METHODS: This study examined the composition purity of PCAV through a decomplexation proteomic approach, applying size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tandem mass spectrometry liquid chromatography-tandem mass spectrometry (LC-MS/MS).
RESULTS: SDS-PAGE and SEC showed that the major protein in PCAV (constituting ∼80% of total proteins) is approximately 110 kDa, consistent with the F(ab')2 molecule. This protein is reducible into two subunits suggestive of the light and heavy chains of immunoglobulin G. LC-MS/MS further identified the proteins as equine immunoglobulins, representing the key therapeutic ingredient of this biologic product. However, protein impurities, including fibrinogens, alpha-2-macroglobulins, albumin, transferrin, fibronectin and plasminogen, were detected at ∼20% of the total antivenom proteins, unveiling a concern for hypersensitivity reactions.
CONCLUSIONS: Together, the findings show that PCAV contains a favorable content of F(ab')2 for neutralization, while the antibody purification process awaits improvement to minimize the presence of protein impurities.
Materials and Methods: The coagulometer for factor VIII assay is Sysmex CS-5100. All data were expressed as mean ± standard deviation (SD).
Results: A total of 135 cases were studied. Of these, 100 cases were of mild hemophilia A diagnosed by molecular genetics and, 15 cases were positive for LAC, which were confirmed by dilute Russell Viper venom test. Clot-based one-stage APTT assay showed 65% sensitivity and 80% specificity in diagnosing mild hemophilia A cases and out of 15 LAC cases, it showed false positivity in five cases. Chromogenic assay showed 85% sensitivity and 90% specificity in diagnosing mild hemophilia cases and was 100% specific in excluding LAC cases.
Conclusions: One-stage APTT assay is the most commonly used test for determining factor VIII levels but chromogenic assay are considered as the gold standard and recommended as the reference method by European Pharmacopoeia and ISTH subcommittee. Mild hemophilia A patients with missense mutations show discrepancy between the one-stage clot-based APTT assay and chromogenic assays for determination of factor VIII level and this can lead to misdiagnosis or misclassification of mild hemophilia A. Therefore, it is recommended that both the assays should be used in the evaluation of mild hemophilia cases.
METHODS: This study examined the immunological binding and neutralization capacity of PCAV against the two cobra venoms using WHO-recommended protocols.
RESULTS: In mice, both venoms were highly neurotoxic and lethal with a median lethal dose of 0.18 and 0.20 µg/g, respectively. PCAV exhibited strong and comparable immunoreactivity toward the venoms, indicating conserved venom antigenicity between the two allopatric species. In in vivo assay, PCAV was only moderately effective in neutralizing the toxicity of both venoms. Its potency was even lower against the hetero-specific N. samarensis venom by approximately two-fold compared with its potency against N. philippinensis venom.
CONCLUSION: The results indicated that PCAV could be used to treat N. samarensis envenomation but at a higher dose, which might increase the risk of hypersensitivity and worsen the shortage of antivenom supply in the field. Antivenom manufacturing should be improved by developing a low-dose, high-efficacy product against cobra envenomation.