JPS6255103B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an immunological test method. More particularly, the present invention relates to an immunological test method for the determination of immunologically active substances in physiological fluids, cell extracts and tissue extracts. Immunological testing methods are based on antigen-antibody reactions. However, conventionally developed immunological test methods
Sensitivity is relatively low and gives only qualitative or semi-quantitative results in the case of simple test methods (agglutination or precipitation tests) or very expensive (immunofluorescence tests, radioimmunotests,
Enzyme immunoassay). Now, according to the present invention, an immunoassay method has been discovered which has very high sensitivity (magnitude order: ng/ml) and gives quantitative results, while at the same time being simple and rapid to carry out. More particularly, according to the invention, an immunological reaction between an active substance and a partner of the immunological reaction is made measurable with the aid of sensitized polymeric carrier particles. The kinetics, i.e., the rate of progress of the antigen-antibody interaction, is measured as the increase in absorbance in the wavelength range from 400 nm to 600 nm, and the concentration of the immunologically active substance is measured as the rate of this immunological reaction. Provided is a method for quantifying an immunologically active substance in an analytical sample, characterized in that the amount of immunologically active substances is calculated from the following. By "immunologically active substances" we may mention all components of physiological fluids, cell extracts or tissue extracts in which they are present or capable of forming partners in an immunological reaction. They include primary amines, amino acids, peptides, proteins, fatty proteins, glycoproteins, throls, steroids, lipoids, nucleic acids, enzymes, hormones, vitamins, polysaccharides and alkaloids. Preferred immunologically active substances or their immunologically reactive partners are summarized in Table I below: Table I Antigens produced by microorganisms Bacteria 1 Gram-positive cocci Streptococci [Streptococcus pyogenes ( pyogenes), faecalis fecalis, and viridans; staphylococci (aureus and albus); pneumococci (D. pneumoniae). 2 Gram-negative cocci Neisseria (gonorrhoeae and meningitidis) 3 Gram-positive aerobic bacilli
bacilli) Bacillus anthracis Corynebacterium
diphtheriae) Erysipelothrix Monocytosis Listeria (Listeria diphtheriae)
monocytogenes) 4 Gram-positive anaerobic bacilli (anaerobicbailli) Clostridium (Clostridium botulinum, Clostridium perfringens type A, Welchii and tetani) 5 Gram-negative anaerobic bacilli ( anaerobicbacilli) Bacteroides 6 Gram-negative intestinal bacilli Escherichia Klebsiella Enterobacter Proteus Pseudomonas Salmonella Shigella 7 Gram-negative nonintestinal bacilli (nonintestinal
bacilli) Pasteurella (pestis and tularensis) Haemophilus influenzae
influenzae) Brucella [Melitensis, Abortus and suis] Bordetella
pertussis) Malleomyces 8 Spirochetae Treponema pallidum Leptospira Borrellia 9 Mycoplasma 10 Mycobacteria 11 Vibrio 12 Actinobacteria ( Actinomyces) Protozoa 1 Intestinal Protozaoa Amoeba 2 Flagellates Trichomonas Leishmania Trypanosomes Toxoplasma 3 Sporozoa Plasmodium Genus (Plasmodia) (vivax, Plasmodium falciparum)
(falciparum), malariae, and ovale] Fungi (Fungi) 1. Sporotrichum 2. Cryptococcus 3. Blastomyces 4. Histoplasma genus (Histoplasma) 5 Coccidioides 6 Candida Viruses and Rickettsia 1 Rickettsia 2 Canin
Shope papilloma (Shope hepatitis)
papilloma) Influenza A and B Fowl plague Herpes simplex Adenoviruses Polyoma Rous sarcoma Vaccinia Polio virues Measles German measles) Canine Day Temper (German measles)
distemper) Leukemia Mumps New castle
disease) Sendai ECHO Foot and mouth disease
disease) Psitta-cosis Rabies Extromelia Arbor viruses II Heterologous antigens Polysaccharides Hyaluronic acid degrading enzyme Tetanus toxin Egg albumin Sheep serum albumin Human plasma gamma albumin Human serum albumin III Natural antigens 1 Hormones Pituitary hormones Insulin Glucagon Thyroid hormone Chorionicgonadotropin
Chorionic growth hormone
hormone-prolactin〕 2 Enzyme Pancreatic chymotrypsinogen (Pancreas
chymotrypsinogens) Procarboxypeptidases Deoxyribonuclease Ribonuclease Catalase Creatine Phosphokinase 3 Organ specific antigens Kidney Liver Skin Heart (myoglobin) Gastrointestinal tract Prostate Emprio antigen (e.g. CEA antigen) Swelling antigen 4 Components of connective tissue Muscle Collagen Amyloid 5 Blood cell antigens, blood group substances and other alloantigens Platelets Megakaryocytes Alpha leucocytes Red blood cells Blood group substances Forsman antigen 6 Plasma proteins Fibrin and fipurinoids Plasminogen and plasmin 7 Pathology Glypurin Proteins in myeloma, polyglobulinemia and globulin disorders Lithomatoid factor C-reactive protein IV Natural antibodies 1 Natural gamma glopurin Natural antibodies - nephrotoxic antibody component 2 Autoantibodies Antinuclear factor Thyroid autoantibodies Adrenal autoantibodies Pernicious anemia autoantibodies of gastric cavity parietal cells autoantibodies of sperm autoantibodies of nervous tissue autoantibodies against fibrous tissue and vascular components autoantibodies against platelets and megakaryocytes autoantibodies against trophoplasts 3. Induced autoantibodies against: Bropurin classes IgG, IgM, IgA or variant V Hapten compounds Opium alkaloids (morphine) Antipyrine Valpituric acid Particularly preferred immunologically active substances are human chorionic gonadotropin, rheumatoid factor, Toxoplasma gondii, Candida, Trypanosoma, CEA antigen and myoglobin. "Sensitized polymeric carrier particles" include immunologically inactive polymeric carrier particles carrying the immunologically active substance to be quantified or its immunologically reactive partner. Immunologically inert polymeric carriers consist of water-inert, finely divided polymers whose specific gravity is approximately equivalent to that of water, which are immunologically inert, and which are immunologically inert. The biologically active substance or its immunologically reactive partner (antigen or antibody) can be bound physically and/or chemically. Carrier particles having a particle size of about 0.05-0.9μ are particularly preferred for the purposes of the present invention. Particularly suitable carrier particles are latex suspensions, for example: particle size 0.05-0.9μ.
polymerized styrene resins, polymethacrylic resins with a particle size of 0.07-0.9μ, polystyrene with a particle size of 0.05-0.9μ, copolymers of butadiene and styrene, carboxylated copolymers of styrene and butadiene,
