Several Commonly Used Antibody Screening Methods v1
Authors: Smith Bella 
Published: December 2018

I Immunoenzymatic technique Immunoenzymatic technology is an immunological technology which combines the specificity of antigen-antibody reaction and the high-efficiency catalytic effect of enzyme on the color reaction of substrates. Because of its high specificity and sensitivity, it has been widely used to screen and identify monoclonal antibodies. Equipment and reagents Coating buffer: Ø Carbonate buffer: 0.2 mol/L Na2CO3 8 ml, 0.2 mol/L NaHCO 3 17 ml, 75 ml distilled water, adjust PH to 9.6. Ø Tris-HCl buffer (PH8.0, 0.02 mol/L): 0.1 mol/L_Tris_100 ml, 0.1 mol/L_HCl_58.4 ml, mixed with distilled water to 1000 ml. Washing Buffer (PBS of PH7.2) Ø Washing Buffer (PBS of PH7.2): KH2PO4_0.2g, KCl_0.2g, Na2HPO4&S226; 12H2_2.9g, NaCl Diluting and sealing fluids Ø Bovine serum albumin (BSA) 0.1g, add detergent to 100ml, or use detergent to prepare calf serum for 5-10% use. Enzyme termination solution (2mol/L_H2SO4) Ø Distilled water was 178.3 ml and concentrated sulfuric acid (98%) was added 21.7 ml. Substrate buffer (PH5.0, Phosphate-Citric acid buffer) Ø Take 0.2mol/L Na2HPO4 25.7ml, 0.1ml/L citric acid 24.3ml, and add 50ml distilled water. The citric acid solution and the prepared substrate buffer are unstable and easy to form precipitation, so it is not suitable to prepare too much at one time. Substrate use liquid: Ø OPD Substrate Use Solution (OD value of 490 nm measured): OPD_5 mg, Substrate Buffer 10 ml, 3%H_2O_2_0.15 ml. TMBS or TMB Substrate Use Solution (OD at 450 nm): TMBS or TMB (1mg/ml) 1.0 ml, Substrate Buffer 10 ml, 1% H2O2 25ul. ABTS Substrate Use Liquids (Measuring OD at 410 nm) Ø ABTS_0.5 mg, 1 ml of substrate buffer, 3%H_2O_2_2ul Antibody control Ø Soluble antigen: purify as much as possible to obtain high specificity. Antigen Ø Soluble antigen: purify as much as possible to obtain high specificity Enzyme-labeled anti-mouse antibodies or enzyme-labeled SPA or other similar reagents. Cell stationary fluid Ø Acetone at - 20 C; or acetone - formaldehyde stationary solution: Na2HPO4 100mg, KH2PO4 500mg, distilled water 150 ml, acetone 225ml, formaldehyde 125 ml; or acetone - formaldehyde solution (1; 1); or methanol at - 20 C. Polystyrene microporous board Ø 40 holes, 96 holes, or strip holes; hard board or soft board can be used. Ø Enzyme-linked immunoreader; or light microscopy. Ø Pipette, Sampler and Water Bath Tank, Centrifuge, etc. Ø Enzyme-linked immunosorbent assay (ELISA) of soluble antigens Purified antigens were diluted to 1-20 ug/ml with coating solution. Ø A kind of B. Add 50-100ul/pore volume into the pore of the enzyme label plate, and place it at 4 C overnight or 37 C for 2 hours. Ø Discard the liquid in the hole and wash it three times with detergent, 3-5 minutes each time, pat dry. Ø Each pore is sealed at 4 or 37 for 2 hours with 200 UL blocking solution; this step can be omitted for some antigens. Ø Wash the washing liquid three times; at this time, the cladding plate can be stored at - 20 C or 4 C for reserve. Ø Each hole was added 50-100ul culture supernatant of hybridoma cells to be examined, and positive, negative and blank control were set up at the same time; incubated at 37 for 1-2 hours; washed and patted dry. Ø Add the second antibody labeled with enzyme, incubate for 1-2 hours at 37 50-100 UL per pore, wash and pat dry. Ø Substrate solution added, each hole with fresh preparation of the substrate using liquid 50-100ul, 37 10-30 minutes. Ø Terminated the reaction with 2mol/L_H2SO4 and read OD value on ELISA. Ø Result: P/N (> 2.1) or P (> N + 3D) were positive. If the negative control hole is colorless or nearly colorless, and the positive control hole is clearly colored, the results can be directly observed with the naked eye. ELISAS of whole bacterial antigen Ø Fresh cultured bacteria were suspended in distilled water or PBS, and the concentration of bacteria was adjusted to 1*108/ml. It must be pointed out that for