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Anal Biochem. 2015 Oct 5. pii: S0003-2697(15)00462-5. doi: 10.1016/j.ab.2015.09.020. [Epub ahead of print]
Specific Determination of Influenza H7N2 Virus Based on Biotinylated Single-Domain Antibody from A Phage-Displayed Library.
Gong X1, Zhu M2, Li G2, Lu X3, Wan Y4.
Author information
Abstract
The unpredicted spread of avian influenza virus subtype H7N2 in the world is threatening animals and humans. Specific and effective diagnosis and supervision are required to control the influenza. However, the existing detecting methods are laborious, time consuming and required appropriate laboratory facilities. To tackle this problem, we isolated VHH antibodies against the H7N2 avian influenza virus (AIV) and performed an enzyme-linked immunosorbent assay (ELISA) to detect the H7N2 virus. To obtain VHH antibodies with high affinity and specificity, a camel was immunized. A VHH antibody library was constructed in a phage-display vector pMECS with diversity of 2.8?109. Based on phage-display technology and periplasmic extraction ELISA, H7N2 specific VHH antibodies were successfully isolated. According to pairing test, two VHH antibodies (Nb79 and Nb95) with good thermal stability and specificity can recognize different epitopes of H7N2 virus. The capture antibody (Nb79) was biotinylated in vivo and the detection antibody (Nb95) was coupled with horseradish peroxidase (HRP). Based on biotin-streptavidin interaction, a novel sandwich immune ELISA was performed to detect H7N2. The immunoassay exhibited a linear range from 5 to 100 ng/mL. From the above, the newly developed VHH antibody-based double sandwich-ELISA (DAS-ELISA) offers an attractive alternative to other diagnostic approaches for the specific detection of H7N2 virus.
Copyright ? 2015. Published by Elsevier Inc.
KEYWORDS:
Influenza H7N2 virus; Phage library; Sandwich ELISA; VHH antibody
PMID: 26450565 [PubMed - as supplied by publisher]
Specific Determination of Influenza H7N2 Virus Based on Biotinylated Single-Domain Antibody from A Phage-Displayed Library.
Gong X1, Zhu M2, Li G2, Lu X3, Wan Y4.
Author information
Abstract
The unpredicted spread of avian influenza virus subtype H7N2 in the world is threatening animals and humans. Specific and effective diagnosis and supervision are required to control the influenza. However, the existing detecting methods are laborious, time consuming and required appropriate laboratory facilities. To tackle this problem, we isolated VHH antibodies against the H7N2 avian influenza virus (AIV) and performed an enzyme-linked immunosorbent assay (ELISA) to detect the H7N2 virus. To obtain VHH antibodies with high affinity and specificity, a camel was immunized. A VHH antibody library was constructed in a phage-display vector pMECS with diversity of 2.8?109. Based on phage-display technology and periplasmic extraction ELISA, H7N2 specific VHH antibodies were successfully isolated. According to pairing test, two VHH antibodies (Nb79 and Nb95) with good thermal stability and specificity can recognize different epitopes of H7N2 virus. The capture antibody (Nb79) was biotinylated in vivo and the detection antibody (Nb95) was coupled with horseradish peroxidase (HRP). Based on biotin-streptavidin interaction, a novel sandwich immune ELISA was performed to detect H7N2. The immunoassay exhibited a linear range from 5 to 100 ng/mL. From the above, the newly developed VHH antibody-based double sandwich-ELISA (DAS-ELISA) offers an attractive alternative to other diagnostic approaches for the specific detection of H7N2 virus.
Copyright ? 2015. Published by Elsevier Inc.
KEYWORDS:
Influenza H7N2 virus; Phage library; Sandwich ELISA; VHH antibody
PMID: 26450565 [PubMed - as supplied by publisher]