Applications of Bivalent HPV Virus-like Particles in Neutralizing Antibody Detection and Yolk IgY Antibody Production

doi:10.19586/j.2095-2341.2025.0052

Current Biotechnology ›› 2025, Vol. 15 ›› Issue (5): 913-920.DOI: 10.19586/j.2095-2341.2025.0052

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Applications of Bivalent HPV Virus-like Particles in Neutralizing Antibody Detection and Yolk IgY Antibody Production

Gaijuan LIU¹(), Wei LIU², Tingbao YAN², Lijing ZHOU¹, Yaxin QIANG¹, Longlong XING¹, Xianjian CHEN², Zhenmin XIA¹()

^1.Hebei Jianhai Medical Laboratory Co. ，Ltd. ，Shijiazhuang 050000，China
^2.Hebei Jianhai Biochip Technology Co. ，Ltd. ，Shijiazhuang 050000，China

Received:2025-04-18 Accepted:2025-05-30 Online:2025-09-25 Published:2025-11-11
Contact: Zhenmin XIA

Abstract

Abstract:

Human papillomavirus （HPV） infection is associated with various diseases. This study aimed to prepare bivalent HPV pseudovirus， establish a neutralizing antibody detection system， and investigate the preparation method and neutralizing efficacy of yolk IgY antibodies. Eukaryotic expression plasmids of HPV16/18-L1/L2 were constructed by homologous recombination. Pseudovirus particles were prepared and observed under transmission electron microscopy. Yolk IgY antibodies were extracted， and a dual-color fluorescence detection system was established. The prepared HPV virus-like particles （VLPs） had a titer of 4.0×10? to 5.7×10? IU·mL^-1. Electron microscopy showed icosahedral symmetry with a diameter of 55 ± 5 nm， consistent with natural HPV. The neutralizing antibody detection system exhibited high specificity， with ID₅₀ values of 177 and 463 for HPV16 and HPV18 antibody standards， respectively， and no reaction was observed with negative serum. The dual-color and single-color fluorescence systems showed good agreement. After immunizing chickens with the pseudovirus， yolk IgY antibodies with approximately 90% purity were obtained. The neutralizing titers peaked at week 6 post-immunization， reaching 1∶5 424 for HPV16 and 1∶3 304 for HPV18. The successful preparation of HPV pseudovirus， establishment of the neutralizing antibody detection system， and production of yolk IgY antibodies provide important support for vaccine evaluation and development of novel antibody-based drugs.

Key words: bivalent HPV virus-like particles, neutralizing antibodies, yolk IgY antibodies, vaccine evaluation

摘要：

人乳头状瘤病毒（human papillomavirus, HPV）感染与多种疾病相关，制备了二价HPV假病毒并建立了中和抗体检测体系，并探究了卵黄IgY抗体的制备方法及中和效力。采用同源重组构建HPV16/18-L1/L2表达质粒，制备假病毒通过透射电镜观察形态；制备卵黄IgY抗体，建立双色荧光检测体系。结果发现，制备的HPV病毒样（virus-like particles，VLPs）颗粒滴度为4.0×10?~5.7×10? IU·mL^-1，电镜观察呈二十面体对称，直径55±5 nm，与天然HPV一致。中和抗体检测特异性好，HPV16和HPV18抗体标准品ID₅₀分别为177和463，阴性血清无反应。双色与单色荧光检测体系一致性好。假病毒免疫鸡群后，制备的卵黄IgY抗体纯度约90%，中和效价在免疫后第6周达峰值，分别为1∶5 424（HPV16）和1∶3 304（HPV18）。HPV假病毒和中和抗体检测体系的建立及卵黄IgY抗体的制备，为疫苗效果评价和新型抗体药物的开发提供了重要支撑。

关键词: 二价HPV病毒样颗粒, 中和抗体, 卵黄IgY抗体, 疫苗评价

CLC Number:

Q939.4

Gaijuan LIU, Wei LIU, Tingbao YAN, Lijing ZHOU, Yaxin QIANG, Longlong XING, Xianjian CHEN, Zhenmin XIA. Applications of Bivalent HPV Virus-like Particles in Neutralizing Antibody Detection and Yolk IgY Antibody Production[J]. Current Biotechnology, 2025, 15(5): 913-920.

刘改娟, 刘薇, 颜廷宝, 周立敬, 强亚欣, 邢龙龙, 陈鑫健, 夏振民. 二价HPV病毒样颗粒在血清中和抗体检测与卵黄IgY抗体制备中的应用[J]. 生物技术进展, 2025, 15(5): 913-920.

Figures/Tables 9

Fig. 1 Transfection efficiency of HPV16/HPV18 recombinant plasmids

Table 1 Titration results of different batches of pseudoviruses

假病毒型别	假病毒滴度（TCID₅₀/·mL^-1）			CV/%
假病毒型别	第1批	第2批	第3批	CV/%
HPV16	5.62×10⁷	4.33×10⁷	5.62×10⁷	11.74
HPV18	4.33×10⁷	4.64×10⁷	5.62×10⁷	11.33

Fig. 2 Transmission electron microscopy observation of HPV16 and HPV18 pseudovirus particles

Fig. 3 Fluorescence acquisition of HPV pseudovirus neutralization test at different dilution gradients

Fig. 4 Detection of mixed HPV16/18 neutralizing antibodies

Fig. 5 Bland-Altman analysis of the average values of single test and mixed test

Fig. 6 Purification and detection of yolk IgY

Fig. 7 Evaluation of neutralizing titer of egg yolk IgY antibodies

Table 2 Bland-Altman descriptive statistics

项	HPV16值	HPV18值
有效样本量	47	47
均值（第1种方法-单检）	2 046.766	2 046.766
均值（第2种方法-混合检）	2 031.787	2 021.149
均值（差值）	14.979	25.617
标准差（差值）	109.876	122.691
95% CI（差值均值）	-17.282~47.239	-10.406~61.641
95% CI（差值）	-200.374~230.331	-214.854~266.088
t值（H0：差值的平均值=0)	0.935	1.431
P值（H0：差值的平均值=0)	0.355	0.159

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Applications of Bivalent HPV Virus-like Particles in Neutralizing Antibody Detection and Yolk IgY Antibody Production

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