Optimization of a Human Papillomavirus-Specific Enzyme-Linked Immunosorbent Assay

Karem, Kevin L.; Poon, Alysia C.; Bierl, Cynthia; Nisenbaum, Rosane; Unger, Elizabeth R.

doi:10.1128/cdli.9.3.577-582.2002

Cited by 18 publications

(19 citation statements)

References 15 publications

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“…While many studies have employed ELISA techniques for the detection of specific HPV antibodies in epidemiological studies, only a few have focused on assay validation and optimization (6,8,13). We expand on this topic by reporting the advantages of using a control serum pool to derive NAR values that control for the inherent variation in ELISA performance in a molecular epidemiological study in which serological testing was performed in many different runs over a period of several months.…”

Section: Discussionmentioning

confidence: 99%

“…Being mindful of the above-described issues and based on our previous experience (9) and the experiences of others (6,8,13), we set out to standardize the VLP ELISA protocol that we have used in our long-standing cohort study of HPV infection and cervical neoplasia. We report herein our use of normalized absorbance ratios (NARs) as a technical refinement to control for the sources of variation that can affect the validity of seroreactivity in a study whose laboratory measurements spanned a period of nearly 10 years.…”

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confidence: 99%

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Use of the Normalized Absorbance Ratio as an Internal Standardization Approach To Minimize Measurement Error in Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Papillomavirus Infection

et al. 2010

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The serological detection of antibodies against human papillomavirus (HPV) antigens is a useful tool to determine exposure to genital HPV infection and in predicting the risk of infection persistence and associated lesions. Enzyme-linked immunosorbent assays (ELISAs) are commonly used for seroepidemiological studies of HPV infection but are not standardized. Intra-and interassay performance variation is difficult to control, especially in cohort studies that require the testing of specimens over extended periods. We propose the use of normalized absorbance ratios (NARs) as a standardization procedure to control for such variations and minimize measurement error. We compared NAR and ELISA optical density (OD) values for the strength of the correlation between serological results for paired visits 4 months apart and HPV-16 DNA positivity in cervical specimens from a cohort investigation of 2,048 women tested with an ELISA using HPV-16 virus-like particles. NARs were calculated by dividing the mean blank-subtracted (net) ODs by the equivalent values of a control serum pool included in the same plate in triplicate, using different dilutions. Stronger correlations were observed with NAR values than with net ODs at every dilution, with an overall reduction in nonexplained regression variability of 39%. Using logistic regression, the ranges of odds ratios of HPV-16 DNA positivity contrasting upper and lower quintiles at different dilutions and their averages were 4.73 to 5.47 for NARs and 2.78 to 3.28 for net ODs, with corresponding significant improvements in seroreactivity-risk trends across quintiles when NARs were used. The NAR standardization is a simple procedure to reduce measurement error in seroepidemiological studies of HPV infection.Serological testing for circulating anti-human papillomavirus (HPV) antibodies has potential clinical utility in measuring cumulative exposure to HPV infection and as a marker of HPV-associated disease, particularly for the anogenital tract (10). Long-term persistent HPV infection tends to yield polyclonal immunoglobulin G (IgG) serological reactivity, which can be measured by using enzyme-linked immunosorbent assay (ELISA) techniques based on type-specific L1-only (7) or L1-plus-L2 virus-like particles (VLPs) as antigens (8, 11). Serological testing for HPV antibodies cannot replace direct HPV DNA measurements or cytological and histological examinations of the target tissue, but it can serve as an adjunctive tool, particularly in molecular epidemiology studies of the natural history of HPV infection and cervical precancerous lesions (3,16,17,19). Thus far, however, HPV serology has not been used in the clinical setting because of a variety of technical and biological limitations (8,10). Although the specificity of HPV VLP ELISA is reasonably adequate, at over 90%, its sensitivity is relatively low (50% to 60%) (14).Among the main technical issues that affect its clinical utility are the lack of cross-laboratory standardization and reproducibility. Aside from continued improveme...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Use of the Normalized Absorbance Ratio as an Internal Standardization Approach To Minimize Measurement Error in Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Papillomavirus Infection

et al. 2010

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“…Recently, we have shown that a prototype HPV 16 vaccine was 100% efficacious in preventing acquisition of HPV 16 infection and cervical disease among women who were HPV 16 naïve at baseline (19). These results have led to phase II and III studies of a quadrivalent vaccine to HPV 6,11,16,and 18. Early results from a randomized, placebo-controlled, phase II trial have shown this quadrivalent vaccine to be 90% efficacious against HPV 6-, 11-, 16-, and 18-related infection or disease (36).…”

