JPS62151759A - Detection of antigen material - Google Patents

Abstract

PURPOSE:To make easy detection, concentration, sepn. and refining of an antigenic material by using the solubilized protein A which is fixed to a carrier consisting of slide glass or the like and is bound with an antibody for a specific antigenic material. CONSTITUTION:The antigenic material is bound with the carrier made of glass, plastic or paper via the antibody for the antigenic material and the protein A and the antigenic material is qualitatively or quantitatively analyzed, by which the antigenic material is detected. The protein A-carrier conjugate is cultured in a medium as necessary to propagate the antigenic material and the bound or propagated antigenic material is recovered, by which the antigenic material is concd., separated or refined. The carrier of a plane or bar shape is used; for example, slide glass, glass bar, etc., are used. The protein A may be used alone or in the form of the cells bonding with the cells to produce. The eucaryotic cells, procaryotic cells and virus are used for the antigenic material and a polyclonal antibody and monoclonal antibody are used for the antibody for the antigenic material.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for analyzing, concentrating, separating, and purifying antigenic substances using a conjugate of protein A and a carrier made of glass, plastic, or paper. I will provide a. Therefore, the present invention is applicable not only to the field of clinical laboratory medicine but also to a wide range of medical fields.
Can be used in the field of biology.

Conventional technology Protein A has been used to detect and separate cell surface antigens including IgG [Immunology
nol, ), 26.1081-1(15)1.19
74. Scandinavian Journal of Immunology (Scand, J. Immunol), 3
．． 405-411. 1974].

In addition, detection of surface antigens by rosette formation reaction using red blood cells coated with protein A [Procedures of the National Academy of Sciences (
Proc, Natl, Acad, Sci,),
USA 71°4831-4835. 1974)
, Fluorescein Isothiocyanate) (FITC
) Detection of tissue-specific antigen using protein Δ labeled with protein Δ, purification of TgG antibody using protein A-Sepharose immobilized with protein A [FεOS Letters, 28
．． 73-76, 1972], protein A is widely used.

As a clinical reagent, the coagglutination method, which is a serological diagnostic method for pneumococci and other bacteria [Journal of Medical Microbiology 4 (J9Med
, Microbiol, ), 16. 187
-190. 1973) is known. Sepharose 6MBC journal with protein A binding
of Immunological Methods (J, Immunol
lMeth,), 24.305-3(15)゜197
8) and cell separation methods using magnetic particles are known.

However, it is made of flat or rod-shaped glass.

Protein A using paper or plastic carriers
There is no known method for detecting, isolating, and purifying antigenic substances using immobilized A.

Problems to be Solved by the Invention and Means for Solving Conventionally, in order to identify antigenic substances, especially microorganisms, in clinical tests, a part of the test material containing the microorganisms is smeared and cultured on a selective medium to grow the target bacteria, and then biological Identification methods have been carried out by analyzing various properties and using serological diagnostic methods. In this case, it is necessary to use a unique culture medium depending on the microorganism, and separating the target bacteria from the overwhelming majority of mixed bacteria takes time and effort, and the determination is also troublesome, so a simple and reliable method is desired. It was rare.

Furthermore, when handling animal cells, if only the desired cells can be selectively grown on a culture medium, it is thought that not only materials but also labor and time can be saved.

The present inventors believe that if solubilized protein A is immobilized on a carrier such as a slide glass and an antibody against a specific antigenic substance is used, the detection of the antigenic substance can be carried out.
The present invention was completed by discovering that separation and purification can be carried out simply and efficiently.

Structure of the Invention The present invention involves binding an antigenic substance to a carrier made of glass, plastic, or paper via an antibody against the antigenic substance and protein A, and qualitatively or quantitatively analyzing the antigenic substance. A method for detecting an antigenic substance by binding the antigenic substance to a carrier made of glass, plastic, or paper via an antibody against the antigenic substance and protein Δ, and culturing it in a medium as necessary to detect antigenicity. A method for concentrating or purifying antigenic substances by propagating the substance and recovering the bound or proliferated antigenic substances, as well as methods for the isolation or purification of antigenic substances by binding Protein A to glass, plastic or paper carriers. Provided is a protein A-carrier conjugate for the detection, concentration, separation or purification of substances.

As the carrier, a flat or rod-shaped carrier made of glass, plastic, or paper is used.

