Method for detecting human serum IgG antibody by immunofluorescence (IFA) and its significance
experiment material:
(1) Antigen tablets: The standard strains of house mouse and squirrel type Hantavirus are infected with Vero E6 or Vero cells, and stored at low temperature;
(2) positive and negative controls: monoclonal antibody or confirmed positive patients with recovery period serum (positive control), negative serum;
(3) goat anti-human (or rabbit anti-human) IgG fluorescent antibody;
(4) Commonly used diluents: pH 7.2 ~ 7.4 PBS, Evanslan, etc.;
(5) Fluorescence microscope.
Detection steps:
(1) The serum to be tested was diluted with PBS at pH 7.4, 0.02 mol/L, and serial dilutions were made from 1:20 to a desired dilution. (2) The antigen sheet was taken out, rinsed with distilled water, and dried by cold air.
(3) Add the diluted serum to be tested one by one from high dilution to low dilution with the sampler, and the amount added is to cover the antigen surface of the cell (if it is double serum, the upper row is preferably the acute phase serum, The lower row is the recovery period serum), incubated in a 37 ° C water bath wet box for 30 to 40 minutes (simultaneous control is set at the same time for each test).
(4) Washing with PBS for 3 times, each time for 5 minutes, and then washing once with distilled water for desalting, and blowing with cold air.
(5) Dilute the fluorescent conjugate with a concentration of 1:3 million Evans PBS at the working concentration, add each well (to cover the antigen surface of the cell), incubate in a wet box at 37 °C for 30 minutes, then the same ( 4) Washing, rinsing, and drying.
(6) Results observed by fluorescence microscopy. Results: The intracellular virus-specific fluorescence was yellow-green particles distributed in the cytoplasm of infected cells. The immunofluorescence reaction can be roughly divided into 1 to 4 "+" according to the specific fluorescent particles, the fluorescence brightness, and the proportion of positive cells in the total number of cells. Refer to the number of positive cells: <25% for "+", 25% to 50% for "++", 51% to 75% for "+++", >75% for "++++"; no specific The person who is sexually fluorescent is "-" (negative). When the antibody titer is detected, it is expressed as the reciprocal of the highest serum dilution at which a significant specific fluorescence response (> "+") can be observed.
Detecting human serum significance: Positive results indicate that Hantavirus infection has been used, and >1:20 has diagnostic reference significance; serum antibody titer in recovery period is 4 times or more than 4 times higher than acute phase antibody titer.
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