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Development and validation of a broadly cross-reactive polyclonal antibody as capture antibody for pan human β
Qiang Wu1, Zhan-Zhao Wang1, Zhen-Ming Wang1
1Clinical Laboratory, Weifang People's Hospital, Shandong Second Medical University, Weifang, 261000, Shandong, China.
Background:
Human β-coronaviruses are a group of viruses that infect humans and cause upper respiratory tract diseases. Antigen detection could be used as an auxiliary diagnostic method for viral infection. The study aimed to develop a broadly cross-reactive antibody capable of simultaneously recognizing the nucleocapsid (N) proteins of five human β-coronaviruses, for use as a capture antibody for viral antigen detection.
Methods:
The homologous sequence peptides of N proteins from five human β-coronaviruses were synthesized, coupled to carrier proteins, and used as immunogens to immunize New Zealand white rabbits. The antisera were purified using ammonium sulfate precipitation and antigen affinity chromatography. The capture performance of the NPA antibody for the N proteins of the five β-HCoVs was verified by sandwich ELISA and competitive ELISA. Throat swab specimens from SARS-CoV-2-infected patients were used to verify the capture performance of the NPA antibody against the N proteins in real-world clinical samples.
Results:
The synthetic peptides were >95% pure as determined by high-performance liquid chromatography and their molecular weights were confirmed by mass spectrometry to match the theoretical molecular weights. The antibody titer of rabbit antiserums was determined to be 1:8000 and the purity of purified polyclonal antibody (NPA) was higher than 90%. The NPA antibody was capable of capturing the N proteins of the five β-HCoVs. When the NPA antibody was tested for N proteins capture in SARS-CoV-2 patient specimens, six of eight nucleic acid-positive specimens with cycle threshold (Ct) values < 30 tested positive.
Conclusion:
The broadly cross-reactive antibody obtained in the study could be used as capture antibody in human β-coronavirus antigen detection and provides a solid technical foundation for other newly emerging β-coronavirus subspecies in the future.
