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An immunoassay for the assessment of total alternative pathway activity
Abstracts / Molecular Immunology 46 (2009) 2818–2871
A negative correlation was found between MM2A and attack number recorded in the year of blood sampling (Spearman’s r = −0.2212, p = 0.0241). Spontaneous activation of MBL-MASP2 is not substantial in HAE patients, because MM2A is not decreased. The consumption of C4 results from the activation of C1, primarily. Importantly, the capability of the AA and AB genotypes of MBL-MASP2 is identical for cleaving C4. doi:10.1016/j.molimm.2009.05.264 OP79 Mannose-binding lectin complement pathway plays a key role in complement activation by Paracoccidioides brasiliensis夽 Renan G. Toledo a,∗ , Wilmar D. da Silva a , Vera L.G. Calich b , Thereza L. Kipnis a a
Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, RJ, Brazil b Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, SP, Brazil Paracoccidioides brasiliensis (Pb) accounts for paracoccidioidomycosis. We have previously reported that Pb activates complement through the alternative pathway (AP) and becomes opsonized. We, herein, demonstrate that dispersed Pb18 (high virulence), when incubated with Normal human serum (NHS) induces consumption of hemolytic complement and, when immobilized, promotes binding of C4b, C3b and C5b-C9. Consumption of components C4, C3 and mannose-binding lectin (MBL) was also verified in MBL-sufficient, but not in MBL-deficient, serum pretreated with dispersed Pb265 (low virulence) or Pb18 (high virulence). When residual complement components were quantified, by deposition on immune-complexes or mannan, consumption of C4, C3 and mannose-binding lectin (MBL) were observed in MBL-sufficient, but not in MBL-deficient, serum pretreated with dispersed Pb265 (low virulence) or Pb18 (high virulence). Higher complement component consumption was observed in serum pretreated with Pb265, as compared with Pb18. The higher complement-activating property exhibited by low virulent strains, was further investigated by comparing the complement activating properties of Pb265 and Pb18(94), two low virulence strains, with the high virulence strain, Pb18. After treating samples of MBL-sufficient serum with various amounts of dispersed fungus, residual C4, the key component of the classical pathway (CP) and lectin pathway (LP) was evaluated by C4c deposition on coated human IgG. Higher C4 consumption was observed in serum samples pretreated with low virulence Pb strains. In addition, data demonstrate that, although attenuated, Pb18 acquires a substantial ability to activate complement, as also evaluated by C4c deposition on coated human IgG. Pb18’s reactivated counter-part simultaneously reacquires virulence, while loosing its complement activating property. Results suggest that non-virulent Pb strains interact more efficiently with MBL, activating MASPs and organizing C4bC2b complexes, with consequent cleavage of C3 and release of C3a and C3b. 夽 “In
Memoriam” to Thereza L. Kipnis*, a dedicated Immunology Professor who developed active research programs that engaged students. doi:10.1016/j.molimm.2009.05.265
2847
OP80 An immunoassay for the assessment of total alternative pathway activity N. de Forest ∗ , J. DeTorres, D. Baker, C. Duncan, N. Nasser Specialty Products Group (SPG), Quidel Corporation, San Diego, CA, United States The binding of C3 or C3b to activating substances triggers the alternative complement pathway. This activation results in a cascade of enzymatic and non-enzymatic reactions, culminating in the formation of terminal complement complexes (TCC). The level of TCC that can be generated in a serum is a quantitative expression of the serum’s total alternative pathway activity. The traditional method for measuring the alternative complement pathway activity in serum is the AH50 hemolytic assay, which uses rabbit erythrocytes (Er) as the activator of the alternative complement pathway and various dilutions of the test serum to determine the amount required to give 50% lysis. The traditional, hemolytic AH50 test is an indirect measure of TCC, since the TCC themselves are directly responsible for the hemolysis being measured. The AH50 Eq