Development and Application of Broad-Spectrum Detection Monoclonal Antibodies Against HIV-1 P24 Antigen

Objective To enhance the accuracy and coverage of early HIV diagnosis, this study aimed to develop anti-p24 monoclonal antibodies with broad-spectrum recognition capability.Methods Mice were immunized subcutaneously with multi-subtype recombinant HIV-1 P24 antigens. Hybridoma technology was employed to screen and produce P24-specific mAbs. Cross-reactivity was evaluated using a double-antibody sandwich chemiluminescence immunoassay against recombinant P24 proteins and viral lysates from 10 common HIV-1 genotypes, including subtypes A, B, C, D, and CRF01_AE. The affinity of coated antibodies was measured by surface plasmon resonance (SPR). A saturation mutagenesis library targeting key antigen-binding pockets of the coated antibody was constructed. Phage display technology was used to screen the library and enrich high-affinity mutants.Results Three positive hybridoma clones stably secreting antibodies were obtained, all demonstrating strong binding activity against all 10 tested HIV-1 genotypes. The affinity-matured mutant M295# exhibited a dissociation constant (KD) of 1.16×10⁻¹¹ M for subtype B P24 antigen, representing an approximately 40-fold improvement compared to the parental antibody. The limit of detection (LOD) for the WHO international P24 standard antigen decreased from 0.76 IU/mL to 0.66 IU/mL. The detection rate across 10 HIV-1 genotypes reached 59.69%, outperforming commercial assays from Abbott (56.59%), Roche (40.31%), Snibe (13.95%), and Mindray (36.43%). Furthermore, the detection results in clinically suspected HIV-infected patients were fully consistent with nucleic acid test results.Conclusion A panel of monoclonal antibodies capable of efficiently recognizing P24 antigens across diverse HIV-1 subtypes was successfully developed. These antibodies provide superior core materials for constructing highly sensitive detection systems and lay a foundation for more reliable HIV diagnostic reagents.

【研究目的】为提升人类免疫缺陷病毒（HIV，Human Immunodeficiency Virus）早期诊断的准确性与覆盖范围，本研究旨在开发具备广谱识别能力的抗p24单克隆抗体。【研究方法】本研究采用多亚型重组HIV-1 P24抗原对小鼠进行皮下免疫；借助杂交瘤技术筛选并制备P24特异性单克隆抗体。采用双抗体夹心化学发光免疫分析法，针对10种常见HIV-1基因型（包括A、B、C、D亚型及CRF01_AE）的重组P24蛋白与病毒裂解液，评估抗体的交叉反应性。通过表面等离子体共振（SPR）测定包被抗体的亲和力。构建针对包被抗体关键抗原结合口袋的饱和诱变文库，并利用噬菌体展示技术筛选该文库，富集高亲和力突变体。【研究结果】本研究共获得3株稳定分泌抗体的阳性杂交瘤克隆，所有克隆对全部10种受试HIV-1基因型均表现出较强的结合活性。经亲和力成熟的突变体M295#对B亚型P24抗原的解离常数（KD）为1.16×10⁻¹¹ M，较亲本抗体提升约40倍。世界卫生组织（WHO）国际P24标准抗原的检测限（LOD）从0.76 IU/mL降至0.66 IU/mL。10种HIV-1基因型的总检出率达59.69%，优于Abbott（56.59%）、Roche（40.31%）、Snibe（13.95%）及Mindray（36.43%）的商用检测试剂盒。此外，临床疑似HIV感染者的检测结果与核酸检测结果完全一致。【研究结论】本研究成功开发出一组可高效识别不同HIV-1亚型P24抗原的单克隆抗体。此类抗体可作为构建高灵敏度检测体系的优质核心原料，为研发更可靠的HIV诊断试剂奠定基础。