buffer diluted.

Carbodiimide: N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide-HCl. The material is available from K & K Laboratories and becomes a solution immediately prior to use processed at a concentration of 20 mg / ml.

Sodium Taurocholate: Available from the Nutritional Company Biochemicals.

Triton X-100: Available from Rohm & Haas.

PSA buffer: (phosphate buffered saline, sodium azide): 0.01 M K ₂ HPO ₄ solution, 0.15 M NaCl solution and 0.1% sodium azide solution, which is adjusted to pH 7.0 with hydrochloric acid 7.1 is set.

PSA + 0.02% NaTC + 0.02% TX-100: This is the PSA buffer that contains 0.02% sodium taurocholate and 0.02% Triton X-100. _A " _{t Λ Α Λ} "

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Procedural measures:

(A) Charge 1.5 ml of the 5% polystyrene emulsion to a test tube. 1.8 ml of the phosphate buffer are then added, 1.2 ml of the tetanus toxoid solution are added, 1.9 ml of the carbodiimide solution are added and the mixture is incubated for 10 minutes at 50 ^° C. The mixture is then diluted to 10 with the phosphate buffer ml and centrifuged for 15 minutes at 20,000 rpm. The supernatant liquid is discarded. The centrifuged material is resuspended in 10 ml of the PSA buffer. The product is centrifuged three more times and resuspended in the PSA buffer. The material finally obtained is suspended in 10 ml PSA + 0.02 NaTC + 0.02% TX-100.

(B) All measures of method (A) are used, with the difference that no carbodiimide solution is added.

(C) A test tube is charged with 1.8 ml of the phosphate buffer, 1.2 ml of the tetanus toxoid solution are added, 1.0 ml of the carbodiimide solution is added and the mixture is incubated for 10 minutes at 50 ^° C., after which it is left overnight Dialyzed against the phosphate buffer at 5 ° C. 0.15 ml of the 50% strength polystyrene emulsion are added and the mixture is diluted to 5.5 ml with the phosphate buffer, after which it is heated ^{to 50 ° C. for 10 minutes.} The mixture is diluted to 10 ml with the PSA buffer and centrifuged for 15 minutes at 20,000 rpm. The centrifuged material is resuspended in 10 ml of the PSA buffer. The product is centrifuged three more times and resuspended in the PSA buffer. The material finally obtained is suspended in 10 ml of the PSA buffer containing 0.02% NaTC and 0.02% TX-100.

(D) A test tube is charged with 1.8 ml of the phosphate buffer, 1.2 ml of the tetanus toxoid solution are added, the mixture is incubated for 10 minutes at 50 ^° C. and then dialyzed overnight according to the procedure of method C. Then there are

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0.15 ml of the 50% polystyrene emulsion is added and then crosslinked with 20 mg of the carbodiimide and then with 1.1 ml of distilled water. The mixture is diluted with the phosphate buffer to 5.5 ml and heated to 50 ^° C. for 10 minutes. The mixture is diluted to 10 ml with the PSA buffer and centrifuged at 20,000 rpm for 15 minutes and then again in 10 ml of the PSA buffer suspended. The product is centrifuged three more times and resuspended in the PSA buffer. The finally centrifuged material is suspended in 10 ml of the PSA buffer which contains 0.02% NaTC and 0.02% TX-100.

investigation :

All four end products (A to D) are individually tested against various dilutions of human serum with a known tetanus toxoid antibody titer (7.5 units / ml) tested. The results are given in Table II below, in which the extent of agglutination is indicated with the aid of evaluation numbers, which range from (-) for no agglutination to to extend to (4+) for strong agglutination.

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O O

O O

O O CN

O O

O) il ri

(U CO

β φ

cd α) pi

ι ι ι ι

<m ο Q

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The results given in Table II above show that using methods A, C and D Tetanus toxoid latex reagents made a crosslinking reagent is a vastly superior diagnostic reagent compared to that made by Method B, which does not use such a crosslinking reagent. Since there is no noticeable difference in the level of sensitivity between reagents A, C, and D, the crosslinking agent (the carbodiimide) only reacts with the protein molecules (tetanus toxoid) and does not lead to a chemical coupling of the protein to the polystyrene latex, as might be the case with reagent A. Furthermore is a chemical reaction between the protein and the polystyrene via the carbodiimide is very unlikely because that Polystyrene has no reactive groups (free carboxyl groups or amino groups). The results are to be interpreted in such a way that that the crosslinking agent reacts with the protein to form a protein polymer, which is then essential is more firmly adsorbed (as in the case of reagent C shown in Table II) on the polystyrene surface than is the case with the simple one Adsorption in the case of reagent B occurs.

