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Characterization of selected clones

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Phage ELISA

  1. 40 single colonies are usually picked up from the 4th-5th panning eluate titration plate, and inoculate in 2 mL LB medium with 100ug/mL AmpR. Shake the 2 mL tubes at 250 rpm for 5-8 hr at 37 °C.
  2. Add 40 μL of helper phage to each culture, and shake for another 2 hr at 37 °C.
  3. Add 2 μL of kanR to each culture, and shake the cultures overnight at 250 rpm and 30 °C.
  4. Centrifuge the cultures at 12,000 rpm for 15 min, and take the supernatant.
  5. Dilute the supernatant containing phage with equal volume of 5% milk. Incubate for 10 min at room temperature.
  6. Add 100 μL of the dilute phage to each well, and incubate for 2 hr at 37 °C.
  7. Wash the plate 5 times.
  8. Add 50 μL per well of diluted secondary antibody conjugate at 1:3,000 (HRP-conjugated anti-M13 antibodies, diluted in 5% milk), and incubate for 1 hr at 37 °C.
  9. After 5 times washing, 50 μL/well TMB substrate was added for detection, and incubate for 30 min at RT. 50 mL of 1M H2SO4 was added to stop the reaction. Absorbance was read at 450 nm.

Recombinant human Fab expression and soluble Fab ELISA

  1. Prepare phagemid DNA from E.coli cells containing Fab clones of interest, and transform phagemid DNA into competent E.coli TOP10F’.
  2. Expression of soluble Fab fragments from individual clones is induced on a 500-ml scale in SB medium containing 50μg/ml Ampicillin and 1% glucose.
  3. The culture is grown at 30°C to an OD600 of 1.5, and the cells are pelleted (1,500 g, 10 min) and re-suspended in an equal volume of fresh SB medium containing 50μg/ml Ampicillin, 20 mM MgCl2, and 1 mM IPTG. Induction is continued at 25 °C for 18–20 h.
  4. Collect the overnight culture, spin at 10,000 rpm for 5 min, and transfer the soluble Fab-containing supernatant for ELISA.
  5. Dilute the supernatant containing soluble human Fab with equal volume of 5% milk. Incubate for 10 min at room temperature.
  6. Add 100 μL of the dilute Fab to each well coated with target antigen, and incubate for 2 hr at 37 °C.
  7. Wash the plate 5 times.
  8. Add 50 μL per well of diluted secondary antibody conjugate at 1:2,0000 (HRP-conjugated second antibody, Fab specific, diluted in 5% milk), and incubate for 1 hr at 37 °C.
  9. After 5 times washing, 50 μL/well TMB substrate was added for detection, and incubate for 30 min at RT. 50 μL of 1M H2SO4 was added to stop the reaction. Absorbance was read at 450 nm.

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