As the glass or plastic carrier, a slide glass, a glass rod, a petri dish, a flask, a funnel, etc. are used. The size of the carrier is determined as appropriate depending on the amount of sample to be processed and the purpose of use.

In the method of the present invention, protein A is generally immobilized on a carrier made of glass, plastic, or paper, and the antigenic substance is bound to the protein A to which an antibody against the antigenic substance is bound. Antigenic substances can be detected by qualitatively or quantitatively analyzing the antigenic substances.

In addition, it is possible to concentrate, separate, and purify the antigenic substance by culturing the bound antigenic substance in a medium as necessary to multiply the antigenic substance, and then collecting the bound or proliferated antigenic substance. can. carrier, protein A,
The binding of antibodies and antigenic substances is usually carried out according to the above procedure, but it can also be carried out in a different order.

As protein A, Staphy, Staphylococcus aureus
(Journal of Immun
ol, ) 103: 828-833.1969), recombinant DNA techniques [Journal.
Op Bacteriology (J.

Bacteriol, 159: 713.1984)
The one produced by is used. Commercially available Protein A manufactured by Pharmacia Fine Chemicals may also be used. Protein A can also be produced as a single product:
It can also be used in cell-bound form for bacteria.

For example, Staphylococcus aureus Cowan I strain is known as a protein A-containing strain, and can be used without isolating protein A from this strain.

Journal of Immunology
1,) 135, 2589. Protein G, which was described in 1985, is a protein that has the same activity as protein A, so protein G can be used in place of protein Δ.

Antigenic substances include eukaryotic cells, prokaryotic cells, and viruses. Eukaryotic cells include animal and plant cells, such as sheep red blood cells, human white blood cells, Ehrlich cancer cells, and yeast such as Candida albicans; prokaryotic cells include bacteria such as Salmonella typhi, Salmonella typhimurium, Vibrio parahaemolyticus, Escherichia coli, and Shigella shigella; Examples include rickettsia, chlamydia, actinomycetes, and molds.

As antibodies against antigenic substances, polyclonal antibodies or monoclonal antibodies are used.

Polyclonal antibodies are obtained by administering an antigenic substance to dogs, Guinea pigs, rabbits, etc., and antiserum is obtained.
&Milstein's method [: Nature, 2
56°495-497.1975) can be used.

As a method for immobilizing protein A onto the carrier, the po I717 zinc-glutaraldehyde method (solubilized protein A), the ovalbumin-geltaraldehyde method (Staphylococcus aureus cells), etc. are used. These methods can be performed according to the methods described in the following documents.

Biochem, Biophys, Res, Commu
n, 36. 235-242. 1969 Arch,
Biochem, Biophys, 129.221-
227. 1969Biochem, Biophys,
Res, Commun, 45. 1574-158
0. 1971εxperImentia 29.
958-959. 1973Proc, Natl, Ac
ad, Sci] SA, 70. 2534-2538
．． Binding of the antibody to the 1973 protein Δ immobilized carrier was
This is carried out by immersing the protein A-immobilized carrier in a solution containing the antibody and leaving it at room temperature for 10 to 60 minutes. The antibody-bound carrier is washed with physiological saline, immersed in a solution containing the antigenic substance, and left at room temperature for 10 to 60 minutes. The carrier bound to the antigenic substance is washed with physiological saline, and the antigenic substance is assayed. The method for assaying an antigenic substance may be according to a qualitative or quantitative analysis method specific to the antigenic substance. If the antigenic substance is bacteria, use Durham staining method, etc.

In the method for concentrating, separating, and purifying an antigenic substance, the carrier, antigenic substance, protein Δ, etc. are the same as in the above method for detecting an antigenic substance, and the steps up to the binding of the antigenic substance are also carried out in the same manner. If the bound antigenic substance is a bacterial cell, cellulose, plant cell, etc., the bound antigenic substance or the grown antigenic substance is recovered by culturing the bound substance in a medium to proliferate the cells, if necessary. do. In this way, antigenic substances can be efficiently and easily separated and purified.

゛Examples of the present invention will be shown below.

Example 1 One end of a glass rod with a diameter of 311,110° was rounded by flame. Meanwhile, add egg whites to 1. (16) Staphylococcus aureus Cowan 1 strain ID975 (The Institute of Medical Science, The University of Tokyo) was suspended in a solution diluted 0 times at a concentration of 5 x 1 (15)/ml. Dip the glass rod into this suspension,
I immediately pulled it out and dried it with cold air using a hair dryer.

