基于生物膜干涉技术检测PDL1抗体与PDL1抗原的亲和力

doi:10.19586/j.2095-2341.2021.0003

生物技术进展 ›› 2021, Vol. 11 ›› Issue (6): 795-801.DOI: 10.19586/j.2095-2341.2021.0003

基于生物膜干涉技术检测PDL1抗体与PDL1抗原的亲和力

胡妍(), 陈玲()

福建医科大学免疫治疗研究院，福州 350122

收稿日期:2021-01-11 接受日期:2021-03-29 出版日期:2021-11-25 发布日期:2021-11-26
通讯作者: 陈玲
作者简介:胡妍 E-mail：huyan0027@163.com；
基金资助:
福建省教育厅中青年教师教育科研项目(JT180169);福建省科技厅项目-省自然科学基金青年项目(2019J05074)

Detection the Affinity Between PDL1 Antibodies and PDL1 Antigen Based on Biolayer Interferometry

Yan HU(), Ling CHEN()

Institute of Immune Therapy，Fujian Medical University，Fuzhou 350122，China

Received:2021-01-11 Accepted:2021-03-29 Online:2021-11-25 Published:2021-11-26
Contact: Ling CHEN

摘要/Abstract

摘要：

生物膜干涉（biolayer interferometry，BLI）技术可对抗体与抗原的相互作用进行亲和力、动力学的全面分析。在抗体克隆筛选、动力学常数测定中对链霉亲和素（streptavidin，SA）生物传感器的需求量较大，但目前鲜有关于SA传感器重复利用的报道。基于BLI技术、再生SA生物传感器建立一种使用再生后的传感器检测PDL1抗体与PDL1抗原亲和力的方法。通过将生物素化的PDL1抗原偶联至SA生物传感器上，再与单链抗体、双价单链抗体、完整抗体和双特异性抗体这4类PDL1抗体结合，计算抗原抗体的亲和力常数，利用甘氨酸（10 mmol·L^-1，pH 1.7）再生SA传感器，再次进行分子间相互作用力分析。结果显示，重复性相对标准偏差（relative standard deviation，RSD）均值为6.87%，批间重复性RSD为0.82%，稳定性RSD均值为6.13%，说明运用甘氨酸再生后的SA生物传感器测分子间的亲和力数据可靠、重现性好、稳定性高，再生后的传感器可继续用于本样品的实时、无标记的抗原抗体相互作用力分析。BLI技术可节省检测成本，为SA传感器的重复利用提供理论依据。

关键词: 生物膜干涉技术, PD?L1, 生物传感器, 亲和力常数, 再生

Abstract:

Biolayer interferometry （BLI） technology can conduct a comprehensive analysis of affinity and kinetics of the interaction between antibody and antigen. There is a large demand for streptavidin （SA） biosensors in antibody clone screening and determination of kinetic constants， but there are few reports on the reuse of SA sensors. Based on BLI technology and regenerative SA biosensor， a method for detecting the affinity of PDL1 antibody and PDL1 antigen using the regenerated sensor was established. By coupling the biotinylated PDL1 antigen to the SA biosensor， and then combining with the four types of PDL1 antibodies， single?chain antibody， bivalent single?chain antibody， intact antibody， and bispecific antibody， to calculate the affinity constant of the antigen?antibody， using glycine （10 mmol·L^-1， pH 1.7） regenerated the SA sensor， and analyzed the intermolecular interaction force again. The result showed that the mean repeatability relative standard deviation （RSD） was 6.87%， inter?batch repeatability RSD was 0.82%， and the mean stability RSD was 6.13%， the data showed that the SA biosensor regenerated by glycine had reliable data， good reproducibility and high stability， and reused SA sensor could still continue to be used for real?time， lable?free antigen?antibody interaction analysis of previous sample. BLI technology can greatly save the detection cost and provide the experimental basis for the repeated use of SA sensor.

