Solid Phase Screening Related Questions-Nano Antibody Special Topic-Tekbiotech-Yeast Display Service,Phage display technology

Solid Phase Screening Related Questions

The solid phase screening method is to coat the purified or sufficient antigen on the solid phase medium (such as ELISA plate, affinity chromatography column and immune test tube), and then add the screened phage antibody library for incubation. After multiple "adsorption-washing-dissociation-amplification" processes, wash away the non-specific binding or weak binding phage antibodies, and recover the bound phage antibodies. The following are some solid phase screening experimental processes:

一．Selection of Antigen and Antibody Binding Time

In the ELISA plate wells coated with antigen, add antigen phage antibody and non-specific phage antibody respectively. The binding time can be set to 1, 10, 20, 30, 45min and 1, 1.5, 2h. After washing with PBST 5 times, add 1:5000 HRP-sheep anti-M13 antibody, add PBST and wash 5 times, add HRP substrate colorimetric solution and stop, and measure the A value.

二．Selection of Washing Conditions

Add the above-mentioned types of phage antibodies to the ELISA plate coated with antigens, react for 2 hours, and then wash with different washing conditions, wash 5 times with PBST, wash 5 times with PBS and PBST, wash 5 times with PBS and then 10 times with PBST, wash 10 times with PBS and PBST, and then perform ELISA detection of phages as above.

三．Selection of Buffer pH

Add the above-mentioned types of phage antibodies to the wells of the ELISA plate coated with antigens, and incubate at 37℃ for 2 hours. After washing for 10 minutes with acetic acid/sodium acetate buffer at pH 3, 4, 5, and 6, perform ELISA detection of phage antibodies as above.

四．Effect of Antigen Coating Concentration on Screening Results

Use antigen coating plates with concentrations of 1, 2, 4, 6, and 8 mg/L, add 100μL of mixed phage antibodies, and incubate at 37℃ for 2 hours. Then wash the plate 20 times with PBST and PBS, add 100μL E coli TG1, and incubate at 37℃ for 1h. Then dilute 105 and apply to the plate, and incubate at 30℃ overnight. Pick 96 single colonies from the plates obtained under different screening conditions for competitive ELISA detection of phage antibodies.

五．Competitive ELISA Detection of Phage Antibodies

The phage antibody supernatant was mixed with an equal volume of MPBS (PBS containing 40 mLL milk) and allowed to stand at room temperature for 30 min. Similarly, the sample was mixed with an equal volume of MPBS containing antigen and allowed to stand at room temperature for 30 min. Take 100μL of each and add it to the plate coated with antigen for reaction for 1 h, and wash 5 times with PBST. Add 1:5 000 HRP-sheep anti-M13 antibody and incubate at 37℃ for 1h; after washing as above, add HRP substrate colorimetric solution for 15 min, terminate the reaction with 2 mol/L H2SO4, and measure the A value at a wavelength of 450/620nm. The clones with more than 50% antigen competition are considered positive, that is, the clones with A value with antigen competition less than 50% of the A value without antigen competition are positive.

六．Impact of the Washing Process

If the stringency is too high, some clones with low expression may be screened out, and these clones may contain target phages with extremely high affinity; if the stringency is too low, the phage enrichment process may be too slow and the number of screening rounds may increase. Consider reducing the washing intensity (no soil temperature is added to the PBS washing solution) and reducing the number of washing times.

七．Determination of Antigen Concentration

It is generally believed that low concentrations of antigens should be used to screen high-affinity antibodies first, while high concentrations of antigens are conducive to the screening of low-affinity antibodies. It should be noted that there is an effective concentration of antigen for screening. Not all antigens used for coating can be effectively used for screening. The effective concentration of antigen must reach a certain value. However, the effective antigen concentration cannot be higher than 10^-6 mol/L, which will reduce the enrichment effect of specific antibodies.

Tek Biotech has established a complete and mature phage antibody display technology platform. Based on the phage display technology platform, Tek Biotech can provide major experimental links including antigen design, alpaca immunization, library construction and screening (solid/liquid screening), and active function verification, and screen camelid VHH antibodies with high specificity and high affinity for scientists around the world.

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