Carboxylated polystyrene, carboxylated polystyrene containing amino groups, acrylic acid polymers, methacrylic acid polymers, acrylonitrile, mixed polymers of butadiene and styrene, polyvinyl acetate acrylate, polyvinyl pyridine, vinyl chloride acrylate, etc. Generally, 0.1-15.0% by weight of antigen or antibody is bound to the immunologically inert polymeric carrier. However, each individual immunologically active substance is associated with the carrier in the ratio most appropriate to the particular requirements. In the test method of the present invention, the sensitized polymeric carrier particles act as an indicator of the immunological reaction. Therefore, after mixing suitably sensitized polymeric carrier particles with the analytical sample, very small amounts of the immunologically active component will increase the absorbance, and this increase in absorbance can be photometrically measured, and from these It has been found that it is possible to calculate the concentration of immunologically active substances in an analytical sample. The sensitized polymeric carrier particles can be present in the reaction mixture in varying amounts. However, the concentration of the carrier is advantageously chosen such that the increase in absorbance during the immune reaction can be accurately measured photometrically. When latex particles are used, the concentration of carrier in the reaction mixture is preferably between 0.06 and 0.9 mg/ml, especially between 0.3 and 0.5 mg/ml. Immunological reactions are carried out in an appropriate buffer system while observing the optimal Ω value. The optimum Ω value can be between 5 and 10, preferably between 7 and 8.5. Examples of preferred buffers are phosphate buffer, Tris buffer [tris(hydroxymethyl)aminomethane], triethanolamine buffer, borate buffer or glycine buffer. Preferably, the immunological reaction is carried out at a temperature of 0-40°C. Temperatures between 25 and 37°C are particularly advantageous. Further care can be taken to stabilize the sensitized carrier particles and to eliminate unspecified reactions. According to the invention, the determination of immunologically active substances can be carried out not only by direct test methods, but also by indirect (inhibition) test methods. In direct test methods for the determination of immunologically active substances, the sample to be analyzed is mixed with carrier particles sensitized with the corresponding immunological reaction partner, and the rate of the immunological reaction is determined in a suitable manner. Quantitated by measuring the increase in absorbance of the mixture. In the case of indirect (inhibition) test methods for the determination of immunologically active substances, the sample to be analyzed is mixed with a certain amount of the corresponding immunologically reactive partner, and then the immunologically active substance is added to this mixture. and add carrier particles. From the increase in absorbance of the reaction mixture,
Quantify the rate of immunological response. In the test method according to the invention, the immunological reaction itself shows an increase in light absorption, which can be photometrically measured. The concentration of the immunologically active substance to be quantified in the analysis sample is calculated from this increase in absorbance. To determine the rate of the immunological reaction, the test method according to the invention makes it possible to measure the increase in absorbance at the beginning of the reaction, preferably during the first 30 seconds to 5 minutes, especially 1 to 2 minutes. At least two measurement time points must be chosen within this period. In the case of direct test methods, the increase in absorbance per time interval at the onset of the immunological reaction is directly proportional to the concentration of immunologically active substance in the analytical sample. In the case of indirect (inhibition) test methods, the rate of the immunological reaction decreases with increasing immunologically active substance in the analyzed sample, and per time interval, measured during the initial phase of the immunological reaction. The increase in absorbance of is inversely proportional to the concentration of immunologically active substance in the sample to be analyzed. In an automatic test method, the increase in absorbance and thus the course of the reaction can be followed continuously, and the concentration of the immunologically active substance can be calculated, for example, by a computer. Absorption measurements are carried out in the wavelength range from 400 nm to less than 600 nm. The test method according to the invention is preferably performed using an optical sample container, and the increase in absorbance can be quantified using a colorimeter, spectrophotometer, nephelometer, etc. Immunological tests can be performed using manual or automated analyzers. Any device capable of detecting the rate of immunological reactions is suitable for the purposes of the present invention. As analytical samples, body fluids such as serum, plasma, body fluids, urine, saliva or cell and tissue extracts can be used. The optical kinetics test method according to the invention can be used for: - qualitative control of sensitized carrier particles, - rapid establishment of optimal reaction conditions in immunological test systems, - for indirect immunoassays. - Quantitative standardization of antiserum - quantification of immunologically active substances in physiological fluids, cell extracts and tissue extracts, thereby replacing the traditional titration of analytical samples with a single test sample. quantitation is carried out using a method to obtain quantitative results, - quantification of immunologically active substances present in an analytical sample at such low concentrations that they would not be detected by conventional agglutination tests, - Quantification of active substances in immunological drops by automatic analyzer applied to reaction rate measuring device. The following examples illustrate the invention. Example 1 2.0 ml (0.1 mol/l) of Tris (HCI (Ω
7.5)) containing 13 mgl/ml of carboxylated latex from styrene and butadiene.
human chorionic gonadotropin (HCG) is covalently bound to it, and then 0.10 ml of HGC antiserum (rabbit) is added to it. The HCG antiserum was diluted to give the following titer for HGC antiserum in the test sample (1:
4000, 1:8000, 1:16000, 1:32000, 1:
64000, 1:128000). The increase in absorbance per minute (ΔE) for each test sample was determined at 37°C and wavelength.
Measure immediately at 578nm. The results are summarized in Table II below:

