Antibody Enzyme-linked Immunosorbent Assay Kit Research Articles

Comparative Analysis of Different ELISA Methods for the Serodiagnosis of Przhevalskiana silenus Infestation in Goats

Przhevalskiana silenus (warble fly) grubs cause myiasis in goats, in mountainous and semi-mountainous areas and different regions in Pakistan, and cause substantial losses to livestock. The palpation method for detecting warble flies generally neglects the infestation intensity; therefore, the development of a reliable and efficient diagnostic technique is extremely necessary. This study compared three indirect enzyme-linked immunosorbent assay (ELISA) methods for detecting anti-P. silenus antibodies using the hypodermin C (HyC) purified from Hypoderma spp. Larvae collected in cattle (local isolate, Microbiology Laboratory, PMAS-Arid Agriculture University, Rawalpindi), the crude antigen from the first instar stage of P. silenus, and a commercial Bovine Hypodermosis Antibody ELISA kit (IDEXX Laboratory), for accurately estimating the seroprevalence of goat warble fly infestation (GWFI) in the Pothwar plateau, Punjab, Pakistan. The ELISA with the crude antigen of P. silenus proved very sensitive and specific, 91% and 93%, respectively. The optical density exhibited a monthly variation, and the antibody titer began increasing from June, continually increased from July to December, and gradually decreased thereafter until March. The study confirmed the endemic status of GWFI in the Pothwar region and identified that ELISA based on the crude antigen of P. silenus was a more sensitive and specific immunodiagnostic method for determining seroprevalence, and could be employed for initiating nationwide eradication campaigns.

Development of an indirect ELISA based on glycoprotein B gene for detecting of Feline herpesvirus type 1.

The study was aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA), which can detect specifically Feline herpesvirus type 1 (FHV-1). The primers were designed based on the conserved sequence of FHV-1 glycoprotein B gene. The recombinant protein with reactogenicity was purified as coating antigen of the assay. The indirect ELISA, characterized by high sensitivity showed no cross-reaction with two types of feline virus, had detection limit at 1:2000 dilution. The positive rate of the assay, according to the determined cutoff value (0.25), was basically consistent with Feline Herpes Virus Antibody ELISA kit. In conclusion, the indirect ELISA with high repeatability and reproducibility can be used for detecting FHV-1, and can provide necessary support to related research.

Giardiasis: Serum antibodies and coproantigens in brown rats (Rattus norvegicus) from Grenada, West Indies.

Aim:Giardia is a serious zoonotic parasite, which causes diarrheal disease in humans and animals including rodents. The purpose of this study was to estimate the prevalence of Giardia spp. in brown rats (Rattus norvegicus) in Grenada.Materials and Methods:Intestinal contents from 99 and serum samples from 169 brown rats (R. norvegicus) from Grenada were collected. These samples were examined for the Giardia coproantigens using Cryptosporidium/Giardia Quik Chek assay (Tech lab® Inc., USA), and the serum was screened through an enzyme-linked immunosorbent assay (ELISA) test kit for Giardia antibody (anti-GD) ELISA kit (MyBioSource, San Diego, CA, USA).Result:Giardia coproantigens were positive in 17.17% (95% confidence interval [CI]; 10.33-26.06%) rats, whereas 55% (95% CI: 47.20-62.68) were positive with serum antibodies (anti-GD) to Giardia.Conclusion:The prevalence of Giardia spp. in brown rats in Grenada was moderate based on the presence of coproantigens in the intestinal contents and antibody in serum. The findings of Giardia infections and prevalence in brown rats will help veterinarians and physicians to better plan diagnostic and preventative strategies. This is the first report of prevalence of Giardia in brown rats in Grenada.

Seroprevalence of selected viral pathogens in pigs reared in organized farms of Meghalaya from 2014 to 16.

