Indirect Enzyme-linked Immunoassay Research Articles

Use of recombinant malate dehydrogenase (MDH) and superoxide dismutase (SOD) [Cu[sbnd]Zn] as antigens in indirect ELISA for diagnosis of bovine brucellosis

The objective of this study was to validate an indirect enzyme-linked immunoassay (iELISA) using the recombinant proteins, malate dehydrogenase (MDH) and superoxide dismutase (SOD) [CuZn], as antigens and to evaluate its ability to discriminate antibodies produced by vaccination from those induced by infection in the diagnosis of bovine brucellosis. Sera from six groups were evaluated: G1 – culture-positive animals (52 serum samples) (naturally infected); G2 - non-vaccinated animals (28 serum samples) positive in RBT (Rose Bengal test) and 2ME (2-mercaptoethanol test) selected from brucellosis-positive herds; G3 - animals from a brucellosis-free area (32 serum samples); G4 - S19 vaccinated heifers (114 serum samples); G5 – RB51 vaccinated heifers (60 serum samples); G6 - animals inoculated with inactivated Yersinia enterocolitica O:9 (42 serum samples). Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were estimated using the frequentist approach and the confidence interval (CI) (95%) calculated by the Clopper-Pearson (exact) method. The DSe for iELISA_MDH in the G1 group was 71.7% (CI 95%: 57.6–83.2%) and for the G2 100.0% (CI 95%: 87.7–100.0%), whereas the DSp was 84.4% in the G3 (CI 95%: 67.2–94.7%). For the iELISA_SOD the DSe was estimated 67.3% for the G1 (CI 95%: 52.9–79.7%) and 71.4% for G2 (CI 95%: 51.3–86.8%), while the DSp for G3 was 87.5% (CI 95%: 71.0–96.5%). iELISA_MDH and iELISA_SOD showed potential to be used in the diagnosis of infected animals, increasing the range of serological tests available for the diagnosis of bovine brucellosis, with the advantage of being S-LPS-free. However, none of the tests could differentiate between infection and vaccination.

Performance of clinical signs and symptoms, rapid and reference laboratory diagnostic tests for diagnosis of human African trypanosomiasis by passive screening in Guinea: a prospective diagnostic accuracy study

BackgroundPassive diagnosis of human African trypanosomiasis (HAT) at the health facility level is a major component of HAT control in Guinea. We examined which clinical signs and symptoms are associated with HAT, and assessed the performance of selected clinical presentations, of rapid diagnostic tests (RDT), and of reference laboratory tests on dried blood spots (DBS) for diagnosing HAT in Guinea.MethodThe study took place in 14 health facilities in Guinea, where 2345 clinical suspects were tested with RDTs (HAT Sero-K-Set, rHAT Sero-Strip, and SD Bioline HAT). Seropositives underwent parasitological examination (reference test) to confirm HAT and their DBS were tested in indirect enzyme-linked immunoassay (ELISA)/Trypanosoma brucei gambiense, trypanolysis, Loopamp Trypanosoma brucei Detection kit (LAMP) and m18S quantitative PCR (qPCR). Multivariable regression analysis assessed association of clinical presentation with HAT. Sensitivity, specificity, positive and negative predictive values of key clinical presentations, of the RDTs and of the DBS tests for HAT diagnosis were determined.ResultsThe HAT prevalence, as confirmed parasitologically, was 2.0% (48/2345, 95% CI: 1.5–2.7%). Odds ratios (OR) for HAT were increased for participants with swollen lymph nodes (OR = 96.7, 95% CI: 20.7–452.0), important weight loss (OR = 20.4, 95% CI: 7.05–58.9), severe itching (OR = 45.9, 95% CI: 7.3–288.7) or motor disorders (OR = 4.5, 95% CI: 0.89–22.5). Presence of at least one of these clinical presentations was 75.6% (95% CI: 73.8–77.4%) specific and 97.9% (95% CI: 88.9–99.9%) sensitive for HAT. HAT Sero-K-Set, rHAT Sero-Strip, and SD Bioline HAT were respectively 97.5% (95% CI: 96.8–98.1%), 99.4% (95% CI: 99.0–99.7%) and 97.9% (95% CI: 97.2–98.4%) specific, and 100% (95% CI: 92.5–100.0%), 59.6% (95% CI: 44.3–73.3%) and 93.8% (95% CI: 82.8–98.7%) sensitive for HAT. The RDT’s positive and negative predictive values ranged from 45.2–66.7% and 99.2–100% respectively. All DBS tests had specificities ≥ 92.9%. While LAMP and m18S qPCR sensitivities were below 50%, trypanolysis and ELISA/T.b. gambiense had sensitivities of 85.3% (95% CI: 68.9–95.0%) and 67.6% (95% CI: 49.5–82.6%).ConclusionsPresence of swollen lymph nodes, important weight loss, severe itching or motor disorders are simple but accurate clinical criteria for HAT referral in HAT endemic areas in Guinea. Diagnostic performances of HAT Sero-K-Set and SD Bioline HAT are sufficient for referring positives to microscopy. Trypanolysis on DBS may discriminate HAT patients from false RDT positives.Trial registration The trial was registered under NCT03356665 in clinicaltrials.gov (November 29, 2017, retrospectively registered https://clinicaltrials.gov/ct2/show/NCT03356665)Graphical

