Immunoglobulins and FLC molecules are highly polymorphic (Chapter 3). Therefore, an important feature of any FLC assay must be the ability to recognise all the various forms of FLC molecules with comparable affinity. This is necessary to ensure that the assay is not only effective for diagnosing all patients, but is also quantitatively consistent, allowing comparisons of protein production between patients.
All routine assays for the quantification of total immunoglobulins (IgG, IgA and IgM) use polyclonal antisera, and historically, polyclonal antisera raised in rabbits or sheep have also been used in the majority of FLC assays . This probably reflects the difficulty of producing monoclonal antibodies that are specific for free but not bound light chains, but also recognise all the variations of shape that different monoclonal FLCs can exhibit (Chapter 3).
8.3.1. Monoclonal antibody productionFigure 8.1).
8.3.2. Polyclonal antisera production
To produce polyclonal antisera, animals (typically rabbits or sheep) are immunised with a particular antigen (e.g. FLCs). As a result, multiple B-cells are activated to multiply, differentiate and produce antibodies, which each target a specific epitope on the antigen (Figure 8.1). As a result, serum collected from the immunised animal contains polyclonal antibodies that collectively, will demonstrate a range of different specificities and epitope affinities (Figure 8.2). For Freelite assays, the repertoire of polyclonal antibodies is further increased through blending a large array of polyclonal antisera that have been raised in different sheep and against a wide variety of different monoclonal FLCs (Chapter 5). Prior to use in the assays, the polyclonal sheep antibodies are purified by positive- and negative-affinity chromatography against a diverse range of FLCs and intact immunoglobulins. This is necessary to produce the high-specificity, high-titre and high-affinity antibodies that are required for Freelite sFLC assays.
8.3.3. Requirements for anti-FLC antibodies for use in FLC immunoassays
Regardless of whether an anti-FLC antibody is monoclonal or polyclonal in nature, it should have a number of features for optimum performance in turbidimetric/nephelometric FLC immunoassays. These are summarised in Table 8.2.
|Desired feature||Monoclonal antibody||Binding Site polyclonal antisera|
|Specificity||+++ or 0*||+++|
|Immune complex formation||+#|
Small soluble complexes
Large insoluble complexes
|Recognition of all polymorphic FLCs?||No||Yes|
Table 8.2. A summary of desired features of a FLC immunoassay, and the characteristics of monoclonal and polyclonal antisera. *: Whilst monoclonal antibodies are highly specific for a given epitope, if this epitope is distorted, hidden or absent, they will not recognise the protein. #: As a single monoclonal antibody is unable to form large immune complexes (unless the target antigen has repeated epitopes), a mixture of monoclonal antibodies is required in turbidimetric/nephelometric assays. However, it is difficult to create a mixture of monoclonal antibodies with balanced reactivity against a broad range of FLC molecules.
For any FLC assay, the antibodies used must be highly specific as FLC assays are required to discriminate “free” from “bound” light chains (in intact immunoglobulins), which may be 1000-fold more abundant in serum. Even minor cross reactivity with intact immunoglobulins can cause significant overestimation, and should be avoided. The antibodies must also be of high affinity, to produce FLC immunoassays that are suitably sensitive. In order to maximise light scattering in turbidimetric/nephelometric assays, the antibodies must form large immune complexes. Analytical sensitivity can be enhanced using latex-conjugated antibodies. Polyclonal antibodies are ideally suited for latex-enhanced turbidimetric/nephelometric assays due to their ability to cross-link via the recognition of different epitopes on an antigen. In contrast, large immune complexes cannot be formed with a single monoclonal antibody, unless the relevant epitope is repeated on the antigen. To overcome this limitation, more than one monoclonal antibody can be used; both N Latex FLC κ and λ assays use two monoclonal antibodies . However, it can be difficult to produce multiple antibodies that provide balanced reactivity against the full range of FLC molecules.
Immunoglobulins and FLC molecules are highly polymorphic (Chapter 3). Therefore, an important feature of a FLC assay must be the ability to recognise all the various forms of FLC molecules with comparable affinity. This is necessary to ensure that the assay is not only effective for diagnosing all patients, but is also quantitatively consistent, allowing comparisons of protein production between patients. All routine assays for the quantification of total immunoglobulins (IgG, IgA and IgM) use polyclonal antisera and the issues discussed above suggest that polyclonal antibodies would be the best choice for measuring polymorphic FLCs.