Aim:A pilot study was carried out to find out the seroprevalence of Porcine circovirus 2 (PCV2), classical swine fever virus (CSFV), and Porcine respiratory and reproductive syndrome virus (PRRS) in pig population of Meghalaya.Materials and Methods:Serum samples were collected from piglets of 40–45 days age group, growers, and sows reared under organized and unorganized management in 11 districts of Meghalaya situated in the Khasi, Jaintia, and Garo hills divisions in the time period of 2014-2016 from apparently healthy and suspected pigs. Seroprevalence of PCV2, CSFV, and PRRS specific antibodies was detected by enzyme-linked immunosorbent assay (ELISA).Results:A total of 1899 serum samples were collected and screened using antibody ELISA kits specific for PCV2, CSFV, and PRRS. The highest antibody prevalence during the selected time periods was detected for PCV2 (80.8% in 2014, 79.1% in 2015, and 96.2% in 2016) followed by CSFV (76.4% in 2014, 66.09% in 2015, and 25.5% in 2016) and PRRS (2.8% in 2014, 2.7% in 2015, and 3.62% in 2016). The result indicates high seroprevalence for PCV2, which can be considered as an inducement factor due to the immunosuppressive nature of the virus, for animals being susceptible to other pathogens in farms where airborne transmission of PCV2 and postweaning multisystemic wasting syndrome among animals reared in close pens can be a major possibility.Conclusions:The data from this study indicates ubiquitous prevalence of PCV2 antibodies in the farm animals along with the endemic presence of swine fever and emergence of PRRS in an organized farm. There are few reports regarding PCV2 infections/outbreaks in pigs associated with reproductive failure from northern and southern part of India, but till date, there are no reports regarding concomitant infection of CSFV and PCV2 from India. Considerable high seropositivity of PCV2 indicates the need for high impact hygiene practice in farms, routine seromonitoring and implementation the vaccination program. To the author’s best knowledge, this is the first documented report on the seroprevalence of PCV2, CSFV, and PRRS from pig population of Meghalaya.

Preparation and evaluation of Salmonella Enteritidis antigen conjugated with nanogold for screening of poultry flocks.

Aim:The present work aimed to develop lateral flow immunochromatographic strip (ICS) test for detection of Salmonella Enteritidis (SE) specific antibodies in chicken sera.Materials and Methods:A rapid lateral flow immunochromatographic test (LFIT) has been developed, in which SE Group D antigen labeled with the gold chloride molecules laid on the conjugate pad. Staphylococcus aureus protein A was used as capture antibody at the test line (T) of a nitrocellulose (NC) membrane and anti-SE antigen-specific rabbit antibodies were used as capture antibody at the control line (C) of the NC strip in the lateral flow layout device.Results:Using the developed LFIT, the minimal amount of SE-specific antibodies that can be detected in chicken serum sample was 1427 enzyme-linked immunosorbent assay (ELISA) unit/100 µl that was equal to 0.1 µg (Ab)/100 µl sample. 100 suspected serum samples collected from a poultry flock were tested with the prepared SE-LFIT kits and the locally prepared stained Salmonella antigen, and the results were compared with those obtained from examination of these samples with Salmonella Group D antibody ELISA kit as the gold standard test. The sensitivity, specificity, and accuracy of the prepared SE-LFIT antigen kits were 94.4%, 90%, and 94%, respectively, while those obtained with stained Salmonella antigen were 88.8%, 90%, and 89%, respectively.Conclusion:The developed test is a simple field rapid test of high sensitivity, specificity, and accuracy that can improve and facilitates rapid field surveillance of salmonellosis among chickens.

High False Positive Rate Of An ELISA Screen For The Detection Of Anti-Factor VIII Antibodies In Congenital Hemophilia A