Designing and developing a sensitive and specific SARS-CoV-2 RBD IgG detection kit for identifying positive human samples

More than 3 y into the coronavirus 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to undergo mutations. In this context, the Receptor Binding Domain (RBD) is the most antigenic region among the SARS-CoV-2 Spike protein and has emerged as a promising candidate for immunological development. We designed an IgG-based indirect enzyme-linked immunoassay (ELISA) kit based on recombinant RBD, which was produced from the laboratory to 10L industry scales in Pichia pastoris. A recombinant-RBD comprising 283 residues (31kDa) was constructed after epitope analyses. The target gene was initially cloned into an Escherichia coli TOP10 genotype and transformed into Pichia pastoris CBS7435 muts for protein production. Production was scaled up in a 10L fermenter after a 1L shake-flask cultivation. The product was ultrafiltered and purified using ion-exchange chromatography. IgG-positive human sera for SARS-CoV-2 were employed by an ELISA test to evaluate the antigenicity and specific binding of the produced protein. Bioreactor cultivation yielded 4g/l of the target protein after 160h of fermentation, and ion-exchange chromatography indicated a purity>95%. A human serum ELISA test was performed in 4 parts, and the ROC area under the curve (AUC) was>0.96 for each part. The mean specificity and sensitivity of each part was 100% and 91.5%, respectively. A highly specific and sensitive IgG-based serologic kit was developed for improved diagnostic purposes in patients with COVID-19 after generating an RBD antigen in Pichia pastoris at laboratory and 10L fermentation scales.

Very Low Seroprevalence of Porcine Reproductive and Respiratory Syndrome among Backyard Pigs in Leyte Province and Factors associated with S/P Ratios

orcine Reproductive and Respiratory Syndrome (PRRS) is a viral disease that causes significant production and economic losses to swine raisers. To estimate the seroprevalence of PRRS in pigs from the backyard and small-hold farms in the province of Leyte, Philippines, a total of 384 pigs were sampled at random from 11 localities and their sera were tested for PRRS antibody using indirect enzyme-linked immunoassay. Univariable and multivariable regression analyses were performed to determine the factors associated with the S/P ratios. Results revealed that the true seroprevalence for PRRS in backyard pigs was 0.28% (0.0001 to 0.0155, 95% CI) and the true herd-level seroprevalence was 1.02% (0.0005 to 0.1588, 95% CI). Factors significantly associated with the S/P ratios were: Large White (breed) (adjusted β = 0.22, p = 0.0014), the presence of goats (adjusted β = -0.63, p < 0.0001) in farm vicinity, disposing wastes to bodies of water (adjusted β = 0.27, p < 0.0001) and separating sick animals (adjusted β = 0.34, p < 0.0001). The very low seroprevalence in the backyard and small-hold pig farms may indicate a low prevalence of PRRS in the province. Practices in backyard farms like disposing of pig wastes to water bodies and separating or moving sick animals were present and may promote the spread of the virus and pose higher risks when future disease outbreaks occur. It is recommended that the government impose proper waste management on backyard swine farms to prevent the spread of PRRS and other economically important swine diseases.

