[Production and preliminary application of monoclonal antibodies against structural proteins of human parainfluenza virus type 3].

Abstract

To screen the optimal target for the antigen detection of human parainfluenza virus type 3 (HPIV-3), this study systematically compared the immunoreactivity and diagnostic potential of four major structural proteins [hemagglutinin-neuraminidase (HN), fusion protein (F), nucleoprotein (NP), and phosphoprotein (P)] of HPIV-3. On this basis, developing high-sensitivity, high-specificity antibodies is the premise for establishing the detection method. A baculovirus-insect cell system was used to express four major structural proteins (HN, F, NP, and P) of HPIV-3, and SDS-PAGE suggested that the protein purity reached 80%. Corresponding monoclonal antibodies (mAbs) were generated via the hybridoma technology and 133 clones of mAbs were obtained. The production and immunoreactivity of mAbs against NP were significantly better than those against HN, F, and P. The prepared mAbs were screened with natural antigens through methods such as orthogonal matrix, detection limit evaluation, and surface plasmon resonance (SPR, indicating antibody affinity) determination. Multiple high-quality clones of mAbs were screened out, including 18#, 121#, and 84#, among which 18# showed high affinity to the recombinant antigen, with the K_D value reaching 8.92×10^-11. Moreover, all the three antibody clones demonstrated excellent stability under various buffer systems, temperature conditions, and repeated freeze-thaw cycles. Immunofluorescence assays confirmed that the 84# antibody specifically recognized viral antigens within infected cells. On this basis, a double-antibody sandwich chemiluminescent immunoassay (CLIA) system was developed and evaluated for sensitivity and specificity via clinical samples. The results showed that among the screened antibody pairs, 18#+HRP-84# demonstrated good detection performance in clinical samples, with a positive detection rate of 20% and low cross-reactivity. The pair 121#+HRP-84# demonstrated strong specificity in clinical samples, with no cross-reactivity, though its positive detection rate was 18%. This study demonstrates that NP is the optimal target antigen for HPIV-3 detection. The developed NP-based double-antibody sandwich CLIA system shows high sensitivity, good stability, and low cross-reactivity, exhibiting promising potential for the development of diagnostic kits.