Evaluation of enzyme-linked immunosorbent and alternative assays for detection of HIV antibodies using panels of Brasilian sera

Sera from 472 Brazilian subjects, confirmed to be either positive or negative for HIV antibodies and comprising the total clinical spectrum of HIV infection, were utilized in the evaluation of six commercially available enzyme-linked immunosorbent assays (ELISA), as well as of four alternative assays, namely indirect immunofluorescence (IIF), passive hemagglutination (PHA), dot blot and Karpas AIDS cell test. The sensitivities ranged from 100% (Abbott and Roche ELISA) to 84.2% (PHA) and the specificities ranged from 99.3% (IIF) to 80.2% (PHA). The sensitivity and specificity of the PHA and the sensitivity of the Karpas cell test were significantly lower than those of the other tests. Although the IFF and dot blot had good sensitivities and specificities, the six ELISA were more attractive than those tests when other parameters such as ease of reading and duration of assay were considered.

Evaluation of Leptospiral Recombinant Antigens MPL17 and MPL21 for Serological Diagnosis of Leptospirosis by Enzyme-Linked Immunosorbent Assays

Leptospirosis is a zoonosis of multisystem involvement caused by pathogenic strains of the genus Leptospira. In the last few years, intensive studies aimed at the development of a vaccine have provided important knowledge about the nature of the immunological mechanisms of the host. The purpose of this study was to analyze the immune responses to two recombinant proteins, MPL17 and MPL21 (encoded by the genes LIC10765 and LIC13131, respectively) of Leptospira interrogans serovar Copenhageni in individuals during infection. The recombinant proteins were expressed in Escherichia coli as six-His tag fusion proteins and were purified from the soluble bacterial fraction by affinity chromatography with Ni2+ -charged resin. The recombinant proteins were used to evaluate their ability to bind to immunoglobulin G (IgG) (and IgG subclass) or IgM antibodies in serum samples from patients in the early and convalescent phases of leptospirosis (n = 52) by enzyme-linked immunosorbent assays. The prevalences of total IgG antibodies against MPL17 and MPL21 were 38.5% and 21.2%, respectively. The titers achieved with MPL17 were statistically significantly higher than those obtained by the reference microscopic agglutination test. The specificity of the assay was estimated to be 95.5% for MPL17 and 80.6% for MPL21 when serum samples from individuals with unrelated febrile diseases and control healthy donors were tested. The proteins are conserved among Leptospira strains that cause human and animal diseases. MPL17 and MPL21 are most likely new surface proteins of leptospires, as revealed by liquid-phase immunofluorescence assays with living organisms. Our results demonstrate that these recombinant proteins are highly immunogenic and, when they are used together, might be useful as a means of diagnosing leptospirosis.

Epitope-blocking enzyme-linked immunosorbent assays for the detection of serum antibodies to West Nile virus in multiple avian species

We report the development of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) for the rapid detection of serum antibodies to West Nile virus (WNV) in taxonomically diverse North American avian species. A panel of flavivirus-specific monoclonal antibodies (MAbs) was tested in blocking assays with serum samples from WNV-infected chickens and crows. Selected MAbs were further tested against serum samples from birds that represented 16 species and 10 families. Serum samples were collected from birds infected with WW or Saint Louis encephalitis virus (SLEV) and from noninfected control birds. Serum samples from SLEV-infected birds were included in these experiments because WNV and SLEV are closely related antigenically, are maintained in similar transmission cycles, and have overlapping geographic distributions. The ELISA that utilized MAb 3.11126 potentially discriminated between WW and SLEV infections, as all serum samples from WNV-infected birds and none from SLEV-infected birds were positive in this assay. Assays with MAbs 2132 and 6B6C-1 readily detected serum antibodies in all birds infected with WNV and SLEV, respectively, and in most birds infected with the other virus. Two other MAbs partially discriminated between infections with these two viruses. Serum samples from most WNV-infected birds but no SLEV-infected birds were positive with MAb 3.676, while almost all serum samples from SLEV-infected birds but few from WNV-infected birds were positive with MAb 6B5A-5. The blocking assays reported here provide a rapid, reliable, and inexpensive diagnostic and surveillance technique to monitor WNV activity in multiple avian species.

Performance indexes of a dot-enzime-linked immunosorbent assay (dot-ELISA) and an ezyme-linked immunosorbent assay (IgG-ELISA) for field surveys of new world leishmaniasis

Diagnostic performance indexes of sensitivity, specificity, positive predictive value and efficiency were determined for dot-ELISA and IgG-ELISA tests in 340 leishmaniasis sera. Sensitivity of the dot-ELISA was significantly lower than IgG-ELISA's; the two tests had indexes of specificity and positive predictive value of the same magnitude. Seventy-eight sera gave a negative dot-ELISA test result and a positive IgG-ELISA test result. When sera were classified according to different criteria as how to interpret this diversity, the kappa statistic did not corroborate the classification indicating that the two tests display a substantial strength of agreement. The results presented indicate that performance indexes accrued in a survey where variables arc well known may be extrapolated to other population studies if the disease presents itself as highly prevalent (due to a selection bias or not) and may be expected to discriminate a disease status among test positives.