Example 2

Use of the cross-linked tetanus toxoid adsorbed on polystyrene particles in a slide agglutination test to determine the titer of human tetanus antibodies.

The slide agglutination test using the latex reagent is carried out as follows. The sample to be examined is diluted without and with different dilutions applied. For example, serial dilutions can be carried out (1:10, 1: 100 etc. or 1: 2, 1: 4, 1: 8 etc.). Man Place a drop (approx. 0.05 ml) of the sample or the diluted sample on a (glass) slide, after which a

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Add drops of the latex reagent. After rotating the slide for 2 minutes, the agglutination occurs rated with the help of an evaluation standard ranging from (no agglutination) to a value of 1+ to 4+ (approx up to 100% agglutination). The end point is the last dilution that gives a 1+ value. Dependent on on the sensitivity of the latex reagent, a 1+ value corresponds to a titer of 0.01 to 0.02 tetanus antibody units. Of the The titer of the undiluted sample is determined by multiplying by the dilution factor of the last dilution which results in a 1+ value calculated with the sensitivity of the latex reagent. For example, if the latex reagent has a sensitivity of 0.015 the titer of the serum with an end point (1+ agglutination) at a dilution of 1:20 is 0.015 χ 20 = 0.3 Tetanus antibody units / ml. The samples that can be tested in this way are serum, plasma, and whole Blood.

With minor modifications to the above procedure, the latex reagent can be used for diagnostic purposes to: to determine whether an individual has a level of antibodies protective against tetanus infection. It is believed, that a tetanus antibody titer of only 0.01 units / ml already gives a certain level of protection (B. Brown, J.A.M.A. 204, (1968) 152). The latex slide test used for diagnostic purposes is designed to test individuals with levels less than 0.1 units / ml (no protection to partial protection) from fully protected individuals (Contents of more than 0.1 units / ml) can differentiate. It is recommended that those falling into the first category Individuals receive a booster injection or a series of tetanus toxoid injections and / or a tetanus immunoglobulin injection received if tetanus infection is suspected or is to be feared.

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Example 3

Process for the production of crosslinked adsorbed on polystyrene particles! Tetanus toxoid.

Using the reagents described in Example 1, mix the following ingredients: 100 ml of diluted tetanus toxoid, 150 ml of phosphate buffer, 125 ml of polystyrene latex (12.5 ml of 50% latex + 112.5 ml of phosphate buffer) and 125 ml of the carbodiimide solution (2, 5 g dissolved in 125 ml of the phosphate buffer). The mixture is incubated immediately for 10 minutes at 50 ^° C., cooled and left to stand for one hour at room temperature. The mixture is then diluted to approximately 800 ml with the PSA buffer, after which the latex particles are separated by centrifugation at high speed (by centrifuging in an ultracentrifuge with a # 20 centrifuge head or equivalent centrifuge at 15,000 rpm for 15 to 20 minutes The resuspension and centrifugation operations are repeated several times until the correct sensitivity is achieved. The sensitivity of the preparation is checked by titrating a serum known titer. If the sensitivity is not less than 0.01 units / ml and not more than 0 .02 units / ml, the preparation is centrifuged once more and finally resuspended in 800 to 1000 ml of the PSA buffer, which contains 0.02% sodium taurocholate and 0.02% Triton X-100 Range from 0.01 to 0.02 units / ml.

Example 4

Preparation of cross-linked human albumin adsorbed on polystyrene particles. - ■. .

Reagents:

Polystyrene latex: Bacto-latex, available from Difco Laboratories, average particle size = 0.81 μια. .

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Human albumin: About 10 mg / ml in 1% sodium carbonate-bicarbonate buffer (pH = 9.5).

Crosslinking Reagents :

FNPS: 4,4'-difluoro-3,3'-dinitro-diphenylsulfone, 10 mg / ml in acetone,

CDI: N-ethyl-N ¹ - (3-dimethylaminopropyl) -carbodiimide hydrochloride, 20 mg / ml in water.