EIA provides a direct measure of the alternative complement activity in serum by quantifying the amount of TCC generated. The specimen is activated with the activator and then the amount of TCC generated is measured. The AH50 Eq EIA uses a monoclonal antibody specific to a unique neoantigen on C9 to capture the TCC analyte. The assay has a lower limit of quantitation of 3.0 AH50 U Eq/ml, a lower limit of quantitation of 5.03 AH50 U Eq/ml and an upper limit of quantitation of 306 AH50 U Eq/ml. Within-run and between-run precision was determined to have a coefficient of variation (%CV) is 5.2% and 11.7%, respectively. The correlation with the traditional hemolytic assay has been shown to be >97%. The AH50 Eq EIA is an improvement on the traditional hemolytic assay. It eliminates the need to prepare standardized red blood cells and the shelf life and inter-lot performance are limited and unpredictable in the traditional method. The results generated by the AH50 Eq EIA are sensitive, standardized and quantitative. doi:10.1016/j.molimm.2009.05.266 OP81 Cleavage of kininogen and subsequent bradykinin release by MASP-1 József Dobó a,∗ , Balázs Major a , Katalin A. Kékesi b , István Szabó a , Gábor Juhász c , Péter Závodszky a , Péter Gál a a
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina út 29, H-1113, Budapest, Hungary b Department of Physiology and Neurobiology, Eötvös Loránd University, Pázmány Péter sétány 1C, H-1117, Budapest, Hungary c Laboratory of Proteomics, Institute of Biology, Eötvös Loránd University, Pázmány Péter sétány 1C, H-1117, Budapest, Hungary Mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1) is an abundant component of the lectin pathway of complement. The related enzyme, MASP-2 is capable of activating the complement cascade alone. Though the concentration of MASP-1 far exceeds that of MASP-2 only a supporting role of MASP-1 has been identified regarding lectin pathway activation. Several noncomplement substrates, like fibrinogen and factor XIII, have also been reported. We have investigated the action of activated MASP-1 on plasma proteins using a recombinant truncated form (rMASP-1) encompassing the last three domains (CCP1–CCP2–SP), and human
A negative correlation was found between MM2A and attack number recorded in the year of blood sampling (Spearman’s r = −0.2212, p = 0.0241). Spontaneous activation of MBL-MASP2 is not substantial in HAE patients, because MM2A is not decreased. The consumption of C4 results from the activation of C1, primarily. Importantly, the capability of the AA and AB genotypes of MBL-MASP2 is identical for cleaving C4. doi:10.1016/j.molimm.2009.05.264 OP79 Mannose-binding lectin complement pathway plays a key role in complement activation by Paracoccidioides brasiliensis夽 Renan G. Toledo a,∗ , Wilmar D. da Silva a , Vera L.G. Calich b , Thereza L. Kipnis a a
Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, RJ, Brazil b Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, SP, Brazil Paracoccidioides brasiliensis (Pb) accounts for paracoccidioidomycosis. We have previously reported that Pb activates complement through the alternative pathway (AP) and becomes opsonized. We, herein, demonstrate that dispersed Pb18 (high virulence), when incubated with Normal human serum (NHS) induces consumption of hemolytic complement and, when immobilized, promotes binding of C4b, C3b and C5b-C9. Consumption of components C4, C3 and mannose-binding lectin (MBL) was also verified in MBL-sufficient, but not in MBL-deficient, serum pretreated with dispersed Pb265 (low virulence) or Pb18 (high virulence). When residual complement components were quantified, by deposition on immune-complexes or mannan, consumption of C4, C3 and mannose-binding lectin (MBL) were observed in MBL-sufficient, but not in MBL-deficient, serum pretreated with dispersed Pb265 (low virulence) or Pb18 (high virulence). Higher complement component consumption was observed in serum pretreated with Pb265, as compared with Pb18. The higher complement-activating property exhibited by low virulent strains, was further investigated by comparing the complement activating properties of Pb265 and Pb18(94), two low virulence strains, with the high virulence strain, Pb18. After