Anti-protective antigen IgG enzyme-linked immunosorbent assay for diagnosis of cutaneous anthrax in India

Anthrax caused by Bacillus anthracis is a public health problem in several developing countries whose main source of income is farming. Anthrax is a disease of herbivorous animals, and humans can be infected by handling infected animals or contaminated animal products. Specific diagnostic tests are unavailable in India for the detection and confirmation of cutaneous anthrax in humans. Here, we describe the development of an enzyme-linked immunosorbent assay (ELISA) for detection of serum antibodies against Bacillus anthracis protective antigen in the Indian population. A total of 405 serum samples collected from different groups were tested by the developed ELISA. The assay provided a specificity of 99.41% (95% confidence interval [CI], 97.89 to 99.93) and a sensitivity of 100% (CI, 94.4 to 100) using a cutoff value of 0.29 ELISA unit (EU). The positive predictive value (PPV) and negative predictive value (NPV) of the assay were 97% and 100%, respectively. The efficiency and J index for the reliability of the assay were 99.5% and 0.994, respectively. The assay can be a very useful tool for surveillance as well as for diagnosis of cutaneous anthrax cases in India.     

Development of antibodies to protective antigen and lethal factor components of anthrax toxin in humans and guinea pigs and their relevance to protective immunity.

A competitive inhibition enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies in serum to the protective antigen (PA) and lethal factor (LF) components of anthrax toxin. Current human vaccination schedules with an acellular vaccine induce predictable and lasting antibody titers to PA and, when present in the vaccine, to LF. Live spore vaccine administered to guinea pigs in a single dose conferred significantly better protection than the human vaccines (P less than 0.001), although they elicited significantly lower (P less than 0.0005) anti-PA and anti-LF titers at time of challenge with virulent Bacillus anthracis. Substantial anti-PA and anti-LF titers may not, therefore, indicate solid protective immunity against anthrax infection. The ELISA system was also shown to be capable of detecting anti-PA and anti-LF antibodies in the sera of individuals with histories of clinical anthrax. The advantage of ELISA over the Ouchterlony gel diffusion test and indirect microhemagglutination assay are demonstrated. There was a highly significant degree of correlation between ELISA and the indirect microhemagglutination assay (P less than 0.0005); but ELISA was markedly superior in terms of reproducibility, reliability, specificity, and simplicity in performance and stability of the bound antigen.     

Detection of hepatitis C virus antibodies in oral fluid specimens for prevalence studies

Within the framework of hepatitis C virus (HCV) prevalence monitoring, we evaluated oral fluid (OF), which is richer in IgG than whole saliva, as a possible alternative to serum for the detection of HCV antibodies. Paired OF and serum samples were collected from 90 individuals, including 45 HCV-positives and 45 HCV-negatives. The detection of HCV antibodies in both serum and OF was performed using the Ortho HCV 3.0 SAVe enzyme-linked immunosorbent assay (ELISA) (Ortho-Clinical Diagnostics, Inc., Raritan, NJ), but a modified, more sensitive protocol was used to process OF. The sensitivity and specificity of this assay were 86.67% (95% confidence interval (CI): 72.51–94.46%) and 100% (95% CI: 90.20–99.80%) in OF and 100% in serum. The correlation obtained between both types of clinical specimens was excellent (k: 0.87, 95% CI: 0.66–1.07). However, the negative predictive value (NPV) of the assay in OF decreased with the prevalence of HCV infection in the population studied. Our results suggest that the modified Ortho HCV 3.0 SAVe ELISA is suitable for the detection of HCV antibodies in OF for epidemiological studies. Using this assay, we observed an unadjusted anti-HCV prevalence of 78.6% among a population of intravenous drug users; when adjusted to account for assay sensitivity, this prevalence may be closer to 90%.     

