Evaluation of a genus-specific rGroEL 1-524 IgM-ELISA and commercial ELISA kits during the course of leptospirosis in Thailand

In the present study, we developed a genus-specific rGroEL1-524 IgM-ELISA assay for use in screening diagnosis of suspected leptospirosis among acute undifferentiated febrile illness patients during acute fever. The diagnostic accuracies of the rGroEL1-524 IgM-ELISA, commercial Panbio IgM-ELISA, and Virion-Serion Classic IgG-ELISA were evaluated using 133 Thai leptospirosis sera and 210 controls. Sensitivities were 91.7%, 59.6%, and 17.7% for acute infection, and the specificities were 92.6%, 90.2%, and 88.3% for the non-leptospirosis control, respectively. The rGroEL1-524 IgM-ELISA had high sensitivity, at 92.3% and 91.7%, among culture-positive and MAT-negative cases at 1-3 days post-onset of symptoms (DPO1-3), respectively. Impaired specificity on scrub typhus was found, possibly from antibody cross-reaction to ortholog GroEL.
Commercial Panbio IgM-ELISA had sensitivities at DPO1-3 of 30.8% and 41.7% for culture-positive and MAT-negative cases whereas Virion-Serion IgG-ELISA showed sensitivities of 5.9% and 13.3%, respectively. The rGroEL1-524 IgM-ELISA could be useful as a screening test for early diagnosis. The performance of the commercial ELISA suggests the applicability of IgM-ELISA for diagnosis, while IgG-ELISA is useful for seroprevalence surveys. However, confirmation by reference tests is recommended.

Development of Human Toxo IgG ELISA Kit, and False-Positivity of Latex Agglutination Test for the Diagnosis of Toxoplasmosis

Toxoplasma gondii is an intracellular zoonotic parasite that causes infection in a wide range of warm-blooded animals and humans. The main aim of this study was to assess the diagnostic value of the recombinant SAG1 antigen (rSAG1) for T. gondii-IgG screening through the Human Toxo IgG ELISA Kit (K). The rSAG1 was expressed in E. coli (DE3), and it was purified through metal-affinity chromatography. The rSAG1 was confirmed by immunoblotting, and it had a band on 35 kDa. Total of 400 human sera were tested by LAT and K. One hundred and twenty-two (30.5%) sera were found positive by LAT and eighty-nine (22.25%) sera were found positive by K. Out of 400 samples, 80 were selected to evaluate the performance of K through commercial Toxoplasma gondii IgG ELISA Kit (C). Out of 80 human sera, 55 (68.75%) were found positive, 25 (31.25%) were found negative by K and C, respectively.
The cut-off value for K was 0.398 and it was calculated through the receiver operator characteristic curve. The ELISA plates were coated at optimized concentration of rSAG1 = 0.125 µg/mL, and the test was performed by diluting the sera at 1:50. The sensitivity and specificity of K were observed to be 98.5% and 100%, respectively. The six sera (KL+) were found positive through LAT and these human sera were later evaluated by Western blot analysis. These sera did not produce a band equivalent to 35 kDa on WB analysis thus, LAT produced false-positive results.

Sero-diagnostic efficacy of various ELISA kits for diagnosis of infectious bovine rhinotracheitis (IBR) in cattle and buffaloes in India

Bovine alphaherpesvirus-1 (BoHV-1), the causative agent of infectious bovine rhinotracheitis (IBR), is an economically important viral pathogen affecting cattle and buffaloes. Serological assays are mostly used for detection of the antibodies, but variation has been detected in the diagnostic performances of the individual assay. In the present study, four commercially available ELISA kits {two indirect ELISA (kits A and B) and two blocking ELISA (kits C and D)} were evaluated for the detection of antibodies against BoHV-1 in Indian cattle and buffaloes (fitness of purpose). The diagnostic sensitivity (dsn) and specificity (dsp) of these kits were determined by three ways; considering virus neutralization test (VNT) as gold standard test, using pre-test information of the samples, and majority of tests. Screening of 200 known negative sera (124 cattle, 76 buffaloes) sourced from IBR free farms revealed gB based ELISA kits are more specific than the indirect ELISA kits.
Testing of 125 known positive sera (81 cattle, 44 buffaloes) suggests kit B be most sensitive followed by kit C, A and D. Interestingly, kit D was found to be most sensitive for detection of vaccination-induced BoHV-1 antibodies followed by kit B. Similar trend were also observed in the limit of dilution experiment performed using known infected and vaccinated sera. VNT was found to be the most specific test and its use as the gold standard test revealed all kits to have more than 99 % sensitivity. All the ELISA kits could detect BoHV-1 specific antibodies in the IBR vaccinated calves as early as 11 days post-vaccination. In Kappa statistics, an almost perfect agreement between the ELISA kits was recorded. The overall performance of the kits in serodiagnosis of IBR as determined by the area under curve in ROC analysis was good.

