JPS60259965A - Immunological reaction apparatus - Google Patents

Abstract

PURPOSE:To obtain an apparatus for continuous immunological analysis by providing a mechanism wherein an antigen or antibody is supported on an immunologically inert polymer carrier, a fixed amount of a sample is injected into an optical sample container and then, the absorbance increasing speed of a reactive mixture is measured to calculate the density of the antibody or antigen. CONSTITUTION:An antigen formed by an microorganism such as gram-positive or gram- negative coccus, protozoan, fungi, virus, polysaccharide, serum alubmin, hormone, enzyme, natural gamma globulin and auto-antibody or antigen or antibody of a hapten compound or the like supported and fixed on an immunologically inert polymer particle of polystyrene or the like is placed into an optical sample container from a vessel while a sample such as serum is put into the optical sample container from an analysis sample container to react. A light with a specified waveform from a light source is made to irradiate the optical sample container and the absorption increasing speed of a reaction mixture is determined with an absorption measuring device by measuring absorbance at two points in the initial reaction based on a command from a time measuring mechanism and the density of the antibody or antigen in the sample is calculated with a computer based on the absorbance increasing speed to be displayed on a density recorder. Thus, an analysis can be done continuously and quickly with a high densitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a device for immunological reactions. More particularly, the present invention relates to an immunological reaction device 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 immunological reaction can be performed to perform an immunological test that has very high sensitivity (magnitude order: ng/wt) and gives quantitative results, and at the same time can be performed easily and rapidly. A device was discovered and used.

More specifically, according to the present invention, for causing an immunological reaction between an immunologically active substance supported on immunologically inert polymeric carrier particles and an immunologically reactive partner. for immunological reactions, consisting of a colorimeter, spectrophotometer or nephelometer, equipped with an optical sample container, and a mechanism capable of calculating the concentration of the immunological reaction partner by measuring the rate of increase in absorbance of the reaction mixture. Equipment 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 I 1 Antigens produced by microorganisms Bacteria 1, Gram-negative cocci Streptococci [Streptococcus pyogenes] pyogenes), Enterococcus faecalis (fe-e
alie) and viridans
] Staphylococcus (5taphy1ococci) (Staphylococcus aureus (aurθus) and Staphylococcus aureus (al)
bus)] Pneumococci [P, pneumoniae]2 Gram-negative cocci Neisseria [Gonococcus
norrhoeae) and Clostridium meningi (meningi)
-tidis) ] 5 Gram-positive aerobic bacillus (aerobicbacillus)
illi ) Bacillus anthracis Oorynebacterium dip
h-theriae) Erysipelo-th
rix) Listeria monocytosis (Li5teria monocy-
togenes) 4, Gram-positive anaerobic bacillus (anaerobic
bacilli) Clostridium (Clostridia) [(Clostridium botulinum), Clostridium Welch A
perfringens, wel
lchii) and Clostridium tetani [5]
Gram-negative anaerobic bacillus (aBaerobicba
ci'lli) Bacteroiaes & Gram-negative intestinal bacilli
li) Escherichia (Eachrichia) Klebsiella (Klebsiella) Enterobacter (
-Enterobacter) Proteus (Proteus)
teus) Pseudomonas genus (PaeudOmOna8) ゛Salmonella genus (Sa1mone'l'la) Shigella genus (
Shigella) l Gram-negative nonintestinal bacillus (noninte-et
inal bacilli) Pasteurella spp. (Pa5teurel'la) C. pestis (pestis)
n-sis)] Haemophilus influenzae
-engae ) Brucella spp.
melitensis), bovine abort tube (abort)
us) and Pig Abortion Tube (5uis) ] Bordetella verrussis (Bordθtel'll!
Lpertussis ) Mal'leomyces a Spirochetae Treponema pallidum
) Leptospira (Borrell, ia) 9 Mycoplasma (My
coplasma) 10, Mycobacterium (My
cobacteria ) 11, Vibrio spp.
iO) 12, Actinomyces protozoa (
Protozoa) 1. Intestinal Protozoa
zoa ) Ameba θ 2 Flagellates Trichomonas Leishmania Trypanoso-r (T
r7panO8Om68 ) Toxoplasma gondii (Toxo
Plasma ) Five sporozoa Plasmodium [vivax, Plasmodium falciparum
ciparum), malari-aθ
) and Plasmodium ovale ] fungi (F
ungi) 1, Sporotrichum (8porotrichum)
)Z Cryptococcus ((liryptococ
us) B'lastomyces
4. Histoplasma
5. C!occlioidea
& Candida (0an41da) Viruses and Rickettsia (R1-
ckettsia) 1, Rickettsia Z. Can1ne hepatitis
) 5hops papilloma
) Influenza A and B Fowl plague ) Herpes simplex (H
erpes simplex ) adenovirus (Ad
enoviruses) Polyoma
) Rous sarcoma Vaccinia Polio viruses Hemp
(German measles) Canine dis-tsmper Leukemla Mumps New castle disease
ee) Sendai (8endai) ECHO foot and mouse disease (FQOt ancL mouth disease)
) Psitta-cosis Rabies
ies ) Ectromelia Arbor viruses ■, xenoantigens polysaccharide hyaluronic acid degrading enzyme tetanus toxin egg albumin sheep serum albumin human plasma comma albumin human serum albumin ■, natural antigen 1, hormone ptosis hormone ( Pituitary hormones
) ink nillin lucagon thyroid hormone chorionic gonadotropin (Ohorionicgonaa
otropin) chorionic growth hormone [Chorionic g, ro
-wth hormone-prolactin (prola
ctin)] Z enzyme pancreatic chymotrypsinogen (Pancrθaschymo
trypsinogen) procarboquine peptidaes (Proaar-boxypeptidaes)
) Deoxidase 7 ribonuclease ribonuclease catalase creatine phosphokinase Quintessential antigens of organs Kidney liver skin Heart (myoglobin) Gastrointestinal tract Prostate embryonic antigen (e.g. OEA antigen) Swelling antigen 4, component of connective tissue Muscle collagen amyloid platelets (plateletθ) ) Megakaryocytes (Mθgakaryocytes) Alpha leukocytes (Leucocytes) Erythrocytes Blood group substances Forsman antigen
) & plasma proteins fibrin and fibrinoid plasminogen and plasmin Pathology globulin Proteins of myeloma, polyglobulinemia and globulinopathy Rheumatoid factor C-reactive protein ■, natural antibodies 1, natural gamma globulin Natural antibodies - nephrotoxic antibody component Z Autonomic anti-cough factor Thyroid linear antibody Adrenal autoantibodies Autonomic of gastric lumen parietal cells Autonomic of spermatozoa Continuous of nervous tissue Fibrous tissue and vascular components Induced autoantibodies against: immunoglobulin 221180116M1gA or variant opium alkaloid (morphine) antipyrine barbituric acid, especially preferred immunology The sexually active substances are human chorionic gonadotropin, rheumatoid factor, Toxoplasma gondii, Candida, trypanosomes, OEA 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 polymer carrier is made of a water-insoluble fine polymer, and its specific gravity is approximately equivalent to that of water, and is immunologically inert and immunologically inactive. The active substance or its immunologically reactive partner (antigen or antibody) can be bound physically and/or chemically. About aOS~
Carrier particles with a particle size of 0.9μ are particularly preferred for the purposes of the invention.