【table】

TABLE As Table II shows, the increase in absorbance per minute is directly proportional to the anti-HCG concentration at serum dilutions of 1:4000 to 1:128000. Example 2 In each case 1.0 ml of HCG antiserum (dilution 1:2000)
Add 1.0 ml of HCG solution with the following HCG content: 0 ng/ml, 10 ng/ml, 15 ng/ml, 30 ng/ml.
After incubating the resulting mixture for 2 minutes at 37°C, each test sample was treated with a 75 μl solution containing 13.5 mg/l of carboxylated latex from styrene and butadiene.
1 of latex suspension is added. HCG is covalently bonded to this latex. The increase in absorbance per minute for each test sample was measured at 37℃ and wavelength.
Measure immediately at 578nm. The results are summarized in Table III:

[Table] As Table III shows, even 2 ng/ml HCG can be detected reliably. Example 3 To 1.5 ml (0.1 mol/l) of Tris/HCl is added 0.5 ml of a latex suspension containing an acrylonitrile latex to which modified gamma-alpmin has been covalently bound (R _f latex I). Dilute 0.1 ml of rheumatoid factor serum (R _f serum) to 0.1
After mixing ml of R _f serum with the latex suspension, try to give the following titers for R _f serum in the test sample: 1:200, 1:400, 1:800,
1:1600. The increase in absorbance per minute in each test sample is immediately measured at 37° C. and a wavelength of 578 nm. The results are summarized in Table IV:

[Table] Example 4 1.0 ml (0.1 mol/l) Tris/HCl (Ω8.2)
contains a carboxylated latex from styrene and butadiene to which morphine is co-bound.
Add 0.1 ml of latex suspension. 5, 10, 20 and 50 μl of antimorphine serum (dilution 1:60) are added to this latex suspension and the increase in absorbance per minute of each test sample is measured at 37° C. and a wavelength of 578 nm. The results are summarized in Table V:

【table】

Claims (1)

[Claims] 1. The rate of an immunological reaction between an immunologically active substance and a partner of the immunological reaction made measurable with the aid of sensitized polymeric carrier particles. Immunological activity in an analytical sample, characterized in that the increase in absorbance is measured in the wavelength range from 400 nm to 600 nm, and the concentration of the immunologically active substance is calculated from the rate of this immunological reaction. A method for quantifying substances. 2. The analytical sample is combined directly with an immunological agent containing sensitized polymeric carrier particles, the carrier particles being an immunologically inert polymer having an immunologically reactive partner of the substance to be quantified. 2. A method as claimed in claim 1, in which the increase in absorbance is measured, and the concentration of the immunologically active substance to be quantified is determined therefrom. 3. The analytical sample is prepared by mixing the immunological substance to be quantified with its immunologically reactive partner, and then adding loose particles of an immunologically inert carrier containing the immunologically active substance to be quantified. Claim 1 wherein the concentration of the immunologically active substance to be quantified is calculated by combining it with an immunizing agent, measuring the increase in absorbance and calculating therefrom the concentration of the immunologically active substance to be quantified, contained as carrier particles of a sensitized polymer. the method of. 4. The method according to claim 1, wherein a latex suspension is used as a carrier.