complement binding assay
complement binding assay
Complement fixation test (CFT) is one of the antigen-antibody reactions of the classical pathway. This method is generally used for: ① diagnosis of clinical viral diseases; ② epidemiological investigation of viral infectious diseases; ③ detection of viral antigens and corresponding antibodies; ④ identification of viral subtypes and so on.
Principle
The basic principle of complement binding test is based on: 1. There are 3 systems ① reaction system, i.e., known antigen (or antibody) and antibody (or antigen) to be tested; ② complement system; ③ indication system, i.e., sheep red blood cell (SRBC) and the corresponding hemolysin, to form a sensitized red blood cell.
2. If there is an antibody (or antigen) to be tested in the reaction system. then the antigen-antibody reaction can bind complement; when the indicator system is added again, without hemolysis, it is a positive test.
3. If the antibody (or antigen) to be tested does not exist in the reaction system, there is still free complement in the liquid, and hemolysis occurs when the indicator system is added, it is a negative complement binding test.
Appliance
Complement binding test is generally used for: ① clinical diagnosis of viral diseases; ② epidemiological investigation of viral infectious diseases; ③ detection of viral antigens and corresponding antibodies; ④ identification of viral subtypes.
Operation method
complement binding assay
Principle
The basic principle of complement binding test is based on: 1. There are 3 systems ① reaction system, i.e. known antigen (or antibody) and antibody (or antigen) to be tested; ② complement system; ③ indication system, i.e. sheep red blood cell (SRBC) and the corresponding hemolysin, to form a sensitized erythrocyte. 2. If there is antibody (or antigen) to be tested in the reaction system, then the antigen-antibody reaction can bind complement; and then when the indication system is added without hemolysis, it is a positive test. 3. If there is no antibody (or antigen) to be tested in the reaction system, then the liquid can bind complement after the reaction. If there is an antibody (or antigen) in the reaction system, the antigen and antibody can bind complement after the reaction; when the indication system is added, no hemolysis occurs, which is a positive test. 3. If there is no antibody (or antigen) in the reaction system to be examined, there is still free complement in the liquid, and hemolysis occurs when the indication system is added, which is a negative test for the binding of complement.
Materials and Instruments
Equipment: Move The basic procedure of the complement binding test can be divided into the following steps:
① centrifuge
② Incubator
③ Syringe
④ bacterial filter
Reagents:
①Materials: virus antigen
② guinea pig fresh serum
③4.1% SRBC
④Alsever preservation solution
⑤ Glycerol
⑥Diluent solution
⑦Hemolysin
(i) Material preparation
1. Viral antigen obtained from tissue culture, chicken embryos and animal tests should be appropriately purified, the higher the purity, the greater the specificity. If the use of crude antigen, must be the same treatment of normal tissue as antigen control, in order to identify the serum to be examined in the possible existence of non-specific reaction to normal tissue components.
2. It is preferable that the viral antigen used for antibody immunization is not the same cell or animal to avoid cross-reactivity. Serum samples should be heat-inactivated to eliminate complement activity and nonspecific anticomplement activity. Antibodies from human and guinea pig should be inactivated at 56 ℃; antibodies from rabbit and horse should be inactivated at 65 ℃.
Complement detection of human and mammalian serum, the application of guinea pig fresh serum complement, prepared before use.
4.1% SRBC preparation from the jugular vein of sheep aseptically operated blood collection, placed in a triangular flask containing Alsever preservation solution (Alsever liquid and sheep blood ratio of 1:1), immediately shaking evenly 4 ℃ refrigerator for storage and spare, can be used for 2 ~ 3 weeks. When used, wash with 10 times the volume of saline three times, each time at 2000 r/min centrifugation for 10 minutes, take up the centrifugal deposition of erythrocytes 1 mL, and then add 99 mL of saline, i.e., 1% of erythrocyte suspension.
5. hemolysin with sheep erythrocytes immunized rabbits, the potency of 1:4000 can be obtained; inactivated at 56 ℃ for 30 minutes, plus glycerol for long-term preservation.
6. Diluent saline containing calcium and magnesium ions, can maintain the stability of complement.
(B) the titration of the reagents used
1. Titration of hemolysin Firstly, dilute hemolysin into 1:1000~1:100 dilution, then add hemolysin 0.1 mL, calcium and magnesium saline 0.2 mL, 1:60 complement 0.2 mL, 1% sheep red blood cells 0.1 mL, mix well and then water bath.
Results: the liquid of the test tube was red and transparent, and a few cells were seen after centrifugation; the liquid of the test tube without hemolysis was turbid, and erythrocyte precipitation was seen after placing, and the supernatant was colorless and transparent.
Determination of hemolysin potency: the highest dilution of complete hemolysis was determined as 1 unit.
2. Exact unit: The minimum amount of complement for complete hemolysis is taken as an "exact unit". 1:60 Complement minimum of 0.12 mL produces complete hemolysis, which is 1 exact unit. For formal testing, 2 units of certainty are used. (2x0.12):60 = 0.2:X, resulting in X = 50, i.e., the 2 units of complement in practice are 0.2 mL of 1:50 complement.
Titration of Complement: Complement is unstable and needs to be titrated before each test.
(iii) Titration of antigen and antibody
1. Find out the optimal ratio of antigen and antibody, and use the square array titration method.
(1) Inactivate the antigen and antiserum by 56 ℃ for 30 minutes, and then dilute the antigen and antibody with calcium and magnesium saline (1:521～1:8).
(2) Add 0.1 mL of different dilutions of antigen to the first 7 rows of horizontal tubes; add 0.1 mL of different dilutions of antiserum to the first 7 columns of vertical tubes; add 0.1 mL of calcium-magnesium saline to the eighth row of tubes and the eighth column.
(3) Add 0.2 mL of complement containing two real units to each tube.
(4) Refrigerate at 4 ℃ overnight. Because in general, overnight in 4 ℃ refrigerator can fix more complement than 37 ℃ for 1 hour, and it is more sensitive and convenient, so most of the 4 ℃ refrigerator overnight.
(5) Finally, add sensitized SRBC.
(6) Result determination: choose the highest dilution of antigen and antibody that shows a strong positive reaction (completely non-hemolytic) as the potency of the antibody; from Table 4-6, we can see that the antigen of 1:64 and the antiserum of 1:32 are 1U respectively; in the formal test, the antigen is generally used for 2-4U (1:32-1:16), and the antiserum is used for 4U (1:8).
2. Formal test
(1) Prepare various reagents.
(2) Negative and positive controls should be clearly hemolyzed and unhemolyzed, respectively.
(3) Complement in the control tube 2U should be completely dissolved, if incomplete hemolysis indicates that the amount of complement is not enough, 0.5U should not be dissolved, if complete hemolysis indicates that the amount of complement is too much.
Caveat
(1) The purity and titer of antigen and antibody should be sufficient; complement should be stored at low temperature without repeated freezing and thawing; and the content of complement is usually 0.5-2U.
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