Chapter 9

Inevitably, one of the most demanding aspects of HLC assay production is to ensure specificity. A panel of antigens is used to test specificity; this panel includes sera containing monoclonal immunoglobulins of each class, subclass, and light chain type.

Initial immunisation and tolerisation procedures result in antisera that are strongly reactive against each HLC molecule, with a degree of cross-reaction with other immunoglobulin specificities. Cross-reacting antisera are recycled through affinity and adsorption columns until specificity is satisfactory. Antisera with subclass bias are purified against immobilized monoclonal immunoglobulins of other subclass types. Antisera are considered specific when they react in a balanced manner with the appropriate panel of immunoglobulins, and no cross-reactivity occurs with other molecules, including FLCs. The final purified reagents are produced using positive affinity chromatography against the target HLC immunoglobulin. Antisera specificity is the most important aspect of the HLC immunoassays, and is evaluated using several techniques, described below.

To test specificity by radial immunodiffusion (RID), HLC antisera are immobilised in an agarose gel and a panel of antigens are applied to wells cut into the gel. The diameters of the resulting immunoprecipitates are used to determine specificity.

Neat or latex-conjugated HLC antisera are tested for specificity by turbidimetry/nephelometry. Known concentrations of purified potential interfering substances are added to a normal serum containing concentrations of immunoglobulins within the reference interval for each assay. Results for IgG and IgA HLC assays are shown in Figure 9.3. Overall, the specificity assessments show that HLC antisera have minimal reactivity with intact immunoglobulins of the alternate light chain type or class. HLC antisera also have no significant reactivity with FLCs or other potentially interfering substances.