PSA buffer: (see example 1) Procedure:

To show that another protein and another crosslinking agent may be used in place of the carbodiimide in the preparation of the latex diagnostic reagent, the following becomes Experiment carried out. There are five reaction mixtures of the composition given in Table III below manufactured.

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Table III

Crosslinking agents

Tube No. Bacto-Latex Albumin-Lo- H ₀ O (ml) 0.2m

2 ^V

FNPS CDI

(ml) solution (ml) NaH ₃ PO ₄ * ™ ^

(ml)

1.0 1.0 1.9 - 0.1

1.0 1.0 1.5 - 0.5

S ³ 1 / ° 1 / ° 0.5 0.5 - _1/0

co ¹ '°' - '0.5 1, 0 _w

S 5 1.0 1.0 0.5 0.5 _ * °

co: '' '-

In each case, the latex and albumin solution are first incubated for 10 minutes at room temperature. Then water, with 0.2m NaH ₃ PO ₄ solution and the crosslinking agent are added in the amounts given in the table, after which the tubes are incubated again for 15 to 20 minutes at room temperature. The mixture is left to stand for 2 hours at room temperature and the latex particles are then recovered by filtration (0.45 μm Millipore filter). The filter cake is washed several times with the NaHCOo-Na ₂ CO ₃ buffer, then washed several times with the PSA buffer. Each filter cake is finally suspended in 2.0 ml of the PSA buffer.

The latex preparations formed on which the crosslinked human albumin is adsorbed are then subjected to a slide agglutination test used, which uses horse serum containing antibodies to human albumin. To carry out of the test, 1 drop of the diluted horse serum and one drop of the latex reagent are placed on a (glass) slide mixed and the slide rotated for 6 minutes. The results are given in Table IV below, where (+) stands for agglutination, (+) for partial agglutination and (-) for no agglutination.

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Table IV

Dilutions of the anti-human horse serum

Tubes undiluted 1: 0 1: 100 1: 1000 1: 2000 1: 4000

S ^{4 +} ±

CX) ^D

OO ^ o

O co

U)

K)

cn

CO CO

The above results show that the two crosslinking agents are approximately equally effective (tube # 2 and # 3), and that their combination (tube 4) does not increase the sensitivity to the crosslinking agents used individually. Farther a decrease in the amount of crosslinking agent (tube # 1) leads to a decrease in sensitivity. If the crosslinking agent is not added to the reaction mixture (tube # 5), the product formed shows none Reactivity to anti-human sera, i.e. undiluted horse anti-human serum does not lead to agglutination of the latex preparation made without the use of CDI. The results illustrate a more than 1000-fold increase in sensitivity through the use of the crosslinking agent.

Example 5

Manufacture of cross-linked human chorionic gonadotropin (HCG) adsorbed on polystyrene particles

Reagents:

HCG (human chorionic gonadotropin) with a titer of 8600 units / mg, available from Organon, Inc. of West Orange, New Jersey.

BSA (bovine serum albumin), crystallized, available from Pentex Inc., Kankakee, Illinois. Polystyrene latex: see example 1.
CDI: see example 1.
Phosphate buffer: see example 1.
Sodium taurocholate: see example 1. Triton X-100: see example 1.
PSA buffer: see example 1.

Procedure:

Using these reagents, mix the following ingredients: 200 ml polystyrene latex (20 ml of the 50% latex plus 180 ml of phosphate buffer), 100 ml of the HCG-BSA solution (i.e.

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30 mg HCG + 300 mg BSA dissolved in 100 ml phosphate buffer), 200 ml CDI (4 g dissolved in 200 ml water). The mixture is kept at 50 ^° C. for 12 minutes, cooled to room temperature and then left to stand at room temperature for at least 1 hour. The reaction mixture is then treated in a manner similar to that described in Example 3, except that more suspending and centrifuging operations may have to be carried out. The final preparation is satisfactory if the supernatant liquid obtained last contains less than 1 unit / ml free HCG. The finally centrifuged material is then suspended in 800 to 1000 ml of the PSA buffer which contains 0.02% sodium taurocholate and 0.02% Triton X-100.

