When using purified antigen for selection, the best way to evaluate the selected antibody is to perform an enzyme-linked immunosorbent assay (ELISA) in a high-throughput titration mode. Although this may not have anything to do with the final use of the antibody, it provides an initial screening protocol that will allow subsequent analysis to focus on promising antibodies. Most phagemid display vectors are designed to allow two ELISA modes:
One is phage ELISA, which uses labeled anti-phage secondary antibody reagents to detect bound phages;
The other is a soluble scFv ELISA, which detects soluble scFv by means of an epitope tag attached to the N-terminus or C-terminus. In both cases, antibody expression is driven by the lacZ promoter. Phage display depends on leaky expression in the absence of glucose, and in order to induce the expression of soluble antibody fragments, IPTG is also required. The labels used for testing include: 9E10, SV5 and FLAG.
Overall, although we also found that some antibodies can signal in the phage mode and disappear in the soluble mode, the results of the two ELISA modes are similar. This may be caused by the frame shift of the antibody. When these antibodies are displayed on the surface of phage, a large amount of scFv will be detected; but not when expressed as soluble scFv. This situation is as previously described in the peptide library. Most currently used phagemid vectors allow simple switching from phage-binding scFv to soluble scFv. This is achieved by including an inhibitory amber codon (TAG) between the scFv gene and gIII. In a non-suppressed strain (such as HB2151), the amber codon is read as a terminator, so only soluble scFv is produced. In a non-suppressed strain (such as TGi, DHSaF '), the TAG codon is read as glutamine and a stop codon, so soluble scFv and scFv-pIII fusion proteins will be generated. Perhaps the ideal situation is to switch from a phage-binding scFv to a soluble scFv through an infection-suppressing strain, but this is actually not necessary. The expression of soluble scFv is easily accomplished in the inhibitory strain, so the inhibition is usually no more than 50%. Crude phage or scFv samples are suitable for most pre-analysis experiments. However, simple antibody purification is advantageous for subsequent analysis. It is best to use a phage vector that contains a hexahistidine tag, or to subclone the scFv gene into a vector that fuses the hexahistidine tag to the C-terminus of scFv.
In different rounds of selection, the proportion of positive clones will depend on the quality of the antibody library, the nature of the antigen and the physical selection process, including antigen density, whether soluble or solid phase antigens are used, and the rigor of washing. We found that, according to the immune tube selection procedure, generally 10% to 100% of clones were positive after the second round. It is best to assess diversity as soon as possible, because as selection progresses, diversity will decrease. In this way, a larger antibody library can be provided to facilitate the selection of suitable antibodies.
Although binding screening methods are useful, these often do not use this method for final antibody selection. The ability to generate large amounts of antibodies relatively quickly can allow for the establishment of screening methods other than binding methods. Various selection methods for cell internalization, receptor activation, ligand antagonism, virus inactivation, induction or inhibition of apoptosis can be expected. Moreover, in some cases, such as internalization, it may even be possible to directly select antibodies with these functions.
With ELISA, phage antibodies can be prepared in 96-well microtiter plates. Single clones were picked into the wells of the plate, cultured and rescued as helper phage as phagemids. In the microplate mode, the production efficiency of phage is not as good as that of larger volume culture. This is because the growth rates of different clones are too different. As a result, some clones are infected with helper phage at the optimal OD value, while other clones are infected when the OD value is too high or too low. This leads to very different phage titers in different wells.
Shanghai Hengyuan Biological Laboratory now has multiple detection systems:
1. ELISA double antibody sandwich method detection system
2. ELISA indirect detection system
3. ELISA competition method detection system
4. ELISA capture coating detection system
5. ABS-ELISA detection system
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