JPS60259964A - Assay of immunologically active substance in analysis sample - Google Patents

Abstract

PURPOSE:To assay reacting components in a sample quickly at a high accuracy by bringing immunologically active substance supported on a polymer particle into contact with a sample containing the components intended to react immunologically upon the substance to perform calculation by reaction dynamics based on increase in absorption measured in a specified wavelength range. CONSTITUTION:An immunologically active substance such as antigen generated by a micro-organism, serum alubmin, hapten and antibody is supported on an immunologically inert polymer particle of polystyrene or the like, for example, a latex particle with the particle size of about 0.05-0.9mum. A biological liquid sample such as serum and urea containing components intended to react upon the antigen, antibody such as hapten or the like and a sample such as cell extraction liquid is brought into contact with the supporting latex particle suspension to measure absorption increase in a wavelength range of 400nm-600nm after the start of the contact. Thus, based on calculation by immunological reaction dynamics, the concentration of reacting components in the sample can be assayed in a short time with a high sensitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immunological test method. For more details,
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, previously developed immunological test methods have relatively low sensitivity and only give quantitative or semi-quantitative results in the case of simple test methods (agglutination or precipitation tests), or only give very It has the disadvantage of being expensive (immunofluorescence test, radiation immunoassay, enzyme immunoassay).

Now, according to the present invention, an immunoassay method has been discovered which has a very high sensitivity (magnitude order: nf/ml) and which gives quantitative results and at the same time is simple and rapid to carry out.

More particularly, according to the invention, the immunological relationship between an immunologically active substance and an immunologically reactive partner is made measurable with the aid of sensitized polymeric carrier particles. The kinetics of the reaction is defined as the increase in absorbance from 400 nm to 600 nm.
A method for quantifying an immunologically active substance in an analytical sample, characterized by photometry in the sub-nm wavelength range and calculating the concentration of the immunologically active substance from the kinetics of this immunological reaction. is provided.

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,
Includes proteins, fatty proteins, glycoproteins, sterols, steroids, lipoids, nucleic acids, enzymes, hormones, vitamins, polysaccharides and alkaloids. Preferred immunologically active substances or their immunologically reactive partners are summarized in Table 1 below: Table 1. Antigens produced by microorganisms Bacteria 1, Gram-positive cocci Streptococcus pyogenes [Streptococcus pyogenes] pyogenes), Enterococcus faecalis (feca
lis) and Streptococcus aureus (vir, 1aan-8)
] Staphylococcus (5 taphylococci) [: Staphylococcus aureus (aureus) and Staphylococcus albicans (al)
bu day)] Pneumococci (D, pneumoniae)] 2,
Gram-negative cocci Neisseria [Gonococcus
norrhoeae) and meningococcus (meningi)
-tiai8) ] 5 Gram-positive aerobic bacillus (aerobicbacillus)
i'lli) Bacillus anthracia Oorynebacterium diph
therl, ae) Erysipelothrix (xr78ipelo-tb
rix) Listeria monocytosis (Li5teria monocy-
togenθS) 4, Gram-positive anaerobic bacillus (anaerobic
bacilli) Clostridium (Clostridia) [(Clostridium botulinum, Perfringens, Vre
llch'ii) and Clostridium tetani
] 5, Gram-negative anaerobic bacillus (anaerobi
cbacilli) Bacteroiaes & Gram-negative intestinal bacilli (1ntestinalbacil)
li) Escherichia, Klebaiella, Enterobacter (
Enterobacter) Proteus (Prot.
eus) Pseudomonas, Salmonella (Ba1m□He1la) Shigella (Shigella)
el/la) 1 Gram-negative non-intestinal bacillus (nonintθB-t
ina'l'bacilli) Pasteurella spp.
sis)) Haemophilus influenzae
1nf1u-enzae) Bruce'lla
melitensis), Bovine Abortion Bacteria (abort
us) and Swine Abortus (5uis) 3 Bordetella pertusns (, βOrdθtellap
θrtussis) MalleomycθB a Spirochetae Trebonne pallidum
1 (Treponema pallid-um) genus Leptospira genus Borrelia 9 Mycoplasma 10,
Mycobacteria 1
1. Vibrio 12. Actinomyces protozoa (
Protozoa) 1. Intestinal Protozoa
zoa ) Amebae Z Flagellates Trichomonas Leishmania Trypanonoma
panosomea ) toxopraz-z (Toxo
plasma) & sporozoans (5poro, zoa
) Plasmoaia [vivax, falciparum
'lciparum), vivax protozoa (malari-
aθ) and Plasmodium ovale (ova1=)] Fungi (Fungi) 1, Sporotrichum (8porotrichum,
)λ Cryptococcus
s) 5 BlaatomycθB 4,
Histoplasma (H1etop'lasma, ) 5,
Coccidioides (0occiaioiaes) &
Candida 1, Rickettsia 2 Can1ne hepatitis
) Elhope papilloma
a) Influenza A and B, Fowl plague, Herpes simplex (H
erpes simplex ) adenovirus (Aa
enoviruses) polyol
ma ) Rous family chicken M CRous day arcoma ) Vaccinia ) Polio viruses Measles (
German measles) Canin distemper (Leukemia) Mumps (Mumps) New casteθaisea
se ) 5enaai Ecno Foot and mouth disease
) Psitta-cosis Rabies (Rab
iθB) Extromelia, Arbor viruses, xenoantigen, polysaccharide, hyaluronic acid degrading enzyme, tetanus toxin, egg albumin, sheep serum albumin, human plasma gamma albumin, human serum albumin, natural antigen 1, hormone ptosis hormone y (Pituitary hormone
s) insulin lucagon thyroid hormone chorionic gonadotropin
otropin) chorionic growth hormone
hormone-prolactin (prola-cti)
n)] Z enzyme pancreaschymo
trypsinogens) procarboxypeptidases
) Deoxyribonuclease Ribonuclease Catalase Creatine Phosphokinase Quintessential organ antigens Kidney Liver Skin Heart (myoglobin) Gastrointestinal tract Prostate embryo antigen (e.g. CEA antigen) Swelling antigen 4, Components of connective tissue Muscle Collagen Amyloid 5 Blood cell antigens, Blood group substances and other alloantigenic platelets (p
'1ateleta) Megakaryocytes (Megakaryocytes) Alpha leukocytes (Leucocytes) Erythrocytes Blood group substances Forsman antigen (Forsman antigen)
) & plasma proteins fibrin and fibrinoid plasminogen and plasmin Pathological globulin Proteins of myeloma, polyglobulinemia and globulinopathy Rheumatoid factor C-reactive protein ■, natural antibodies 1, natural gamma globulin Natural antibodies - Nephrotoxic antibody component Z Autoantibodies Anti-cough factor Thyroid linear antibodies Adrenal autoantibodies Autoantibodies of gastric cavity parietal cells in pernicious anemia Autoantibodies of spermatozoa Autoantibodies of nerve tissue Autoantibodies against fibrous tissue and vascular components Small autoantibodies Autoantibodies against platelets and megakaryocytes Autoantibodies against trophoblasts Five Induced autoantibodies against: Immunoglobulin class 180118M1 EnggA or variant ■, hapten compound Opium alkaloids (morphine) Antipyrine Barbyric acid Preferred immunology The sexually active substances are human chorionic gonadotropin, rheumatoid factor, Toxoplasma gondii, Candi da, trypanosomes, CFiA antigen and myoglobin.