The product obtained is used as a diagnostic tool for detecting early pregnancy in women in whom have suspended one or more menstrual cycles. The test is based on the fact that the HCG separation over the Urine sets in at a very early stage of pregnancy. By an indirect method using the HCG latex diagnostic reagent, HCG can be easily detected in the urine and used to prevent pregnancy of a patient. A drop of an appropriately diluted antibody is used to carry out the test for HCG mixed with a drop of the HCG latex reagent on a (glass) slide. After mixing and Strong agglutination occurs when the slide is rotated. On the other hand, when a pregnant woman's urine contains the antibody is mixed before adding the HCG latex reagent, the HCG present in the urine neutralizes the antibody, so that agglutination does not occur when the HCG latex reagent is added. Thus in the latter case indicates the non-occurrence agglutination suggests that the patient is pregnant. If the HCG latex reagent is used, a known one To titrate solution of HCG, the sensitivity of the Preparation must be at least large enough that 1 to 2 units of HCG / ml inhibit agglutination (an inhibition of 25% or more) and higher levels cause complete in-

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effect (no agglutination of the HCG latex reagent by the HCG antibody).

Example 6 Tetanus Toxoid Latex Reagent Diagnostic Equipment

The Tetanus Toxoid Latex Reagent Diagnostic Kit provides a 2-minute slide latex agglutination test for determination the tetanus immunity by determining the content of tetanus antitoxin in the serum. The diagnostic equipment is available as a Multiple test equipment packaged, each equipment being sufficient for 50 determinations. This test to determine the active Tetanus immunity is based on the agglomeration of the latex particles coated with the crosslinked tetanus toxoid by tetanus antitoxin. The antigen (cross-linked tetanus toxoid) is on Polystyrene latex particles (Lytron 615, mean particle size = 0.25 μΐη, Monsanto Company) adsorbed and becomes in the presence of the corresponding antibody (tetanus antitoxin) agglutinated. The agglutination can quickly be determined macroscopically, since large, easily visible clumps of latex particles result.

Equipment components (50 provisions)

1 ampoule - 2 ml tetanus latex reagent.

1 squeeze vial - 2 ml tetanus test diluent (phosphate-buffered Saline PSA).

1 ampoule - 1 ml positive tetanus control serum (human). 1 glass slide with 6 oval wells. 60 micropipettes.
1 micropipette holder.
60 application pens.

In order to ensure a suitable distribution and mixing of the reagents and the test samples, only drop devices, Disposable micropipettes and applicator sticks used in the equipment. A new, disposable micropipette is used for each test and a new applicator stick. When filling the micropipettes care must be taken that the sample does not fall into the rubber

5 09887/0817

suction bladder is pulled. If overcrowding is to be feared, the rubber bladder is rinsed in tap water, allowed to air dry and the test is continued. One uses just a clean, dry slide washed with a mild detergent and rinsed well with water.

Sampling and preparation

The test should only be performed on serum obtained from clotted blood. Hemolyzed specimens should be avoided. The test sera are stored at refrigerator temperature (2 to 8 ^° C.) or frozen if longer periods of time have elapsed before testing. Repeated freezing and thawing of the samples should be avoided. Likewise heating.

Test procedure

The test reagents are brought before the test is carried out and the serum samples to room temperature. With the help of the disposable micropipette transfer the entire contents of the serum (0.004 ml) into the oval recess on the slide. This stands out you fill the pipette through the hole in the white rubber cap, hold the black rubber bubble between your thumb and middle finger the pipette by capillary action and squeezes out the material by covering the hole of the black bubble with the index finger and squeeze out the bladder. Then add one drop of the tetanus test diluent (0.04 ml) from the squeeze vial into the oval well containing the serum. Mix well with the applicator stick. Then add 1 drop of the good suspended tetanus latex reagent from the squeeze vial. Mix and distribute the diluted serum and tetanus latex reagent with the other end of the applicator stick in the entire oval recess. Then you rotate the slide violent for 2 minutes. Then you check with reflected light against a black background whether a Agglutination has occurred.

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calibration

When used in the manner indicated above, the test is for this purpose suitable for tetanus antitoxin in amounts from 0.01 to 0.2 units / ml in the case of undiluted serum and in amounts of 0.1 to 2.0 units for sera diluted 1:10.

control

A positive tetanus control serum (human) is provided to confirm the positive test results. Control is in treated in the same way as the serum sample.

Results Positive test

A strong agglutination that occurs within two minutes on at least 25% of the latex particles represents one positive test for tetanus immunity.

