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Human Zonulin ELISA Kit

Citations(10)Uniprot : P00738

  • Cat.No.:E-EL-H5560

  • Reactivity: Human
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  • 96T
  • 48T
  • 24T
  • 96T*5
  • 96T*10
Price: $609
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Product Details

Properties

Assay type Sandwich-ELISA
Format 96T/48T
Assay time 3.5h
Detection range 0.78-50 ng/mL
Sensitivity 0.47 ng/mL
Sample type &Sample volume serum, plasma and other biological fluids; 100μL
Specificity This kit recognizes Human Zonulin in samples. No significant cross-reactivity or interference between Human Zonulin and analogues was observed.
Reproducibility Both intra-CV and inter-CV are < 10%.
Application This ELISA kit applies to the in vitro quantitative determination of Human Zonulin concentrations in serum, plasma and other biological fluids.

Test Principle

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human Zonulin. Standards or samples are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human Zonulin and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human Zonulin, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human Zonulin. You can calculate the concentration of Human Zonulin in the samples by comparing the OD of the samples to the standard curve.

Kit components & Storage

An unopened kit can be stored at 2-8℃ for 1 month. If the kit is not supposed to be used within 1 month, store the components separately according to the following conditions once the kit is received.
ItemSpecificationsStorage
Micro ELISA Plate(Dismountable) 96T: 8 wells ×12 strips
48T: 8 wells ×6 strips 		-20℃, 6 months
Reference Standard 96T: 2 vials
48T: 1 vial
Concentrated Biotinylated Detection Ab (100×) 96T: 1 vial, 120 μL
48T: 1 vial, 60 μL
Concentrated HRP Conjugate (100×) 96T: 1 vial, 120 μL
48T: 1 vial, 60 μL 		-20℃(Protect from light), 6 months
Reference Standard & Sample Diluent 1 vial, 20 mL 2-8°C, 6 months
Biotinylated Detection Ab Diluent 1 vial, 14 mL
HRP Conjugate Diluent 1 vial, 14 mL
Concentrated Wash Buffer (25×) 1 vial, 30 mL
Substrate Reagent 1 vial, 10 mL 2-8℃(Protect from light)
Stop Solution 1 vial, 10 mL 2-8°C
Plate Sealer 5 pieces
Manual 1 copy
Certificate of Analysis 1 copy

Technical Data

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human Zonulin were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human Zonulin were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample 1 2 3 1 2 3
n 20 20 20 20 20 20
Mean (ng/mL) 2.46 5.57 22.64 2.31 6.06 23.45
Standard deviation 0.14 0.27 1.23 0.12 0.28 1.02
CV (%) 5.69 4.85 5.43 5.19 4.62 4.35

Recovery

The recovery of Human Zonulin spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum(n=8) 91-103 95
EDTA plasma (n=8) 87-99 91
Cell culture media (n=8) 91-99 95

Linearity

Samples were spiked with high concentrations of Human Zonulin and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

Target Information

Database LinksSwissProt:   P00738
Research AreaCancer, Cardiovascular

Assay Procedures

elisa assay procedure 1

1. Add 100μL standard or sample to the wells. Incubate for 90 min at 37°C
elisa assay procedure 2

2. Discard the liquid, immediately add 100μL Biotinylated Detection Ab working solution to each well. Incubate for 60 min at 37°C
elisa assay procedure 3

3. Aspirate and wash the plate for 3 times
elisa assay procedure 4

4. Add 100μL HRP conjugate working solution. Incubate for 30 min at 37°C. Aspirate and wash the plate for 5 times
elisa assay procedure 5

5. Add 90μL Substrate Reagent. Incubate for 15 min at 37°C
elisa assay procedure 6

6. Add 50μL Stop Solution
elisa assay procedure 7

7. Read the plate at 450nm immediately. Calculation of the results

Citations

From now on, if you have published a paper by using any of our products since 1/1/2019, fill out the “Elabscience Publication Reward Application Form”carefully and send it to orders@elabscience.com, we will get back to you with the reward after we confirm it ASAP!