Preparation of Monoclonal Antibody against Pyrene and Benzo [a]pyrene and Development of Enzyme-Linked Immunosorbent Assay for Fish, Shrimp and Crab Samples

Polycyclic aromatic hydrocarbons (PAHs) are significant environmental and food pollutants that can cause cancer. In this work, a specific monoclonal antibody (mAb) to identify pyrene (PYR) and benzo [a]pyrene (BaP) was prepared, and an indirect competitive enzyme-linked immunoassay (ic-ELISA) was established to detect PYR and BaP residues in living aquatic products for the first time. The effects of complete antigens with different coupling ratios on the production of high-sensitivity mAb was explored. Under the optimal conditions, the IC50 value was 3.73 ± 0.43 µg/L (n = 5). The limits of detection (LODs) for PYR and BaP in fish, shrimp, and crab ranged from 0.43 to 0.98 µg/L. The average recoveries of the spiked samples ranged from 81.5–101.9%, and the coefficient of variation (CV) was less than 11.7%. The validation of the HPLC-FLD method indicated that the ELISA method set up in this experiment provided a trustworthy tool for PAHs residues detection in aquatic products.

West Nile virus in the Republic of Serbia-Diagnostic performance of five serological tests in dog and horse sera.

West Nile virus (WNV) is a zoonotic mosquito-borne virus classified as family Flaviviridae and genus Flavivirus. The first WNV outbreak in humans in the Republic of Serbia was recorded in 2012. Equids and dogs can show clinical symptoms after WNV infection and are often used as sentinels. This study aimed to (i) give insight into seropositivity for WNV in clinically healthy dog and horse sera in different regions of Serbia and (ii) compare diagnostic value of 'in-house' and commercially available indirect immunofluorescence (IFA) and enzyme-linked immunoassay (ELISA) tests to 'gold standard' virus neutralization test (VNT). Due to cross-reactivity, sera were tested for Usutu virus and tick-borne encephalitis virus in VNT based on the epidemiological data of field presence. Blood sera of dogs (n=184) and horses (n=232) were collected from 2011 to 2013. The seropositivity was confirmed by VNT in 36.9 % tested dog sera and 34.9% tested horse sera with highest positivity in regions near two big rivers, while in four dog and seven horse sera, positivity resulted from Usutu virus infection. Comparative results of diagnostic tests in dogs ranged from 18.7 % seropositivity by 'in-house' ELISA to 31.9% by commercially available ELISA. In horses, seropositivity ranged from 36.2% by 'in-house' IFA to 32.5% by commercially available IFA and from 26.3% by 'in-house' IgG ELISA to 20.9% by commercially available ELISA. There were no statistically significant differences according to the McNemar test between 'in-house' and commercially available IFA and ELISA test in horse sera, while the same was not true for two ELISAs used in dog sera (χ2 =8.647, p=.003). Established seropositivity in dogs and horses was in accordance with the epidemiological situation and WNV spread in the Republic of Serbia and proven Usutu virus co-circulation. 'In-house' tests remain a valuable tool in early diagnostics of WNV.

Accuracy of multitarget indirect enzyme-linked immunoassay assay for detection of tuberculosis antibody.