Negative test

An agglutination of less than 25% in 2 minutes means a negative test for tetanus immunity.

Titer

The antitoxin titer can be determined by titrating positive sera. If desired, the antitoxin equivalent can be used specified (AE).

Application Limitations

If the undiluted serum sample gives positive results, dilute 1:10 for greater accuracy.
Expected values

Although it cannot be precisely stated, it has been reported that the minimal protective level of tetanus antitoxin

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probably between 0.01 and 0.1 units / ml of serum lies. In order to allow a safety margin from the specified minimum protective content of tetanus antitoxin, the includes Immuno-tetanus test a 1:10 dilution of the test serum. Antitoxin content is required to achieve positive test results of at least 0.1 to 1.2 units / ml of serum required.

Specific characteristics of behavior sensitivity

The sensitivity of this test for the detection and determination of tetanus antitoxin was compared with that of the accepted mouse neutralization test compared to reference antitoxins. In this test, for human antitoxins, as shown in Table V, 0.01 to 0.02 antitoxin units / ml detected.

Specificity and Reliability

The correlation of this test with the recognized mouse neutralization test for the detection and determination of tetanus antitoxin was carried out on 250 human serum samples from Costa Rica, Arizona and New York determined. There is a correlation of 95.2% with 1.2% false positive results and 3.6% false negative results. The results obtained are shown in Table VI.

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Table V

Determination of the sensitivity of the tetanus latex test. Correlation of slide-latex agglutination titers and mouse test with US standard antitoxin and comparison substances available from Lederle be used.

Test sample dilution factor AU / ml experiment

Comparative material no. TlG 40056-67RF (human) ' (180 AU / ml)

CTi O CO

Comparative material No. 2 ^ v ^^ «- * -., - ...... _U1

Plasma 9035B-46 (Human) 200 0.037 + + + + + + or

^ ³ (7.5 AU / ml)

Reference material no. Plasma (human) (<0.004 AU / ml)

undiluted 180 4000 0.045 8000 0.022 10,000 0.018 15000 0.012 20000 0.009 30000 0.006 undiluted 7.5 200 0.037 400 0.018 500 0.015 1000 0.007 undiluted 0.004 1:10 0.0004

Positive slide latex test = 1+ (a latex agglutination of 25% or more in the course of of 2 minutes).

(JH CjJ CO

Table VI

Comparison of tetanus antitoxin titers obtained with the mouse test and by Latex agglutination were determined (250 sera from Costa Rica, Arizona and

New York)

Mouse neutralization titer AU / ml

Number of sera

Latex titre AU / ml *

negative positive

<0.02 0.01-0.06 0.1-0.24 1.0-2.0 4.0-8.0 16-32 64

CO OO OO

CD CX?

negative

positive 0.02-0.06 0.1-0.5 1.0-2.0 4.0-8.0 16

67

16 8th 8th 24 10 21 3 59 1 24 19th 32 17th 7th 79 4th 2 1 1 27 2

* The latex titers are given as mouse AU / ml and are based on a latex test sensitivity of 0.01 to 0.02 AU / ml calculated.

Ca) CO

Example 7. ·,,. -

HCG latex reagent diagnostic equipment

The HCG latex reagent diagnostic kit is a 2-minute slide latex reagent inhibition test, the fast detection of human chorionic gonadotropin (HCG) used in the urine of pregnant women. This diagnostic equipment is packaged as multiple test diagnostic equipment, each equipment is sufficient for 50 determinations. The reagents supplied with this diagnostic kit include crosslinked Antigen in the form of purified HCG hormone deposited on spherical polystyrene latex particles (Lytron 615, middle Diameter = 0.25 Jim, Monsanto Company) is adsorbed, and rabbit or goat derived HCG antiserum hyperimmunized with HCG. Mix the HCG latex reagent on a slide with the HCG antiserum, rapid agglutination occurs to form large, visible clumps. However, if the antiserum is mixed with the urine of a pregnant woman that contains HCG, the antiserum will be neutralized, and it. there is no agglutination, which is a positive result of the pregnancy test means. Urine from non-pregnant women that does not contain HCG may contain the antiserum do not neutralize, so that agglutination of the latex particles occurs. This is a negative result of the pregnancy test.

