Chapter 5

Specificity is the most important aspect of the immunoassays and was evaluated using several techniques.

5.3.1. Immunoelectrophoresis

Polyclonal antiserum was purified until it showed no cross-reactions by immunoelectrophoresis with the alternate FLC and intact immunoglobulin molecules (Figure 5.2)

5.3.2. Western blot analysis

Western blot analysis is a sensitive technique used to assess the reactivity of the antisera against immunoglobulin fragments and FLC polymers. The results showed that both κ and λ FLC antisera reacted strongly, with two closely migrating bands at 25 - 30 kDa, and weakly with several larger and smaller molecular weight fragments. Similar staining patterns were observed using monoclonal antibodies. The FLC antisera were readily able to detect monomers and dimers of both κ and λ molecules (Figure 5.3).

5.3.3. Haemagglutination assays

Haemagglutination assays are far more sensitive than immunoelectrophoresis, and provide better assessment of specificity. Sheep red blood cells were sensitised with individual FLCs and purified IgG, IgA and IgM, and tested against the FLC antisera. The results showed that κ and λ FLC antibodies reacted with the appropriately labelled cells at >1:16,000 dilution and at <1:2 against cells coated with the alternate FLCs or intact immunoglobulins (Figure 5.4).

5.3.4. Nephelometry

Latex-conjugated FLC antisera were tested for specificity by nephelometry. Potentially interfering substances were added to serum containing known concentrations of FLCs and the changes in values indicated the effect on the assays (Figure 5.5). Nephelometric assays demonstrated that FLC antisera had minimal reactivity with light chains on intact immunoglobulins and other potentially interfering substances (0.2 - 0.01%). These values are within the purity specification for FLC contamination in the tested interfering materials.

There have been no published independent specificity analyses of the nephelometric Freelite latex reagents. Nakano et al. [148] reported an evaluation but, in error, only tested FLC antisera that were manufactured for immunofixation electrophoresis (IFE), where specificity requirements are less demanding.