"Sensitized polymeric carrier particles" include immunologically inactive polymeric carrier particles that carry the immunologically active substance to be quantified or its immunologically reactive partner.

The immunologically inert polymeric carrier is composed of a water-insoluble fine polymer whose specific gravity is approximately equivalent to that of water, is immunologically inert, and has an immunologically The active substance or its immunologically reactive partner (antigen or antibody) can be bound physically and/or chemically. Approximately a, 05
Support particles with a particle size of ~α9μ are particularly preferred for the purposes of the invention.

Particularly suitable carrier particles are latex suspensions, for example: polymerized styrene resins with a particle size of 005 to 0.9 μ, polymethacrylic resins with a particle size of LL 07 to 0.9 μ, particle sizes of 0.05 to 0.9 μ. 0.9μ polystyrene, copolymer of butadiene and styrene, carboxylated copolymer of styrene and butadiene, carboxylated polystyrene, carboxylated polystyrene containing amino groups, acrylic acid polymer, methacrylic acid polymer, acrylonitrile, butadiene and mixed polymers of styrene, polyvinyl acetate acrylate, polyvinyl pyridine, vinyl chloride acrylate, etc.

Generally, an a1 to 15.0 weight scale antigen or antibody is bound to an 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, from which the analytical sample It turns out that it is possible to calculate the concentration of immunologically active substances in

Sensitized polymeric carrier particles can be present in the reaction mixture in varying amounts. However, the concentration of carrier is advantageously
Select so that the increase in light absorption during the immune reaction can be accurately measured.

When latex particles are used, the concentration of carrier in the reaction mixture is preferably between α06 and α9av/-1, especially l1l.
L3~α5Ilv/-.

Immunological reactions are carried out in a suitable buffer system while observing the optimum pH value. Optimum pH value is 5-10, preferably 7-10
It can be a5.

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 of 25 DEG to 37 DEG C. are particularly advantageous.

Further care can be taken to stabilize the sensitized carrier particles and to eliminate unspecified reactions.

According to the invention, quantitative assays for immunologically active substances can be carried out not only with direct test methods, but also with indirect (inhibition) test methods.

