Qudsia Bibi

Pakistan

Development and Evaluation of an Indigenous ELISA Kit Prototype for the Detection of Human Antimitochondrial Antibodies

Qudsia Bibi1, Perveen Akhtar1, Muhammad Usman1, Waqar Khalid Saeed1*
1Biological and Health Sciences, Pak-Austria Fachhochschule: Institute of Applied Sciences and Technology (PAF-IAST), Haripur, Pakistan

Abstract

Background

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by antimitochondrial antibodies (AMAs) that target the bile duct epithelium. This study aimed to develop a cost-effective indigenous human AMA ELISA kit prototype and to assess the diagnostic value of the recombinant human Dihydrolipoyl transacetylase (DLAT) antigen for the diagnosis of PBC.

Methods

The Human DLAT was used as a coating antigen; other combinations of antigens, including mitochondria and submitochondrial particles, were also tested. Development and optimization were carried out using a pilot set of 21 samples. Seven confirmed positive samples (validated by immunofluorescence assay) and 14 samples from healthy volunteers were used. Serial dilutions of secondary antibody (ranging from 1:500 to 1:6000) were tested. The kit was optimized by adjusting incubation times, temperature, washing frequency, modifying the chemical composition, and adjusting the concentration of solutions at each step.

Results

A one-way ANOVA was performed to analyze the differences in mean optical densities among positive, negative, and blank groups, p < 0.0001, indicating a significant difference among the groups. For further confirmation of reproducibility and accuracy, intra- and inter-assay variability tests were conducted. Based on these results, expressed as the Coefficient of Variation (CV%), all CV% values were within the acceptable range. Similarly, a cutoff value of 0.8 was calculated using the receiver operating characteristic curve (ROC). Conclusions The indigenous human AMA ELISA kit prototype is a cost-effective and reliable option for diagnosing PBC. Further validation is required for a larger population to establish its clinical utility.