Evaluation of the affinity measurement of anti-HIV-1 p17 monoclonal antibody by BIAcore.
The methods of measuring the affinity constants of anti-HIV-1 p17 monoclonal antibodies (MAbs) using the double antibody methods in the liquid phase and the biomolecular interaction analysis by BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden) were compared. MAbs, HyHIV1-6, recognizing residues 12-29 (P12-29) of p17 and the naive protein, p17, were used. The kinetic association constants (KAs) obtained using the double antibody method were 2.40 x 10(7) - 1.40 x 10(8)M(-1) for P12-29, and 4.80 x 106 - 1.80 x 10(7) M(-1) for p17. In the BIAcore system where P12-29 or p17 was used as immobilized antigens onto the sensorchip, the KAs were 1.57 x 10(9) - 4.81 x 10(9) M(-1) for P12-29, and 1.52 x 10(9) - 1.21 x 10(10) M(-1) for p17. On the other hand, when MAbs were immobilized onto the sensorchip and P12-29 or rp17 was used as analyte, the KAs for P12-29 and p17 were in the region 3 x10(8) - 3 x 10(9), 1 x 10(8) - 3 x 10(9) M(-1), respectively. These data show that the KAs were higher than those obtained using the double antibody method, however, no significant difference could be observed. Moreover, the KAs obtained for p17 using MAbs as ligand were similar for BIAcore and the double antibody method except for HyHIV2. Therefore, the BIAcore system can be used for the affinity measurement instead of the double antibody method.
Ota
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Department of Neurovirology, Research Institute for Microbial Diseases, Osaka University, Suita City, Japan.
Ueda
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D015683:Gene Products, gag / Q000276:immunology*; D015488:HIV Antigens / Q000276:immunology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D000911:Antibodies, Monoclonal; D015683:Gene Products, gag; D015488:HIV Antigens; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1