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[Study on CH50 levels in factor D-depleted serum]
Emi Uchibori1, Etsuko Kitano, Takao Tsuji
1Department of Clinical Laboratory Science, Osaka Prefecture College of Health Sciences, Habikino 583-8555.
Insights
Serum whole complement activity (CH50) assays may not be influenced by alternative pathway (AP) activation. This study demonstrates that CH50 levels remain unaffected even when AP activation is inhibited during the assay.
Area of Science:
- Immunology
- Biochemistry
Background:
- Serum whole complement activity (CH50) assays typically measure classical pathway (CP) function using sensitized sheep erythrocytes.
- Simultaneous activation of the alternative pathway (AP) during CH50 assays has been proposed, potentially confounding results.
Purpose of the Study:
- To investigate the influence of AP activation on CH50 levels during the assay.
- To determine if CH50 measurements are solely reflective of CP activity or if AP contributes.
Main Methods:
- Developed factor D-depleted serum using polystyrene beads with polyanion (PAMPS-beads).
- Assayed CH50 levels in normal human serum (NHS) before and after PAMPS-beads treatment.
- Utilized immunoblot analysis to confirm factor D depletion.
Main Results:
- PAMPS-beads treatment successfully depleted factor D, rendering the serum incapable of AP activation (ACH50 undetectable).
- CH50 levels remained unchanged in serum treated with PAMPS-beads, indicating no significant AP contribution.
- Re-addition of factor D restored ACH50 levels, validating the depletion method.
Conclusions:
- The CH50 assay is not significantly affected by alternative pathway activation.
- CH50 levels accurately reflect classical pathway activity, independent of simultaneous AP activation.
Abstract:
It is generally accepted that levels of serum whole complement activity (CH50) reflect the activities of complement (C) components of the classical C pathway (CP), since CH50 is assayed by use of sensitized sheep erythrocytes (EA). However, the alternative C pathway (AP) is considered to be also activated simultaneously in the process of activation of serum CP by EA. Thus, serum CH50 levels may possibly reflect not only CP but also AP activation in CH50 assay. We studied on the influence of AP activation during CH50 assay on CH50 levels, by comparison of CH50 levels in serum samples before and after treatment of factor D depletion. Polystyrene beads carrying polyanion, poly (2-acrylamide 2-methylpropane sulfonate) (PAMPS-beads), on the surface were prepared and used for preparation for factor D-depleted serum. After treatment of pooled normal human serum (NHS) with PAMPS-beads (2.5 mg/ml of serum), serum ACH50 level decreased to be undetectable, indicating that AP activation is prohibited in PAMPS-beads-treated serum. When isolated factor D was added to this PAMPS-beads-treated serum, ACH50 level recovered to that of before treatment. Immunoblot analysis revealed that factor D band observed in NHS disappeared completely after PAMPS-beads treatment. From these results, it is clear that factor-D deficient serum is prepared by PAMPS-beads treatment. Besides, since serum CH50 level was not decreased by PAMPS-beads treatment, it may be concluded that CH50 level is not affected by AP activation during CH50 assay.