Development and Performance Evaluation of a Rapid Microplate-Based Bovine Serum Albumin Protein Denaturation Assay for Anti-Inflammatory Screening

Abstract

The bovine serum albumin (BSA) protein denaturation assay is one of the most widely used in vitro methods for preliminary screening of anti-inflammatory activity because of its simplicity and low cost. However, the conventional tube-based assay requires relatively large reagent volumes, prolonged analytical time, and intensive manual handling, limiting its application for high-throughput screening. Therefore, the development of a rapid and reagent-saving analytical platform with comparable analytical performance is highly desirable. This study aimed to develop and evaluate a microplate-based BSA protein denaturation assay for anti-inflammatory screening. The conventional protocol was adapted to a 96-well microplate format by optimizing the experimental conditions. Diclofenac sodium was employed as the reference anti-inflammatory drug to compare the analytical performance of the developed method with the conventional assay. The applicability of the proposed method was further demonstrated using ethanolic extracts of Zingiber montanumand Etlingera pavieana. The optimized microplate assay exhibited comparable anti-inflammatory activity to the conventional assay with no statistically significant difference in IC₅₀ values (p > 0.05). In addition, the developed method reduced reagent consumption by approximately 15-fold and shortened the analytical time by approximately 2.8-fold. The assay was successfully applied to evaluate the anti-inflammatory activities of the selected medicinal plant extracts, demonstrating its applicability for routine laboratory analysis. The developed microplate-based BSA protein denaturation assay provides a rapid, reliable, and cost-effective alternative to the conventional tube-based method. The proposed protocol is suitable for routine and high-throughput preliminary screening of anti-inflammatory compounds and offers an efficient analytical platform for natural product research.