Determination of ELISA Titre

The titre of an antibody preparation is a measure of its concentration under a defined set of conditions. Thus, there will be a different measured titre for each assay system used. Indeed, some monoclonal antibodies, while of high titre in one assay system, may not react at all in another system.

In order to determine titre, serial dilutions of antibody preparation (e.g. pooled ascites, supernatant or IgG) are allowed to react with a know amount of antigen. The antibody titre is usually defined as the lowest dilution to bind significantly to antigen, or, alternatively the dilution of antibody that gives a half-maximal binding to antigen. These definitions are illustrated in Figure 7.1.1, which shows the results of an ELISA titre test as described in Protocol 7.1.1.

Protocol 7.1.1: ELISA: Antibody Titre Test

Materials:

* Microtitre plate for ELISA;
* Antigen for coating plate;
* Irrelevant protein (i.e. 1% BSA) for negative control coating;
* Coating buffer;
* Wash buffer;
* Anti-mouse or rat Ig-enzyme conjugate;
* Enzyme substrate;
* Substrate stopping solution;
* Blocking solution;
* Monoclonal antibody (ascitic fluid, Ig preparation or culture supernatant) in serial 10-fold dilutions to zero;
* Negative control antibody as above (e.g. raised to an irrelevant cell line or to myeloma cells);
* Multichannel pipette;
* Plate washer or wash bottle.

Method:

1. Adsorb predetermined maximal concentration of antigen to four rows (48 wells) of the microtitre plate. Dilute in coating buffer and incubate 100 µl per well overnight at 4°C. Also coat two rows (24 wells) with irrelevant protein (i.e. 1% BSA in coating buffer) to serve as negative control.
2. Aspirate antigen solution and block unbound sites on the plastic by incubating 100 µl of blocking solution per well for 15 min.
3. Wash off unbound antigen and block protein three times with wash buffer.
4. Add 10-fold dilutions of ascitic fluid (or Ig or culture supernatant) across the plate to two of the antigen-coated rows and to the two negative control rows. Add the same dilutions of negative ascitic fluid to the other two antigen-coated rows as negative control. Incubate 100 µl per well for one hour at ambient temperature.
5. Wash off unbound antibody three times with wash buffer.
6. Add enzyme-labeled anti-mouse Ig (or rat or human as appropriate) at the recommended or predetermined dilution. Incubate 100 µl per well for one hour at ambient temperature.
7. Wash off unbound label three times with wash buffer.
8. Add enzymesubstrate. Incubate 100 µl per well at ambient temperature until colour develops sufficiently (ideal approx. 30 min).
9. Do not wash off. Add 50 µl of the appropriate stopping solution and assess either visually or quantitatively in a spectrophotometer designed to read microtitre plates.

For more detailed information, see A Practical Guide to Monoclonal Antibodies written by J. Eryl Liddell and A. Cryer.