JPS5942262B2 - Insolubilized antibodies and antigens for enzyme immunoassay - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to insolubilized antibodies and insolubilized antigens used in enzyme immunochemical measurements.

In recent years, various immunochemical methods have been used to measure blood and urine concentrations of physiologically active substances in living organisms and drugs administered to living organisms.

Among them, radioimmunoassay (Radioimmunoassay) uses radioisotope as a labeling agent for antigen or antibody.
assay is hereinafter abbreviated as RIA. ) is widely used because of its high measurement sensitivity and excellent quantitative performance. However, since RIA uses radioactive isotopes, its handlers are required to have a high level of knowledge and skill, and it also requires expensive equipment and equipment, and there are many restrictions such as environmental pollution. Recently, enzyme immunoassay (hereinafter abbreviated as EIA) using enzymes instead of radioactive isotopes has been developed.

) has been developed, but this EIA has measurement sensitivity and accuracy comparable to RIA, and can be measured more easily, so it has a wide range of applications. The RIA and EIA have the same measurement principle in common, and there are two conventional methods called the competitive method and the sandwich method, and the measurement principle will be briefly explained below.

1. Competitive method: When an unknown amount of unlabeled antigen to be measured and a fixed amount of labeled antigen labeled with a labeling agent are competitively reacted with a fixed amount of the corresponding antibody, the unlabeled antigen and the labeled antigen are respectively The amount of labeled antigen that binds to the antibody increases or decreases in proportion to the amount of antigen present, and the amount of labeled antigen that binds to the antibody increases or decreases in inverse proportion to the increase or decrease of unlabeled antigen.

Next, the labeled antigen bound to the antibody and the labeled antigen not bound to the antibody are separated by an appropriate method, and the activity of the labeling agent of the labeled antigen bound to the antibody or the labeled antigen not bound to the antibody is measured. The unknown amount of unlabeled antigen is measured using a standard curve prepared in the same manner using a standard substance. 2. Sandwich method: When an unknown amount of unlabeled antigen to be measured is reacted with an insolubilized antibody against the antigen, the two bind to form an antigen-antibody complex.

One part of this complex is separated from the reaction mixture, and it is reacted with a certain amount of labeled antibody, which is an antibody bound to a labeling agent, and the labeled antibody that is bound to the antigen-antibody complex is mixed with the labeled antibody that is not bound to the antigen-antibody complex. The activity of the labeling agent in one of the fractions is measured, and at the same time, the unknown amount of unlabeled antigen is measured using a standard curve prepared in the same manner using standard substances with known concentrations. In the competitive method, chromatography, gel filtration method, dextran charcoal method, double antibody method, solid phase method, etc. are used to separate labeled antigen bound to antibody and labeled antigen not bound to antibody. However, a solid-phase method using an insoluble antibody, in which the antibody is bound to an insoluble carrier, is advantageous for actual measurements due to its ease of operation.

In addition, in the Cydeutsch method, the antibody is usually bound to an insoluble carrier to make it insolubilized to serve as a partner for the reaction with the antigen, and a means for separating the antigen-antibody complex generated by the reaction with the antigen from the reaction solution. It is said that

As carriers for insolubilizing the antibodies, conventionally used are cellulose powder, Fuchi paper, carboxymethyl cellulose, ion exchange resins, dextran, Cephadex, plastic films, plastic tubes, synthetic fibers such as nylon and silk, natural fibers, etc. It is being

Each of these carriers has advantages and disadvantages; for example, plastic film, plastic tubes, etc. are easy to operate during measurement, but because their surface area is small, the amount of antibody or antigen that binds to them is small, and the slope of the standard curve is low. Since it is small, it is difficult to improve measurement accuracy. On the other hand, powder or particulate materials such as cellulose powder and Cephadex have a large surface area and can increase the slope of the standard curve, but they tend to adhere to test tube walls, etc., so during measurement Some of it will adhere to the vessel wall, etc., reducing measurement accuracy. As a result of repeated research into carriers that do not have these drawbacks, the present inventors found that blood cells have a large surface area and are easy to handle, and that blood cells treated with a binding agent have a particularly high ability to bind antibodies or antigens. It was discovered that it exhibits excellent properties as a carrier, and the present invention was completed.