BackgroundDiagnosis of tuberculosis (TB) is still difficult. The development of rapid and sensitive laboratory tools for the diagnosis of tuberculosis is a priority. This study aimed to develop an indirect enzyme-linked immunoassay (ELISA) assay for detection of TB antibody and explore its diagnostic value in patients with pulmonary tuberculosis (PTB) via a multi-center clinical evaluation.MethodsThe specific antigen, fusion antigen, and specific antibody peptide were obtained using molecular cloning and phage peptide library screening. An indirect ELISA assay was developed using multiple target materials. Further, the assay was validated in six institutions with clinically confirmed TB patients, non-TB patients with pulmonary disease, and healthy controls as research subjects.ResultsAn indirect ELISA assay was developed with 16 kD antigen, 11,488 (CFP10-MPT48-TB8.4) fusion antigen, and TB18 and pl2 as target antigens against TB antibody. The results of this multicenter study showed that the sensitivity, specificity, and the area under the receiver operating characteristic curve (AUC) of the assay were 48.25% [95% confidence interval (CI): 45.5–51.1%], 92.20% (95% CI: 90.7–93.5%) and 0.724 (95% CI: 0.707–0.741), respectively, and the cut-off value was 0.119. According to the meta-analysis, the combined ROC was 0.736 (95% CI: 0.692–0.779), I2=83.73%. The sensitivity of the sputum-positive PTB group (culture or smear positive) was 58.75% (95% CI: 52.96–65.00%); the sensitivity in sputum-negative group (culture or smear negative) was 37.38% (95% CI: 32.71–42.52%), respectively; the sensitivity of the sputum-positive group was significantly higher than that of sputum-negative group (OR =1.57, 95% CI: 1.29–1.92, P<0.001).ConclusionsMultitarget indirect ELISA assay based on specific-TB antigen, fusion antigen, and antibody peptide is of value for the diagnosis of PTB and can be used as an auxiliary rapid diagnostic tool to improve the sensitivity of sputum-negative TB.

Development of the test kit for detection of SARS-CoV-2 antibodies in sera of susceptible animals

The novel coronavirus infection COVID-19, caused by the SARS-CoV-2, has triggered a pandemic, and has also been reported in animal populations – in farm minks, dogs and felines: domestic cats, lions and tigers. The susceptibility of some animal species to the SARS-CoV-2 has been proven by experimental infection. Serological methods are effectively used to detect the infection in animals. Currently, methods such as neutralization test, immunofluorescence assay and enzyme-linked immunoassay are used to detect antibodies to coronaviruses. Thanks to these studies, a test kit was developed based on an indirect enzyme-linked immunoassay to detect the SARS-CoV-2 antibodies in sera of susceptible animals. The use of a purified concentrated inactivated virus as an antigen allows the detection of antibodies to various SARS-CoV-2 immunodominant proteins (S and N). The reaction conditions were optimized, and a positive-negative threshold was established by testing of 154 negative sera from animals of six species (ferrets, minks, foxes, arctic foxes, cats and dogs). The method reproducibility analysis showed that the average value of the variation coefficient did not exceed 7%, which is an acceptable value. The specificity and sensitivity of the neutralization test, when testing 30 sera from ferrets was 100 and 92.6%, respectively. The high diagnostic sensitivity and specificity shown by testing of 50 serum samples from minks, foxes, cats and dogs with different immune status, allow us to recommend the developed test kit for screening and monitoring tests and post-vaccination immunity control.

