Serological selection for cloning of new antigens or genes

Summary

E.coli/ phage lysate adsorption serum removes antibodies in serum that immunoreact with bacterial and phage lysin, this reduces the false positive rate, the serum can be either patient serum or immune serum, in the case of patient serum a minimum of 10 patient sera are required for mixing.

Operation method

Serological selection for cloning of new antigens or genes

Materials and Instruments

Serum
E.coli phagelysate TBST NZY agar plates MgSO4 IPGT TBS BCIP-NBT SM buffer chloroform LB medium
nitrocellulose membranes filter paper constant temperature incubator -80℃ low temperature storage box spectrophotometer low temperature centrifuge constant temperature water bath tweezers

Move

I. E.coli/phage lysate pre-adsorbed serum1. Dilute E.coli /phage lysate 1:10-20 in TBST solution.2. 4 sheets of 82 mm nitrocellulose membranes (NC) were immersed in the diluted E.coli/phage lysate and shaken horizontally for 30 minutes at room temperature, the NC were removed and the membranes were allowed to drain.3. The membrane was washed three times with 50 ml of TBST solution for 10 minutes each time.4. Gently remove the liquid from the membrane with filter paper.5. Place the membrane in 50 ml of blocking solution and shake horizontally for at least 30 minutes at room temperature.6. Remove the membrane from the blocking solution and wash the membrane with 50 ml of TBST solution three times for 10 minutes each time.7. Dilute serum 1:5 in TBST solution, place one membrane in the solution and gently shake horizontally at 37°C for 10 minutes.8. Remove the membrane from the serum dilution solution and discard, add another new membrane and gently shake horizontally at 37°C for 10 minutes.9. Repeat step 8 until all 4 membranes have been processed.10. Remove the last membrane, collect the serum (prima ry antibody), and store in small portions at -80°C in the refrigerator until ready to use.Note: This procedure is to remove the antibodies in the serum that can react with bacterial and phage lysin, so as to reduce the rate of false positives; the primary antibody should not be frozen or thawed repeatedly, and should not be refrozen after thawing, but can be stored at 4℃ for a short period of time; it can be either the patient's serum or the immune serum, and in the case of the patient's serum, it is necessary to mix at least 10 patient's serums;

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