Validation of a modified commercial enzyme-linked immunoassay for detection of human immunodeficiency virus type 1 immunoglobulin G antibodies in saliva

This study was performed to evaluate the performance of a saliva collection device (OmniSal) and an enzyme-linked immunoassay (EIA) designed for use on serum samples (Detect HIV1/2) to detect human immunodeficiency virus type 1 (HIV-1) antibodies in the saliva of high-risk women in Mombasa, Kenya. The results of the saliva assay were compared to a "gold standard" of a double-EIA testing algorithm performed on serum. Individuals were considered HIV-1 seropositive if their serum tested positive for antibodies to HIV-1 by two different EIAs. The commercial serum-based EIA was modified to test the saliva samples by altering the dilution and lowering the cutoff point of the assay. Using the saliva sample, the EIA correctly identified 102 of the 103 seropositive individuals, yielding a sensitivity of 99% (95% confidence interval [CI], 94 to 100%), and 96 of the 96 seronegative individuals, yielding a specificity of 100% (95% CI, 95 to 100%). In this high-risk population, the positive predictive value of the assay was 100% and the negative predictive value was 99%. We conclude that HIV-1 antibody testing of saliva samples collected with this device and tested by this EIA is of sufficient sensitivity and specificity to make this protocol useful in epidemiological studies.     

Multisite Clinical Evaluation of a Rapid Test for Entamoeba histolytica in Stool

Rapid point-of-care detection of enteric protozoa in diarrheal stool is desirable in clinical and research settings to efficiently determine the etiology of diarrhea. We analyzed the ability of the third-generation E. histolytica Quik Chek assay developed by Techlab to detect amebic antigens in fecal samples collected from independent study populations in South Africa and Bangladesh. We compared the performance of this recently released rapid test to that of the commercially available ProSpecT Entamoeba histolytica microplate assay from Remel and the E. histolytica II enzyme-linked immunosorbent assay (ELISA) from Techlab, using real-time and nested-PCR for Entamoeba species to resolve any discrepant results. After discrepant resolution, The E. histolytica Quik Chek assay exhibited sensitivity and specificity compared to the E. histolytica II ELISA of 98.0% (95% confidence interval [CI], 92.9% to 99.8%) and 100% (95% CI, 99.0% to 100%), respectively. Compared to the ProSpecT microplate assay, the E. histolytica Quik Chek (Quik Chek) assay exhibited 97.0% sensitivity (95% CI, 91.5% to 99.4%) and 100% specificity (95% CI, 99.0% to 100%). Our results indicate that the Quik Chek is a robust assay for the specific detection of E. histolytica trophozoites in unfixed frozen clinical stool samples.     

Sensitivity of an anti-HCV core peptide ELISA.

A newly developed antibody assay based on a synthetic peptide of 15 amino acids derived from the core region of the hepatitis C virus (HCV) genome was evaluated in serum and plasma panels of (A) 225 haemophiliacs and (B) 44 patients with chronic non-A, non-B (NANB) hepatitis, and in (C) sequential serum samples of 9 patients with transfusion transmitted HCV infection. The new anti-core peptide ELISA was compared with the anti-C100 ELISA. For confirmation of HCV infection, samples were tested in a 4-antigen recombinant immunoblot assay (4-RIBA) and samples of panels B and C were also assayed in cDNA-polymerase chain reaction (PCR). In two panels with a high prevalence of HCV infection (88.4 and 70.5% in haemophilia and NANB hepatitis patients, respectively), the sensitivity of the anti-core peptide ELISA did not differ significantly from the sensitivity of the anti-C100 ELISA. The sensitivity of the new assay as compared with the anti-C100 assay was found to be 0.84 [95% confidence interval (CI): 0.78-0.89] versus 0.92 (95% CI: 0.87-0.95) in haemophilia patients and 0.71 (95% CI: 0.52-0.86) versus 0.84 (95% CI: 0.66-0.95) in NANB hepatitis patients. In sequential serum samples of patients with transfusion-transmitted HCV infection antibodies to the core peptide (in 6/9 patients) appeared later than antibodies to C100 (in 7/9 patients): 168 (range: 70-322) and 143 (range: 59-365) days after transfusion, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of HIV antibody and antigen/antibody combination ELISAs for use in an alternative confirmatory HIV testing strategy in Dar es Salaam, Tanzania

