Monoclonal antibody recognizing human papillomavirus (HPV) L2 protein and method for measuring HPV-neutralizing antibody titer using the same
The present invention relates to the development of a monoclonal antibody which has binding activity to many high-risk types of HPV, etc. The present invention also provides a simple and high-throughput method for measuring cross-neutralizing antibody titers, which is used for assay of cross-neutralizing antibody against HPV in serum samples from subjects, etc. The method of the present invention for measuring cross-neutralizing antibody titers comprises the steps of: preparing a monoclonal antibody against a peptide having a specific amino acid sequence common to high-risk types of HPV; and assaying cross-neutralizing antibody using this monoclonal antibody.
1. A method for measuring the antibody titer of a cross-neutralizing antibody against human papillomavirus (HPV), which comprises the steps of:
(a) bringing a test sample into contact with an HPV antigen to establish binding of cross-neutralizing antibodies in the sample to the antigen;
(b) adding a monoclonal antibody comprising the amino acid sequences shown in SEQ ID NO: 11 (CDRH1), SEQ ID NO: 12 (CDRH2), SEQ ID NO: 13 (CDRH3), SEQ ID NO: 14 (CDRL1), SEQ ID NO: 15 (CDRL2) and SEQ ID NO: 16 (CDRL3) or a monoclonal antibody comprising the amino acid sequences shown in SEQ ID NO: 17 (CDRH1), SEQ ID NO: 18 (CDRH2), SEQ ID NO: 19 (CDRH3), SEQ ID NO: 20 (CDRL1), SEQ ID NO: 21 (CDRL2) and SEQ ID NO: 22 (CDRL3) to the reaction system in step (a);
(c) contacting the monoclonal antibody bound to the antigen with a labeled secondary antibody recognizing the monoclonal antibody to determine the amount of the monoclonal antibody bound to the antigen; and
(d) measuring signal intensity arising from the labeled secondary antibody bound to the monoclonal antibody in the presence and absence of the test sample, thereby determining the antibody titer of the cross-neutralizing antibody in the test sample.
2. A method for measuring the antibody titer of a cross-neutralizing antibody against human papillomavirus (HPV), which comprises the steps of:
(a) bringing a test sample into contact with an HPV antigen to establish binding of cross-neutralizing antibodies in the sample to the antigen;
(b) bringing residual epitopes, which remain unbound to the antibodies in step (a), into contact with a monoclonal antibody comprising the amino acid sequences shown in SEQ ID NO: 11 (CDRH1), SEQ ID NO: 12 (CDRH2), SEQ ID NO: 13 (CDRH3), SEQ ID NO: 14 (CDRL1), SEQ ID NO: 15 (CDRL2) and SEQ ID NO: 16 (CDRL3) or a monoclonal antibody comprising the amino acid sequences shown in SEQ ID NO: 17 (CDRH1), SEQ ID NO: 18 (CDRH2), SEQ ID NO: 19 (CDRH3), SEQ ID NO: 20 (CDRL1), SEQ ID NO: 21 (CDRL2) and SEQ ID NO: 22 (CDRL3);
(c) contacting the monoclonal antibody bound to the residual epitopes with a labeled secondary antibody recognizing the monoclonal antibody to determine the amount of the monoclonal antibody bound to the epitopes; and
(d) measuring signal intensity arising from the labeled secondary antibody bound to the monoclonal antibody in the presence and absence of the test sample, thereby determining the antibody titer of the cross-neutralizing antibody in the test sample.
3. A method for measuring the antibody titer of a cross-neutralizing antibody against human papillomavirus (HPV), which comprises the steps of:
(a) bringing a monoclonal antibody comprising the amino acid sequences shown in SEQ ID NO: 11 (CDRH1), SEQ ID NO: 12 (CDRH2), SEQ ID NO: 13 (CDRH3), SEQ ID NO: 14 (CDRL1), SEQ ID NO: 15 (CDRL2) and SEQ ID NO: 16 (CDRL3) or a monoclonal antibody comprising the amino acid sequences shown in SEQ ID NO: 17 (CDRH1), SEQ ID NO: 18 (CDRH2), SEQ ID NO: 19 (CDRH3), SEQ ID NO: 20 (CDRL1), SEQ ID NO: 21 (CDRL2) and SEQ ID NO: 22 (CDRL3) in admixture with a test sample into contact with an HPV antigen to form antibody/antigen conjugates;
(b) contacting the conjugates formed between the monoclonal antibody and the antigen in step (a) with a labeled secondary antibody recognizing the monoclonal antibody to determine the amount of the monoclonal antibody used to form antibody/antigen conjugates among those obtained in step (a); and
(c) measuring signal intensity arising from the labeled secondary antibody bound to the monoclonal antibody in the presence and absence of the test sample or before and after addition of the test sample, thereby determining the antibody titer of the cross-neutralizing antibody in the test sample.
4. The method according to claim 1 , wherein the HPV antigen is immobilized on a solid support.
5. A kit for determining the presence of cross-neutralizing antibody against HPV in a test sample, which comprises (a) an HPV antigen immobilized on a solid support, (b) a monoclonal antibody comprising the amino acid sequences shown in SEQ ID NO: 11 (CDRH1), SEQ ID NO: 12 (CDRH2), SEQ ID NO: 13 (CDRH3), SEQ ID NO: 14 (CDRL1), SEQ ID NO: 15 (CDRL2) and SEQ ID NO: 16 (CDRL3) or a monoclonal antibody comprising the amino acid sequences shown in SEQ ID NO: 17 (CDRH1), SEQ ID NO: 18 (CDRH2), SEQ ID NO: 19 (CDRH3), SEQ ID NO: 20 (CDRL1), SEQ ID NO: 21 (CDRL2) and SEQ ID NO: 22 (CDRL3), and (c) a labeled secondary antibody recognizing the monoclonal antibody.
6. The method according to claim 2 , wherein the HPV antigen is immobilized on a solid support.
7. The method according to claim 3 , wherein the HPV antigen is immobilized on a solid support.