zoonotic pathogens, attention should be paid to safe operation, preferably inactivation treatment. Ø Add 100 ul_5% glutaraldehyde solution (0.1 mol/L_NaHCO 3_95 ml+25% glutaraldehyde solution 5 ml) to each pore, and wash it with distilled water for 2 hours at 37 add the bacterial suspension 50ul/pore mentioned above; dry it at 37-56 add 200 UL sealing solution 4 overnight or 37 2 hours to seal each pore. Ø Step B can also be dried at 37 56 with 50 UL bacterial suspension added to each hole, then washed with distilled water for 15 minutes at room temperature with anhydrous methanol pre-cooled at - 20 and sealed at 4 or 37 2 hours with 200 UL sealing liquid added to each hole. Ø Wash the washing liquid three times. At this time, the cladding plate can be stored at - 20 C or 4 C for reserve. Ø The following steps are the same as the above. ELISA Using Whole Cell Antigen Ø Cells were cultured by routine methods, inoculated with virus, infected and uninfected cells were harvested, cell counts were carried out, and PBS suspension was made into appropriate concentration. Ø The preparation of lymphocyte suspension was anticoagulated with fresh peripheral blood and heparin, then dripped on the lymphocyte separation solution, centrifuged at 1500rpm for 30 minutes, and washed the interface cells twice, that is, fresh lymphocyte suspension. If the cell suspension is still mixed with red blood cells, add 0.83% ammonium chloride solution after centrifugation and wash once at room temperature for 10 minutes. Ø Cell suspension of 100 UL a or B was added to each pore to make each pore contain 5 *104 cells; 1 500 r/min 15 minutes to remove the supernatant; after drying or blowing at room temperature, the supernatant was fixed for 10 minutes at acetone-methanol (1:1) 4 and stored at 4 or - 20 for reserve. Ø The following steps are the same as the above. ABC-ELISA experiment ABC-ELISA is based on the principle of conventional ELISA, which adds the amplification between biotin and avidin. The avidin has four subunits and has a high affinity for biotin. Biotin is easily covalent with protein. Therefore, the reaction between avidin binding enzyme and biotin binding antibody plays a multipolar amplification role. The operation steps are as follows: Ø Encapsulation of known antigens and McAb samples to be tested, same as indirect ELISA test Ø Add biotinylated anti-mouse Ig antibody, 100 UL per pore, 37 1 hour; wash. Ø Enzyme labeled avidin was added and washed at 37 30 minutes per pore. Substrate colour was added to determine the result. Dot-ELISA test Dot-ELISA (Dot-ELISA) is an immunoassay method for antigen-antibody reaction using cellulose nitrate membrane or cellulose acetate membrane as solid carrier. The method uses insoluble substrates (such as DAB, 4-chloronaphthol or AgNO3) and reacts with the corresponding markers (HRP, AP, colloidal gold) to form insoluble products, which show spot-like coloration, thus easy to determine the results. According to the different markers used, it can be divided into HRP_immunodot test, AP immunodot test and immunogold and silver dot test. The operation steps are as follows: Ø 2-5ul point of antigen solution was added to cellulose membrane and dried at room temperature 37 C. Ø Immerse the fibrous membrane in the sealing solution at 37 30 minutes. Ø Wash twice with detergent, add McAb samples to be tested after drying, 37 1 hour; wash three times with detergent oscillation, 5 minutes each time, add HRP or AP or colloidal gold labeled anti-mouse Ig antibody, 37 30 minutes. Ø The same method of washing, drying, coloring with the corresponding new substrate solution, and then washing to stop the reaction, observe the results. Immunohistochemical staining This method is mainly used to detect MCAb for cell antigen components. The commonly used methods are indirect immunoperoxidase test (IIP) and APAAP technology. The results were examined by light or inverted microscopy. Related service: screening antibodies screening library what is antibody screening