Particularly suitable carrier particles are latex suspensions, for example: polymerized styrene resins with particle sizes [L05 to α9μ, polymethacrylic resins with particle sizes 0.07 to 0.9μ, particle sizes 0.05 to 0.9μ polystyrene, copolymer of butadiene and styrene, carboxylated copolymer of styrene and butadiene, carboxylated polystyrene, carboxylated polystyrene containing amine groups, acrylic acid polymer, methacrylic acid polymer, acrylonitrile, butadiene and mixed polymers of styrene, polyvinyl acetate acrylate, polyvinyl pyridine, vinyl chloride acrylate, etc.

Generally, a 1-15.0 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.

The polymeric carrier particles used in the reaction device of the present invention act as indicators for immunological reactions. Therefore, after mixing suitably sensitized polymeric carrier particles with the sample to be analyzed, very small amounts of immunologically active components will increase their absorbance, and this increase in absorbance can be photometrically measured. The concentration of immunologically active substances in a sample can be calculated.

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 using latex particles, the concentration of carrier in the reaction mixture is preferably from 0.06 to α9/d1, especially from 03 to
It is 0.511v/-.

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

Examples of preferred buffers are diphosphate 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.

The determination of immunologically active substances using the device according to the invention can be carried out not only by direct test methods, but also by 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. 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 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 using the device of the present invention, the immunological reaction itself shows an increase in light absorption, which is measured photometrically. The concentration of the immunologically active substance to be quantified in the analysis sample is calculated from this increase in absorbance.

In quantifying the kinetics of the immunological reaction in the test method using the device of the invention, the increase in absorbance can be measured at the beginning of the reaction, preferably during the first 30 seconds to 5 minutes, especially during the first 1 to 2 minutes. It will not last until at least two measurement points are selected within this period.

In the case of a direct test method, 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 analyzed sample, and the time, measured during the initial phase of the immunological reaction, increases. The increase in absorbance over time 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 can be performed in the wavelength range of 4 Q Q nm to I Q OO nm. However, this measurement is preferably carried out at longer wavelengths, ie 500-750 nm. The reaction apparatus of the present invention includes an optical sample container for carrying out an immunological reaction, a colorimeter and a spectrometer equipped with a mechanism capable of calculating the concentration of an immunologically active substance by measuring the rate of increase in light absorption of the reaction mixture. It consists of a photometer and a nephelometer.

