Simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types 6, 11, 16, and 18 by a multiplexed luminex assay

Abstract

Several different methods have been developed to quantitate neutralizing antibody responses to human papillomaviruses (HPVs), including in vivo neutralization assays, in vitro pseudoneutralization assays, competitive radioimmunoassays (cRIAs), and enzyme-linked immunosorbent assays. However, each of these techniques possesses one or more limitations that preclude testing large numbers of patient sera for use in natural history studies and large vaccine clinical trials. We describe here a new multiplexed assay, by using the Luminex Laboratory MultiAnalyte Profiling (LabMAP3) assay system, that can simultaneously quantitate neutralizing antibodies to human papillomavirus types 6, 11, 16, and 18 in 50 micro l of serum. The HPV-Luminex competitive immunoassay measures titers of polyclonal antibodies in serum capable of displacing phycoerythrin-labeled detection monoclonal antibodies binding to conformationally sensitive, neutralizing epitopes on the respective virus-like particles. This competitive Luminex immunoassay was found to be as sensitive, accurate, and precise as the currently used cRIAs. An effective HPV vaccine will most likely require several distinct genotypes to protect against multiple cancer causing papillomaviruses. The HPV-Luminex immunoassay should prove to be a useful tool in simultaneously quantitating antibody immune responses to multiple HPV genotypes for natural history infection studies and for monitoring the efficacy of prospective vaccines.

FIG. 1.

HPV-Luminex multiplex competitive immunoassay. The immunoassay quantitatively measures the ability of HPV type-specific antibodies in patient serum to prevent the binding of fluorescently labeled (with PE), HPV type-specific detection MAbs. The fluorescent signal from bound HPV-specific, detection MAbs is inversely proportional to the patient’s neutralizing antibody titer.

FIG. 2.

Stability of HPV-VLP-16 coupled to Luminex microspheres. The stability of HPV-6, -11, -16, and -18 VLPs coupled to Luminex microspheres was examined for a period of 6 months. VLP-microspheres were stored at 4°C in the dark in PBS, Column A buffer (COL.A), or histidine buffer (HIST) in the presence or absence of 1% BSA. MFI values for H16.V5-PE binding to VLP-16 (A) and %CV values for MFI values of 32 replicate samples (B) measured at 1-month intervals. Open symbols represent storage in the absence of 1% BSA, and closed symbols represent storage conditions in the presence of 1% BSA, respectively. Representative data for VLP-16 coupled to microsphere 38 is shown. The MFI for 32 replicate samples was averaged, and the %CV determined. The %CV was <10% for all four HPV-VLP types stored in buffers containing 1% BSA.

FIG. 3.

Simplex and multiplex standard curves for HPV-6, -11, -16, and -18. Representative standard curves for a 12-point dilution series of different African green monkey sera are presented for HPV-VLP-6 (A), HPV-VLP-11 (B), and HPV-VLP-18 (D) and of chimpanzee sera for HPV-VLP-16 (C). A simplex curve depicts a single standard serum run in the assay with only one VLP-microsphere type present, and multiplex curves depict a quadrivalent standard with all four VLP-microspheres present in a single well. Error bars represent the standard deviation of duplicate samples.

FIG. 4.

Effect of serum and different assay buffers on the HPV-Luminex assay. A standard curve serum from a VLP-11-immunized African green monkey was analyzed in various assay diluent buffers. Twofold serial dilutions of a stock standard into HPV-negative NHS was performed to create a 12-point standard curve. The standard serum was analyzed in PBS-1% BSA or HPV-negative NHS. Standard serum in an NHS diluent was analyzed in both a wash format and a no-wash format. Error bars represent the standard deviation of duplicate samples.

FIG. 5.

Calculated HPV type-specific, antibody titers. (A) MFI values for two representative HPV-negative, -low-positive, or -seropositive samples. (B) mMU/milliliter values for the same two representative samples shown in panel A. Error bars represent the standard deviation of duplicate samples. (C) Comparison of titers determined in the HPV-16 cRIA with HPV-16 titers from a multiplex Luminex competitive immunoassay. HPV type-specific antibody titers for 45 samples were determined by cRIA and Luminex immunoassays. Antibody titers were determined for 15 HPV-negative and 15 low-, 10 medium-, and 5 high-titer serum samples. The cRIA and Luminex assays were both performed in duplicate, and the average of the duplicate runs were compared for intra-assay precision and interassay accuracy. Average mMU/milliliter values for the HPV-16 cRIA and Luminex assays are shown. Titers were calculated from standard curves by using a four-parameter logistic curve fit.

FIG. 5.

Calculated HPV type-specific, antibody titers. (A) MFI values for two representative HPV-negative, -low-positive, or -seropositive samples. (B) mMU/milliliter values for the same two representative samples shown in panel A. Error bars represent the standard deviation of duplicate samples. (C) Comparison of titers determined in the HPV-16 cRIA with HPV-16 titers from a multiplex Luminex competitive immunoassay. HPV type-specific antibody titers for 45 samples were determined by cRIA and Luminex immunoassays. Antibody titers were determined for 15 HPV-negative and 15 low-, 10 medium-, and 5 high-titer serum samples. The cRIA and Luminex assays were both performed in duplicate, and the average of the duplicate runs were compared for intra-assay precision and interassay accuracy. Average mMU/milliliter values for the HPV-16 cRIA and Luminex assays are shown. Titers were calculated from standard curves by using a four-parameter logistic curve fit.