Measurement of progesterone in sheep using a commercial ELISA kit for human plasma

Determination of serum or plasma progesterone (P4) concentrations is important to recognize pregnant and non-pregnant ewes, and also to predict the number of carried lambs. The 2 most common methodologies for the detection of plasma P4 are radioimmunoassay (RIA) and enzyme immunoassay (EIA). RIA is very expensive, and not all laboratories are equipped to perform this test; EIA is commercially available for human use, but only a few companies produce species-specific kits, which are expensive.
We verified for ovine plasma a less expensive and easily available ELISA kit (DiaMetra) designed to quantify P4 in humans. Pools of ovine and human plasma were used to compare repeatability, accuracy, sensitivity, and stability of P4 measured by the DiaMetra kit. Repeatability data were within 15%, and accuracy values were ~90% for both plasma matrices. Stability data showed a loss of <20% for freeze-thaw and <30% for 30-d storage. All parameters were acceptable under international guidelines for method validation. The human ELISA kit was used successfully to quantify plasma P4 in 26 ewes during pregnancy until delivery. P4 concentrations were also correlated with the number of carried lambs.

T-Pro Plasmid Mini Kit (100)

RB94-YPD100 T-Pro Biotechnology 100preps/Kit 193.2 EUR

T-Pro Plasmid Mini Kit (250)

RB94-YPD250 T-Pro Biotechnology 250preps/Kit 266.4 EUR

T-Pro Plasmid Midi Kit (20)

RB94-YPI020 T-Pro Biotechnology 20preps/Kit 255.6 EUR

T-Pro Plasmid Maxi Kit (10)

RB94-YPM010 T-Pro Biotechnology 10preps/Kit 266.4 EUR

T-Pro BCA Protein Assay kit

JB04-D001 T-Pro Biotechnology 500assay/KIT 244.8 EUR

T-Pro Cell Counting Kit (CCK8)

JK95-C008L T-Pro Biotechnology 5ml*20/BT 1000 EUR

T-Pro Cell Counting Kit (CCK8)

JK95-C008M T-Pro Biotechnology 5ml*2/BT 120 EUR

T-Pro Cell Counting Kit (CCK8)

JK95-C008S T-Pro Biotechnology 1ml*5/BT 80 EUR

T-Pro LumiDura Chemiluminescence Detection Kit (ECL Kit)

JT96-K006M T-Pro Biotechnology 250ml*2/Kit 200 EUR

T-Pro LumiDura Chemiluminescence Detection Kit (ECL Kit)

JT96-K006S T-Pro Biotechnology 100ml*2/Kit 100 EUR

T-Pro Genomic DNA Mini Kit (100)

RB94-NGS100 T-Pro Biotechnology 100preps/kit 255.6 EUR

T-Pro Genomic DNA Midi Kit (20)

RB94-NGM020 T-Pro Biotechnology 20preps/kit 266.4 EUR

T-Pro Bradford Protein Assay kit(1X)

JB04-D002 T-Pro Biotechnology 500ml/BT 193.2 EUR

T-Pro Bradford Protein Assay kit (5X)

JB04-D003 T-Pro Biotechnology 500ml/BT 120 EUR

T-Pro LumiFast Plus Chemiluminescence Detection Kit (ECL Kit)

JT96-K002M T-Pro Biotechnology 250ml*2/Kit 244.8 EUR

T-Pro LumiFast Plus Chemiluminescence Detection Kit (ECL Kit)

JT96-K002S T-Pro Biotechnology 100ml*2/Kit 182.4 EUR

T-Pro LumiLong Plus Chemiluminescence Detection Kit (ECL Kit)

JT96-K004M T-Pro Biotechnology 250ml*2/Kit 286.8 EUR

T-Pro LumiLong Plus Chemiluminescence Detection Kit (ECL Kit)

JT96-K004S T-Pro Biotechnology 100ml*2/Kit 204 EUR

T-Pro Plant Genomic DNA Mini Kit (100)

RB94-PGS100 T-Pro Biotechnology 100preps/kit 266.4 EUR

T-Pro Gradient Gel Solution kit 4-12%

JB02-B110M T-Pro Biotechnology 250ml+100ml/kit 60 EUR

T-Pro Plant Genomic DNA Midi Kit (20)

RB94-PGM020 T-Pro Biotechnology 20preps/kit 266.4 EUR

T-Pro Endotoxin Removal Plasmid Midi kit (25)

RB94-EPI020 T-Pro Biotechnology 20preps/Kit 297.6 EUR

T-Pro Endotoxin Removal Plasmid Maxi kit (10)

RB94-EPM010 T-Pro Biotechnology 10preps/Kit 318 EUR

T-Pro Gel/PCR DNA Purification Mini Kit (100)

RB94-NES100 T-Pro Biotechnology 100preps/Kit 193.2 EUR

T-Pro Gel/PCR DNA Purification Mini Kit (250)

RB94-NES250 T-Pro Biotechnology 250preps/Kit 266.4 EUR

T-Pro Gel/PCR DNA Purification Maxi Kit (10)

RB94-NEL010 T-Pro Biotechnology 10preps/Kit 193.2 EUR

A leptin sandwich ELISA kit unusable for domestic animals

An instance of hormone assay method flaw is reported. In this journal Chronobiology International, two papers appeared in which an ELISA method for human serum or plasma was utilized for blood serum of horse and sheep, respectively. From our testing, it is resulted that such method does not work at all for equine, sheep and other animal species.
The use of commercial hormone assay kits for heterologous species always needs a careful validation procedure. First, the same hormone molecule by different species could not share enough homology to be regognized by and react with antibodies utilized in the method. Furthermore, even with a full overlap of the molecules, possible interferences by other components of the sample (matrix effect) have to be considered.

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