Прогнозирование тяжести остеопороза у пациентов с ревматоидным артритом путем определения уровня фетуина-А

Aim of study. To investigate the level of dependence of osteoporosis (OP) progression severity on the level of fetuin-A (FA) in order to predict the OP progression severity in RA patients. Material and methods. We have examined a total of 110 RA patients (mean age 54.2+11.97). Th e diagnosis was established based on ACR/EULAR criteria 2010. A total of 30 conventionally healthy subjects were enrolled serving as a control group. Evaluation of bone mineral density (BMD) was performed using DXA (Lunar DPX-NT GE densitometer). The FA level was determined through indirect enzyme-linked immunoassay (Human Fetuin-A ELISA test-system). Results. Mean serum FA in healthy subjects amounted to 653.55-972.19μg/ml. We formed 2 groups of RA patients: with blood serum FA levels &lt;653.55μg/ml (23 patients) and with normal FA values: 653.55μg/ml or higher (87 patients). A lowered FA level was revealed in 20 (86.96 %) subjects in the group of OP patients and only in 3 (13.04 %) patients with RA without OP (р&lt;0.001). Osteoporotic bone fractures were diagnosed in 12 (13.79 %) patients in the group with normal FA levels and were absent in 75 (86.21 %) patients (р&lt;0.001). Apart from that, RA patients with lowered FA had reliably lower mean BMD in the area of femoral neck and vertebral column as well as lower BMD total value (р&lt;0.001). In order to determine the diagnostic accuracy of laboratory tests, the characteristic curve (ROC curve) was applied. Th e area under the ROC-curve was 0.663 with the cut-off point matching the FA level of 660.3μg/ml. Th is value corresponds with 84.72 % specifi city and 47.37 % sensitivity. Conclusion. We have revealed reliable correlations between the serum FA level in RA patients, the frequency of OP development, osteoporotic bone fractures and BMD values (p&lt;0.001). Laboratory evaluation of FA for OP progression severity prediction for RA patients has good quality with the specifi city of 84.72 % and the sensitivity of 47.37 %.

Development and Optimization of In-house ELISA for Detection of Human IgG Antibody to SARS-CoV-2 Full Length Spike Protein.

The ongoing coronavirus disease 19 (COVID-19) pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a threat to human health. Despite this, many affected countries are now in the process of gradual lifting of COVID-19 restrictions that were initially implemented in response to the pandemic. The success of the so-called “exit strategy” requires continued surveillance of virus circulation in the community and evaluation of the prevalence of protective immunity among population. Serology tests are valuable tools for these purposes. Herein, SARS-CoV-2 full-length spike (S) recombinant protein was utilized to develop and optimize an indirect enzyme-linked immunoassay (ELISA) that enables a reliable detection of virus-specific IgG antibody in human sera. Importantly, the performance of this assay was evaluated utilizing micro-neutralization (MN) assay as a reference test. Our developed ELISA offers 100% sensitivity, 98.4% specificity, 98.8% agreement, and high overall accuracy. Moreover, the optical density (OD) values of positive samples significantly correlated with their MN titers. The assay specifically detects human IgG antibodies directed against SARS-CoV-2, but not those to Middle East respiratory syndrome coronavirus (MERS-CoV) or human coronavirus HKU1 (HCoV-HKU1). The availability of this in-house ELISA protocol would be valuable for various diagnostic and epidemiological applications.

Development of indirect competitive enzyme-linked immunoassay of colistin for milk and egg analysis

ABSTRACT Polyclonal antibodies were obtained by immunizing rabbits with polymyxin B (PMB) conjugated to bovine serum albumin. Based on the heterologous coating conjugate of PMB with periodate-oxidized gelatin and the resulting antibodies, an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect residual colistin (COL), a structural analogue of PMB in livestock products. The developed analysis was characterized by close specificity towards PMB (100%) and COL (88%), sensitivity (IC50 = 5.7 ng/mL), dynamic range (IC20-IC80 = 1.0–80 ng/mL) and the detection limit of COL (IC10 = 0.4 ng/mL). It was possible to quantify the antibiotic in milk and eggs without special and labour-intensive sample pretreatment using a simple 10- and 50-fold dilution of the corresponding samples. The developed analysis system made it possible to record the maximum residue limit of COL in dairy products (50 μg/kg) and eggs (300 μg/kg) with 89–104% recovery of analyte.

