Indirect erythrocyte agglutination test

Indirect hemagglutination test

Hemagglutination test (hemagglutinationtest) is the abbreviation of red blood cell agglutination test. The indirect hemagglutination test is an indirect agglutination test using red blood cells as a carrier. It is widely used in clinical testing. The operation process is briefly described below.

(1) Carrier

Red blood cells are carrier particles of uniform size, the most commonly used are red blood cells of sheep, rabbits, chickens and O-type human red blood cells. Fresh red blood cells can adsorb polysaccharide antigens, but the ability to adsorb protein antigens or antibodies is poor. The sensitized fresh red blood cells have a short storage time and are brittle, hemolytic, and contaminated, and can only be used for 2 to 3 days. For this reason, erythrocytes are generally hydroformylated before sensitization, which can be stored for a long time without hemolysis. Commonly used aldehydes include formaldehyde, glutaraldehyde, and pyruvaldehyde. The red blood cells are slightly increased in volume after aldification, and the protrusions on both sides are disc-shaped.

Aldehyde red blood cells have a strong ability to adsorb protein antigens or antibodies, and the effect of hemagglutination is basically similar to that of fresh red blood cells. It is better to use two different aldehyde treatments. It can also be treated with glutaraldehyde first and then with tannic acid. Aldehyde erythrocytes can withstand heating at 60 ℃, and can be frozen and thawed repeatedly without breaking, and can be stored at 4 ~ C for 3 to 6 months, and at -20 ℃ for more than 1 year.

(2) Sensitization

The antigen or antibody used for sensitization is required to have high purity and maintain good immune activity. There are direct and indirect methods for sensitizing red blood cells with protein. The direct method only needs to be directly adsorbed with alded red blood cells at low pH and low ion concentration. The indirect method requires a coupling agent to bind the protein to the red blood cells. Commonly used coupling agents are bidiazotizedbenzidine (BDB) and chromium chloride. The former uses a covalent bond, and the latter uses a metal cation to electrostatically bind the protein to the surface of the red blood cell to achieve the purpose of sensitization.

(3) Blood coagulation test

The hemagglutination test can be carried out in a microtiter plate or test tube. The specimen is diluted in multiples, usually 1:64, and the dilution solution without specimen is set as a control well. Add 1 drop of 0.5% sensitized erythrocyte suspension to the well (or test tube) containing 1 drop of diluted specimen, mix thoroughly, and let the room temperature stay for 1 to 2 hours to observe the result. Where erythrocytes are deposited at the bottom of the pores, those that are concentrated in a circle are not agglomerated (1). If red blood cells agglutinate, they are distributed around the bottom of the well. Judging the strength of the positive reaction according to the degree of erythrocyte agglutination, the +10 agglutination well was used as the end point of titer.