The Complete Guide to Antibody Detection by Immunoenzyme Technique: Whole Bacteria and Whole Cell Antigen-based ELISA
The immunoenzyme technique is an immunological technique that combines the specificity of the antigen-antibody reaction and the efficient catalytic action of the enzyme on the substrate’s color reaction. Because of its specificity and high sensitivity, it has been widely used for screening and identification of monoclonal antibody.
Whole Bacteria Antigen-based ELISA
a. Suspend the freshly cultured bacteria with distilled water or PBS, and adjust the bacterial concentration to 1 x 108/ml. It must be noted that zoonosis pathogens must be treated carefully, and inactivation is the best choice.
b. Add 100ul 5% glutaraldehyde solution (95ml 0.1mol/L NaHCO3 and 5ml 25% glutaraldehyde solution) into each hole. Incubate for 2 hours at 37 ℃, and wash for 3 times with distilled water. Add 50ul bacterial suspension from the last step into each hole, and dry the late at 37–56℃. Then, add 200ul blocking solution into each hole, and maintain overnight at 4℃ or seal up for 2 hours at 37℃.
c. Step b can also be replaced by this way. Add 50ul bacterial suspension into each hole. Dry at 37 ℃-56 ℃, then maintain at room temperature for 15 minutes with -20 ℃ pre-cooled anhydrous methanol and wash for 3 times with distilled water. Add 200ul blocking solution, and maintain at 4℃ overnight or at 37℃ for 2 hours.
d. Wash 3 times with washing solution. Maintain the coated plate at -20℃ or 4℃ for later use.
e. The following steps are the same with steps introduced in B.
Whole Cell Antigen-based ELISA
a. Culture the cells with a conventional manner. Inoculate virus, harvesting infected cells and uninfected cells. Count obtained cells. Make suspension of appropriate concentration with PBS.
b. The preparation of lymphocyte suspension. After adding peripheral blood into heparin anticoagulation, drop it on lymphocytes separation medium and centrifuge. Then add 0.83% ammonium chloride solution, maintain at room temperature and wash for one time. Dilute the cell suspension to appropriate concentration.
c. Add 100ul cell suspension from step a or step b to each hole, making the whole cell number of each hole 5×104. Separate the supernatant at 1500r/min for 15 minutes. Dry at room temperature or blow-dry, and fix for ten minutes with acetone — methanol (1: 1) at 4℃. Store at 4℃ or -20℃ for later use.
d. The following steps are the same with steps introduced in B.
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