IntroductionAnti-factor VIII antibodies are a serious complication of factor replacement therapy in hemophilia A patients. Neutralizing antibodies could previously only be detected via the functional and gold-standard Bethesda assay with or without the Nijmegen modification. The introduction of an enzyme-linked immunosorbent assay (ELISA) screening test provides a less laborious alternative to the Bethesda assay and also has a high sensitivity. Unlike the Bethesda assay, the ELISA can potentially detect non-neutralizing antibodies, which raise the possibility of false positive screens. As it is important that both clinicians and the coagulation laboratory understand the clinical performance of ELISA screening, this study evaluated the sensitivity and specificity of ELISA and Bethesda assays used in both a laboratory validation study and in subsequent clinical experience. MethodsResults from all active adult and pediatric congenital hemophilia A patients who underwent both Bethesda assay and ELISA in British Columbia, Canada as of July 2013 were included in this study. The sensitivity and specificity were compared to the provincial coagulation laboratory validation study of the GTI Factor 8 Antibody ELISA kit against the classical Bethesda assay conducted in both hemophilia A and normal controls in 2010. Optical density (OD) readings were retrieved for eligible samples and from the laboratory validation study. Results35 samples from 26 different patients were used in the validation study performed in 2010. Of 147 adult and 86 pediatric hemophilia A clinic patients active during the study period, 56 samples from 16 adults and 17 children underwent concurrent Bethesda assay and ELISA screen between November 2010 and June 2013. Since ELISA implementation, 389 ELISA screens and 187 Bethesda assays were performed. ELISA screens resulted in the avoidance of Bethesda assays in 85% (330/389) of samples submitted during that period. Specificities and positive predictive values were lower in the clinical sample due to a larger number of false positives (n=18; 32%) relative to the validation study (n=3; 9%), while sensitivity and negative predictive values remained at 100% (Table 1). Interestingly, 67% (2/3) of false positive ELISA screens in the laboratory setting had Bethesda positive histories; however, in the clinical sample, only 6% (1/18) had a distant history of inhibitors. ODs were available for all validation study samples and for 54 clinical samples. Redefinition of our OD cutoff to improve specificity was prevented by false positive patients with strongly positive ODs.Table 1ELISA pre-clinical and clinical performance summaryData sourceSample sizeSensitivitySpecificityPositive predictive valueNegative predictive valueLaboratory validation study351009057100Clinic561005547100Pediatric331004350100Adult231006840100 ConclusionsThe ELISA screen used in this setting is highly sensitive for anti-factor VIII antibody detection in congenital hemophilia A. However, compared to pre-clinical data, our 2.5 years of clinical experience reveals a high incidence of false positives resulting in a significantly lower specificity. Our approximate cost savings in British Columbia as a result of avoided Bethesda assays due to ELISA screens (n=330) is $90 per test, and may exceed $200 per test in the United States. Additional testing and follow-up on positive ELISA results can be an inconvenience in both time and blood sampling, especially in the pediatric population where inhibitor status is more closely monitored. The cost ramifications of this follow up testing could not be quantified in this study. The potential implications for the detection of non-neutralizing anti-FVIII antibodies in hemophilia A need to be further explored and long-term study and monitoring of these discrepant patients is warranted. Disclosures:No relevant conflicts of interest to declare.

A second-generation ELISA (STRATIFY JCV™ DxSelect™) for detection of JC virus antibodies in human serum and plasma to support progressive multifocal leukoencephalopathy risk stratification

BackgroundJC virus (JCV) infection is a prerequisite for development of progressive multifocal leukoencephalopathy (PML). The development and validation of a two-step enzyme-linked immunosorbent assay (ELISA) that detects JCV antibodies in human serum or plasma and its clinical utility for stratification of PML risk have been described. ObjectiveTo develop a second-generation JCV antibody ELISA kit with improved assay performance characteristics. Study designThe assay design was optimized by pre-coating the JC virus-like particles (VLP) on microtiter plates. Assay cut-points were statistically established using sera from >1300 multiple sclerosis patients from natalizumab clinical studies. The assay was analytically validated and then used to determine the presence of JCV antibodies in both treatment-naïve and natalizumab-treated MS patients, as well as in natalizumab-treated PML patients. ResultsAn improved assay for detection of JCV antibodies in human serum and plasma was developed. Key enhancements included improved delineation and reproducibility of low JCV antibody responses and assay ease of use. The assay was validated, demonstrating good agreement with the original two-step JCV antibody ELISA, and similar seroprevalence of 50%–60%. Samples from 63 natalizumab-treated PML patients collected 6–180 months prior to PML diagnosis tested JCV antibody positive. One patient tested JCV antibody negative 15 months prior to PML diagnosis but JCV antibody positive 2 months prior to PML diagnosis. ConclusionsThe validated second-generation JCV antibody ELISA offers improved assay design as a kit and enhanced performance characteristics that advance routine clinical use of the assay as a PML risk stratification tool.

