Human papillomavirus multiplexed assay
AssigneeMerck & Co., Inc.
InventorsMark T. Esser, David W. Opalka, Victor Goetz, James E. Drummond, Michael W. Washabaugh
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Abstract
The present invention relates to an immunoassay for simultaneously measuring the presence of antibodies to a plurality of HPV types that utilizes particle-based flow cytometric analysis. The presence and/or titre of neutralizing antibodies in a test sample are determined in a competitive format, where known, type-specific, fluorescently labeled neutralizing monoclonal antibodies compete with antibodies within a test sample for binding to conformationally sensitive, neutralizing epitopes on specific HPV-VLPs. The invention also provides a microsphere complex comprising a microsphere coupled to an HPV VLP.
Claims14
A method for detecting antibodies to neutralizing epitopes on a plurality of human papillomavirus (HPV) types in a sample from a subject comprising:
- (a) providing a plurality of microsphere sets, wherein each of the plurality of microsphere sets comprises:
- a plurality of microsphere complexes, each complex comprising a microsphere coupled to a HPV virus-like particle (VLP) and a type-specific monoclonal antibody (mAb) that binds to the VLP;
- wherein the mAb is detectably tagged with a fluorescent reporter molecule;
- and wherein the HPV VLP is of a different HPV type relative to the HPV type of the VLP of a different microsphere set;
- and
- wherein microspheres of one microsphere set differ from microspheres of another microsphere set in a fluorescence emission property that distinguishes the microspheres of one set from those of another set;
- (b) contacting the plurality of microsphere sets with the sample so that antibodies within the sample can bind to the VLPs;
- and
- (c) for each microsphere set, determining either
- (i) the amount of detectably tagged monoclonal antibodies that bind to the VLP;
- or
- (ii) the amount of detectably tagged monoclonal antibodies that do not bind to the VLP;
- (d) comparing the amount obtained in (c) to a control, wherein a reduction in the amount of binding of the detectably tagged monoclonal antibodies to the HPV VLPs indicates that antibodies to HPV neutralizing epitopes are present in the sample of the subject which are specific to that HPV type.
A microsphere complex comprising a microsphere coupled to a human papillomavirus (HPV) virus-like particle (VLP).
A method for detecting antibodies to neutralizing epitopes on human papillomavirus (HPV) types -6, -11, -16 and -18 in a sample from a subject comprising:
- (a) providing a first, a second, a third, and a fourth microsphere set, wherein:
- (i) the first microsphere set comprises a plurality of micro sphere complexes, each complex comprising a microsphere coupled to an HPV6 virus-like particle (VLP) and a H6.B10.5 monoclonal antibody (mAb) that binds to the VLP;
- wherein the mAb is labeled with a fluorescent reporter molecule;
- (ii) the second microsphere set comprises a plurality of microsphere complexes, each complex comprising a microsphere coupled to an HPV11 VLP and a H11.B2 mAb that binds to the VLP;
- wherein the mAb is labeled with a fluorescent reporter molecule;
- (iii) the third microsphere set comprises a plurality of microsphere complexes, each complex comprising a microsphere coupled to an HPV16 VLP and a H16.V5 mAb that binds to the VLP;
- wherein the mAb is labeled with a fluorescent reporter molecule;
- and
- (iv) the fourth microsphere set comprises a plurality of microsphere complexes, each complex comprising a microsphere coupled to an HPV18 VLP and a H18.J4 mAb that binds to the VLP;
- wherein the mAb is labeled with a fluorescent reporter molecule;
- and, wherein the microspheres of each microsphere set are labeled with distinguishing proportions of two or more fluorescent dyes, allowing each microsphere set to be distinguished from a different set based on a unique spectral property;
- (b) contacting the four microsphere sets with the sample so that antibodies within the sample can bind to the VLPs;
- and
- (c) for each microsphere set, determining the amount of detectably tagged monoclonal antibodies that bind to the VLP;
- and
- (d) comparing the amount obtained in (c) to a control, wherein a reduction in the amount of binding of the monoclonal antibodies to the HPV VLPs indicates that antibodies to HPV neutralizing epitopes are present in the sample of the subject which are specific to HPV6, -11, -16, or 18.
Dependencies are inferred from the claim text.