Contents of the diagnostic equipment

1 Auspreßampulle - 2 ml HCG-latex ^{reagent; !} -

1 squeeze vial - 2 ml Ariti-HCG-Serum, '' -

1 ampoule - 1 ml HCG-positive control urine, freeze-dried,

1 glass slide with 3 oval wells; 60 disposable pipettes and

55 applicator sticks. . ■. ·. :. _; ■ ■ '· ·

To ensure proper application and mixing, the reagents and of the test samples, only the drip

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tongues, disposable pipettes and applicator sticks that come with this equipment. A new disposable pipette and a new applicator stick are used in each test. One uses only clean, dry slides that have been washed with a mild detergent and rinsed well with water. At each stage, the test components are mixed thoroughly. The HCG latex reagent must not be added to the urine / antiserum be added before an incubation period of 30 seconds has expired. Known positive and negative female urine samples are used. The freeze-dried HCG-positive control urine supplied with this diagnostic kit should be mixed with 1.0 ml of distilled water.

Sampling and preparation

Urine taken at any time can be used, although the first morning urine is preferred as it is generally has the greatest concentration of HCG and the greatest test sensitivity enables. Serum cannot be used in place of urine. Filtration of the urine is not necessary. The urine is stored at refrigerator temperature or frozen if waiting a long time before performing the test will. Repeated freezing and thawing of the samples should be avoided.

Test procedure

Bring the test reagents and urine samples before testing to room temperature. With the help of the disposable pipettes supplied, 1 drop of the urine is placed in the oval recess of the glass slide. Hold the pipette near the closed end between your thumb and forefinger. then the pipette is inserted under the surface of the sample, pressed firmly and then released. The pipette is removed from the sample, holds it vertically over the oval recess on the slide and lets a drop fall onto the slide by pressing

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presses gently on the pipette. Then add a drop of anti-HCG serum reagent from the squeeze vial to the same oval depression that contains the urine. Mix well with the applicator stick and slowly rotate the slide during 30 seconds. Then 1 drop of the well-suspended crosslinked HCG latex reagent is added from the squeeze-out ampoule. Use the applicator stick to mix the urine / antiserum mixture and the crosslinked HCG latex reagent and spreads it over the entire oval well. You rotate the Slide vigorously for 2 minutes and at the same time observed the occurrence of macroscopic agglutination The help of reflected light against a black background. The urine of non-pregnant women is used as a negative control.

Results Positive test

If no agglutination occurs within 2 minutes, this means a positive pregnancy test. That in that HCG present in the urine of pregnant women inhibits the agglutination of the latex particles and leads to a uniform, milky appearance.

Negative test

Agglutination occurring within 2 minutes means a negative pregnancy test. The absence of HCG in the urine of non-pregnant women allows the latex particles to agglutinate, resulting in the latex particles readily visible macroscopic lumps result.

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Claims (12)