treating samples of MBL-sufficient serum with various amounts of dispersed fungus, residual C4, the key component of the classical pathway (CP) and lectin pathway (LP) was evaluated by C4c deposition on coated human IgG. Higher C4 consumption was observed in serum samples pretreated with low virulence Pb strains. In addition, data demonstrate that, although attenuated, Pb18 acquires a substantial ability to activate complement, as also evaluated by C4c deposition on coated human IgG. Pb18’s reactivated counter-part simultaneously reacquires virulence, while loosing its complement activating property. Results suggest that non-virulent Pb strains interact more efficiently with MBL, activating MASPs and organizing C4bC2b complexes, with consequent cleavage of C3 and release of C3a and C3b. 夽 “In
Memoriam” to Thereza L. Kipnis*, a dedicated Immunology Professor who developed active research programs that engaged students. doi:10.1016/j.molimm.2009.05.265
2847
OP80 An immunoassay for the assessment of total alternative pathway activity N. de Forest ∗ , J. DeTorres, D. Baker, C. Duncan, N. Nasser Specialty Products Group (SPG), Quidel Corporation, San Diego, CA, United States The binding of C3 or C3b to activating substances triggers the alternative complement pathway. This activation results in a cascade of enzymatic and non-enzymatic reactions, culminating in the formation of terminal complement complexes (TCC). The level of TCC that can be generated in a serum is a quantitative expression of the serum’s total alternative pathway activity. The traditional method for measuring the alternative complement pathway activity in serum is the AH50 hemolytic assay, which uses rabbit erythrocytes (Er) as the activator of the alternative complement pathway and various dilutions of the test serum to determine the amount required to give 50% lysis. The traditional, hemolytic AH50 test is an indirect measure of TCC, since the TCC themselves are directly responsible for the hemolysis being measured. The AH50 Eq EIA provides a direct measure of the alternative complement activity in serum by quantifying the amount of TCC generated. The specimen is activated with the activator and then the amount of TCC generated is measured. The AH50 Eq EIA uses a monoclonal antibody specific to a unique neoantigen on C9 to capture the TCC analyte. The assay has a lower limit of quantitation of 3.0 AH50 U Eq/ml, a lower limit of quantitation of 5.03 AH50 U Eq/ml and an upper limit of quantitation of 306 AH50 U Eq/ml. Within-run and between-run precision was determined to have a coefficient of variation (%CV) is 5.2% and 11.7%, respectively. The correlation with the traditional hemolytic assay has been shown to be >97%. The AH50 Eq EIA is an improvement on the traditional hemolytic assay. It eliminates the need to prepare standardized red blood cells and the shelf life and inter-lot performance are limited and unpredictable in the traditional method. The results generated by the AH50 Eq EIA are sensitive, standardized and quantitative. doi:10.1016/j.molimm.2009.05.266 OP81 Cleavage of kininogen and subsequent bradykinin release by MASP-1 József Dobó a,∗ , Balázs Major a , Katalin A. Kékesi b , István Szabó a , Gábor Juhász c , Péter Závodszky a , Péter Gál a a
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina út 29, H-1113, Budapest, Hungary b Department of Physiology and Neurobiology, Eötvös Loránd University, Pázmány Péter sétány 1C, H-1117, Budapest, Hungary c Laboratory of Proteomics, Institute of Biology, Eötvös Loránd University, Pázmány Péter sétány 1C, H-1117, Budapest, Hungary Mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1) is an abundant component of the lectin pathway of complement. The related enzyme, MASP-2 is capable of activating the complement cascade alone. Though the concentration of MASP-1 far exceeds that of MASP-2 only a supporting role of MASP-1 has been identified regarding lectin pathway activation. Several noncomplement substrates, like fibrinogen and factor XIII, have also been reported. We have investigated the action of activated MASP-1 on plasma proteins using a recombinant truncated form (rMASP-1) encompassing the last three domains (CCP1–CCP2–SP), and human