The aim of this study was to evaluate the performance of two antibody enzyme-linked immunosorbent assays (ELISAs) [Vironostika Uni-Form II plus O and Enzygnost anti-HIV-1/2 Plus], and two antigen/antibody combination ELISAs [Murex and Vironostika HIV Uni-Form II] for use in an alternative confirmatory HIV diagnostic testing strategy in Dar es Salaam, Tanzania. Altogether, 1380 serum samples were included. All ELISA reactive samples were tested using the Inno-Lia antibody assay and discrepant samples were tested on the Innotest p24 antigen assay. Three hundred and one (21.8%) samples were confirmed HIV-1 antibody positive by Inno-Lia including 27/508 (5.3%) from blood donors, 65/511 (12.7%) from pregnant women and 209/361 (57.9%) from hospital patients. The sensitivity at initial testing was 100% (95% CI; 98.8-100%) for all assays except Vironostika Uni-Form II plus O (99.7%; 95% CI; 98.2-99.9%) which showed one false negative sample at initial testing but 100% sensitivity after repeat testing. The final specificity at repeat testing was 100% (95% CI; 99.7-100%) for Enzygnost anti-HIV-1/2 Plus, 99.4% (95% CI; 98.8-99.8%) for each of the antigen/antibody combination ELISAs and 97.9% (95% CI; 96.8-98.6%) for Vironostika plus O ELISA. An alternative confirmatory HIV testing strategy based on initial testing on any of the two antigen/antibody assays followed by testing of reactive samples on the Enzygnost anti-HIV-1/2 Plus assay gave 100% specificity (95% CI; 99.7-100%).     

Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n = 36) were analyzed. Both of these parameters were found to have coefficients of variation of <3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n = 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n = 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n = 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies against B. burgdorferi.     

Antibodies to small ubiquitin-like modifier activating enzyme are associated with a diagnosis of dermatomyositis: results from an unselected cohort

The aim of this study was to examine the frequency and significance of antibodies targeting the small ubiquitin-like modifier 1 activating enzyme (SAE) in patients under serologic evaluation for idiopathic inflammatory myopathies. Patient sera (n?=?17) recognizing bands at approximately 40 (SAE1) and 90 (SAE2) kDa were identified in 6445 consecutive samples for myositis autoantibody evaluation by immunoprecipitation (IP) of S³⁵-labeled K562 cell lysate. All 17 positive samples, 176 disease, and 67 healthy controls were evaluated for SAE1 antibodies using a line immunoblot assay (LIA). Clinical data of SAE antibody-positive patients were obtained by retrospective chart review. Positivity with both methods was associated with a diagnosis dermatomyositis with characteristic skin manifestations of varying severity and muscle involvement. Majority of the patients were female (73.7%), mean age of 55.0 (range 12.0–82.0) years at the time of testing. Using the IP as reference, the SAE1 LIA had a sensitivity of 100% (95% CI 82.4–100%), specificity of 99.6% (95% CI 97.7–100%), positive predictive value of 95.0% (95% CI 75.1–99.9%), and negative predictive value of 100% (95% CI 98.5–100%). This study confirms the association of SAE antibodies in patients with dermatomyositis. A combination of IP and the LIA specific for SAE1 may be useful in antibody detection.     

Comparison of chemiluminescent immunoassay and ELISA for measles IgG and IgM

In the context of measles elimination, the identification of recent infections is important for clinical laboratories. Serological diagnosis is achieved by detecting specific IgG and IgM. Recently an automated chemiluminescent immunoassay (CLIA) (Liaison, DiaSorin, Italy) has been used to quantify the measles antibody. The aim of this study was to compare this assay with Enzygnost ELISA (Siemens, Germany), with final classification of discrepancies by indirect immunofluorescence (Euroimmun, Germany). For measles IgM, 204 sera were analyzed: 50 IgM‐positive, 104 IgM‐negative/IgG‐positive, and 50 from other viral infections (B19V, rubella, mumps, CMV, and EBV). For the measles IgG assay, 162 samples were tested: 106 were positive and 56 were negative. For measles IgM, the sensitivity and specificity of CLIA against ELISA were 94% (95% CI: 83.2–98.6) and 100% (95% CI: 97.1–100), respectively; the corrected figures after the final classification of discrepancies were 100% (95% CI: 91.0–100) and 99.4% (95% CI: 96.1–100), respectively. In relation to IgG, the sensitivity and specificity of CLIA against ELISA were, respectively, 97.2% (95% CI: 91.7–99.4) and 92.9% (95% CI: 82.5–97.7), and 95.5% (95% CI: 89.5–98.3) and 100% (95% CI: 91.8–100) after the final classification. CLIA showed excellent sensitivity and specificity in detecting measles IgG and IgM antibodies, eliminating the need to aliquot specimens before carrying out the assay.     