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Optimization and Validation of a Multiplexed Luminex Assay To Quantify Antibodies to Neutralizing Epitopes on Human Papillomaviruses 6, 11, 16, and 18

Dias

Doren

Schlottmann

et al. 2005

Clin Vaccine Immunol

233

194

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Optimization increased both the analytical sensitivity and the clinical specificity of the assay to more effectively discriminate the low-titer antibody response of HPV-infected persons from noninfected individuals. The characteristics of the assay that were optimized included monoclonal antibody (MAb) specificity, scaling up the conjugation of virus-like particles (VLPs) to microspheres, VLP concentration, MAb concentration, sample matrix, sample dilution, incubation time, heat inactivation of sample sera, and detergent effects on assay buffer. The assay was automated by use of a TECAN Genesis Workstation, thus improving assay throughput, reproducibility, and operator safety. Following optimization, the assay was validated using several distinct serum panels from individuals determined to be at low and high risk for HPV infection. The validated assay was then used to determine the clinical serostatus cutoff. This high-throughput assay has proven useful for performing epidemiology studies and evaluating the efficacy of prophylactic HPV vaccines.Cervical cancer is the second most common cancer in women worldwide. Every year, 450,000 women are diagnosed with cervical cancer and 220,000 succumb to this disease (27). Current approaches to cervical cancer control involve lifelong screening using the Papanicolau (Pap) test (13). The goal of screening is to detect precancerous lesions so that they can be removed prior to the development of cancer. Despite widespread Pap testing, there were an estimated 10,520 new cases of cervical cancer and nearly 4,000 cervical cancer-related deaths in the United States in 2004 (1). The national health care burden of current screening systems combined with direct costs of treating precancerous and cancerous lesions is in excess of 3.5 billion U.S. dollars per annum (7).Infection with human papillomavirus (HPV) is the first and obligate step in the development of cervical cancer (3, 4). Infection of the cervical epithelium with HPV results in expression of the E6 and E7 proteins, which have been shown to be potent oncogenes. More than 35 different HPV types are capable of infecting the human genital tract (2, 4, 28). Of these, four types cause the majority of the HPV-related cervical pathology. HPV 16 and 18 together account for 74.6% of all cervical cancers (23), whereas HPV6 and -11 cause a significant fraction of precancerous lesions which rarely develop into cervical cancer but morphologically are indistinguishable from lesions from more dangerous HPV types (37). HVP 6 and 11 are responsible for approximately 90% of all genital wart cases (37).The HPV LI capsid protein, when expressed recombinantly, assembles into empty viral capsids or "virus like particles" (VLPs) (12,15,16,29). Several prophylactic vaccines based on HPV LI VLPs are currently in phases II and III clinical development (14,17,36). The VLPs in the vaccine present the immune system with the conformational, neutralizing epitopes found on the natural virus and prime the immune system to generate antibodies that neutra...