Ring test evaluation of the repeatability and reproducibility of a Porcine reproductive and respiratory syndrome virus oral fluid antibody enzyme-linked immunosorbent assay

The precision of a Porcine reproductive and respiratory syndrome virus (PRRSV) oral fluid antibody enzyme-linked immunosorbent assay (ELISA) was evaluated by calculating reliability coefficients for assay repeatability (within laboratory) and assay reproducibility (between laboratories). Randomly ordered oral fluid samples of known (n = 39) and unknown (n = 224) PRRSV antibody status were tested in 12 diagnostic laboratories. Each laboratory tested the samples twice, first using an antibody ELISA kit and reagents provided to them (phase 1) and then using an ELISA kit and reagents configured in their respective laboratory (phase 2). Repeatability (within laboratory) reliability coefficients calculated using results from samples of known PRRSV antibody status ranged from 0.724 to 0.997 in phase 1 and from 0.953 to 0.998 in phase 2. Reproducibility (between laboratories) reliability coefficients were calculated for 3 conditions: case 1--samples of unknown status (n = 224); case 2--samples of known status (n = 39), and case 3--all samples (n = 263). Among the 3 cases, reliability coefficients ranged from 0.937 to 0.964 in phase 1 and from 0.922 to 0.935 in phase 2. For case 3, it was estimated that 96.67% of the total variation in phase 1 and 93.21% in phase 2 could be attributed to the oral fluid samples themselves. Overall, the PRRSV oral fluid antibody ELISA was highly repeatable and reproducible. The current study supports the routine use of this test in laboratories providing diagnostic service to pig producers.

The increasing prevalence of serologic markers for syphilis among Chinese blood donors in 2008 through 2010 during a syphilis epidemic

In China, the growing syphilis epidemic parallels the spread of human immunodeficiency virus (HIV) in the general population. This study evaluated the prevalence and incidence of serologic markers for syphilis among donors at five Chinese blood centers. We examined whole blood and apheresis donations collected from January 2008 through December 2010. Postdonation testing of syphilis was conducted using two different Treponema pallidum antibody enzyme-linked immunosorbent assay kits. The prevalence of serologic markers for syphilis (%), and the rate of coinfection with HIV-1/2, hepatitis B virus (HBV), and hepatitis C virus (HCV) were calculated. A multivariable logistic regression analysis was conducted examining donor characteristics associated with positive syphilis serology. Seroconversion rate and syphilis incidence were estimated. Of 801,511 donations, 60% were from first-time donors and 40% were from repeat donors. There was a significant increase in syphilis serologic markers among first-time donors with 0.41, 0.45, and 0.57% positivity over 3 years (p < 0.001). Approximately 2.8, 0.8, and 0.5% of HIV-1/2-, HBV-, and HCV-positive donations also tested reactive for syphilis. Logistic regression results suggest that first-time donors were nine times more likely to be syphilis positive than repeat donors. Higher syphilis positivity was associated with donors older than 25 years and with less education. Estimated incidence among repeat donations was 33 (95% confidence interval, 29-39) per 100,000 person-years. The increase in syphilis serologic prevalence reflected the syphilis epidemic in the general population. Without screening, most of these syphilis-positive donations would get into the blood supply. Thus, during a syphilis epidemic, continued syphilis screening of blood donations may be important to maintain blood safety and public health.

A new ELISA kit which uses a combination of Plasmodium falciparum extract and recombinant Plasmodium vivax antigens as an alternative to IFAT for detection of malaria antibodies

