Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol

ABSTRACT Johne's disease (JD), or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Control of JD could be accomplished by diagnosis and good animal husbandry, but this is currently not feasible because commercially available diagnostic tests have low sensitivity levels and are incapable of diagnosing prepatent infections. In this study, a highly sensitive and subspecies-specific enzyme-linked immunosorbent assay was developed for the diagnosis of JD by using antigens extracted from the surface of M. avium subsp. paratuberculosis. Nine different chemicals and various intervals of agitation by vortex were evaluated for their ability to extract the surface antigens. Various quantities of surface antigens per well in a 96-well microtiter plate were also tested. The greatest differences in distinguishing between JD-positive and JD-negative serum samples by ethanol vortex enzyme-linked immunosorbent assay (EVELISA) were obtained with surface antigens dislodged from 50 μg/well of bacilli treated with 80% ethanol followed by a 30-second interval of agitation by vortex. The diagnostic specificity and sensitivity of the EVELISA were 97.4% and 100%, respectively. EVELISA plates that had been vacuum-sealed and then tested 7 weeks later (the longest interval tested) had diagnostic specificity and sensitivity rates of 96.9 and 100%, respectively. In a comparative study involving serum samples from 64 fecal culture-positive cattle, the EVELISA identified 96.6% of the low-level fecal shedders and 100% of the midlevel and high-level shedders, whereas the Biocor ELISA detected 13.7% of the low-level shedders, 25% of the mid-level shedders, and 96.2% of the high-level shedders. Thus, the EVELISA was substantially superior to the Biocor ELISA, especially in detecting low-level and midlevel shedders. The EVELISA may form the basis for a highly sensitive and subspecies-specific test for the diagnosis of JD.

Download Full-text

Association between Enzyme-Linked Immunosorbent Assay-Measured Kidney Injury Markers and Urinary Cadmium Levels in Chronic Kidney Disease

Journal of Clinical Medicine ◽

10.3390/jcm11010156 ◽

2021 ◽

Vol 11 (1) ◽

pp. 156

Author(s):

Kai-Fan Tsai ◽

Pai-Chin Hsu ◽

Chien-Te Lee ◽

Chia-Te Kung ◽

Yi-Chin Chang ◽

...

Keyword(s):

Chronic Kidney Disease ◽

Kidney Disease ◽

Immunosorbent Assay ◽

Renal Injury ◽

Kidney Injury ◽

Cadmium Exposure ◽

Enzyme Linked Immunosorbent Assay ◽

Urinary Cadmium ◽

Low Level ◽

Renal Biomarkers

Cadmium exposure is associated with chronic kidney disease (CKD), but the optimal biomarker for early cadmium-associated nephrotoxicity in low-level exposure has not yet been established. We conducted a cross-sectional investigation involving 167 CKD patients stratified according to tertiles of urinary cadmium levels (UCd), in which enzyme-linked immunosorbent assay (ELISA)-measured novel renal biomarkers were utilized to assess the extent of renal injury associated with cadmium burden. In the analyses, urinary kidney injury molecule-1 (KIM-1) levels and age were the independent factors positively correlated with UCd after adjusting for covariates in non-dialysis-dependent CKD patients (high vs. low UCd, odds ratio (95% confidence interval), 1.0016 (1.0001–1.0032), p = 0.043, and 1.0534 (1.0091–1.0997), p = 0.018). Other conventional and novel renal biomarkers, such as serum creatinine, estimated glomerular filtration rate, CKD staging, urinary protein/creatinine ratio, urinary 8-hydroxy-2-deoxyguanosine (8-OHdG), and urinary epidermal growth factor (EGF) were not independently correlated with UCd in the analyses. In conclusion, our study found that the ELISA-measured urinary KIM-1 level could serve as an early renal injury marker in low-level cadmium exposure for non-dialysis-dependent CKD patients. In addition, age was an independent factor positively associated with UCd in this population.

Download Full-text

Improvements to the immunoassay for detection of Rathayibacter toxicus in hay

Crop and Pasture Science ◽

10.1071/cp10337 ◽

2011 ◽

Vol 62 (6) ◽

pp. 523 ◽

Cited By ~ 3

Author(s):

A. M. Masters ◽

B. Samarasinghe ◽

M. J. Kalkhoven ◽

G. L. den Hollander ◽

D. G. Palmer

Keyword(s):

Monoclonal Antibody ◽

Horseradish Peroxidase ◽

Immunosorbent Assay ◽

Cost Effective ◽

Enzyme Linked Immunosorbent Assay ◽

Export Industry ◽

Large Numbers ◽

Assay Protocol ◽

Very High ◽

Peroxidase Conjugate

An improved protocol for the previously described enzyme-linked immunosorbent assay for Rathayibacter toxicus in hay is described. The improvements were driven mainly by the export hay industry requirement of same-day turnaround for testing of hay extracts. The preparation of hay extracts was shortened by 8 h. The time for adding samples to the enzyme-linked immunosorbent assay plates was shortened by the use of sample tubes with penetrable stoppers combined with specially designed racks. The monoclonal antibody used in the original protocol was purified and conjugated to horseradish peroxidase. This eliminated the need for a secondary step with an anti-mouse horseradish peroxidase conjugate and thereby shortened the assay by over 1 h. Results with the improved assay protocol showed a very high correlation with results obtained with the original protocol (r = 0.98). The assay is still sensitive enough to detect antigen equivalent to less than 1 average gall per kg of hay. These cost-effective changes have streamlined the testing of large numbers of samples for the presence of R. toxicus, in support of the hay export industry.

Download Full-text

Analysis of the effectiveness of the use of enzyme-linked immunosorbent assay for the diagnosis of leukemia in cattle during health-improving activities

Bulletin of NSAU (Novosibirsk State Agrarian University) ◽

10.31677/2072-6724-2020-57-4-95-102 ◽

2020 ◽

pp. 95-102

Author(s):

S. I. Loginov

Keyword(s):

Enzyme Immunoassay ◽

Immunosorbent Assay ◽

Serological Test ◽

Leukemia Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Agricultural Enterprises ◽

Low Level ◽

Speed Up ◽

Disease Indicators ◽

Low Sensitivity

The aim of the work is to analyze the efficiency of using enzyme-linked immunosorbent assay in the diagnosis of cattle leukemia when carrying out health-improving measures in livestock enterprises that are unfavorable for this disease. Indicators of infection of cattle with leukemia virus on 6 farms of agricultural enterprises of the Tomsk region are presented. For serological diagnostics, the immunodiffusion reaction and enzyme immunoassay were used. It has been established that while carrying out health-improving measures in enterprises unfavorable for leukemia in cattle, the use of enzyme-linked immunosorbent assay for serological diagnosis of the disease enabled to identify a greater number of seropositive animals in comparison with a low-sensitivity immunodiffusion reaction. With a decrease in the herd infection rate to single cases of detection of animals infected with the leukemia virus at the final stages of rehabilitation of the enterprise, the number of additional seropositive animals detected by enzyme immunoassay increases. In the final period of herd recovery, the number of animals with a low level of antibodies to the leukemia virus, inaccessible to detection by immunodiffusion, increases. The use of an expensive and labor-consuming delivery of an enzymelinked immunosorbent assay to detect antibodies to the cattle leukemia virus is advisable at the final stages of an enterprise’s recovery. This enables to identify animals with a low level of antibodies to the leukemia virus, to speed up the negative result of a serological test of the entire herd and to exclude repeated outbreaks of infection of animals with this virus in a rehabilitated enterprise.

Download Full-text