This glass rod was mixed with a 2.5% gel taraldehyde solution (
0,2M phosphate buffer (pH 7,4): l at 37°C
After soaking in water for 1 hour, it was washed with physiological saline. The glass rod was immersed in a solution containing a rabbit antibody against Salmonella Typhimurium (a 10-fold dilution of an antiserum with an agglutination titer of 2(16)0) at 37° C. for 15 minutes to bind the antibody. The antibody was produced as follows.

Salmonella typhimurium type S was collected on a heart infusion agar plate (DIFCOLABORATORIBS DIETR).
Manufactured by OITMICHIGAN USA', Un heart effusion 5 (16) g/ρ.

Tryptose 10g/β, sodium chloride 5g, #! .

Culture 15 g of agar at 37°C for 15 hours and suspend in physiological saline (5 x 1 Q9cfu/ml) L, 1 (
16) Heated at ℃ for 2 hours. lQ'cf in terms of viable bacteria
It was diluted with physiological saline to a ratio of υ/n+1 and used as an immunogen. This immunogen was intravenously injected into rabbits (manufactured by Nichizai Co., Ltd., female weighing 2 kg) five times at weekly intervals. The injection amount was 1st Q, 5ml, 2nd injection 1, Qml,
3rd 2. Qml, 4th 3.3+nl, 5th 3, Q
It was ml. Test blood was drawn one week after the fifth injection, and the O antibody titer was 1. (16) Whole blood was collected from those with 0 or more. This was used as an antibody.

The antibody-conjugated glass rods were immersed in a bacterial suspension containing 105 Salmonella typhimurium/ml and 108 E. coli/ml for 5 minutes at 25°C, and then washed three times with 1 Qml of physiological saline. When this glass rod was smeared onto a BTB plate medium and cultured overnight at 37°C, the number of Salmonella typhimurium and Escherichia coli colonies was 40 and 4, respectively. The ratio of Salmonella typhimurium to E. coli in the initial bacterial suspension is 1.
:1(16)0, the ratio on the BTB plate at the final stage is 10:1, so Salmonella typhimurium is i
o, oo.

It can be said to be twice as concentrated.

6hler and Mils monoclonal antibodies
An antiserum prepared using the Salmonella Typhimurium immunogen described above according to the method of Tein and obtained by immunizing a rabbit with mouse immunoglobulin in parallel was first bound to the glass rod bound to the Staphylococcus aureus cells described above. After washing with physiological saline, an anti-Salmonella typhimurium monoclonal antibody was bound to the rod and the rod was thoroughly washed again with physiological saline.The glass rod was placed in the same suspension of Salmonella typhimurium E. coli as mentioned above at 25°C.
When the cells were soaked in water for 5 minutes, washed with physiological saline, and plated on a BTB agar plate, the number of Salmonella typhimurium and Escherichia coli colonies was 124 and 3, respectively.

Example 2 Poly L-lysine hydropromide (manufactured by Sigma, No. 1524) was added to a slide glass.
ml of the solution was dissolved in deionized water and 10 μβ was applied onto a slide glass in the same manner as for preparing a smear. Immediately dry it with cold air, put it in a humidity box, layer it with 2.5% geltaraldehyde solution (0.2M phosphate buffer pH 7.4), leave it at 37°C for 1 hour, and add 0.2M! Wash with I phosphate buffer (pH 7,4), and remove
Overlay 5 (16) μl of protein A solution and
It was left at ℃ for 1 hour and washed with physiological saline.

1 ml of anti-sheep red blood cell rabbit antiserum diluted 10 times with physiological saline was overlaid on the above slide glass at 37°C.
After leaving it for a minute, it was washed with physiological saline.

As a negative control for this, normal rabbit serum was used instead of sheep red blood cells and bound to the slide glass in the same manner.

Both slide glasses were overlaid with 1 ml of a 2×10 5 /ml sheep red blood cell suspension and left at 25° C. for 15 minutes.

The glass slide was immersed in physiological saline at a 45° angle with the reaction side facing down.

After 15 minutes, the slide glass was taken out, covered with a cover glass, and observed under a microscope. When the number of red blood cells was counted in a 4 (16) magnification field of view, the number of red blood cells was 4. (16)
0 red blood cells were detected, and 20 red blood cells were detected on the control side.