The Aptima HPV Assay Fulfills the Cross-Sectional Clinical and Reproducibility Criteria of International Guidelines for Human Papillomavirus Test Requirements for Cervical Screening

The Aptima HPV assay (Hologic Gen-Probe, San Diego, CA) is an FDA-approved assay for detecting human papillomavirus (HPV) E6/E7 mRNA from 14 high-risk HPV types. This study evaluated the clinical performance of the Aptima HPV assay for cervical intraepithelial neoplasia of grade 2 or worse (CIN2+), relative to the high-risk HPV GP5+/GP6+ PCR, in a cross-sectional clinical equivalence analysis using the noninferiority score test with cervical samples from population-based screening, i.e., 69 cervical scraping samples from women with CIN2+ and 843 from women without evidence of CIN2+. In addition, intralaboratory reproducibility over time and interlaboratory agreement of the Aptima HPV assay results were assessed with another set of 548 cervical samples. The Aptima HPV assay showed a clinical sensitivity for CIN2+ of 94.2% (95% confidence interval [CI], 85.5 to 97.8%) and a clinical specificity for CIN2+ of 94.5% (95% CI, 92.8 to 95.9%); by comparison, these figures were 97.1% (95% CI, 89.1 to 99.3%) (67/69 samples) and 93.6% (95% CI, 91.7 to 95.0%) (785/839 samples), respectively, for GP5+/GP6+ PCR. The clinical sensitivity and specificity of the Aptima HPV assay were noninferior to those of GP5+/GP6+ PCR (P = 0.039 and 0.00016, respectively). In addition, high reproducibility of the Aptima HPV assay, as reflected by the intralaboratory reproducibility over time of 96.0% (95% CI, 94.4 to 97.3%) (526/548 samples; kappa = 0.89) and interlaboratory agreement of 96.7% (95% CI, 95.4 to 98.1%) (531/548 samples; kappa = 0.91), was found. Altogether, these data show that the Aptima HPV assay meets the cross-sectional clinical and reproducibility criteria of the international guidelines for HPV test requirements for cervical screening. Longitudinal data are needed to ensure that the long-term negative predictive value of this mRNA assay is similar to those of validated HPV DNA tests.     

Comparison of Enzyme-Linked Immunosorbent Assay, Western Blotting, Microagglutination, Indirect Immunofluorescence Assay, and Flow Cytometry for Serological Diagnosis of Tularemia

The serodiagnostic efficiencies of five different approaches to detecting antibodies (immunoglobulins G, A, and M) developed in clinically proven infections with Francisella tularensis have been assessed. Fifty serum samples from patients suffering from tularemia during an outbreak in Sweden were compared with samples from 50 healthy blood donors (controls) by using an enzyme-linked immunosorbent assay (ELISA), microagglutination (MA), Western blotting (WB), an indirect immunofluorescence assay (IIFA), and flow cytometry (FC). ELISA, WB, and FC were based on the use of preparations of lipopolysaccharides (LPS) of the live vaccine strain of Francisella tularensis subsp. holarctica (ATCC 29684) as a capture antigen. Whole methanol-fixed bacteria were used for IIFA and MA. Optimized protocols yielded a diagnostic sensitivity and specificity of 100% for WB, MA, and FC, 98% for ELISA, and 93% for IIFA. A total of 6,632 serum samples from individuals between the ages of 18 and 79 years, representatively recruited from all regions of Germany, were screened to estimate and confirm the positive predictive value (PVpos) of the ELISA. Serum samples from 15 (0.226%) individuals tested positive for F. tularensis-specific antibodies by ELISA and confirmatory WB. The resulting prevalence-dependent PVpos of 10.2% and specificity of 98.1% were consistent with our findings for tularemia patients and controls. We conclude that the combined usage of a screening ELISA and a confirmatory WB based on LPS as a common antigen, as well as the MA, is a suitable serodiagnostic tool, while the quality of the IIFA is hampered by subjective variations of the results. FC is a promising new approach that might be improved further in terms of multiplex analyses or high-throughput applications.     

Co-infection with hepatitis C virus and human immunodeficiency virus among patients with sexually transmitted diseases in Pondicherry, South India

Hepatitis C virus (HCV), which is usually transmitted by blood and blood products is emerging as an important agent in the list of sexually transmitted diseases (STDs). The present study was undertaken to document the burden of HCV infection in individuals with STDs in this tertiary care hospital in South India. One hundred serum samples collected from individuals with STDs were tested for antibodies to HCV by a third generation ELISA. All the samples were also screened for HIV infection. Six out of 100 individuals were found to possess antibodies against HCV (95% confidence interval [CI=1.3-10.7%). Fourteen out of 100 samples were positive for HIV (95% CI=7-20.9%). The seroprevalence of HCV in HIV positive individuals was 21.4% (3/14) whereas the corresponding figure for HIV negative individuals was only 3.5% (3/86). The difference was found to be statistically significant (p<0.01).     