In a direct test method 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 kinetics of the immunological reaction is determined in a suitable manner. It is quantified 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 this mixture is then added with the immunologically active substance. Sensitize and add carrier particles. The kinetics of the immunological reaction is quantified from the increase in absorbance of the reaction mixture.

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.

For quantification of the kinetics of the immunological reaction in the test method according to the invention, the initial phase of the reaction, preferably the first 30 minutes of the reaction.
The increase in absorbance can be measured over a period of 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 during the time interval at the onset of the immunological reaction is directly proportional to the concentration of the 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 analytical sample and, measured during the initial phase of the immunological reaction, the time interval ab The increase in absorbance of is inversely proportional to the concentration of immunologically active substance in the sample to be analyzed.

In an automated 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 of 400 nm to 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 kinetics of an immunological reaction 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 kinetic-photometric test method according to the invention can be used for qualitative adjustment of the sensitizing carrier particles, for the quickest achievement of optimal reaction conditions in immunological test systems, for antisera for indirect immunoassays. Quantitative localization of - physiological fluids,
Determination of immunologically active substances in cell and tissue extracts, thereby replacing the traditional titration of analytical samples.
Quantitation can be performed using a single test sample to obtain quantitative results; one is the determination of immunologically active substances present in the analytical sample at such low concentrations that they would not be detected by conventional agglutination tests; Quantification, determination of immunologically active substances by means of an automatic analyzer provided by a kinetic measuring device.

The following examples illustrate the invention.

Example 1 Tris(Hat (p
H7,5) ) containing carboxylated latex from styrene and butadiene of 151191/-
Mix with 5 μt of latex suspension to which human chorionic gonadotropin (HOG) is covalently bound and then add 0.10- HCG antiserum (rabbit). H.C.
G antiserum was diluted to give the following titers for HOG antiserum in the test sample (1:4000, 1:8000, 1
:16000. 1:32000, 1:6400
0°1:128000). The increase in absorbance (△E) for 1 minute in each test sample was determined at 37°C and at a wavelength of 578°C.
Measure immediately in nm.

The results are summarized in Table ■ below: Table... As Table ■ shows, the increase in absorbance for 1 minute increases with the dilution of serum from 1:40DD to 1:, 128,000 anti-HO.
Directly proportional to G concentration.

Example 2 In each case 1. Otllt's HOG antiserum (1:2 dilution)
000'), HOG of 1.0 barges with the following HCG content:
Add solution: Ongz”s 10 ng/d, 15
ng/IRt, 30 ng/+d. After incubating the resulting mixture at 37°C for 2 minutes, each test sample was
Add 75 PL of a latex suspension containing 9/l of carboxylated latex from styrene and butadiene. HOG is covalently bonded to this latex.

The increase in absorbance for 1 minute in each test sample was measured at 37°C.
and immediately measured at a wavelength of 578 nm.

The results are summarized in Table ■: Table ■ As Table ■ shows, 2ng/scl! Even HOGs of

Example 3 1.5m7! (0.1 mol/l) of Tris/HCt,
Latex suspension containing acrylonitrile latex to which denatured gamma albumin is covalently bound (Rf
Add latex 1) 05-.

After diluting 0.1 μg of rheumatoid factor serum (Rf serum) and mixing 0.1 − μg of Rf serum with the latex suspension, the following titers for Rf serum were obtained in the test sample: Do: 1: 200.1: 400.1:
800.1:1600° The increase in absorbance for 1 minute in each test sample is immediately measured at '57°C and a wavelength of 578 nm.

The results are summarized in Table ■: Table ■ Example 4 1.0m! (a1 mol/z)), X 7 acz
(pH & 2) is added a 0,1- latex suspension containing a carboxylated latex of morphine-bound styrene and butadiene.

5.10, 20. and 50μ
t of anti-morphine serum (dilution 1:60) is added and the increase in absorbance for 1 minute in each test sample is measured at 37° C. and a wavelength of 578 nm.

The results are summarized in table ■: Table ■ 4

Claims (1)

[Claims] 1. 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. An analytical sample characterized in that the kinetics of the immunological reaction is measured as an increase in absorbance in a wavelength range of 400 nm or more and less than 600 nm, and the concentration of an immunologically active substance is calculated from the kinetics of this immunological reaction. A method for quantifying immunologically active substances in 2. The analytical sample is combined directly with an immunological agent containing sensitized polymeric carrier particles, the carrier particles being an immunologically inert material 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 photometrically on discrete particles of the polymeric carrier and the concentration of the immunologically active substance to be quantified is determined therefrom. (5) 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 inactive carrier containing the immunologically active substance to be quantified. Claim 1: "Containing as sensitized polymeric carrier particles" together with an immunological agent, measuring the increase in absorbance and calculating therefrom the concentration of the immunologically active substance to be quantified. 4. The method according to claim 1, wherein a latex suspension is used as a carrier.