PhD student Mangalore University Mangalore, Karnataka, India
Disclosure(s):
Ghodke Sandu: No financial relationships to disclose
Introduction/Rationale: C-reactive protein (CRP) is an acute-phase protein synthesized in the liver. IL-6 primarily regulates responses to inflammatory signals and serves as a marker and mediator of inflammation. Elevated CRP levels in the blood reflect acute or chronic inflammation, and recent research suggests that CRP is a potent cardiovascular marker for risk management. However, detection methods are not sensitive enough. Hence, a sensitive competitive enzyme-linked assay for CRP is needed.
Methods: Following the CDI activation method, the anti-CRP antibody was coupled to magnetizable cellulose particles to facilitate the separation. CRP was conjugated with HRP using the sodium periodate oxidation method. Assay parameters, including recovery, cross-reactivity, parallelism, precision, inter-assay, and intra-assay variation, were evaluated for analytical validation. Optimized assay protocol: addition of standard 50 µL to the well, 50 µL solid phase, and 25 µL HRP-CRP added to the wells and incubation for two hours at RT with Shaking. Then, 200 µL wash buffer was added and placed over the magnetic rack for separation for 10 minutes. Afterwards, 50 µL TMB was added to the wells and 0.2M sulphuric acid to stop the reaction and measured at 450 nm for absorption to plot the standard graph.
Results: The prepared standard ranged from 3.12 to 100 ng/mL, which covers both normal and sensitive CRP levels, with a sensitivity of 2 ng/mL. Assay incubation time is standardized for 2 hours at RT with continuous shaking. Asaay standards were calibrated with the WHO international standards. Measured intra-assay variation for the assay was 6% and inter-assay variation was below 4% which is found within the permissible limit. %recovery for assay ranged between 92-109% and other validation parameters were found within the permissible limit.
Conclusion: The optimized assay procedure is useful for CRP level detection in human serum. The formulated kit can be used for clinical sample analysis and CRP levels can be quantified