BackgroundThe methods most commonly used to measure malarial antibody titres are the Indirect Fluorescence Antibody Test (IFAT), regarded as the gold standard, and the Enzyme-Linked ImmunoSorbent Assay (ELISA). The objective here was to assess the diagnostic performance, i.e. the sensitivity and specificity, of a new malaria antibody ELISA kit in comparison to IFAT. This new ELISA kit, the ELISA malaria antibody test (DiaMed), uses a combination of crude soluble Plasmodium falciparum extract and recombinant Plasmodium vivax antigens.MethodsTwo groups were used: 95 samples from malaria patients to assess the clinical sensitivity and 2,152 samples from blood donors, who had not been exposed to malaria, to assess the clinical specificity.ResultsThe DiaMed ELISA test kit had a clinical sensitivity of 84.2% and a clinical specificity of 99.6% as compared with 70.5% and 99.6% respectively, using the IFAT method. The ELISA method was more sensitive than the IFAT method for P. vivax infections (75% vs. 25%). However, in 923 malaria risk donors the analytical sensitivity of the ELISA test was 40% and its specificity 98.3%, performances impaired by large numbers of equivocal results non-concordant between ELISA and IFAT. When the overall analytical performances of ELISA was compared to IFAT, the ELISA efficiency J index was 0.84 versus 0.71 for IFAT. Overall analytical sensitivity was 93.1% and the analytical specificity 96.7%. Overall agreement between the two methods reached 0.97 with a reliability k index of 0.64.ConclusionThe DiaMed ELISA test kit shows a good correlation with IFAT for analytical and clinical parameters. It may be an interesting method to replace the IFAT especially in blood banks, but further extensive investigations are needed to examine the analytical performance of the assay, especially in a blood bank setting.

The use of filter paper-dried blood spots for thyroid-antibody screening in adults

Thyroid-antibody screening is recommended for several adult populations, including the elderly and pregnant and postpartum women, and analysis of filter paper-dried blood spots (DBSs) has been a cost-effective means of screening newborns for hypothyroidism for more than 30 years. The aim of this study was to show that DBS specimens can be used to test adults for thyroid-stimulating hormone (TSH) antibodies and thyroid antibodies during screening for thyroid disease in a community setting. Thyroid peroxidase antibody and thyroglobilin antibody enzyme-linked immunosorbent assay kits for serum have been adapted for use with DBSs. A TSH assay for newborn DBSs was adapted for the screening of adults. Parallel specimens, serum and DBSs, were collected during routine care of patients attending a thyroid clinic. In addition, 962 DBS specimens were collected from volunteers at community centers and nursing homes. Twelve months later, a second specimen was collected from each of 411 original volunteers. Antibody results are reported as normal and positive. Ninety-seven percent of the serum/DBS results correlated exactly. Of 962 volunteers from nursing homes and community centers, testing for antibodies was positive or indeterminate (borderline) in 266 (27%). Eighty percent of the patients with an initially normal TSH reading who were positive on the 12-month collection were positive for at least one antibody in the first collection. The use of DBSs is a convenient and efficient way to screen for thyroid antibody as a means of detecting occult thyroid disease in adults.

Soluble Cytokine Receptors and Receptor Antagonists Are Sequentially Released after Trauma

Cytokine receptors and receptor antagonists (RAs) have been identified in trauma patients. We hypothesized that after traumatic injury, a sequential release of soluble cytokine receptors and RAs may exist that mirrors the release of the primary cytokines themselves. Twenty-two patients were included in the study: 14 males and 8 females. The mean age was 30.1 +/- 12.5 (range, 19 to 71), and the mean Injury Severity Score was 28.7 +/- 12.6 (range, 4 to 57). There were 15 survivors and 7 nonsurvivors. Samples were collected on arrival to the emergency department and at serial intervals for up to 7 days. Monoclonal antibody enzyme-linked immunosorbent assay kits to tumor necrosis factor (TNF), soluble TNF-receptor (sTNF-R) 55 kd and 75 kd, interleukin (IL)-1 and IL-1 RA, and IL-2 and IL-2r were used. Sera from 22 healthy individuals were used as normal controls. No TNF, IL-1, or IL-2 could be detected in any patient sera after injury. Control levels for the soluble cytokine receptors and RAs were as follows: sTNF-R 55 kd, 607 +/- 89 pg/mL; sTNF-R 75 kd, 2,141 +/- 169 pg/mL; IL-1 RA, 291 +/- 35 pg/mL; and IL-2r, 426 +/- 53 U/mL. In trauma patients, both 55 kd and 75 kd sTNF-R were significantly elevated on arrival to the emergency department, with values of 2,441 +/- 506 pg/mL (p < 0.001) and 4,736 +/- 537 pg/mL (p < 0.001), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)