Abstract
Background:
Bovine subclinical mastitis is a significant cause of economic losses in dairy farms, primarily due to its asymptomatic nature and difficulty in early detection. The enzyme-linked immunosorbent assay (ELISA) is a widely used diagnostic tool in bioscience, facilitating antigen detection through antibody binding.

Aim:
The present study designed a gold nanoparticle (AuNPs)-based ELISA, to enhance the sensitivity of conventional ELISA by improving the binding efficiency of capture antibodies to purified antigens. This modified ELISA could enable more accurate detection of bovine subclinical mastitis.

Methods:
A total of 200 milk samples from apparently healthy cows were screened for subclinical mastitis using the California Mastitis Test (CMT). Positive samples were then subjected to bacteriological culture, biochemical testing, and PCR targeting the Nuc2 gene for confirmation of S. aureus. The purified fraction antigen of all 78 confirmed S. aureus isolates was extracted using sepharose 4B affinity column chromatography, described by SDS-PAGE, and assessed for its sensitivity in S. aureus mastitis diagnosis compared with crude antigen and purified fraction antigen-conjugated gold nanoparticles within an indirect enzyme-linked immunosorbent assay (ELISA). Immunoglobulins (IGS) from positive cow serum were extracted and purified from all confirmed S. aureus. Gold nanoparticle-based indirect ELISA was used on 400 samples (200 milk and 200 serum) from the same cows.

Results:
Using CMT, out of 200 examined milk samples from apparently normal cows, 65% (130/200) were sub-clinically infected. Out of these 130 positive milk samples, 60% (78/130) were confirmed to be infected with S. aureus. Purified fraction antigen-conjugated gold nanoparticles achieved the highest sensitivity to ELISA at 97%, 94%, and 92% immediately before storage, 6 months at -20 o C, and 1 year at -20 o C, respectively. Gold nanoparticle-based indirect ELISA detected specific IgG antibodies in 97% (76/78) sera and milk samples related to confirmed S. aureus isolates.

Conclusion:
The utilization of purified fraction antigen with gold nanoparticles enhances the sensitivity of ELISA, increasing it from 83.3% to 97% (p < .01; CI: 99%). The current study establishes a valuable way for S. aureus mastitis diagnosis within the use of purified fraction antigen-conjugated gold nanoparticles instead of the classical way to improve the sensitivity and the specificity of ELISA.

Key words: Indirect ELISA, Gold nanoparticle, Subclinical mastitis, S. aureus, Antigen purification