Antibody Elisa

Inhibition ELISA assay

1) Plate coating: End result: Two plates (96 wells) will be coated with either 1 ug/mL or 0.1 ug/mL KLH-conjugated peptide.

a) Reconstitute KLH protein in 230 uL H2O. b) Dissolve 0.7 mg peptide in 100 uL conjugation buffer. c) Immediately add 70 uL KLH to peptide solution. Incubate at RT for 2 hrs. d) Final concentration is 4.1 mg/mL e) Dilute peptide to 1 ug/mL and 0.1 ug/mL with PBS (1:4,000 and 1:40,000). (e.g., 2 uL into 8 mL PBS, 0.6 mL of this into 5.4 mL PBS) f) Add 50 uL conjugated peptide per well and incubate at 4°C O/N (no shaking).

2) Incubation with primary and secondary antibodies: End result: Conjugated peptide on plates is incubated with primary antibody at 1:500 and 1:2000 dilutions plus 0, 10, or 50 ug/mL of 2 different competitors.

a) Make blocking buffer b) Wash plates with 100 uL/well with PBS. Repeat once. c) Add 100 uL blocking buffer per well. Incubate at 4°C for 1 hr. d) Dissolve competitor peptide (non-conjugated) in PBS-B (e.g., 0.4 mg in 0.4 mL = 1 mg/mL. Dilute with PBS-B to 50 ug/mL and 10 ug/mL. 350 uL into 6.65 mL, 1.1 mL of this into 4.4 mL). e) Dilute antisera to 1:500 and 1:2000 in blocking solution (4 uL into 2 mL and 400 uL of this into 1.2 mL). Need enough for 12 wells each. f) Shake blocking solution from plate. g) Add 50 uL/well competitor peptide and 50 uL/well antisera (or blocking solution for negative) (see diagram below). h) Incubate at 4°C for at least 1 hr (to O/N) i) Wash plate (add 100 uL/well, then remove) with: PBS-T/1 M NaCl 2X PBS-T 2X PBS 2X j) Dilute secondary antibody (anti-Rabbit-HRP, 1:1000, e.g., 10 uL into 10 mL) with blocking buffer, add 50 uL/well, and incubate at 4°C for 1 hr. max. k) Wash plate (100 uL/well) with: PBS-T 3X dH2O (from sink) 4X

3) OPD reaction:

a) Prepare Buffer B. Dispense 100 uL/well and incubate at RT for 10 min. b) Add 100 uL/well 5% H2SO4 (stop solution). c) Read absorbance at 492 nm in plate reader.

Solutions:

10X PBS, pH 7.5 For 400 mL: 1.4 M NaCl 112 mL 5 M NaCl 25 mM KCl 10 mL 1 M KCl 15 mM KH2PO4 6 mL 1 M KH2PO4 81 mM Na2HPO4 32.4 mL 1 M Na2HPO4 pH to 7.5 to 400 mL

PBS-T (PBS + 1% Tween 20) 10 mL 10X PBS 1 mL Tween 20 89 mL H2O

PBS-B (PBS + 0.2 mM 2-mercaptoethanol) 1 uL 14 M 2-ME into 69 uL PBS (200 mM), then 12 uL of this to 12 mL PBS.

Blocking buffer (PBS + 1% BSA) 5 mL 10X PBS 0.5 g BSA 45 mL H2O (DON’T HEAT)

PBS-T/1 M NaCl 10 mL 10X PBS 20 mL 5 M NaCl 1 mL Tween 20 69 mL H2O

Buffer A 0.1 M citric acid: 3.06 g citric acid into 100 mL H2O 0.2 M Na2HPO4: 2.8 g sodium phosphate into 100 mL H2O Add 0.2 M Na2HPO4 to 0.1 M citric acid until pH = 5

Buffer B (prepare fresh) 6.25 mL Buffer A 6.25 mL H2O 5 mg OPD (1 tablet) 5 uL 30% H2O2.

5% H2SO4 1 mL H2SO4 19 mL H2O (Mix in hood)