9780521516358book.pdf

optimal). The magnitude of the response will increase the greater the molecular

weight. If an antibody is to be made to a molecule smaller than 2 000 Da then it has

to be conjugated to a carrier molecule to effectively increase its size above the

threshold for immune surveillance. These small molecules are known ashaptens

and may be peptides, organic molecules or other small chemicals. They are usually

conjugated to a protein such as albumin, keyhole limpet haemocyanin or thyroglo-

bulin and then used to immunise animals for antibody production (Fig. 7.7). If a

polyclonal antibody is being made it is advisable to change the carrier protein at least

once in the immunisation procedure as this favours more antibody being made to the

hapten and less to each of the carrier proteins. If a monoclonal antibody is being made

then the carrier protein can be the same throughout the immunisations. When

screening hybridomas for monoclonal antibody production it is necessary to screen

against the hapten and carrier separately. Any antibodies responding to both should

be discarded as these will be recognising the junction between the hapten and carrier

and will not recognise the native hapten.

Hybridoma

cell line

Clone cells

ELISA test then
choose one
clone to expand

12 simultaneous

freezings for

master cell bank

Thaw last

freezing and

grow cells

50 simultaneous

freezings for

working cell bank

Fig. 7.6Master and working cell banks.

278 Immunochemical techniques