Evaluation of a commercial rubella IgM assay for use on oral fluid samples for diagnosis and surveillance of congenital rubella syndrome and postnatal rubella.

BACKGROUND: Clinical diagnosis (surveillance) of rubella is unreliable and laboratory confirmation is essential. Detection of virus specific IgM in serum is the most commonly used method. However, the use of serum necessitates the drawing of blood, either through venipuncture or finger/heel prick, which can be difficult in young babies. Oral fluid samples have proved useful as an alternative, less invasive sample for virus specific IgM detection however until recently no commercial rubella IgM tests were available, restricting the usefulness of this approach. OBJECTIVES: To evaluate the performance of the Microimmune Rubella IgM capture EIA using oral fluid samples from outbreaks as well as in cases of suspected congenital rubella syndrome (CRS). STUDY DESIGN: Paired serum and oral fluids were collected from cases during a rubella outbreak in three provinces in Turkey. Matched serum and oral fluid samples were collected from children with suspected CRS in an active surveillance programme at the Aravind Eye Hospital in South India. Serum samples were collected as part of the measles surveillance programme in Ethiopia. RESULTS: On serum samples the sensitivity and specificity of the Microimmune Rubella IgM capture EIA compared to Behring Enzygnost rubella IgM test was 96.9% (62/64; 95% CI 94.2-100%) and 100% (53/53; 95% CI 93.2-100%). On oral fluids compared to matched Behring results on serum the sensitivity was 95.5% (42/44; 95% CI 84.5-99.4%). The sensitivity and specificity of Microimmune Rubella IgM capture EIA on oral fluids from suspected CRS cases compared to serum results using Behring Enzygnost IgM assay was 100% (95% CI 84.5-100%) and 100% (95% CI 95.8-100.0%) respectively. CONCLUSION: Microimmune Rubella IgM capture EIA has adequate performance for diagnosis and surveillance of rubella in outbreak using either serum or oral fluid specimens.     

Validation of a Modified Commercial Enzyme-Linked Immunoassay for Detection of Human Immunodeficiency Virus Type 1 Immunoglobulin G Antibodies in Saliva

This study was performed to evaluate the performance of a saliva collection device (OmniSal) and an enzyme-linked immunoassay (EIA) designed for use on serum samples (Detect HIV1/2) to detect human immunodeficiency virus type 1 (HIV-1) antibodies in the saliva of high-risk women in Mombasa, Kenya. The results of the saliva assay were compared to a “gold standard” of a double-EIA testing algorithm performed on serum. Individuals were considered HIV-1 seropositive if their serum tested positive for antibodies to HIV-1 by two different EIAs. The commercial serum-based EIA was modified to test the saliva samples by altering the dilution and lowering the cutoff point of the assay. Using the saliva sample, the EIA correctly identified 102 of the 103 seropositive individuals, yielding a sensitivity of 99% (95% confidence interval [CI], 94 to 100%), and 96 of the 96 seronegative individuals, yielding a specificity of 100% (95% CI, 95 to 100%). In this high-risk population, the positive predictive value of the assay was 100% and the negative predictive value was 99%. We conclude that HIV-1 antibody testing of saliva samples collected with this device and tested by this EIA is of sufficient sensitivity and specificity to make this protocol useful in epidemiological studies.     

Study of Abbott Toxo IMx system for detection of immunoglobulin G and immunoglobulin M toxoplasma antibodies: value of confirmatory testing for diagnosis of acute toxoplasmosis.

