Assay IL-1a aAb test

IL-1a aAb are quantitated by radioimmunoassay using plasma/serum IgG binding to 125-I- labeled, soluble, human recombinant IL-1a. If binding is detected, saturability and cross-reactivity to native IL-1a will be tested using properly validated IL-1a tracers.

Other methods for cytokine Ab and aAb detections are generally not suited for quantitation of IL-1a aAb. For example, we and others have found that detection of cytokine Ab by Western blotting and direct ELISA is hampered by unsatisfactory sensitivities and strong but non-specific immunoglobulin binding to cytokines fixed to solid phases.

Test considerations and pitfalls

Methods used for the detection of Ab to cytokines include cytokine bioassays, immunometric assays and various blotting techniques. Serum Ab, however, do not always bind soluble polypeptides in a specific manner or with any appreciable affinity (1, 2). Thus, heterophilic Ab may give false positive results in ELISA consisting of two or more layers of Ab (3), and immunoblotting techniques and immunometric assays may show some degree of specificity, even though the binding of Ab to ligand is weak and topographically unassociated with the specific binding sites of the aAb; see figure below. For example, we and others have found that Western blotting and detection in direct ELISA are not suited for detection of IL-1a Ab because of strong, but non-specific immunoglobulin binding to IL-1a fixed to solid phases (4, 5).

Without previous knowledge of the binding characteristics, competition in these assays by surplus cytokine in solution may also not discriminate between the native conformation, different degrees of denaturation of the cytokines, or other factors as possible cause of the competitive activity.

Although ELISA techniques can be used for screening purposes, verification of specific cytokine Ab would require ligand binding to the Fab fragments of the Ab, using properly validated cytokine tracers, combined with saturation binding analyses and, at least in the case of natural aAb, demonstration of cross-binding to the endogenous cytokines (6).

Another potential problem is that assays for cytokine Ab often make use of unglycosylated, E.coli derived cytokines. Since many native cytokines are glycosylated, the recombinant forms may possess other or concealed antigenic sites that could jeopardise the interpretation of the assays; see figure below. Furthermore, even though glycosylated forms of some recombinant cytokines are available, their carbohydrate moieties may differ significantly from those of the wildtype cytokines. It is therefore possible that pre-existing Ab to certain polysaccharide structures may mimic a cytokine Ab by binding in vitro to a glycosylated recombinant cytokine (7, 8). These Ab may bind glycosylated cytokines both in direct binding assays, in solution and in solid-phase assays. Whenever binding to Ab (aAb) has been detected with a recombinant cytokine, demonstration of cross-binding with the natural cytokine is always relevant (2, 9-11).

Example:

Only cases #1 and #2 detect specific antibody (Ab) to the native cytokine (CK). These cases should be discriminated from cases #3, #4 and #5 by competition experiments using excess (non-denatured) CK in solution. However, while Ab-binding is displaceable in both cases with the natural CK, a non-glycosylated, non-denatured, recombinant (rec.) CK would displace binding only in case #1. In practice, the native CK used to capture Ab in case #2 will rarely be available, and any carbohydrate-specific Ab will therefore go unnoticed.

The binding in case #4 is displaceable only by the glycosylated rec. CK and, possibly, by relevant saccharides. In case #5, it will be difficult or impossible to displace the binding with soluble CK.

These principles also apply to the detection of Ab to soluble CK by many other methods, particularly those involving solid-phase binding techniques.

Cited references:

1. Hansen MB, Svenson M, Bendtzen K. Serum-induced suppression of interferon (IFN) activity. Lack of evidence for the presence of specific autoantibodies to IFN-alpha in normal human sera. Clin Exp Immunol 1992; 88:559-62.

2. Svenson M, Hansen MB, Bendtzen K. Binding of cytokines to pharmaceutically prepared human immunoglobulin. J Clin Invest 1993; 92:2533-9.

3. Grassi J, Roberge CJ, Frobert Y, Pradelles P, Poubelle PE. Determination of IL1a, IL1b and IL2 in biological media using specific enzyme immunometric assays. Immunol Rev 1991; 119:125-45.

4. Suzuki H, Kamimura J, Ayabe T, Kashiwagi H. Demonstration of neutralising autoantibodies against IL-1a in sera from patients with rheumatoid arthritis. J Immunol 1990; 145:2140-6.

5. Suzuki H, Ayabe T, Kamimura J, Kashiwagi H. Anti-IL-1a autoantibodies in patients with rheumatic diseases and in healthy subjects. Clin Exp Immunol 1991; 85:407-12.

6. Svenson M, Herbrink P. Measurement of cytokine antibodies. Test development. Biotherapy 1997;10:87-92.

7. Sedmak JJ, Grossberg SE. High levels of circulating neutralising antibody in normal animals to recombinant mouse interferon-b produced in yeast. J Interferon Res 1989; 9, Suppl. 1:S61-S5.

8. Hansen MB, Svenson M, Diamant M, Ross C, Bendtzen K. Interleukin-6 (IL-6) autoantibodies and blood IL-6 measurements. Blood 1995; 85:1145.

9. Svenson M, Hansen MB, Bendtzen K. Distribution and characterization of autoantibodies to interleukin 1a in normal human sera. Scand J Immunol 1990; 32:695-701.

10. Hansen MB, Svenson M, Diamant M, Bendtzen K. High-affinity IgG autoantibodies to IL-6 in sera of normal individuals are competitive inhibitors of IL-6 in vitro. Cytokine 1993; 5:72-80.

11. Ross C, Svenson M, Hansen MB, Vejlsgaard GL, Bendtzen K. High avidity IFN-neutralising antibodies in pharmaceutically prepared human IgG. J Clin Invest 1995; 95:1974-8.