As analytical samples, body fluids such as serum, plasma, body fluids, urine, saliva or cell and tissue extracts can be used.

The kinetic-photometric device according to the invention can be used to: qualitatively control the sensitizing carrier particles; to achieve the fastest possible optimal reaction conditions in immunological test systems; Quantitative orientation, - physiological fluids,
Determination of immunologically active substances in cell and tissue extracts, thereby replacing the traditional titration of analytical samples.
Quantification can be performed using a single test sample to obtain quantitative results; one is the detection of immunologically active substances present in the analytical sample at such low concentrations that they would not be detected by conventional agglutination tests; Quantitative.

Next, the present invention will be explained based on the drawings.

An example of the present invention is shown in FIG. 1, which includes an optical sample container to which a light source, an analysis sample container, and a sensitized carrier particle container are combined, a computer to which an absorption measuring device, and a time measuring device are combined; A concentration recorder is connected as shown in the figure, and its operation is explained as follows: While irradiating light of a predetermined wavelength from a light source through an optical sample container, an analytical sample is recorded from an analytical sample container and a sensitized carrier particle container. Introduce the sample and sensitized carrier particles into an optical sample container at a predetermined ratio, and measure the absorbance increase rate of the reaction mixture at two points at the beginning of the reaction based on the command from the time measuring device. The concentration of the immunologically active substance in the analysis sample is calculated by computer based on the rate of increase in light absorption, and is recorded or displayed on the concentration recorder.

An example in which the titer of antiserum was measured using the above device is shown.

Example 1 2, o, t (Q, 1 mol/1) of Tris (Hat (p
H15)) was mixed with a 75 μt latex suspension containing 15 ηt/− of carboxylated latex from styrene and butadiene to which human chorionic gonadotropin (HC!G) was covalently attached, and then 0.
Add 10 d of HOG antiserum (rabbit). The HOG antiserum was diluted to give the following titer for the naG antiserum in the test sample (1:4000.1:8000.1:1
6000.1 :32000.1 :64000.1
:128000). The increase in absorbance (ΔE) for 1 minute in each test sample was determined at 37°C and at a wavelength of 578 nm.
Measure immediately.

The results are summarized in Table 2 below: Table 2 As shown in Table 2, the increase in absorbance per minute is directly proportional to the anti-HOG concentration at serum dilutions of 1:4000 to 1:128000.

Example 2 In each case 1, Od of HOG antiserum (dilution 1:2000)
Add 1.0 d of HCG solution with the following) IOG content: Ong/d, I Q ng/d.

15ng/l117! , 30 ng/do After incubating the resulting mixture at 37°C for 2 minutes, each test sample received 13, 30 ng/do.
Add 75 pL of latex suspension containing carboxylated latex from 5 Ml/L styrene and butadiene. HOG is covalently bonded to this latex. The increase in absorbance for 1 minute in each test sample is immediately measured at 37° C. and a wavelength of 578 nTn.

The results are summarized in Table ■: As Table ■ shows, even 2 ng/d HOG can be reliably detected.

Example 3 t 5 ml (o, 1 mol/zl Tris/Hat
A latex suspension (R
f latex I) 0.5 +l+/! Add.

01 rheumatoid factor serum (Rf serum) is diluted to give the following titer for Rf serum in the test sample after mixing the 0.1-Rf serum with the latex suspension: 1: 200.1:400.1:80
0.1:160[]. The increase in absorbance for 1 minute in each test sample is immediately measured at 37° C. and a wavelength of 578 nm.

The results are summarized in Table ■ Example 4 1, o, 1 (o, 1 mol/l) of Tris/H
At (pHa2) is added a 0,1- latex suspension containing a carboxylated latex of styrene and butadiene co-conjugated with morphine.

5. Add to this latex suspension. io, 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 ■

[Brief explanation of drawings]

FIG. 1 shows a schematic diagram of the immunological reaction device of the present invention. Patent applicant: Mitsubishi Chemical Industries, Ltd. Agent Yoshi Mine Katsura

Claims (1)

[Scope of Claims] 1. An optical sample for causing an immunological reaction between an immunologically active substance supported on an immunologically inert polymer carrier and an immunological reaction partner. An apparatus for immunological reactions consisting of a colorimeter, spectrophotometer or nephelometer, which is equipped with a container and a mechanism capable of calculating the concentration of the partner of the immunological reaction by measuring the rate of increase in light absorption of the reaction mixture. 2. The device according to claim 1, wherein the concentration is calculated by a computer. 5. Continuously track the course of the immunological reaction; 1. 3. The apparatus according to claim 2, wherein the result of the rate of increase in light absorption is evaluated by a computer.