A multiplexed, indirect enzyme-linked immunoassay for the detection and differentiation of E. coli from other Enterobacteriaceae and P. aeruginosa from other glucose non-fermenters

Gram-negative bacteria (GNB) are important causes of community (CA) and hospital (HA)- associated infections. Here we describe the development of an indirect ELISA (I-ELISA), which can be used to detect and differentiate the Enterobacteriaceae Escherichia coli, and glucose non-fermenter Pseudomonas aeruginosa from other GNB species. The I-ELISA utilizes six antibodies for bacterial speciation, which were grouped according to their bacterial targets; Enterobacteriaceae (SL-EntA and CH1810 mAb), Escherichia coli (SL-EcA and 6103–46 mAb), Pseudomonas aeruginosa (SL-PaA and SL-PaB). The six, anti-GNB antibodies were first screened against a panel of well-characterized clinical GNB isolates to optimize assay conditions and to determine individual antibody sensitivity and specificity. When tested against a diverse, blinded panel of 94 GNB clinical isolates, the I-ELISA exhibited the following sensitivity/specificity for each target: Enterobacteriaceae (94.4%/95%), E. coli (82.6%/88.7%), P. aeruginosa (83.3%/96%). An I–ELISA to detect and differentiate the most common GNB pathogens offers advantage in terms of simplicity over diagnostic tests currently used in most clinical settings.

Isolation of Bactrian Camel Single Domain Antibody for Parathion and Development of One-Step dc-FEIA Method Using VHH-Alkaline Phosphatase Fusion Protein.

A heavy chain variable fragment of heavy chain only antibodies derived from camelids termed VHH shows beneficial characteristics for immunoassay in terms of high sensitivity, outstanding stability and ease in expression. In the present study, we isolated six VHHs from phage display library against parathion, which is a widely used organophosphorus pesticide with high toxicity and persistence. One of six selected VHHs named VHH9, showed highest specificity and superior thermo-stability. A VHH9-alkaline phosphatase (AP) fusion was constructed and used to establish a one-step direct competitive fluorescence enzyme immunoassay (dc-FEIA) with a half maximal inhibitory concentration (IC50) of 1.6 ng/mL and a limit of detection of 0.2 ng/mL which was 4-fold or 3-fold higher sensitivity than direct competitive enzyme-linked immunoassay (dc-ELISA) and indirect competitive enzyme-linked immunoassay (ic-ELISA) for parathion. Furthermore, our assay indicated a 50% reduction on operation time compared with the ic-ELISA method. The presented immunoassay was validated with spiked Chinese cabbage, cucumber, and lettuce samples, and confirmed by UPLC-MS/MS. The results indicated that the VHH-AP-based dc-FEIA is a reproducible detection assay for parathion residues in vegetable samples.

Autoantibodies against glucose-regulated protein 78 as serological biomarkers in metastatic and recurrent hepatocellular carcinoma.

PurposeTo identify Heptocellular carcinoma (HCC) associated antigens by proteomics, and validate whether autoantibodies against tumor-associated antigens (TAAs) could be used for diagnosis and conditional monitoring.RESULTSThe 78 kDa glucose regulated protein (GRP78) was selected as a candidate TAA. The titers of autoantibodies against 78 kDa glucose regulated protein (GRP78) from patients with HCC, liver cirrhosis (LC), and chronic hepatitis (CH) were significantly higher than that from normal controls (P<0.05, P<0.001, and P<0.01, respectively). The expression of autoantibodies against GRP78 was associated with clinical stage (P<0.01), portal vein invasion (P<0.05), and metastasis (P<0.05). The expression of anti-GRP78 antibodies was significantly higher 1 month after surgery in recurrent patients who had accepted hepatic resection 1 month after surgery compared to patients who had surgery before surgery or within 1 week after surgery (P<0.01 and P<0.001). Immunohistochemistry (IHC) showed higher expression of GRP78 in HCC compared to the non-HCC liver tissues (P <0.05).Materials and MethodsHCC serum with high titer of autoantibodies against TAAs were screened and used for a proteome-based approach to identify HCC associated antigens. Indirect enzyme-linked immunoassay (ELISA) was used to detect the corresponding autoantibodies against TAAs.ConclusionGRP78 is an autoantigen that could stimulate autoimmune responses and serve as a potential marker for recurrent and metastatic progression in HCC.

