Preliminary Study on Screening and Identification of Lewis a Antigen Mimic Epitope in Alpaca Phage Display Nanobody Library.
10.19746/j.cnki.issn.1009-2137.2022.03.035
- Author:
Xiao-Long ZHONG
1
;
Lu YANG
2
;
Jie ZHANG
1
;
Li-Ping SUN
2
;
Ming-Zi MA
2
;
Bin FAN
1
;
Wei SHANG
2
;
Yuan-Shuai HUANG
3
;
De-Qing WANG
4
Author Information
1. Department of Blood Transfusion, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China.
2. Department of Transfusion Medicine, The First Medical Center, Chinese PLA General Hospital, Beijing 100853, China.
3. Department of Blood Transfusion, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China,E-mail: hys@live.cn.
4. Department of Transfusion Medicine, The First Medical Center, Chinese PLA General Hospital, Beijing 100853, China,E-mail: deqingw@vip.sina.com.
- Publication Type:Journal Article
- Keywords:
alpaca phage display nanobody library;
le;
mimotope
- MeSH:
Animals;
Antibodies, Monoclonal;
Antineoplastic Agents, Immunological;
Bacteriophages;
Blood Group Antigens;
Camelids, New World;
Enzyme-Linked Immunosorbent Assay/methods*;
Epitopes;
Humans;
Lewis Blood Group Antigens;
Peptide Library
- From:
Journal of Experimental Hematology
2022;30(3):877-883
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To establish a new method for synthesizing Lewis blood group antigens, that is, the mimotopes of Lewis blood group antigens were screened by using an alpaca phage display nanobody library.
METHODS:We selected mimotopes of the Lewis a (lea) antigen by affinity panning of an alpaca phage display nanobody library using a monoclonal anti-lea antibody. Enzyme-linked immunosorbent assay (ELISA) was used to test the affinity of the positive clones for the monoclonal anti-lea antibody, and the high-affinity positive clones were selected for sequencing and synthesis. Finally, the sensitivity, specificity and reactivity of the synthesized lea mimotope in clinical samples were verified by ELISA.
RESULTS:A total of 96 phage clones were randomly selected, and 24 were positive. Fourteen positive clones with the highest affinity were selected for sequencing. The result showed that there were 5 different sequences, among which 3 sequences with the highest frequency, largest difference and highest affinity were selected for expression and synthesis. The sensitivity and specificity of lea mimic antigen by ELISA showed that, the minimum detection limit of gel microcolumn assay (GMA) and ELISA method were 25 times different, and the lea mimic antigen had no cross reacted with the other five unrelated monoclonal antibodies(P<0.001). Finally, 30 clinical plasma samples were analyzed. The mean absorbance of the 15 positive plasma samples was significantly higher than that of the 15 negative plasma samples (P=0.02). However, the positive signal values of the clinical samples were much lower than those of the monoclonal antibodies.
CONCLUSION:A new method of screening lea mimic antigen by using alpaca phage nanoantibody library has been established, which is expected to realize the screening of lea mimotopes, thus realizing the application of high-sensitivity detection methods such as ELISA and chemiluminescence in blood group antibody identification.
