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Tissue culture supernatant is often concentrated before purification to reduce the

volume of liquid.Tangential flowdevices andcentrifugal concentratorsmay be used

to reduce the volume to 10% of the starting amount. This makes antibody purification

byaffinity chromatographymuch easier with the smaller volume of liquid (Fig. 7.9).

Antibodies from both polyclonal and monoclonal sources can be purified by similar

means. In both cases the antibody type is IgG which allows purification byprotein

A/Gaffinity chromatography. Proteins A and G are derivatives of bacterial cells and

have the ability to reversibly bind IgG molecules. Binding to the column occurs at

neutral pH and the pure antibody fraction can be eluted at pH 2.0. Fractions are

collected and neutralised back to pH 7.0. Antibody-containing fractions are identified

by spectrophotometry using absorbance at 280 nm (specific wavelength for protein

absorbance) and are pooled. A solution of protein at 1 mg cm^3 will give an absor-

bance reading of 1.4 at 280 nm. This can be used to calculate the amount of antibody

in specific aliquots after purification.

Purified antibody should be adjusted to 1 mg cm^3 and kept at 4C, or 20 C for

long-term storage. It is usual to add 0.02% sodium azide to the antibody solution as

this increases shelf-life by suppressing the growth of adventitious microorganisms.

Antibodies can be stored for several years at 4C and for decades if kept below 20 C

without losing activity.

7.2.10 Antibody modification

Antibodies can be labelled for use in assays such as ELISA by the addition of marker

enzyme such as horse radish peroxidise (HRP) or alkaline phosphatase(AP).

Other enzymes such as urease have been used but HRP and AP are by far the most

Antibody in

medium added

to column

Antibody binds to

protein A/G on

beads in column

Column washed

to remove

contaminants

Elution buffer

added to

release antibody

Fig. 7.9Affinity chromatography.

282 Immunochemical techniques