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Human Zonulin ELISA Kit

Citations(37)Uniprot : P00738

Cat.No.:E-EL-H5560
Reactivity: Human

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Product Details

Properties

Assay type	Sandwich-ELISA
Format	96T/48T/24
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.

Item	Specifications	Storage
Micro ELISA Plate(Dismountable)	96T: 8 wells ×12 strips 48T: 8 wells ×6 strips	-20℃, 12 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), 12 months
Reference Standard & Sample Diluent	1 vial, 20 mL	2-8°C, 12 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 Links	SwissProt: P00738
Research Area	Cancer, Cardiovascular

Assay Procedures

	1. Add 100μL standard or sample to the wells. Incubate for 90 min at 37°C
	2. Discard the liquid, immediately add 100μL Biotinylated Detection Ab working solution to each well. Incubate for 60 min at 37°C
	3. Aspirate and wash the plate for 3 times
	4. Add 100μL HRP conjugate working solution. Incubate for 30 min at 37°C. Aspirate and wash the plate for 5 times
	5. Add 90μL Substrate Reagent. Incubate for 15 min at 37°C
	6. Add 50μL Stop Solution
	7. Read the plate at 450nm immediately. Calculation of the results

Citations

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

PHARMACOLOGICAL RESEARCH (2020) IF: 5.893

Probiotics and fructo-oligosaccharide intervention modulate the microbiota-gut brain axis to improve autism spectrum reducing also the hyper-serotonergic state and the dopamine metabolism disorder

DOI: 10.1016/j.phrs.2020.104784
	Sample: Plasma

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES (2022) IF: 6.208

Macrophage Activation Markers Predict Liver-Related Complications in Primary Biliary Cholangitis

DOI: 10.3390/ijms23179814
PMID: 36077228	Sample: Serum

Biomedicines (2023) IF: 4.757

The Effects of Probiotics on Small Intestinal Microbiota Composition, Inflammatory Cytokines and Intestinal Permeability in Patients with Non-Alcoholic Fatty Liver Disease

DOI: 10.3390/biomedicines11020640
	Sample: Serum

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES (2021) IF: 5.924

Examining the Gastrointestinal and Immunomodulatory Effects of the Novel Probiotic Bacillus subtilis DE111

DOI: 10.3390/ijms22052453
PMID: 33671071	Sample: Plasma

JGH Open (2021)

Intestinal fatty acid-binding protein is a biomarker for diagnosis of biliary tract infection

DOI: 10.1002/jgh3.12644
	Sample: Blood

Turkish Journal of Biochemistry-Turk Biyokimya Dergisi (2021) IF: 0.401

The role of zonulin in the pathogenesis of diabetic retinopathy

DOI: 10.1515/tjb-2020-0575
	Sample: Serum

JOURNAL OF OBSTETRICS AND GYNAECOLOGY (2020) IF: 0.807

Association of adverse outcomes of intrahepatic cholestasis of pregnancy with zonulin levels

DOI: 10.1080/01443615.2020.1820463
PMID: 33228404	Sample: Serum

African Health Sciences (2021) IF: 0.927

Gut permeability is associated with hypertension and measures of obesity but not with Endothelial Dysfunction in South African youth

DOI: 10.4314/ahs.v21i3.26
	Sample: Plasma

NORDIC JOURNAL OF PSYCHIATRY (2020) IF: 1.78

Serum zonulin and claudin-5 levels in children with obsessive–compulsive disorder

DOI: 10.1080/08039488.2020.1715474
	Sample: Serum

Journal of Clinical Research in Pediatric Endocrinology (2020) IF: 1.803

Children with Hashimoto’s Thyroiditis Have Increased Intestinal Permeability: Results of a Pilot Study

DOI: 10.4274/jcrpe.galenos.2020.2019.0186
PMID: 31990165	Sample: Serum

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

JOURNAL OF PAEDIATRICS AND CHILD HEALTH (2022) IF: 1.954

Zonulin and claudin-5 levels in multisystem inflammatory syndrome and SARS-CoV-2 infection in children

DOI: 10.1111/jpc.16033
	Sample: Serum

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

ARCHIVES OF GYNECOLOGY AND OBSTETRICS (2019) IF: 2.199

Serum zonulin level is not elevated in patients with polycystic ovary syndrome without metabolic syndrome

DOI: 10.1007/s00404-019-05345-z
	Sample: Serum

Journal of Cosmetic Dermatology (2022) IF: 2.696

Is there a link between alopecia areata and gut?

DOI: 10.1111/jocd.15095
PMID: 35579378	Sample: Serum

CHRONOBIOLOGY INTERNATIONAL (2022) IF: 2.877

Changes in intestinal parameters and their association with dietary patterns in rotational shift workers

DOI: 10.1080/07420528.2022.2044349
PMID: 35232303	Sample: Serum

HIV MEDICINE (2021) IF: 3.18

Lipopolysaccharide induces platelet activation in HIV patients: the role of different viral load patterns

DOI: 10.1111/hiv.13059
PMID: 33426758	Sample: Serum

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

EUROPEAN ARCHIVES OF PSYCHIATRY AND CLINICAL NEUROSCIENCE (2020) IF: 3.288

Serum zonulin and claudin-5 levels in patients with schizophrenia

DOI: 10.1007/s00406-020-01152-9
	Sample: Serum

International Journal of Environmental Research and Public Health (2021) IF: 3.39

