1. SUMMARY SPECIFICITY of EIA IMMUNOASSAY for COMPLEMENT FACTOR Bb TESTING Igor Pavlov, 1 Nikol deForest 2, Julio C. Delgado 1 1 ARUP Institute for Clinical and Experimental Pathology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 2 QUIDEL Corporations, Santa Clara, CA INTRODUCTION MATERIALS and METHODS RESULTS CONCLUSIONS REFERENCES 1. Pangburn, M. K. 1988. Alternative pathway of complement. Methods Enzymol 162:639-53. 2. Rother, K., Till, G.O., Hansch, G.M., eds. 1998. The Complement System, 2nd revised ed., Springler-Verlag, New York. 3. Thurman, J. M., and V. M. Holers. 2006. The central role of the alternative complement pathway in human disease. J Immunol 176:1305-10. 4. Kolb, W. P., P. R. Morrow, and J. D. Tamerius. 1989. Ba and Bb fragments of factor B activation: fragment production, biological activities, neoepitope expression and quantitation in clinical samples. Complement Inflamm 6:175-204. 5. Hourcade, D.E., L.M. Mitchell, and T.J. Oglesby. 1998. A Conserved Element in the Serine Protease Domain of Complement Factor B. JBC 273: 25996-6000 6. Kobayashi, E., E. Kitano, H. Kondo, and H. Kitamura. 1999. [Complement activation in citrate plasma--inhibitory effect of anticoagulants on serum complement activation]. Rinsho Byori 47:160-4. 7. Mollnes, T. E., P. Garred, and G. Bergseth. 1988. Effect of time, temperature and anticoagulants on in vitro complement activation: consequences for collection and preservation of samples to be examined for complement activation. Clin Exp Immunol 73:484-8. 8. Pfeifer, P. H., M. S. Kawahara, and T. E. Hugli. 1999. Possible mechanism for in vitro complement activation in blood and plasma samples: futhan/EDTA controls in vitro complement activation. Clin Chem 45:1190-9. During the alternative complement pathway activation, factor B is cleaved in two fragments, Ba and Bb. Concentration of those fragments is 2 logs lower than of factor B present in the blood, which makes fragment detection vulnerable because of potential crossreactivity. Immunoaffinity purified factor B demonstrated ~0.15% crossreactivity in Quidel Bb EIA assay. It was shown that Bb concentration in serum is higher than in plasma due to complement activation during clot formation. To investigate the effect of crossreactivity, we run 109 healthy donor plasmas as reference population for Bb assay, and 80 sera samples (representing complement activated state) with factor B immunodiffusion and Bb EIA assays. Our study demonstrates that despite the potential 0.15% crossreactivity between factor B and cleaved Bb molecule, measuring plasma concentrations of factor Bb alone (without factor B assessment) is adequate to evaluate the activation of the alternative complement pathway. Biological functions of the complement system are achieved through the chain of production of activation fragments [1, 2, 3]. During the alternative complement pathway activation, factor B bound to C3 is cleaved by factor D into two fragments, Bb (60 kDa) and Ba (33 kDa) [4, 5]. The Bb fragment bound to C3b initiates formation of the membrane attack complex. In the absence of Bb generation, no alternative pathway activation is possible. Thus, by quantifying the concentration of Bb, the extent of alternative pathway activation at the time of sample collection can be measured. Objective:To investigate the effect of potential crossreactivity of complement factor Bb EIA assay with endogenous factor B. 109 de-identified EDTA plasma samples from ARUP’s healthy donor collection and 80 serum samples were tested for factors B and Bb concentrations to estimate the effect of crossreactivity with endogenous factor B, and to investigate the distribution of factor Bb in normal population. Forty pairs of serum – EDTA plasma samples were run with the Bb Plus assay during matrix evaluation study. Detection of complement factor Bb was performed using the Quidel MicroVue Bb Plus EIA assay. Factor B concentration was measured by The Binding Site radial immunodiffusion assay. Purified factor B sample was developed at Quidel Corp. using immunoaffinity purification The results of B and Bb measurements in EDTA plasmas and sera in 40-paired samples demonstrated that factor Bb concentrations in sera were approximately 5 times higher than in plasma, whereas factor B levels in paired sera and plasma samples were similar. Based on these results, we conclude that the differences in Bb level between paired serum and plasma are not due to the differences in factor B concentrations between those two matrices. As it was shown in number of publications, high levels of serum Bb are most likely due to the activation of factor B cleavage during the clot formation. This finding let us use sera samples as representatives of alternative complement pathway activated state. EDTA plasma samples were used as reference population. Maximum crossreactivity with immunoaffinity purified factor B was measured to be ~0.15% in physiological range of factor B. Reference intervals calculated with and without accounting for the effect of 0.15% crossreactivity provided identical discrimination between normal samples and specimens representing alternative complement pathway activated state. Our study demonstrates that despite the potential cross-reactivity between factor B and cleaved Bb molecule, measuring plasma concentrations of factor Bb alone is adequate to evaluate the activation of the alternative complement pathway. Quidel MicroVue Bb Plus EIA Kit used with human EDTA plasma can be effectively implemented to assess alternative complement pathway activation at the time of sample collection. Factor B, µg/mLMeasured Bb concentrations, µg/mLCross-reactivity 2000.1160.06% 1000.0930.09% 500.0660.13% 250.0380.15% 12.50.0160.13% 6.250.0000.00% 3.1250.0000.00% 1.56250.0000.00% Table 1. Results of cross-reactivity of the Bb assay with purified factor B Figure 1. Complement factors Bb and B in serum / EDTA plasma paired samples Figure 2. Concentrations of factor Bb in sera and EDTA plasmas Legend for Figure 2: open circles: Bb concentration in plasma; open triangles: Bb concentration in serum; grayed circles and triangles represent adjusted Bb values in plasma and serum, respectively; dotted lines represent correspondent upper levels of the reference interval of Bb values in the normal population with 90% confidence limits.