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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

Appendix

REFERENCE NO: AHC/1998/3/3.5/2 Appendix 2

TITLE: QUANTITATIVE ELISA

INTRODUCTION

A simple general purpose ELISA assay has been developed to detect monoclonal antibodies at all stages of production and purification by reaction with anti-species antibody or by reaction with antigen supplied by clients.

REAGENTS

See Procedure AHC/1998/3/3.5/2

METHOD

  1. Dilute appropriate capture agent to required concentration in coupling buffer. Dispense 100ml of this per well. Incubate overnight at 2 to 8oC.

  2. Wash the wells 4 times in PBST. Dip plates into PBST, flick off contents and repeat twice more, at end 3rd wash leave buffer on for ~ 3 min. Repeat once more immediately before next stage. Blot dry.
  3. Add 120ml of blocking buffer to all wells and incubate at room temperature for 1 hour.

  4. Dilute hybridoma culture supernatant and standard to be tested in ELISA diluent and add 200ml to all wells of column 2 with the standard in well H2. Then make doubling dilutions across the plate discarding the last 100ml. To all wells of column 1 add 100ml of ELISA diluent. This acts as a blank. See Plate Plan. Cover plate and incubate for 2 hours at 37 ± 1oC.
    5.

    A1

    A2

    A3

    A4

    A5

    A6

    A7

    A8

    A9

    A10

    A11

    A12

    B

     

     

     

     

     

     

     

     

     

     

     

    C

     

     

     

     

     

     

     

     

     

     

     

    D

     

     

     

     

     

     

     

     

     

     

     

    E

     

     

     

     

     

     

     

     

     

     

     

    F

     

     

     

     

     

     

     

     

     

     

     

    G

     

     

     

     

     

     

     

     

     

     

     

    H

     

     

     

     

     

     

     

     

     

     

     
  5. Wash plate as in 2.
  6. Make a 1/1000 dilution of sheep anti-species peroxidase conjugate and 1:100 dilution of sheep serum both in ELISA diluent (for human quantitative assay add 2% sheep serum). Add 100ml of this to each well. Seal plate again and incubate for 1 hour at 37 ± 1oC.
  7. Wash plate as in 2.
  8. Add 100ml of substrate 'working solution' to each well, leave for 10 to 15 minutes at room temperature.
  9. Stop the reaction by adding 50ml of 2M sulphuric acid to each well.
  10. Measure the OD at 450nm using an ELISA plate reader.

Using values from the positive control, plot a standard curve and calculate antibody concentrations present in the test supernatants.

Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998

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