Claims [1. ^ Diagnostic product for immunochemical determinations, • ^ based on the agglutination phenomenon, characterized in that it comprises chemically and serologically inert polystyrene latex particles which have no reactive groups, the particle size in the range from about 0.05 to about 1.0 ρ and on the surface of which a cross-linked protein with immunological properties is adsorbed. 2. Diagnostic product for immunochemical determinations based on the agglutination phenomenon, characterized in, that it comprises chemically and serologically inert polystyrene latex particles which do not contain reactive groups have, which have an average particle size of 0.25 μΐη have and on the surface of which a cross-linked tetanus toxoid is adsorbed. 3. Diagnostic product for immunochemical determinations based on the agglutination phenomenon, characterized in that that it comprises chemically and serologically inert polystyrene latex particles which have no reactive groups have a mean particle size of 0.25 μια have and on their surface cross-linked human chorionic gonadotropin is adsorbed, 4. Process for the manufacture of the diagnostic product according to Claim 1, characterized in that one a) in a buffered aqueous medium, a protein with immunological properties with a bifunctional one Reagent mixed, b) the mixture is heated for a period of time sufficient for the protein to crosslink or at room temperature lets stand c) the unreacted bifunctional reagent from the Mi- 509887/0817 separation and the crosslinked protein, and d) the crosslinked protein is mixed with chemically and serologically inert polystyrene latex particles which have no reactive groups and which have a particle size in the range from about 0.05 to about 1.0 micrometers, in order to promote the adsorption of the crosslinked protein onto the particles cause,
or alternatively e) a protein in a buffered aqueous medium with immunological properties with a bifunctional Reagent and chemically and serologically inert polystyrene latex particles that do not have reactive groups, and have a particle size in the range from about 0.05 to about 1.0 μm, mixed, f) the mixture for a period of time necessary for crosslinking of the protein and for adsorption of the cross-linked protein to the particles is sufficient, heated or allowed to stand at room temperature, and g) separating the unreacted bifunctional reagent from the mixture. 5. Process for the manufacture of the diagnostic product according to Claim 2, characterized in that one a) in a buffered aqueous medium tetanus toxoid with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride mixed, b) heating or heating the mixture for a period of time which is sufficient for crosslinking of the tetanus toxoid let stand at room temperature, c) the crosslinked tetanus toxoid from the mixture and the separating unreacted carbodiimide, d) the cross-linked tetanus toxoid with chemically and serologically inert polystyrene latex particles that are not reactive Have groups and have an average particle size of 0.25 µm, mixed for adsorption effecting the crosslinked tetanus toxoid on the particles; or alternatively 509887/0817 e) in a buffered aqueous medium tetanus toxoid with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride and chemically and serologically inert polystyrene latex particles that do not contain reactive groups have and have an average particle size of 0.25 μΐη, mixed, f) the mixture for a period of time necessary for crosslinking of the tetanus toxoid and adsorption of the cross-linked tetanus toxoid to the particles is sufficient, heated or allowed to stand at room temperature, and g) separating the unreacted carbodiimide from the mixture . 6. Process for the preparation of the diagnostic product according to claim 3, characterized in that one a) human chorionic gonadotropin mixed with N-ethyl-N ¹ - (3-dimethylaminopropyl) carbodiimide hydrochloride in a buffered aqueous medium, b) the mixture for a period of time sufficient to cause crosslinking of the gonadotropin, heated or left to stand at room temperature, c) separating the unreacted carbodiimide from the mixture and the crosslinked human chorionic gonadotropin, and d) the cross-linked gonadotropin with chemically and serologically inert polystyrene latex particles that are not reactive Have groups and have an average particle size of 0.25 microns, mixed for adsorption effect of the cross-linked gonadotropin on the particles, or alternatively e) in a buffered aqueous medium human chorionic gonadotropin with N-ethyl-N ^T - (3-dimethylaminopropyl) -carbödiimid-hydrachlorid and chemically and serologically inert polystyrene latex particles which have no reactive groups and an average particle size of 0.25 μm own, mixed, 509887/0817 f) the mixture for a period of time for a Cross-linking of the gonadotropin and the adsorption of the cross-linked gonadotropin on the particles is sufficient, heated or left to stand at room temperature, and g) separating the unreacted carbodiimide from the mixture . 7 ". Method for carrying out immunochemical determinations based on the agglutination phenomenon using of the diagnostic product produced according to claim 4, characterized in that a) brings a body fluid into contact with the product and b) the occurrence or non-occurrence of agglutination observed. 8. method for the immunochemical determination of tetanus immunity, which is based on the agglutination phenomenon using the one prepared according to claim 5 diagnostic product, characterized in that one a) brings serum into contact with the product and b) observed the occurrence or non-occurrence of agglutination. 9. Method of immunochemical pregnancy determination based on the agglutination phenomenon using of the immunological diagnostic product prepared according to claim 6, characterized in that a) brings urine into contact with the product and b) observed the occurrence or non-occurrence of agglutination. 10. Diagnostic equipment for performing immunochemical Determinations based on the phenomenon of agglutination, characterized in that they are essentially a) a container containing a diagnostic product produced by the method of claim 4 509887/08 17 and b) a container containing a positive control material and / or one containing an antiserum Containers included. 11. Diagnostic equipment for the immunochemical determination of the Tetanus immunity based on the agglutination phenomenon, characterized in that it is essentially a) an immunological diagnostic product prepared by the method of claim 5 containing Container, b) a tetanus test diluent containing Container and c) comprises a container containing a positive human tetanus control serum. 12. Diagnostic equipment for immunochemical determination of pregnancy based on the agglutination phenomenon, characterized in that it is essentially a) a container containing a diagnostic product produced by the method of claim 6, b) containing a human chorionic gonadotropin antiserum Container and c) comprises a container containing a positive control urea. 509887/0817