View more details about our Publication Reward >>

  1. Hepatology (2020) IF: 14.679
    Increased Liver Localization of Lipopolysaccharides in Human and Experimental NAFLD

    DOI: 10.1002/hep.31056

    PMID: 31808577

    		 Sample: Serum
  2. Antioxidants and Redox Signaling (2018) IF: 6.53
    Does Nox2 over-activate in children with non-alcoholic fatty liver disease?.

    DOI: 10.1089/ars.2018.7596

    PMID: 30019598

    		 Sample: Serum
  3. Oxidative medicine and cellular longevity (2020) IF: 5.076
    Oxidative Stress and Gut-Derived Lipopolysaccharides in Neurodegenerative Disease: Role of NOX2

    DOI: 10.1155/2020/8630275

    PMID: 32089785

    		 Sample: Serum
  4. Nutrients (2020) IF: 4.546
    Intestinal Permeability in Children with Celiac Disease after the Administration of Oligofructose-Enriched Inulin into a Gluten-Free Diet-Results of a Randomized, Placebo-Controlled, Pilot Trial

    DOI: 10.3390/nu12061736

    PMID: 32531982

    		 Sample: Plasma
  5. Biomolecules (2019) IF: 4.694
    Plasma Zonulin Levels as a Non-Invasive Biomarker of Intestinal Permeability in Women with Gestational Diabetes Mellitus

    DOI: 10.3390/biom9010024

    PMID: 30641999

    		 Sample: Plasma
  6. Scientific Reports (2018) IF: 4.259
    Localization Of Lipopolysaccharide From Escherichia Coli Into Human Atherosclerotic Plaque.

    DOI: 10.1038/s41598-018-22076-4

    PMID: 29483584
  7. Endocrine (2020) IF: 3.235
    Could there be a role of serum zonulin increase in the development of hypercalcemia in primary hyperparathyroidism

    DOI: 10.1007/s12020-020-02504-0

    PMID: 32989570

    		 Sample: Serum
  8. Scientific reports (2020) IF: 3.998
    Assessment of haptoglobin alleles in autism spectrum disorders

    DOI: 10.1038/s41598-020-64679-w

    PMID: 32385356

    		 Sample: Serum
  9. International Journal of Psychiatry in Clinical Practice (2020) IF: 2.144
    Serum zonulin and claudin-5 levels in children with attention-deficit/hyperactivity disorder

    DOI: 10.1080/13651501.2020.1801754

    PMID: 32757874

    		 Sample: Serum
  10. BMC pediatrics (2020) IF: 1.909
    Oxidative stress and gut-derived lipopolysaccharides in children affected by paediatric autoimmune neuropsychiatric disorders associated with streptococcal infections

    DOI: 10.1186/s12887-020-02026-8

    PMID: 32188439

    		 Sample: Serum
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Reviews/Q&A

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  • Reviews
  • Q&A
Q

S*eSubmitted [ Sep 13 2022 ]

Asked: In your previous answers to other questions, you said, \"the immunogen sequences of the capture antibody and detection antibody were human haptoglobin 19-406aa (#P00738).\" So that would mean that haptoglobin could produce a false positive or falsely elevated \"zonulin\" result in your test. How do you know you are detecting zonulin/pre-HB2 and not haptoglobin? You also said, \"The antigen is full-length recombinant pre-HP2. The expression host of the antigen is HEK 293 cell.\" Which antigen are you referring to in that second statement, since you already stated that the antigen which the capture antibody is specific for is haptoglobin rather than zonulin/pre-HP2? If the capture and detection antibodies react with both haptoglobin and pre-HP2, how is your assay specific for only pre-HP2? Thank you.

Reply

A

adminSubmitted [ Sep 13 2022 ]

Answered: We applied anti-ZOT antibodies in this kit, ZOT is an enterotoxin elaborated by Vibrio cholera, a zonulin analogue. It has been reported that this antibody specifically recognizes the human zonulin (pre-HP2) instead of the mature Haptoglobin. In addition, we verified that the kit does not have significant cross-reactivity with mature HP protein.

