人副流感病毒3型结构蛋白单克隆抗体制备及初步应用

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

摘要:

为筛选人副流感病毒3型(human parainfluenza virus 3, HPIV-3)抗原检测的最优靶标，本研究系统比较了其4种主要结构蛋白[血凝素神经氨酸酶 (hemagglutinin-neuraminidase, HN)、融合蛋白(fusion protein, F)、核蛋白(nucleoprotein, NP)、磷蛋白(phosphoprotein, P)]作为诊断抗原的免疫反应性与诊断潜力。在此基础上，开发针对HPIV-3的高灵敏度、高特异性抗体是建立检测方法的前提。采用杆状病毒-昆虫细胞表达系统重组表达制备了HPIV-3的4种主要结构蛋白(HN、F、NP、P)，SDS-PAGE纯度达到80%。在此基础上采用杂交瘤技术制备相应的单克隆抗体。通过杂交瘤技术，共获得133株单克隆抗体。针对NP蛋白的单克隆抗体，无论在产生量还是对天然病毒抗原的反应性上，均显著优于针对HN、F、P蛋白的抗体。利用天然抗原通过正交矩阵、检测限评价、表面等离子体共振(surface plasmon resonance, SPR)抗体亲和力测定等方法筛选并评估所制备单克隆抗体性能，最终优选出18#、121#、84#等多株高质量单克隆抗体，其中18#抗体对重组抗原的亲和力解离平衡常数(dissociation constant, K_D)值达到8.92×10^-11，且3株抗体在不同缓冲体系、温度及反复冻融条件下均表现出良好的稳定性。免疫荧光实验验证84#抗体可特异性识别感染细胞内的病毒。以此为材料进一步建立双抗体夹心法HPIV-3抗原化学发光免疫分析(chemiluminescence immunoassay, CLIA)检测体系，并采用HPIV-3病毒稀释液和临床样本检验其灵敏度和特异性，结果显示，抗体配对组合18#+HRP-84#和121#+HRP-84#具有良好的检测效果，75例临床样本阳性检出率为20%，且具有较低的交叉反应；而121#+HRP-84#临床样本阳性检出率为18%，但特异性较强，未出现交叉反应。本研究证实，NP蛋白是HPIV-3抗原检测的显著优势靶标；基于NP蛋白开发的双抗体夹心CLIA检测体系具有较高灵敏度、良好的稳定性和较低的交叉反应，展现了良好的试剂盒开发潜力与应用前景。

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.