Detection of adalimumab and anti-adalimumab antibodies in patients with rheumatoid arthritis: a comprehensive overview of methodology pitfalls and benefits.

About a third of patients with rheumatoid arthritis treated with adalimumab may develop anti-adalimumab antibodies. Anti-adalimumab antibodies are associated with reduced drug levels, loss of drug efficacy, clinical non-response and an increased risk of adverse effects. In case of suspected drug failure and in order to better define clinical efficacy, adalimumab as well as anti-adalimumab antibodies levels should be monitored. Sandwich or indirect enzyme-linked immunoassay is most commonly used for determining adalimumab, while bridging ELISA and antigen-binding test are most useful for determining anti-adalimumab antibodies. Most current assays cannot detect antibodies complexed with the adalimumab; however, methods for dissociation of the complexes using acid/temperature have been developed. The aim of this review is to report on the latest methodology for detecting adalimumab and anti-ADL antibodies, benefits of their detections in clinical practice, as well as expose problematic issues, such as different analytical sensitivity and specificity, standardization and validation. The main problem in measuring adalimumab or anti-ADL antibodies is high drug sensitivity, which can result in false-negative anti-ADL antibodies. Therefore, drug-tolerant assays have been developed. Cell-based assays, such as the reporter gene assay, are recommended for detection of functionally active adalimumab and their neutralizing anti-ADL antibodies.

ANTIDOG IgG SECONDARY ANTIBODY SUCCESSFULLY DETECTS IgG IN A VARIETY OF AQUATIC MAMMALS.

Serological tests play an important role in the detection of wildlife diseases. However, while there are many commercial assays and reagents available for domestic species, there is a need to develop efficient serological assays for wildlife. In recent years, marine mammals have represented a wildlife group with emerging infectious diseases, such as influenza, brucellosis, and leptospirosis. However, with the exception of disease-agent-specific assays or functional assays, few reports describe the use of antibody detection assays in marine mammals. In an indirect enzyme-linked immunoassay (EIA) or an immunofluorescence assay, antibody is detected using an antitarget species secondary conjugated antibody. The sensitivity of the assay depends on the avidity of the binding reaction between the bound antibody and the detection antibody. A commercial polyclonal antidog IgG conjugated antibody was tested in an EIA for its ability to sensitively detect the IgG of seven marine mammals including sea otter ( Enhydra lutris ), polar bear ( Ursus maritimus ), grey seal ( Halichoerus grypus ), harbor seal ( Phoca vitulina ), northern elephant seal ( Mirounga angustirostris ), California sea lion ( Zalophus californianus ), Pacific walrus ( Odobenus rosmarus ) and one freshwater mammal: Asian small-clawed otter ( Aonyx cinerea ). With the exception of Asian small-clawed sea otters, the detection of IgG in these marine mammals either exceeded or was nearly equal to detection of dog IgG. The use of the tested commercial antidog IgG antibody may be a valid approach to the detection of antibody response to disease in sea mammals.

Evaluation of recombinant outer membrane protein C based indirect enzyme-linked immunoassay for the detection of Salmonella antibodies in poultry.