We compared the Abbott Toxo immunoglobulin G (IgG) and IgM IMx assays with the Sabin-Feldman dye test and an IgM enzyme-linked immunosorbent assay (ELISA) in 398 serum samples previously tested in our laboratory (retrospective group) and 1,000 consecutive serum samples, tested as they were received in our laboratory in 1995 (prospective group). In the retrospective group, the IgG IMx had a sensitivity of 100%, specificity of 99.0%, positive predictive value of 99.0%, negative predictive value of 100%, and overall agreement of 99.5%. The percentages for the IgM IMx were 97.8, 99.0, 98.9, 98, and 98.4%, respectively. In the prospective group, the IgG IMx had a sensitivity of 97.8%, specificity of 98.7%, positive predictive value of 97.8%, negative predictive value of 98.7%, and overall agreement of 98.4%. The percentages for the IgM IMx were 88.3, 95.9, 74.7, 98.3, and 95%. A serological profile (IgA and IgE antibodies and the differential agglutination [AC/HS] test) was performed in an attempt to resolve discrepancies. Of 21 serum samples that were positive in the IgM IMx and negative in the IgM ELISA, 19 had serological profiles which were most compatible with an infection acquired in the distant past. Of 8 serum samples which were positive in the IgM ELISA and negative in the IgM IMx, 5 had serological profiles which were most compatible with an infection acquired in the distant past. Of the 55 serum samples that were positive in both IgM tests, 21 were from patients who had serological profiles which were most compatible with an infection acquired in the distal past. In conclusion, our data highlight the importance of confirmatory testing for the diagnosis of recently acquired infection with Toxoplasma gondii. When compared with the dye test and IgM ELISA, the Toxo IgG and IgM IMx assays, respectively, revealed high overall agreement in the retrospective and prospective study.     

Rapid point-of-care test to detect broad ranges of protective antigen-specific immunoglobulin G concentrations in recipients of the U.S.-licensed anthrax vaccine

Currently, there is no routine monitoring of an immune response to the anthrax vaccine. Simple on-site tests are needed to evaluate the antibody response of anthrax-vaccinated individuals in the Armed Forces and others at high risk. Using a prototype lateral flow assay (LFA) (R. E. Biagini, D. L. Sammons, J. P. Smith, B. A. MacKenzie, C. A. F. Striley, J. E. Snawder, S. A. Robertson, and C. P. Quinn, Clin. Vaccine Immunol. 13:541-546, 2006), we investigated the agreement between a validated anthrax protective antigen (PA) immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and the LFA for 335 unvaccinated and vaccinated subjects. We also investigated the performance of the LFA under the following conditions: thermal shock (i.e., thermal cycling between temperature extremes), high temperature/high relative humidity, high temperature/low relative humidity, and low temperature/low relative humidity. With the anti-PA ELISA used as a standard, the LFA was shown to be optimally diagnostic at 11 microg/ml anti-PA-specific IgG. At this concentration, the LFA specificity and sensitivity were 98% (95% confidence interval [CI], 97% to 100%) and 92% (CI, 88% to 97%), respectively. Receiver operating characteristic curve analysis yielded an area under the curve value of 0.988 (CI, 0.976 to 1.00), suggesting that the LFA is an extremely accurate diagnostic test. For < or = 4 or > or = 50 microg/ml PA-specific IgG, the LFA results for each environmental condition were identical to those obtained in the laboratory. These data indicate that this rapid point-of-care test would be a feasible tool in monitoring the serological antibody responses of individuals that have been vaccinated against anthrax.     

Comparison of a Multiplexed Fluorescent Covalent Microsphere Immunoassay and an Enzyme-Linked Immunosorbent Assay for Measurement of Human Immunoglobulin G Antibodies to Anthrax Toxins

Recently, the Centers for Disease Control and Prevention reported an accurate, sensitive, specific, reproducible, and quantitative enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum (C. P. Quinn, V. A. Semenova, C. M. Elie et al., Emerg. Infect. Dis. 8:1103-1110, 2002). The ELISA had a minimum detectable concentration (MDC) of 0.06 μg/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 μg of anti-PA IgG per ml. The reliable detection limit (RDL) was 0.09 μg/ml, while the dynamic range was 0.06 to 1.7 μg/ml. The diagnostic sensitivity of the assay was 97.6% and the diagnostic specificity was 94.2% for clinically verified cases of anthrax. A competitive inhibition anti-PA IgG ELISA was also developed to enhance the diagnostic specificity to 100%. We report a newly developed fluorescence covalent microbead immunosorbent assay (FCMIA) for B. anthracis PA which was Luminex xMap technology. The FCMIA MDC was 0.006 μg of anti-PA IgG per ml, the RDL was 0.016 μg/ml, and the whole-serum equivalent MDC was 1.5 μg/ml. The dynamic range was 0.006 to 6.8 μg/ml. Using this system, we analyzed 20 serum samples for anti-PA IgG and compared our results to those measured by ELISA in a double-masked analysis. The two methods had a high positive correlation (r² = 0.852; P < 0.001). The FCMIA appears to have benefits over the ELISA for the measurement of anti-PA IgG, including greater sensitivity and speed, enhanced dynamic range and reagent stability, the use of smaller sample volumes, and the ability to be multiplexed (measurement of more than one analyte simultaneously), as evidenced by the multiplexed measurement in the present report of anti-PA and anti-lethal factor IgG in serum from a confirmed clinical anthrax infection.     

