1. The rabbit antibodies used for capturing MAbs may also interact with some phage from the library. This might result in selection of many non-MAb specific clones. Thus, the library should be pre-incubated with anti-(mouse Ig) antibodies alone in order to prevent non-specific binding of phage by rabbit Ig.
2. Plates might be coated at the same time with MAbs of different specificity—individually or in combination with others. The principle of MAbs combination depends on whether there is some preliminary information about their mutual relationship, for example, competitive assay or peptide binding assay. Afterwards, the plates would be sequentially incubated with the same library preparation.
3. Phenol red is added to elution buffer, and is yellow at pH 2.2. When 1 M Tris-HCl, pH 9.1 is added, the elution buffer turns red, hence the name "red eluate."
4. Because the phage containing the desired peptides might be present in the main library as only a few particles, it is recommended to concentrate the first "red eluate" to 100 mL using a membrane concentrator (e.g., Vivascience, VSO 132) before amplifying.
5. On further rounds of panning, the MAbs mixture is reacted only with the corresponding amplified eluate.
6. Tentatively, suitable dilutions of "red eluate" (in TBS/gelatin) appear to be A:1 |L in 1000 |L of TBSG, B: 100 |L of A plus 900 |L of TBSG, C:100 |L of B plus 900 |L of TBSG.
7. It is useful to spread the cells remaining after amplification (Subheading 3.2.9.) to obtain single colonies for screening. Add 2 mL of LB broth to the pellet of overnight culture. Make serial dilution (10-, 10-5, 10- should be fine) in LB medium. Use 200 |L to spread on a plate.
8. To fit a 90-mm Petri dish, a NCM might be cut as a rectangle approx 5.0 x 6.5 cm.
9. Hybridoma SN should preferably be used for colony screening because there is always the possibility of false-positive colonies being selected using ascites. Ascites contain a lot of antibodies with unknown specificity, which could bind phage from the library.
10. For some antibodies, positive clones were found even after the first round of panning. It seems that further enrichment is undesirable in these cases because there is the possibility that this might lead to isolation of fewer clones with higher affinity for the antibodies.
11. If quantitative analysis of antibody-phage binding is desirable, it is possible to perform an ELISA procedure using purified phage as antigen.
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