Improvement of an enzyme immunosorbent assay for detecting antibodies against Dioctophyma renale

An available enzyme-linked immunosorbent assay (ELISA) was studied for the detection of anti-Dioctophyma renale antibodies in the sera of dogs using, detection of parasite eggs in urine sediment as a reference test. ELISA uses a soluble antigenic preparation of esophagus of D. renale and the optimal dilutions of the antigen, serum and conjugate were determined by means of checker board titration, using positive (n=13) and negative (n=27) reference serum. The specificity and sensitivity of the ELISA were 93.8% and 92.3% respectively and the kappa index was good (0.76). These results suggest that ELISA described may prove to be an effective serological test for detecting dogs infected and exposed to this parasite mainly dogs that are not eliminating parasite eggs through their urine.

Epitope-blocking enzyme-linked immunosorbent assays for detection of West Nile virus antibodies in domestic mammals

We evaluated the ability of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) to detect West Nile virus (WNV) antibodies in domestic mammals. Sera were collected from experimentally infected horses, cats, and pigs at regular intervals and screened in ELISAs and plaque reduction neutralization tests. The diagnostic efficacies of these techniques were similar.

Improved enzyme-linked immunoadsorbent assay (ELISA) for the study of Trypanosoma cruzi-host cell interaction in vitro

We herein present an improved assay for detecting the presence of Trypanosoma cruzi in infected cultures. Using chagasic human sera (CHS), we were able to detect T. cruzi infection in primary cultures of both peritoneal macrophages and heart muscle cells (MHC). To avoid elevated background levels - hitherto observed in all experiments especially in those using HMC - CHS were preincubated with uninfected cells in monolayers or suspensions prior to being used for detection of T. cruzi in infected monolayers. Preincubation with cell suspensions gave better results than with monolayers, reducing background by up to three times and increasing sensitivity by to twenty times. In addition, the continous fibroplastic cell line L929 was shown to be suitable for preadsorption of CHS. These results indicate that the high background levels observed in previous reports may be due to the presence of human autoantibodies that recognize surface and/or extracellular matrix components in cell monolayers. We therefore propose a modified procedure that increases the performance of the ELISA method, making it an useful tool even in cultures that would otherwise be expected to present low levels of infection or high levels of background

New finding of Giardia intestinalis (Eukaryote, Metamonad) in Old World archaeological site using immunofluorescence and enzyme-linked immunosorbent assays

In this study, nine organic sediment samples from a medieval archaeological site at Pineuilh, France, were examined for Giardia intestinalis using two commercially available immunological kits [enzyme-linked immuno sorbent and immunofluorescence (IFA) assays]. Both techniques detected G. intestinalis in one sample, dated to 1,000 Anno Domini. This is the first time IFA was successfully used to detect protozoa in Old World archaeological samples. Such immunological techniques offer important perspectives concerning ancient protozoa detection and identification.

Serological diagnosis of visceral leishmaniasis by an enzyme immunoassay using protein A in naturally infected dogs

A rapid indirect enzyme-linked immunosorbent assay (ELISA) was developed for measuring antibodies against Leishmania chagasi using total antigen from lysed promastigotes. Fifty symptomatic mixed breed dogs from a region of high incidence of visceral leishmaniasis in Brazil were examined. The results showed that in the positive animals, diagnosed by cytological examination, the ELISA using protein A assay system (mean optical density ± SD / 2.078 ± 0.631) detected more antibodies than the anti-IgG assay (mean optical density ± SD / 1.008 ± 0.437), while in the negative animals, the results by both systems were similar. These results suggest that the ELISA assay using protein A peroxidase conjugated could be useful to detect early infected animals in endemic areas, and thus help to control the spread of the infection.

Serological diagnosis of visceral leishmaniasis by an enzyme immunoassay using protein A in naturally infected dogs

Um ensaio de imunoadsorção enzimática para detecção de anticorpos contra Leishmania chagasi, utilizando antígeno total de formas promastigotas lisados foi desenvolvido. Cinqüenta cães com sintomas clínicos de leishmaniose visceral foram examinados. Esta técnica utilizou anti-IgG de cão conjugado a peroxidase ou proteína A conjugado a peroxidase. Foi verificado que nos animais positivos diagnosticados por exame parasitológico direto o ensaio ELISA utilizando proteína A conjugada a peroxidase (média da densidade óptica ± desvio padrão 2,078 ± 0,631) detecta mais anticorpos do que o sistema utilizando anti-IgG de cão conjugado a peroxidase (média da densidade óptica ± desvio padrão 1,008 ± 0,437), enquanto para os animais negativos o resultado obtido nos dois sistemas de detecção são similares. Esse resultado sugere que o sistema de ELISA utilizando proteína A conjugado a peroxidase pode ser útil na detecção de animais na fase aguda da infecção e desta forma auxiliar na identificação dos animais positivos e no controle desta importante zoonose.