Detection of Dysbiosis and Increased Intestinal Permeability in Brazilian Patients with Relapsing–Remitting Multiple Sclerosis

DOI: 10.3390/ijerph18094621
	Sample: Serum

JOURNAL OF MOLECULAR NEUROSCIENCE (2022) IF: 3.444

Investigation of the Relation between Epithelial Barrier Function and Autism Symptom Severity in Children with Autism Spectrum Disorder

DOI: 10.1007/s12031-021-01954-z
PMID: 34988901	Sample: Serum

NUTRITION METABOLISM AND CARDIOVASCULAR DISEASES (2017) IF: 3.679

Low-grade endotoxemia, gut permeability and platelet activation in patients with impaired fasting glucose

DOI: 10.1016/j.numecd.2017.06.007
	Sample: Serum

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

Scientific Reports (2018) IF: 4.122

Localization of lipopolysaccharide from Escherichia Coli into human atherosclerotic plaque

DOI: 10.1038/s41598-018-22076-4
	Sample: Serum

AIDS (2022) IF: 4.177

Plasma d-amino acids are associated with markers of immune activation and organ dysfunction in people with HIV

DOI: 10.1097/QAD.0000000000003207
PMID: 35212669	Sample: Plasma

Clinical and Translational Gastroenterology (2021) IF: 4.488

Low-Grade Endotoxemia and Thrombosis in COVID-19

DOI: 10.14309/ctg.0000000000000348
PMID: 34092777	Sample: Serum

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
	Sample: Plasma

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

Nutrients (2021) IF: 5.719

Microbiome as Mediator of Diet on Colorectal Cancer Risk: The Role of Vitamin D, Markers of Inflammation and Adipokines

DOI: 10.3390/nu13020363
PMID: 33504116	Sample: Serum

Nutrients (2022) IF: 5.719

Extra Virgin Olive Oil Reduces Gut Permeability and Metabolic Endotoxemia in Diabetic Patients

DOI: 10.3390/nu14102153
PMID: 35631294	Sample: Serum

ANTIOXIDANTS & REDOX SIGNALING (2019) IF: 5.828

Does Nox2 Overactivate in Children with Nonalcoholic Fatty Liver Disease?

DOI: 10.1089/ars.2018.7596
	Sample: Serum

Nutrients (2022) IF: 6.706

Effects of Flavonoid-Rich Orange Juice Intervention on Major Depressive Disorder in Young Adults: A Randomized Controlled Trial

DOI: 10.3390/nu15010145
	Sample: Serum

ANTIOXIDANTS & REDOX SIGNALING (2020) IF: 7.04

Is There an Association Between Atherosclerotic Burden, Oxidative Stress, and Gut-Derived Lipopolysaccharides?

DOI: 10.1089/ars.2020.8109
	Sample: Serum

FOOD CHEMISTRY (2021) IF: 7.514

In vitro digestibility and Caco-2 cell bioavailability of sea lettuce (Ulva fenestrata) proteins extracted using pH-shift processing

DOI: 10.1016/j.foodchem.2021.129683
PMID: 33845254	Sample: Cell

Frontiers in Immunology (2021) IF: 7.561

Detection of Alterations in the Gut Microbiota and Intestinal Permeability in Patients With Hashimoto Thyroiditis

DOI: 10.3389/fimmu.2021.579140
	Sample: Serum

HEPATOLOGY (2020) IF: 17.425

Increased Liver Localization of Lipopolysaccharides in Human and Experimental NAFLD

DOI: 10.1002/hep.31056
PMID: 31808577	Sample: Serum

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Reviews/Q&A

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Reviews
Q&A

A************iSubmitted [ Jul 27 2024 ]

Asked: \"Different target proteins are detected, and the sample content is different. It is recommended that you do a pre-test according to the original solution and dilute 5 times to confirm the concentration of the target protein in the feces samples.\" How much of the diluent should I use each time? Should I use PBS or specimen diluent?

adminSubmitted [ Jul 27 2024 ]

Answered: You can dilute the sample using the Reference Standard & Sample Diluent in the kit. When the sample is not diluted, it is not necessary to use standard product & sample diluent for sample loading. Sample dilution of 5 times requires at least 20ul sample +80ul standard & sample diluent. The loading volume of the sample is 100ul.

A************iSubmitted [ Jul 16 2024 ]

Asked: HI, when we a preparing the samples to the procedure using feces samples the supernates need to be diluted before starting the protocol of the kit?

adminSubmitted [ Jul 16 2024 ]

Answered: Different target proteins are detected, and the sample content is different. It is recommended that you do a pre-test according to the original solution and dilute 5 times to confirm the concentration of the target protein in the feces samples.

A************iSubmitted [ Jan 25 2024 ]

Asked: Hello, can I use this test on stool samples?

adminSubmitted [ Jan 25 2024 ]

Answered: Yes, this ELISA kit can be used to test feces samples.

H**********aSubmitted [ Dec 13 2023 ]

Asked: Hii, i want to know the range of zonulin levels that we will consider as normal and above which we will consider that the zonulin levels are elevated. Thank you.

adminSubmitted [ Dec 13 2023 ]

Answered: For E-EL-H5560 kit, the reference value of normal human serum samples is 40-115ng/ml, according to our internal verification result. However, considering about the individual differences, we recommend that you set up your own normal sample group as control in order to determine whether the zonulin levels are elevated.

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.

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.

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!

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.

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?

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.

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)

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

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.

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

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.