ELISA Kit for Immunoglobulin A (IgA)

ELISA Kit for Immunoglobulin A (IgA)

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 1 hours at 37°C;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.

Specificity

This assay has high sensitivity and excellent specificity for detection of Immunoglobulin A (IgA).
No significant cross-reactivity or interference between Immunoglobulin A (IgA) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Immunoglobulin A (IgA) and the recovery rates were calculated by comparing the measured value to the expected amount of Immunoglobulin A (IgA) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 81-94 88
EDTA plasma(n=5) 89-103 97
heparin plasma(n=5) 96-105 101

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Immunoglobulin A (IgA) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Immunoglobulin A (IgA) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Immunoglobulin A (IgA) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 92-101% 85-92% 94-102% 96-103%
EDTA plasma(n=5) 94-102% 95-105% 89-97% 87-99%
heparin plasma(n=5) 88-96% 92-99% 85-93% 89-103%

Stability

The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Reagents and materials provided

Reagents Quantity Reagents Quantity
Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4
Standard 2 Standard Diluent 1×20mL
Detection Reagent A 1×120µL Assay Diluent A 1×12mL
Detection Reagent B 1×120µL Assay Diluent B 1×12mL
TMB Substrate 1×9mL Stop Solution 1×6mL
Wash Buffer (30 × concentrate) 1×20mL Instruction manual 1
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Magazine Citations
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Poultry Science Effects of Clostridium butyricum on growth performance, immune function, and cecal microflora in broiler chickens challenged with Escherichia coli K88 Pubmed: 24570422
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Pediatrics Oropharyngeal colostrum administration in extremely premature infants: an RCT. Pubmed:25624376
Front Microbiol Fecal microbiota transplantation and bacterial consortium transplantation have comparable effects on the re-establishment of mucosal barrier function in mice with intestinal dysbiosis PubMed: 26217323
Food and Agricultural Immunology Effects of fermented cottonseed meal on growth performance, serum biochemical parameters, immune functions, antioxidative abilities, and cecal microflora in broilers 10.108:09540105.2017.1311308
SYSTEMISCHE ENTZÜNDUNG BEI CHRONISCHER NIERENINSUFFIZIENZ – DIE ROLLE DER DARMFLORA patent:US9763998B2
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Food and Agricultural Immunology Effects of and on growth performance, immune function and volatile fatty acid level of caecal digesta in broilers 10.1080:09540105.2018.1457013
Probiotics Antimicrob Proteins Effect of Multi-Microbial Probiotic Formulation Bokashi on Pro-and Anti-Inflammatory Cytokines Profile in the Serum, Colostrum and Milk of Sows, and in a … Pubmed:29305686
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Nephrology Dialysis Transplantation The gut flora modulates intestinal barrier integrity but not progression of chronic kidney disease in hyperoxaluria-related nephrocalcinosis Pubmed: 31081025
Current Developments in Nutrition Yacon (Smallanthus sonchifolius): Effect on Integrity of Intestinal Barrier, Inflammatory Response and Oxidative Stress in Animal Model of Colon Cancer (P06-052-19) Pubmed: 31225040
Nutrients. Effects of Paramylon Extracted from Euglena gracilis EOD-1 on Parameters Related to Metabolic Syndrome in Diet-Induced Obese Mice Pubmed: 31330894
International Journal of Biological Macromolecules Effects of polysaccharides from Yingshan Yunwu tea on meat quality, immune status and intestinal microflora in chickens Pubmed: 32224178
Effect Long-Term Exposure to Different Doses of Lipopolysaccharides on the Intestinal Mucosal Immune Barrier
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