Aim:To evaluate the efficacy of recombinant outer membrane proteinC (rOmpC) based enzyme-linked immunoassay (ELISA) for the diagnosis of salmonellosis in poultry.Materials and Methods:Three antigens were prepared, and the indirect ELISA was standardized using the antigens and the antiserum raised in chicken against Omp and rOmpC. Sera were collected from a total of 255 apparently healthy field chickens and screened for the presence of Salmonella antibodies by this ELISA.Results:The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Omp revealed major polypeptides at 36, 42 and 52 kDa, and the rOmpC was evident by a single protein band of 43 kDa. The Omp and rOmpC antigen revealed an optimum concentration of 78 and 156 ng, respectively, in the assay, while the whole cell antigen gave an optimum reaction at a concentration of 106 organisms/ml. The test was found to be specific as it did not react with any of the antisera of seven other organisms. The developed ELISA detected Salmonella antibodies from 22 (8.62%) samples with rOmpC antigen, while 24 (9.41%) samples gave a positive reaction with both Omp and whole cell antigens.Conclusion:We suggest rOmpC based indirect ELISA as a suitable screening tool for serological monitoring of poultry flocks.

Evaluation of lateral flow assay as a field test for investigation of brucellosis outbreak in an organized buffalo farm: A pilot study.

Aim:The aim was to evaluate lateral flow assay (LFA) as a field test for investigation of brucellosis outbreak in organized buffalo farm.Materials and Methods:A total of 153 serum samples were tested to detect the presence of brucella antibodies by LFA and three other serological tests i.e. rose bengal plate test (RBPT), protein G based indirect enzyme-linked immunoassay (iELISA), and competitive ELISA (cELISA). The performances of LFA and other serological tests were evaluated using OIE complaint cELISA as the gold standard.Results:Serological tests revealed 50% of the animals were seropositive for Brucella antibodies and correlated with clinical history of abortions, infertility, and productive failures. The newly developed assay showed 87.1% and 92.6% sensitivity and specificity, which was even higher than the specificity of RBPT.Conclusions:The investigation proved the potential usefulness of LFA for field diagnosis of brucellosis in the regions where laboratory facilities are limited.

전북 동부지역 한우에서의 유사산 유발 질병 항체가 조사

Reproductive losses in a herd can be huge. Fetal reabsorption or undefined infertility often remain undetected. Routine herds monitoring for exposure, controlling the introduction of potential agent carriers, appropriate biosafety procedures, and vaccination where possible are together the best security against abortion and stillbirth inducing disease. For biosecurity of local farms, we performed antibody titers of abortion and stillbirth related diseases such as bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), Neospora caninum, Toxoplasma gondii and Campylobacter fetus subsp venerealis. The blood samples were collected from 500 female Hanwoo over 1 year old of 100 farms in Jeonbuk eastern area. Champhylobater serological test was evaluated by the standard tube agglutination test (STAT) and other pathogen's antibodies were detected by indirect-enzyme linked immunoassay (I-ELISA). The seroprevalence of abortion and stillbirth inducing disease were BVDV 72.4%, IBRV 13.0%, N. caninum 1.2%, T. gondii 10.4% and C. venerealis 0.6%, irrespectively.

Development of a monoclonal antibody-based indirect competitive immunosorbent assay for 4(5)-Methylimidazole detection in caramels

In this study, an indirect competitive enzyme-linked immunoassay (ic-ELISA) based on monoclonal antibody for 4(5)-Methylimidazole (4-MI) detection was described. The artificial antigens were prepared by conjugating bovine serum albumin (BSA) or ovalbumin (OVA) with the hapten of 4-MI. And monoclonal antibody, evaluated by ic-ELISA, was obtained by immunizing BABL/c mice. After optimizing, a standard curve for ic-ELISA detection on 4-MI was obtained with the linear detection range of 0.64–20.48mg/L. The cross-reactivity (CR) of all the structural analogues of 4-MI was less than 5.62%. The recoveries of 4-MI in caramels detection were ranged from 88.69% to 114.09%, with relative standard deviation (n=3) below 8.07%. The results suggested that the established ic-ELISA is promising for 4-MI commercial detection in caramels.