The present invention provides insolubilized antibodies and insolubilized antigens for enzyme immunoassay using blood cells as carriers, which have been reacted with a binding agent such as tannic acid after removing the enzymatic activity of the blood cells.

To carry out the present invention, blood cells are usually fixed by reacting with a fixative such as formalin, then treated under acidic conditions with a pH of 3 or less to remove the enzyme activity of the blood cells themselves, and then treated with a binder such as tannic acid. react.

Next, the thus treated blood cells are mixed with a buffer solution of the antibody or antigen and allowed to react, thereby obtaining an insolubilized antibody or an insolubilized antigen. The insolubilized antibody or insolubilized antigen of the present invention can be used in various immunochemical assays based on the conventionally known competitive method and Sand-Deutsch method.

As the blood cells used in the present invention, blood cells of various animals such as mammals such as cows, horses, sheep, domestic rabbits, and humans, and birds such as chickens and turkeys are usually used.
Treat with a fixative such as pyruvaldehyde or hydrogen peroxide. For example, to treat with formalin, equal amounts of a blood cell suspension suspended in physiological saline (approximately 8% concentration) and a formalin solution with a concentration of approximately 3% are mixed and reacted at 37°C for 18 hours. After that, wash and resuspend in physiological saline (concentration 10%)
do. Note that the amount of blood cells is expressed by the apparent volume of blood cells obtained by centrifuging a blood cell suspension.

It is known that various enzyme activities exist in blood cells, but since this enzyme activity becomes an obstacle when measuring the activity of the enzyme that is the labeling agent, at least the enzyme used as the labeling agent and the The activities of enzymes that catalyze the same type of reaction or that share a common substrate need to be removed.

For example, when horseradish peroxidase is used as a labeling agent, at least peroxidase activity and catalase activity of blood cells must be removed. The method for removing enzyme activity varies depending on the type of enzyme to be removed, but generally the enzyme is inactivated by changing the PHL temperature, etc. to an unstable region of the enzyme. For example, to remove peroxidase activity and catalase activity, the enzyme activity can be inactivated and removed by storing the blood cell suspension overnight at 4° C. under acidic conditions with a pH of 3 or less.
In order to bind antibodies or antigens to blood cells from which enzyme activity has been removed, it is necessary to treat them with a binding agent in order to improve their binding to antibodies or antigens.

As a binder, tannic acid, bisdiazobenzidine,
1,3-difluoro-4,6-dinitrobenzene, glutaraldehyde, chromium chloride, etc. can be used. For example, to treat with tannic acid, blood cells that have been treated with a fixative and enzyme activity removed are suspended in a buffer solution, and this is mixed with a tannic acid solution with a concentration of 1/40,000 to 1/50. Mix equal volumes or add 10 to 50 ml of the tannic acid solution per ml of the centrifuged treated blood cells.
and mix, and react at 20 to 56°C for 30 to 90 minutes. With this treatment, 0.3 to 1 m1 of blood cells
500η of tannic acid is bound. To produce an insolubilized antibody or insolubilized antigen using the thus treated blood cells as a carrier, the antibody or antigen to be bound is added to a blood cell suspension in which the binder-treated blood cells are suspended in a buffer solution at an appropriate concentration. are dissolved in the same buffer solution to an appropriate concentration and reacted. The amount of antibody or antigen that binds to binding agent-treated blood cells (ie, carrier) increases as the amount of binding agent bound to the blood cells increases. Using the insolubilized antibody or insolubilized antigen obtained as described above and the enzyme-labeled antigen or antibody, physiologically active substances ranging from high-molecular substances to low-molecular substances can be measured.

For example, to measure an antigen using the insolubilized antibody of the present invention, an insolubilized antibody suspension is added to a test solution diluted to an appropriate concentration, and the antigen and antibody are allowed to react. After the reaction is completed, a labeled antigen solution labeled with an enzyme is added to the reaction mixture to react, and the solid phase is separated by centrifugation and washed thoroughly. Then,
After adding an appropriate substrate solution to this solid phase and causing a reaction, enzyme activity is measured. From the measured values obtained, the amount of antigen in the sample is calculated using a standard curve obtained by performing the same procedure using a standard substance with a known concentration. When performing measurements, various conditions such as the amount of specimen, concentration and amount of reagent used, reaction temperature, time, etc. are determined depending on the type of substance to be measured, the titer of antibody used, and the enzyme used as a labeling agent. Since the conditions differ depending on the type, etc., the most appropriate conditions are determined experimentally for each measurement section.