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“…Most potentially oncogenic, persistent, long-term HPV infections elicit an antibody response which can be detected by using virus-like particles (VLPs) (5,20). In fact, the anti-VLP antibody response is stable; is related to persistent infection, the viral load, and neoplastic lesion development; and is rarely found in patients with transient HPV infections (10,16,25,29).Seropositivity for VLPs seems to be a good indicator of the risk that a patient will have cervical cancer, despite the controversial results regarding its specificity; in fact, an optimized VLP-based enzyme-linked immunosorbent assay (ELISA) that has 93% sensitivity and 98.5% specificity for discriminating between positive and negative control sera has recently been reported (35). Some exposed linear epitopes of the L1 protein that interact with antibodies (21) have been identified by using peptides; i.e., the 473 GLAKPKFTLGKKATPTTS 491 peptide is specifically recognized by serum antibodies from 91% of HPV type 16 (HPV-16)-infected patients, 24% of children, and 66% of HPV-16-negative patients (8).…”

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Specificity of L1 Peptides versus Virus-Like Particles for Detection of Human Papillomavirus-Positive Cervical Lesions in Females Attending Engativa Hospital, Bogota, Colombia

et al. 2008

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A serological test for the detection of human papillomavirus (HPV) infection in females at risk of developing cervical cancer could be based on conserved L1 peptides with low levels of antigenicity specifically recognized by antibodies from patients with cervical lesions infected with high-risk HPV (HR-HPV) types. The aim was to assess the ability of L1 peptides 18283, 18294, and 18301 compared with the ability of virus-like particles (VLPs) to identify these infections in females. A total of 391 HPV-infected female volunteers were interviewed, and peripheral blood and cervical cells were obtained for detection of anti-HPV antibodies and HPV DNA; all of the patients had a Pap smear test; 287 patients were referred for colposcopy or biopsy, according to gynecological criteria. The level of agreement, as determined by the use of the Lin coefficient (rho value), showed that 75 to 83% of females with HR-HPV DNA-positive cervical lesions had antibodies that recognized VLPs and peptide 18283, 18294, or 18301, while 15 to 23% of the HPV DNA-negative females with a normal cytology had antibodies that recognized these three peptides and 45% had antibodies that recognized VLPs. The rate of agreement between peptides and VLPs for antibody detection was higher for patients with HPV DNA-positive cervical lesions. Peptides 18283, 18294, and 18301 showed similar sensitivities for the detection of HR-HPV DNA-positive cervical lesions and were more specific than VLPs. Peptide 18301 might be detecting protective antibodies in HPV DNA-negative females with atypical squamous cells of undetermined significance. These peptides could be useful for the design of a serology test for the detection of HR-HPV infection in females with cervical lesions and at risk of cervical cancer.More than 100 human papillomavirus (HPV) types infect human tissues; 40 of these types of viruses (named high-risk HPV [HR-HPV] types) are able to induce carcinomas such as cervical cancer (3, 23). About 70% of females become infected with HPV within the 2 years following the start of sexual activity (22,37), and 90% of these females have transient HPV infections (2, 13). Cervical lesions caused by persistent HPV infection are detected in about 5% of females; some of these can progress to cervical cancer. Seventy to 90% of HPV-infected females seroconvert 6 to 18 months after HPV DNA is detected, but this rarely occurs in females with transient HPV infection (1,4,25).There is a relationship between a high viral load and the progression of cervical lesions and cervical cancer (13, 27). The anti-HPV antibody response is correlated with the viral load (11, 36). Most potentially oncogenic, persistent, long-term HPV infections elicit an antibody response which can be detected by using virus-like particles (VLPs) (5,20). In fact, the anti-VLP antibody response is stable; is related to persistent infection, the viral load, and neoplastic lesion development; and is rarely found in patients with transient HPV infections (10,16,25,29).Seropositivity for VLPs seems to be a...

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Optimization of a Human Papillomavirus-Specific Enzyme-Linked Immunosorbent Assay

Cited by 18 publications

References 15 publications

Use of the Normalized Absorbance Ratio as an Internal Standardization Approach To Minimize Measurement Error in Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Papillomavirus Infection

Use of the Normalized Absorbance Ratio as an Internal Standardization Approach To Minimize Measurement Error in Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Papillomavirus Infection

Optimization and Validation of a Multiplexed Luminex Assay To Quantify Antibodies to Neutralizing Epitopes on Human Papillomaviruses 6, 11, 16, and 18

Specificity of L1 Peptides versus Virus-Like Particles for Detection of Human Papillomavirus-Positive Cervical Lesions in Females Attending Engativa Hospital, Bogota, Colombia

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