Immunodiagnosis of swine cysticercosis by indirect ELISA employing a heterologous antigen from Taenia crassiceps metacestode

C. Larralde et al. (1990, Arch. Pathol. Lab. Med., 114:926-928) demonstrated that heterologous antigen from the laboratory-adapted murine Taenia crassiceps metacestode may substitute those from Taenia solium in the immunodiagnosis of human cysticercosis by the indirect enzyme-linked immunosorbent assay (IE). ?This antigen is easily obtained at a laboratory level and solves the problem of T. solium cysticerci collection from naturally or experimentally infected swine. In this study an IE employing a heterologous antigen from the T. crassiceps metacestode was evaluated for the immunodiagnosis of swine cysticercosis. Sera from 300 swine free of T. solium cysticerci by post-mortem examination were employed to determine two IE cut- off values: 1) Mean ELISA values + 2 standard deviations (2 sigma cut-off) and 2) - Mean ELISA values + 3 standard deviations (3 sigma cut-off). The specificity of IE was 97% with the 2 sigma cut-off and 100% with the 3 sigma cut-off. When applied to ten sera from swine infected by cysticerci of T. solium by post-mortem examination, the sensitivity of IE was 100% independent of the cut off.

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

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.

Evaluation of two fully automated novel enzyme-linked immunosorbent assays for the determination of human adiponectin in serum

BACKGROUND: In contrast to RIA, recently available ELISAs provide the potential for fully automated analysis of adiponectin. To date, studies reporting on the diagnostic characteristics of ELISAs and investigating on the relationship between ELISA- and RIA-based methods are rare. METHODS: Thus, we established and evaluated a fully automated platform (BEP 2000; Dade-Behring, Switzerland) for determination of adiponectin levels in serum by two different ELISA methods (competitive human adiponectin ELISA; high sensitivity human adiponectin sandwich ELISA; both Biovendor, Czech Republic). Further, as a reference method, we also employed a human adiponectin RIA (Linco Research, USA). Samples from 150 patients routinely presenting to our cardiology unit were tested. RESULTS: ELISA measurements could be accomplished in less than 3 h, measurement of RIA had a duration of 24 h. The ELISAs were evaluated for precision, analytical sensitivity and specificity, linearity on dilution and spiking recovery. In the investigated patients, type 2 diabetes, higher age and male gender were significantly associated with lower serum adiponectin concentrations. Correlations between the ELISA methods and the RIA were strong (competitive ELISA, r=0.82; sandwich ELISA, r=0.92; both p<0.001). However, Deming regression and Bland-Altman analysis indicated lack of agreement of the 3 methods preventing direct comparison of results. The equations of the regression lines are: Competitive ELISA=1.48 x RIA-0.88; High sensitivity sandwich ELISA=0.77 x RIA+1.01. CONCLUSIONS: Fully automated measurement of adiponectin by ELISA is feasible and substantially more rapid than RIA. The investigated ELISA test systems seem to exhibit analytical characteristics allowing for clinical application. In addition, there is a strong correlation between the ELISA methods and RIA. These findings might promote a more widespread use of adiponectin measurements in clinical research.

Performance of commercial enzyme-linked immunosorbent assays (ELISA) for diagnosis of HSV-1 and HSV-2 infection in a clinical setting

Thesis (Master's)--University of Washington, 2016-06

Survey for Tick-Borne Zoonoses in the State of Espirito Santo, Southeastern Brazil

Blood samples collected from 201 humans, 92 dogs, and 27 horses in the state of Espirito Santo, Brazil, were tested by polymerase chain reaction, indirect immunofluorescence assays, and indirect enzyme-linked immunosorbent assay for tick-borne diseases (rickettsiosis, ehrlichiosis, anaplasmosis, borreliosis, babesiosis). Our results indicated that the surveyed counties are endemic for spotted fever group rickettsiosis because sera from 70 (34.8%) humans, 7 (7.6%) dogs, and 7 (25.9%) horses were reactive to at least one of the six Rickettsia species tested. Although there was evidence of ehrlichiosis (Ehrlichia canis) and babesiosis (Babesia cams vogeli, Theileria equi) in domestic animals, no human was positive for babesiosis and only four individuals were serologically positive for E. canis. Borrelia burgdorferi-serologic reactive sera were rare among humans and horses, but encompassed 51% of the canine samples, suggesting that dogs and their ticks can be part of the epidemiological cycle of the causative agent of the Brazilian zoonosis, named Baggio-Yoshinari Syndrome.