Human papillomavirus testing with the hybrid capture 2 assay and PCR as screening tools

The recognition of high-risk human papillomaviruses (HPVs) as etiological agents of cervical cancer has increased the demands to use testing for HPV for the detection of abnormal cervical smears and for cervical cancer screening. The present study compared the performance of the Hybrid Capture 2 (HC2) assay with that of PCR for the detection of significant cervical lesions in 1,511 women with different risks for HPV infections in three New Independent States of the former Soviet Union. The results showed that the level of agreement between the HC2 assay and PCR was substantial, with a kappa (Cohen) value of 0.669 (95% confidence interval [CI], 0.629 to 0.709). Of the 228 samples with discrepant results, 92 were positive by the HC2 assay but negative by PCR, whereas 136 samples were PCR positive but HC2 assay negative. The positive predictive values (PPVs) of the HC2 assay and PCR in detecting high-grade intraepithelial lesions (HSILs) were 4.5% (95% CI, 3.5 to 5.5%) and 3.6% (95% CI, 2.7 to 4.5%), respectively, and the negative predictive values (NPVs) were 99.6% (95% CI, 99.3 to 99.9%) and 99.3% (95% CI, 98.9 to 99.7%), respectively. The sensitivities of the HC2 assay and PCR for the detection of HSILs were 85.2 and 74.0%, respectively, and the specificities were 67.2 and 64.1%, respectively. In receiver operating characteristic (ROC) analysis, the performance of the HC2 assay for the detection of HSILs was excellent (P = 0.0001); the area under the ROC analysis curve was 0.858 (95% CI, 0.811 to 0.905), and the optimal balance between sensitivity (86.5%) and specificity (80%) was obtained with an HC2 assay cutoff level of 15.6 relative light units/positive control. Use of this cutoff would increase the specificity of the HC2 assay to 80.0% without compromising sensitivity. In conclusion, the results of PCR and the HC2 assay were concordant for 85% of samples, resulting in substantial reproducibility. Both tests had low PPVs, equal specificities, and equal (almost 100%) NPVs for the detection of HSILs; but the sensitivity of the HC2 assay was slightly better.     

A sandwich ELISA for properdin in clinical specimens

We have developed a symmetrical sandwich ELISA for measuring human properdin (P) in serum by using the globulin fraction from a commercial antiserum as the capture antibody adsorbed on the plastic. The detecting reagent was a glutaraldehyde conjugate of this Ig fraction with alkaline phosphatase. Two types of inhibition were observed in this study. First, inhibition was observed when greater than 2.5 micrograms/ml of the globulin fraction was used to coat the plates. A second type of inhibition was observed for serum dilutions less than 1/400; it was independent of the concentration of capture Ab and did not occur when purified P was assayed. The data generated with this assay could be fitted in log-log mode by a quadratic equation. The coefficient of the linear term in this equation was found to be the same for serum and purified P, within the limits of experimental error. The results for different samples run on the same plate were expressed in terms of the relative concentration of each sample required to produce an OD405 = 0.2. A sample of pooled normal human serum was run on each plate as a reference; it was assigned a titer of 100 ELISA units/ml (EU/ml). The titers of the unknown samples were expressed in terms of EU/ml by reference to this standard. For purified P, the assay could readily detect 10 ng/ml. By comparing purified P with our reference serum pool, we found that 1 EU equals 0.57 microgram. Day-to-day variation for a group of nine normal sera showed a mean difference of -0.85 EU/ml, SD 5.85 EU/ml. The mean titer for these normal sera was 78.9 EU/ml, SD 15.7 EU/ml. In three recovery experiments in which purified P was mixed with pooled normal serum, the recoveries ranged from 96 to 117%. We conclude that the sandwich ELISA constitutes an adequate immunochemical assay for human P in serum specimens.