Key words: bio?layer interference, PD?L1, biosensor, affinity constant, regeneration

中图分类号:

Q503

胡妍, 陈玲. 基于生物膜干涉技术检测PDL1抗体与PDL1抗原的亲和力[J]. 生物技术进展, 2021, 11(6): 795-801.

Yan HU, Ling CHEN. Detection the Affinity Between PDL1 Antibodies and PDL1 Antigen Based on Biolayer Interferometry[J]. Current Biotechnology, 2021, 11(6): 795-801.

图/表 7

参考文献 23

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程序	传感器结合的缓冲液	运行时间/s
平衡	PBST	60
上样	biotin‑PD‑L1	180
清洗	PBST	120
结合	Ab1~Ab7	300
解离	PBST	600

程序	传感器结合的缓冲液	运行时间/s
平衡	PBST	60
上样	biotin‑PD‑L1	180
清洗	PBST	120
结合	Ab1~Ab7	300
解离	PBST	600

样品	KD/（mol·L^-1）	K_on/（mol·L^-1·s^-1）	K_dis/（·s^-1）	R²
scFv	4.53×10^-9	9.68×10⁵	4.39×10^-3	0.944
diabody	4.34×10^-9	4.61×10⁵	2.00×10^-3	0.956
完整单抗	3.26×10^-10	1.24×10⁶	4.05×10^-4	0.986
BS4Ab	4.95×10^-10	8.21×10⁵	4.07×10^-4	0.995

样品	KD/（mol·L^-1）	K_on/（mol·L^-1·s^-1）	K_dis/（·s^-1）	R²
scFv	4.53×10^-9	9.68×10⁵	4.39×10^-3	0.944
diabody	4.34×10^-9	4.61×10⁵	2.00×10^-3	0.956
完整单抗	3.26×10^-10	1.24×10⁶	4.05×10^-4	0.986
BS4Ab	4.95×10^-10	8.21×10⁵	4.07×10^-4	0.995

初次使用的传感器	与ScFv KD/（mol·L^-1）	与Diabody KD/（mol·L^-1）	与完整单抗 KD/（mol·L^-1）	与Bs4Ab KD/（mol·L^-1）
Sensor‑ScFv传感器	3.62×10^-9	4.03×10^-9	2.65×10^-10	3.48×10^-10
Sensor‑diabody传感器	4.09×10^-9	3.36×10^-9	2.73×10^-10	3.52×10^-10
Sensor‑完整单抗传感器	6.71×10^-9	4.75×10^-9	4.42×10^-10	4.06×10^-10
Sensor‑Bs4Ab传感器	3.61×10^-9	2.20×10^-9	3.02×10^-10	3.78×10^-10
RSD	32.89%	30.17%	25.57%	7.28%

基于生物膜干涉技术检测PDL1抗体与PDL1抗原的亲和力

Detection the Affinity Between PDL1 Antibodies and PDL1 Antigen Based on Biolayer Interferometry

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图/表 7

参考文献 23

相关文章 15

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Metrics

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批次	KD /（mol·L^-1）	K_on /（mol·L^-1·s^-1）	K_dis /（·s^-1）	R²
DA200 #2G1	1.39×10^-10	2.45×10⁶	3.39×10^-4	0.9639
DA200 #2E4	2.66×10^-10	1.55×10⁶	4.11×10^-4	0.9736
DA200 #5G3	2.36×10^-10	1.75×10⁶	4.13×10^-4	0.9701
DA200 #5C6	3.91×10^-10	6.07×10⁶	2.38×10^-4	0.9832
DA200 #5F7	3.40×10^-10	1.26×10⁶	4.29×10^-4	0.9841
RSD	—	—	—	0.82%

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样品	1	2	3	4	5	6
A	B	L	B	Ab1	R	N
G	B	L	B	Ab7	R	N
H	B	L	B	B	R	N

样品	1	2	3	4	5	6
A	B	L	B	Ab1	R	N
G	B	L	B	Ab7	R	N
H	B	L	B	B	R	N