Example 3 The same method as in Example 2 was used, except for using a plastic petri dish for cell culture (Φ5.3 mm) instead of the slide glass.

■ Protein A is immobilized on a plate (manufactured by Terumo), and in this petri dish, serum from a juvenile diabetic patient containing a large amount of antibodies against the membrane antigen of insulin-secreting cells is diluted with phosphate-buffered physiological saline 10x or 1x ( 16) Layer the two-fold diluted mixture, discard the excess liquid, react in a humid cylinder at 37°C for 5 minutes, and then wash with the above-mentioned phosphate buffered physiological saline.
5 x 105 cells/ml JHPI-1 (insulin-secreting cell line derived from human pancreatic islets of Langerhans) (Jik
Eikai! Jed.

J, 28.257-265.1981. Arch
, 1ist, Jap, 45°111-119.19
82) was layered and allowed to stand under the conditions shown in Table 1. After a certain period of time, the petri dish was washed with phosphate-buffered physiological saline, and the cells adhering to the bottom of the dish were counted by placing a glass plate with a scale on top. Judging from these results, the petri dish treated with serum from a juvenile diabetic patient is about 50 to 1 (16
) It was confirmed that cells adhered at twice the density.

Table 1 3 〃2520410415 4 〃2530402417 5 〃2560419408 6 〃375531484 7 〃3710693645 8 Normal adult 37 10 7 4
(Control) According to the present invention, antigenic substances can be efficiently and easily detected, a
It can be separated and purified by condensation.

Claims (17)

[Claims] (1) Antigenicity is determined by binding an antigenic substance to a carrier made of glass, plastic, or paper via an antibody against the antigenic substance and protein A, and analyzing the antigenic substance qualitatively or quantitatively. How to detect substances. (2) The method according to claim 1, wherein the carrier is selected from a glass slide, a glass rod, a petri dish, a flask, and a funnel. (3) The method according to claim 1, wherein the antigenic substance is selected from eukaryotic cells, prokaryotic cells, and viruses. (4) The method of claim 3, wherein the eukaryotic cells are selected from sheep red blood cells, human white blood cells, and Ehrlich cancer cells. (5) The method of claim 3, wherein the prokaryotic cell is selected from Salmonella typhi, Salmonella typhimurium, Vibrio parahaemolyticus, Escherichia coli, and Shigella shigella. (6) The method according to claim 1, wherein the antibody is a polyclonal antibody or a monoclonal antibody having reactivity with an antigenic substance. (7) The antigenic substance was bound to a glass, plastic, or paper carrier via an antibody against the antigenic substance and protein A, and if necessary, cultured in a medium to proliferate and bind the antigenic substance. Or a method of concentrating, separating or purifying antigenic substances by collecting the proliferated antigenic substances. (8) The method according to claim 7, wherein the carrier is selected from a slide glass, a glass rod, a petri dish, a flask, and a funnel. (9) The method according to claim 7, wherein the antigenic substance is selected from eukaryotic cells, prokaryotic cells, and viruses. (10) The method of claim 9, wherein the eukaryotic cells are selected from sheep red blood cells, human white blood cells, and Ehrlich cancer cells. (11) The method of claim 9, wherein the prokaryotic cell is selected from Salmonella enterica, Salmonella typhimurium, Vibrio parahaemolyticus, Escherichia coli, and Shigella shigella. (12) The method according to claim 7, wherein the antibody is a polyclonal antibody or a monoclonal antibody having reactivity with an antigenic substance. (13) Detection and concentration of antigenic substances by binding protein A to glass, plastic or paper carriers;
Protein A-carrier conjugate for separation or purification. (14) The carrier is a slide glass, a glass rod, a petri dish,
Claim 1 selected from flask and funnel
A combination of three terms. (15) The conjugate according to claim 13, wherein the antigenic substance is selected from eukaryotic cells, prokaryotic cells, and viruses. (16) The conjugate according to claim 15, wherein the eukaryotic cells are selected from sheep red blood cells, human white blood cells, and Ehrlich cancer cells. (17) The conjugate according to claim 15, wherein the prokaryotic cell is selected from Salmonella typhimurium, Salmonella typhimurium, Vibrio parahaemolyticus, Escherichia coli, and Shigella shigella.