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Thesis-Neeti -final-09-11-2017 .pdf (6.02 MB)

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Anti-properdin Monoclonal Antibodies: Relevance in Detecting Functional Differences Between Properdin Polymers and in Inhibiting Properdin Function

Galwankar, Neeti
http://rave.ohiolink.edu/etdc/view?acc_num=mco150546773463968

Abstract Details

2017, Master of Science (MS), University of Toledo, Biomedical Sciences (Medical Microbiology and Immunology).
The alternative pathway (AP) of the complement system can be activated excessively in several inflammatory diseases, particularly when there is a defect in regulatory components of the complement system. For instance, defects of complement regulatory protein Factor H are associated with atypical hemolytic uremic syndrome (aHUS), whereas deficiency in glycosylphosphatidylinositol anchored proteins, including complement regulators decay accelerating factor (CD55) and CD59, leads to paroxysmal nocturnal hemoglobinuria (PNH), which causes severe prothrombotic pathologies. Understanding molecular mechanisms involved in complement activity are essential for developing new treatments. Properdin, the positive regulator of complement, is essential for complement amplification by stabilizing enzymatic convertases, and can also initiate complement activation. It exists as head-to-tail associations of dimers, trimers and tetramers (P2:P3:P4) in a 1:2:1 ratio in plasma. Properdin is produced mainly by different leukocytes in response to local stimuli and the ratio of properdin polymers in local proinflammatory microenvironments remains unknown. The convertase-stabilizing function of P4 is greater than P3 and P3 greater than P2. Non-physiological aggregates of properdin (Pn) accumulate in purified properdin preparations, due to prolonged storage and freeze/thaw cycles, and these Pn are artificially highly active (greater than P4) and should be removed before use in research studies. There is no commercially available assay to measure properdin function in biological samples and clinical and research laboratories have been limited to using cumbersome functional assays that require fresh red blood cells. Here we have developed and characterized monoclonal antibodies (MoAbs) against properdin and standardized a functional enzyme linked immunosorbent assay (ELISA) that allows to determine the function of properdin in vitro, in an ELISA format. The data indicate that the functional ELISA can detect differences in how quickly and effectively the different properdin forms (P2-P4 and Pn) activate the AP and can also detect properdin function in normal human serum. We have also developed a highly sensitive sandwich ELISA for measuring properdin concentration, using MoAbs pairs, with a lower properdin detection limit than commercially available kits (20 pg/ml). Finally, when tested in in vitro models of aHUS and PNH, which measure complement-mediated lysis due to defects in complement regulation, the inhibitory MoAbs had lower IC50 values than all other complement inhibitors tested, including Soliris (FDA approved for use to treat patients with these diseases). Further studies aimed at determining the therapeutic value of inhibiting properdin in human inflammatory diseases are warranted.
Viviana Ferreira (Advisor)
138 p.
Biomedical Research

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Citations

  • Galwankar, N. (2017). Anti-properdin Monoclonal Antibodies: Relevance in Detecting Functional Differences Between Properdin Polymers and in Inhibiting Properdin Function [Master's thesis, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=mco150546773463968

    APA Style (7th edition)

  • Galwankar, Neeti. Anti-properdin Monoclonal Antibodies: Relevance in Detecting Functional Differences Between Properdin Polymers and in Inhibiting Properdin Function. 2017. University of Toledo, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=mco150546773463968.

    MLA Style (8th edition)

  • Galwankar, Neeti. "Anti-properdin Monoclonal Antibodies: Relevance in Detecting Functional Differences Between Properdin Polymers and in Inhibiting Properdin Function." Master's thesis, University of Toledo, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=mco150546773463968

    Chicago Manual of Style (17th edition)

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This open access ETD is published by University of Toledo Health Science Campus and OhioLINK.