Multiplexed serologic assay for nine anogenital human papillomavirus types

Abstract

A multiplexed human papillomavirus (HPV) immunoassay has been developed for the detection of human IgG antibodies to HPV type 6, 11, 16, 18, 31, 33, 45, 52, and 58 virus-like particle (VLP) types in serum following natural infection or immunization with VLP-based vaccines. The VLP antigens were covalently conjugated to carboxyl Luminex microspheres (MS) using a carbodiimide chemistry. Antibody (Ab) titers were determined in a direct binding format, in which an IgG1- to -4-specific, phycoerythrin (PE)-labeled monoclonal antibody (MAb) (HP6043) binds to human serum IgG antibodies. Pooled serum samples from rhesus macaques immunized with a 9-valent VLP-based vaccine served as the reference standard. The overall specificity of the assay was >99%, and the linearity (parallelism) of the assay was <7% per 10-fold dilution. Total assay precision was <19% across 3 different VLP-microsphere lots, 2 secondary antibody lots, and 2 different operators over a period of 3 weeks. Three different methods were used to evaluate serostatus cutoffs (SCO): (i) a clinical sensitivity/specificity analysis based on "likely negative" and "likely positive" samples from nonvaccinees, (ii) stringent upper tolerance limits on samples from "likely negatives," and (iii) stringent upper tolerance limits from the same "likely negative" sample set after VLP adsorption. Depending on the method to set the serostatus cutoff, the percentage of seropositive samples at the month 48 time point following vaccination with the HPV 6/11/16/18 quadrivalent vaccine ranged from 70% to 100%. This assay has proven useful for measuring the levels of serum antibody to the nine HPV VLPs following natural infection or administration of VLP-based vaccines.

FIG. 1.

Ruggedness of the HPV 9 IgG serology assay to VLP-microsphere lots, operators, and secondary detection antibody lots. A panel of 56 samples was tested over a period of 3 weeks by 2 analysts using 3 VLP-microsphere lots and 2 different mouse anti-human IgG lots (HP6043). The least-squares (LS) mean antibody (Ab) concentrations (mMU/ml) of the 3 conditions were analyzed for the ruggedness of the assay. Error bars represent the ±1.3-fold design goal. All conditions were within a ±1.3-fold difference, indicating that the assay should be acceptably rugged for use in clinical testing.

FIG. 2.

Precision of the HPV-9 IgG assay. Assay precision was determined by testing 56 samples over a 3-week period by two analysts using three VLP-microsphere lots and two HP6043 secondary antibody lots. The intra-assay precision was <10% relative standard deviation (RSD). The total assay precision for a mix of low, medium, and high HPV titer serum samples was determined to be <18% RSD for all nine HPV types across the dynamic range of the assay. Vertical dashed lines represent the limits of quantitation.

FIG. 3.

Analytical specificity and nonspecificity of the HPV 9 IgG assay. A panel of 11 samples with undetectable or low antibodies (samples 1 to 3), medium antibody levels (4 to 7), and high antibody levels (8 to 11) were tested in the HPV 9 IgG assay following mock adsorption with PBS, adsorption with 2.5 μg of a nonspecific, yeast-derived antigen, IsdB, adsorption with 2.5 μg of the homologous VLP, or adsorption with 22.5 μg of a cocktail of 9 VLPs. The dashed horizontal line in each figure represents the lower limit of quantitation. Results show that the assay is >99% specific for VLP-specific antibodies for samples with antibody titer values of >10 mMU/ml.

FIG. 4.

Antibody level and serostatus of control “likely negative” serum samples and month 48 serum samples from quadrivalent vaccinees. The control and VLP-adsorbed Ab titers are from 32 samples from individuals with 0 or 1 lifetime sex partner. The IgG and cLIA Ab titers are from month 48 samples from 96 females that received the quadrivalent vaccine. The samples were tested at a 1:50 dilution, and data are reported in mMU/ml. The three horizontal lines in the IgG column of data represent the serostatus cutoff values determined by the three different methods. Method 1, maximized number of negative and positive samples in the “likely negative” and “likely positive” groups, respectively; method 2, the 99/99 upper tolerance limit on the likely negative population; method 3, the 99/99 upper tolerance limit on the VLP-adsorbed likely negative samples. The horizontal line in the cLIA data column represents the historical cLIA serostatus cutoff values of 20, 16, 20, and 24 mMU/ml for types 6, 11, 16, and 18, respectively. The percentages represent the percentage of seropositive samples for each type using the different serostatus cutoffs.