Q

p***aSubmitted [ Mar 22 2022 ]

Asked: Good morning, We were thinking of purchasing this kit for an internal study regarding intestinal permeability. We were wondering if you could provide with information regarding the validity of this kit and the accuracy in detecting the pre-haptoglobin-2. Would appreciate quick reply! Thank you for your time and help!

Reply

A

adminSubmitted [ Mar 22 2022 ]

Answered: The zonulin protein is the inactive precursor for HP2 and it is an uncleaved single-chain form. For our kit, it can detect the Precursor of haptoglobin 2, the immunogen sequences of the capture antibody and detection antibody were human haptoglobin 19-406aa (#P00738). The sensitivity report will be shared via email.

Q

h****nSubmitted [ Mar 15 2022 ]

Asked: Hi, I am wondering about the information on the variability of zonulin assay? And what are zonulin and occludin coefficients of variation? of other indexes of test-retest reliability?

Reply

A

adminSubmitted [ Mar 15 2022 ]

Answered: Hi Hicran, thanks for your message. For your question, not only zonulin or occludin kits, but all of our kits need to strictly check the precision evaluation parameters before shipment, that is, the intra-assay and inter-assay coefficients of variation of the kits are both <10%. Please refer to the manuals to obtain the coefficient of variation information of each kit. Thank you.

Q

T***mSubmitted [ Dec 10 2021 ]

Asked: Does your kit not recognize complement C3, as it was said, \"Top matches of the assays\' detected antigen included haptoglobin and complement C3 for the assay manufactured by CUSABIO (Wuhan, China) and complement C3 for the assay manufactured by Immundiagnostik AG (Bensheim, Germany) after the nature of the assays\' detected antigen was investigated using immunoprecipitation followed by mass spectrometric analysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by protein staining. (Ajamian et al., PloS One, 2019)

Reply

A

adminSubmitted [ Dec 10 2021 ]

Answered: Thanks for your message. E-EL-H5560 Human Zonulin ELISA Kit Yes, the kit can recognize zonulin, but cannot recognize complement C3. We also found the document you mentioned in the attachment, these words don't mean one kit can recognize two proteins (zonulin, complement C3), which means the CUSABIO kit can detect serum zonulin levels, and our understanding literature says is human complement C3c, HP1-1, HP2-2 and recombinant zonulin (zon) standards were separated by SDS-PAGE and stained with Coomassie gel stain . If you want to test for C3, we recommend E-EL-H6054 Human C3(Complement Component 3) ELISA Kit for you. https://www.elabscience.com/p-human_c3_complement_component_3_elisa_kit-356179.html

Q

M*************iSubmitted [ Sep 09 2021 ]

Asked: Sorry for disturbing you again, I inquired few minutes ago about the reactivity of antibody to zonulin-recombinant. The another question is about What is the antigen when you make anti-zonulin antibody. I would appreciate your immediate attention to this matter.

Reply

A

adminSubmitted [ Sep 09 2021 ]

Answered: Dear Hayashi san,thanks for your message. This kit is specifically designed to detect Zonulin. The kit can be used to detect natural Zonulin. In theory, the kit also can be used for recombinant Zonulin. However, due to the influence of the immunogen sequence and the expression host of the recombinant protein, some recombinant Zonulin may not match the antibodies in the kit. Therefore, a pre-test is recommended. The antigen is full-length recombinant pre-HP2. The expression host of the antigen is HEK 293 cell. Best Regards, Y

Q

H*************oSubmitted [ Sep 09 2021 ]

Asked: Recently some ELISA kits for zonulin are in controversy because they actually don\'t detect zonulin. I would like to know that you confirm antibodies used in your kits capture zonulin-recombinants because I would like to purchase reliable products. Thank you.

Reply

A

adminSubmitted [ Sep 09 2021 ]

Answered: Dear Hayashi san,thanks for your message. We have replied to you in your second message column, please check it out, thank you! Best regards,

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