The Complete Guide to Antibody Detection by Immunoenzyme Technique: Equipment and Reagents
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.
Equipment and Reagents
a. coating buffer
Carbonate buffer: mix 0.2mol/L Na2CO3 8ml with 0.2mol / L NaHCO3 17ml, and add 75ml distilled water, adjusting the pH to 9.6.
Tris-HCl buffer (pH 8.0, 0.02 mol/L): mix 0.1mol/L Tris 100ml with 0.1mol/L HCl 58.4ml, and add distilled water to 1000ml.
b. Wash Buffer (pH 7.2 PBS): add distilled water to 0.2g KH2PO4, 0.2g KCl, 2.9g Na2HPO4? 12H2O, 8.0g NaCl and 0.5ml Tween-20 to 1000ml.
c. Dilution and blocking solution: add washing solution to 0.1g bovine serum albumin (BSA) to 100ml, or wash the calf serum with the washing solution to 5–10%.
d. Enzyme reaction stop buffer (2mol / L H2SO4): take 178.3ml of distilled water, and drop 21.7ml concentrated sulfuric acid (98%) in it.
e. Take 25.7ml 0.2mol / L Na2HPO4 and 24.3ml 0.1ml / L citric acid, and add 50ml distilled water in it. As citric acid solution and the substrate buffer are unstable and easy to form precipitation, it is not recommended to prepare too much at a time.
f. Substrate solution
OPD substrate solution (measure the OD value of 490nm): 5mg OPD, 10ml substrate buffer, 0.15ml 3% H2O2.
TMBS or TMB substrate solution (measure the OD value of 450nm): 1.0ml TMBS or TMB (1mg / ml), 10ml substrate buffer, 25ul 1% H2O2.
ABTS substrate solution (measure the OD value of 410nm): 0.5mg ABTS, 1ml substrate buffer, 2ul 3% H2O2.
g. Antibody Control: The myeloma cell culture supernatant is used as a negative control. The immunized murine sera are used as positive sera.
Soluble antigen: try to be as pure as possible to achieve high specificity
Virus-infected subcultures or whole-cell antigens
Lymphocytes and other suspensions
i. Enzyme-labeled anti-mouse antibody or enzyme-labeled SPA or other similar reagents
j. Cellulose fixation Solution: -20 ° C acetone
Or acetone-formaldehyde fixative: 100mg Na2HPO4, 500mg KH2PO4, 150ml distilled water, 225ml acetone, 125ml formaldehyde
Or acetone-formaldehyde solution (1:1)
Or -20 ° C methanol
k. Polystyrene microplates: 40 holes, 96 holes, or slot mesh hole. Both hard plate and soft plate can be used.
l. Enzyme-linked immunosensor or light microscope
m. Straw, sampler, water bath, centrifuge, etc.
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