The enzyme used as a labeling agent needs to have stable properties, be able to measure even trace amounts of activity, and be easy to perform measurement operations.

Furthermore, when using blood cells as carriers for antigens or antibodies, as mentioned above, various enzymes present in blood cells may interfere with the measurement of enzyme activity as a labeling agent. It is desirable to select an enzyme that has enzymatic activity that does not exist. However, since it is difficult to obtain an enzyme that satisfies these conditions, the enzyme activity present in blood cells is removed as described above. Taking these conditions into consideration, the enzyme used as the labeling agent is preferably horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, or the like.

The insolubilized antibody or insolubilized antigen according to the present invention has various characteristics as described below. In other words, the blood cells used as carriers have a large surface area, and their ability to bind antibodies or antigens is improved by treatment with a binding agent. In order to retain this, a smaller amount of carrier is required. Therefore, the decrease in reactivity accompanying the solid-liquid phase reaction can be minimized, and the measurement sensitivity can be improved. Furthermore, by adjusting the amount of binding agent used to treat blood cells, the amount of antigen or antibody that binds to blood cells can be adjusted, and measurement sensitivity can be adjusted depending on the purpose of measurement. Furthermore, blood cells are completely sedimented by centrifugation and are not lost by adhering to the vessel wall, etc., and are easy to handle, making it possible to improve measurement accuracy. In this specification, the measurement method using the insolubilized antibody or insolubilized antigen of the present invention has been described mainly as a method for measuring the antigen. Of course, it can be used in the same way when measuring, and even in measuring methods using receptors.

The present invention will be specifically explained below using examples.

Example 1 Production of blood cells from which enzyme activity has been removed a) Formalin fixation Commercially available sheep blood for complement fixation reaction (manufactured by Toshiba Chemical Co., Ltd.)
r. p. m. Centrifuge for 10 minutes to separate blood cell components.

After washing this with physiological saline three times, it is made into a suspension solution with a concentration of 8% in physiological saline. To this, an equal volume of 3% formalin solution was added, and the mixture was incubated at 37°C for 18 hours. Then, it was washed four times with purified water and stored as a 10% suspension until use. b) Production of blood cells from which enzyme activity has been removed Mix equal amounts of the formalin-fixed blood cell suspension from a) above and 055M glycine-hydrochloride buffer (PH2.O), and heat at 4°C for 1 hour.
I left it overnight.

This was washed twice with phosphate buffered saline pH 6.4 (hereinafter abbreviated as PBS) and made into a 10% suspension with the same buffer. Example 2 Measurement of α-phetoprotein a) Preparation of α-phetoprotein standard solution Nishi et al.
cerHes. α-Fetoprotein (hereinafter abbreviated as AFP) was extracted and purified from ascites of a liver cancer patient by the method of , 3O, 27O7, (1970)).

) was diluted with PBS containing 0.5% Tween 20 and 1% bovine serum albumin (hereinafter abbreviated as BSA), and
,150,100,75,50,30,20,10,5
, 0 ng/TfLl were prepared. b
) Production of anti-AFP antibody AFP purified from ascites of a liver cancer patient by the method of Nishi et al. Dissolved in physiological saline to a concentration of 9/ml and its 0
．． 5ml was mixed with Freund's complete adjuvant and rabbits were immunized five or more times to obtain anti-AFP serum.

This antiserum was salted out with sodium sulfate to obtain a globulin fraction, and an anti-AFP antibody was produced. c) Production of anti-AFP antibody-sensitized blood cells 2 ml of blood cells obtained in Example 1 were washed with 30 ml of PBS and resuspended in the same buffer to make a total volume of 60 ml.

