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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/3.5/2.2

TITLE: ELISA FOR TESTING CELL SUPERNATANTS CONTAINING HUMAN IgG

PROCEDURE

COATING MICROTITRE PLATES

  1. Add 100ml capture antibody (goat anti-human IgG Fc) to each well of microtitre plate (2.5ng/ml).
  2. Cover with cling film/sealing tape and incubate overnight at 4°C range 2 - 80C.
  3. Empty wells by turning plate upside-down and tapping onto absorbent surface.

BLOCKING

  1. Pipette 200ml blocking solution into each well. Cover and incubate 37+10C for 1hr.
  2. Empty plates as before.

SAMPLE APPLICATION

  1. Pipette 100ml dilution buffer in all wells from A to G (use row and column markings on side of microtitre plate) leaving row H empty (see plate plan below).

    2. A1

    A2

    A3

    A4

    A5

    A6

    A7

    A8

    A9

    A10

    A11

    A12

    B

     

     

     

     

     

     

     

     

     

     

     

    C

     

     

     

     

     

     

     

     

     

     

     

    D

     

     

     

     

     

     

     

     

     

     

     

    E

     

     

     

     

     

     

     

     

     

     

     

    F

     

     

     

     

     

     

     

     

     

     

     

    G

     

     

     

     

     

     

     

     

     

     

     

    H

     

     

     

     

     

     

     

     

     

     

     
  2. Pipette 100ml of standard antibody (Human IgG/k myeloma protein) at 256ng/ml into wells A1 and A2.
  3. Pipette 100ml of sample in wells A3 to A10, preferably in duplicate, and mix well
  4. Using a multichannel pipette, pipette 100ml from A into row B, mix.
  5. Dilution is continued until row G, from the last 100ml is discarded.
  6. Controls are pipetted into row H.
  7. Incubate 37°C for 1hr.
  8. Empty wells and tap dry
  9. Wash wells 3 times in wash buffer, tapping dry each time.

CONJUGATE APPLICATION

  1. Pipette 100ml conjugate antibody (alkaline phosphatase conjugated goat F(ab)2 anti-human IgG) diluted 1:3000. Cover and incubate 37+1°C at 1 hr.
  2. Empty wells and wash 3 times as before.
  3. Tap plate dry.

SUBSTRATE APPLICATION

  1. Pipette 100ml of substrate solution into each well (alkaline phosphate substrate granules with granule buffer solution).
  2. Incubate room temp. for 15 minutes.
  3. Stop reaction by adding 25ml 2M NaOH to each well.
  4. Measure absorbence using an ELISA reader at 405nm and ref. wavelength 620nm.

WORKING OUT IgG CONCENTRATIONS

  1. Use log scale graph of standard antibody concentration against absorbence reading to obtain a standard curve.

The concentration of IgG in the test sample can then be worked out by reading off the concentration from this standard curve at the various absorbence readings obtained.

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

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