This was mixed with 60 ml of tannic acid solution with a concentration of 1/300, and reacted at 56° C. for 30 minutes. After the reaction is complete, PB
The cells were washed twice with S and resuspended in the same buffer to make a total volume of 2 ml. This blood cell suspension and the anti-AFP antibody (1) from b) above
~/ml) solution and reacted at 56°C for 30 minutes. After washing with 50 ml (7) PBS, wash with 2.5 ml of PBS containing 10% sucrose, 5% BSA and 1% rabbit serum.
Anti-AFP antibody-sensitized blood cells were obtained by suspending the cells at a concentration of 1.5%.

d) Production of anti-AFP antibody/enzyme conjugate 0.3 ml of alkaline phosphatase (Behring Mannheim, grade) solution (5Tf!9/ml) was centrifuged, the supernatant was removed, and 0.1 ml of the solution was centrifuged. 0.5 or more of the anti-AFP antibody in b) above was added.

After dialysis against PBS overnight, 0.01 ml of 4.2% glutaraldehyde solution was added and incubated at room temperature for 2 hours.

Next, the total volume was made up to 1 ml with PBS, and after dialysis against PBS overnight, it was fractionated with a Sepharose 6B column.
An anti-AFP antibody/alkaline phosphatase conjugate was obtained. e) Measurement of AFP 0.1 ml of the standard AFP solution of each concentration prepared in a) above
into a test tube, and add the anti-AFP antibody-sensitized blood cell suspension prepared in c) above diluted to 0.5% with PBS.
．． 4 ml was added and reacted at room temperature for 60 minutes.

After completion of the reaction, add the anti-AFP antibody and the above d) to this reaction mixture.
0.1 TfL1 of alkaline phosphatase conjugate solution was added and reacted at room temperature for 120 minutes. The solid phase was then centrifuged and washed with saline containing 0.005% Sheen 20, which was supplemented with a substrate solution containing 100ヮ/dl P-nitrophenyl phosphate and 1 [TlM magnesium chloride. (PH9.8) was added, and the mixture was reacted at room temperature for 60 minutes. After adding 0.3 ml of 1N sodium monohydroxide to stop the reaction, centrifuge briefly to determine the absorbance of the supernatant (400 ml).
was measured.

An example of the results is shown in FIG. Example 3 Measurement of estriol a) Preparation of estriol standard solution
0.1% BSA at concentrations of 0, 10, 25, 0 ng/ml
was dissolved in PBS containing

)) Manufacturing process of estriol-16,17-dihemisuccinate/BSA conjugate Estriol-16,17-dihemisuccinate (A.J.O.GlO9,897(1
971)) was dissolved in 12 ml of dioxane, 0.3 ml of tri-n-butylamine was added thereto, the temperature was brought to 12°C, and 0.17 ml of isobutyl chlorocarbonate was dissolved.
ml was added and stirred thoroughly.

Add BSAl. After dissolving 79 in 40 ml of purified water, the solution was diluted with 1N sodium monohydroxide to PHL2. O
The solution was mixed with 40 ml of dioxane and kept at 12° C., stirred for 4 hours to react, and unreacted low-molecular substances were separated using Sephadex G25. Next, this was dialyzed against 0.1% sodium azide water and then freeze-dried to obtain a triol-16,17-dihemisuccinate/BSA conjugate. Production of estrone-17-oxime-hemoglobin conjugate Estrone-17-oxime (Erlanger, B.F.,
．． B. C. , 234, lO9O, (1959)) 687
Dissolve η in 20 ml of dioxane and add tri-n-
After adding 0.9 ml of butylamine, the temperature was raised to 11°C, and 0.27 ml of isobutyl chlorocarbonate was added and stirred.

Add 2.0% hemoglobin (hereinafter abbreviated as Hb) to this solution in advance.
After dissolving 429 in 70 ml of purified water, adjusting the pH to 9.5, adding 70 ml of dioxane and keeping the solution at 11°C, the solution was mixed, stirred for 4 hours to react, and then purified with a Sephadex G25 column. The low molecular weight substances of the reaction were separated. This was dialyzed against 0.1% sodium azide water and lyophilized to obtain an estrone-17-oxime/Hb conjugate. d) Production of anti-estriol antibody Dissolve the estriol-16,17-dihemisuccinate/BSA conjugate produced in b) above in physiological saline,
Mix with complete Freund's adjuvant and administer 2η at a time subcutaneously to the back of a rabbit repeatedly.

One week after the final administration, blood was collected and the obtained antiserum was salted out with sodium sulfate to obtain antibody globulin. From this antibody, BS was conjugated to Sepharose using the Bromsian method.
BSA antibodies were removed using A. That is, antibody solution 5
Add BSA/Sepharose conjugate at a ratio of 25 ml to 0 ml, incubate at 37°C for 30 minutes, and then incubate at 4°C.
I left it overnight. This is 3000r. p. m10 minutes (
4C) The liquid phase was separated by centrifugation to obtain an estriol-specific antibody. e) Anti-estriol antibody Fab
Production of fragment The anti-estriol antibody produced in d) above was purified by the method of POrter (BiOchem.J.7).
3, 119 (1959)) and fractionated with a carboxymethyl cellulose column (PH5.5) to obtain an anti-estriol antibody Fab fragment.

f) Production of anti-estriol antibody Fab/enzyme conjugate 5
7n9 horseradish peroxidase (HOrsera
ddishPerOxidase (hereinafter abbreviated as HRP) was dissolved in 1 ml (7) of 0.3M sodium hydrogen carbonate solution, 0.1 ml of 1% 2.4-dinitrofluorobenzene was added, and the mixture was stirred at room temperature for 1 hour.

Add 1 ml of 0.08M sodium periodate solution,
After mixing at room temperature for 0 min, 1.0 ml (7) 0.16 M
Ethylene glycol solution was added and mixed for 1 hour at room temperature.

After dialysis overnight against 0.01M carbonate buffer pH 9.5, anti-estriol antibody F prepared in e) above was added to this.
Add 1.0a of a solution of ab dissolved in 0.01M carbonate buffer pH 9.5 to a concentration of 5ヮ/ml and react at room temperature for 3 hours, then add 5η of sodium borohydride and incubate at 4°C.
The reaction was continued for an additional 3 hours.

After completion of the reaction, the mixture was dialyzed against PBSPH7.2 overnight, and then fractionated and purified using Sephadex G2OO to obtain an anti-estriol antibody Fab/HRP conjugate. g) Production of estrone-17-oxime/Hb sensitized blood cells 0.27M-
Piperazine buffer (PH6.
5) 6.2m11 estrone-17-oxime/Hb physiological saline solution (1 instant/ml) 1.0mZ1 1.0ml of blood cells prepared in Example 1 and 2.25M chromium chloride solution were diluted 400 times with physiological saline. 0.87r1 of the solution
1 was placed in a Maya pot and left to react at room temperature for 5 minutes.

Next, 300 ml of physiological saline was added to this reaction solution to stop the reaction, and the reaction was washed three times with physiological saline.
It was made into a floating liquid. h) Measurement of Estriol 0.1 ml each of the estriol standard solution from a) above and 0.1 ml of the anti-estriol antibody Fab-HRP conjugate solution from f) above were placed in a test tube and incubated at room temperature for 1 hour.

Next, the above g) stolone-17-oxime Hb
0.4 ml of sensitized blood cell suspension was added and incubated at room temperature for 2 hours. After the incubation, the solid phase was collected by centrifugation, washed with physiological saline containing 0.005% Tween 20, and washed with 3 ml of substrate solution (60 ml of 5-aminosalicylic acid).
9/DllO. 3% hydrogen peroxide (1 ml/dl) was added, and the mixture was reacted at room temperature for 60 minutes. After stopping the reaction with sodium azide, the mixture was briefly centrifuged and the absorbance of the supernatant was measured.
An example of the results is shown in Figure 2.

[Brief explanation of the drawing]

FIG. 1 is a graph of the measurement results of Example 2, and FIG. 2 is a graph of the measurement results of Example 3.

Claims (1)

[Claims] 1. In insolubilized antibodies and insolubilized antigens for enzyme immunoassay using enzymes as labeling agents, among the enzymes present in blood cells, at least an enzyme that catalyzes the same type of reaction as the enzyme of the labeling agent or an enzyme that shares the same substrate. An insolubilized antibody and an insolubilized antigen for enzyme immunoassay, which are obtained by removing the activity of the blood cells and then treating the blood cells with a binding agent to bind the antibody or antigen to the blood cells.