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Human Zonulin ELISA Kit (HUES03536)

SKU:
HUES03536
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P00738
Sensitivity:
0.47ng/mL
Range:
0.78-50ng/mL
ELISA Type:
Sandwich
Reactivity:
Human
Sample Type:
Serum, plasma and other biological fluids
  • Human Immunology ELISA Kits 13 Human Zonulin ELISA Kit HUES03536
  • Human Zonulin ELISA Kit (HUES03536)
  • Human Zonulin ELISA Kit (HUES03536)
€699
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Description

Human Zonulin ELISA Kit

Specific Specifically detects Zonulin (pre-Haptoglobin 2)
Cross-Reactivity Does not cross-react with Haptoglobin 2
Sensitive 0.47 ng/mL level of sensitivity
Sample Type This kit can be used for cell culture media samples, and we have tested the HepG2 cell culture media sample.
Cited Development of an Inflammation-Triggered In Vitro “Leaky Gut” Model Using Caco-2/HT29-MTX-E12 Combined with Macrophage-like THP-1 Cells or Primary Human-Derived Macrophages

Zonulin is a protein produced by the immune system that is known to cause tight junctions to open between epithelial cells. This opening of tight junctions can cause gut permeability, meaning Zonulin may be used as a biomarker for 'leaky gut syndrome'. Zonulin levels have also been found to be elevated in certain individuals with autoimmune conditions, most notably celiac disease, where they are known as being 'epitope-grabbing Zonulin-binding Zones' (abbreviated as EGA Zonulin-binding Zones). Furthermore, Zonulin levels have also been found to be elevated in certain individuals with Type 1 diabetes, arthritis, and other autoimmune conditions. Zonulin ELISA kits are important for both the diagnosis of Zonulin-related diseases and Zonulin's role in autoimmune disease.

system_update_alt Datasheet system_update_alt MSDS system_update_alt Validation Data

Key Features

Save Time Pre-coated 96 well plate
Quick Start Kit includes all necessary reagents
Publication Ready Reproducible and reliable results

Overview

Product Name:

Human Zonulin ELISA Kit

Product Code:

HUES03536

Size:

96 Assays

Assay Time:

4.5 hours

Detection Method:

Colorometric

Sample Volume:

100µL

Sensitivity:

0.47 ng/mL

Range:

0.78-50 ng/mL

Storage:

4°C for 6 months

Note:

For Research Use Only

Kit Principle

This ELISA kit uses Sandwich-ELISA as the method. 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 appropriate 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 to each micro plate well successively 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 adding 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. The concentration of Human Zonulin in samples can be calculated by comparing the OD of the samples to the standard curve.

Typical Data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (ng/ml) O.D Average Corrected

50

2.558

2.416

2.487

2.425

25

1.672

1697

1.685

1.623

12.5

0.991

0.96

0.976

0.914

6.25

0.464

0.486

0.475

0.413

3.13

0.275

0.272

0.274

0.212

1.57

0.176

0.179

0.178

0.116

0.78

0.127

0.119

0.123

0.061

0

0.06

0.063

0.062

-

Additional Information

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human Zonulin ELISA Kit 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 ELISA Kit 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

Matrices listed below were spiked with certain level of Zonulin and the recovery rates were calculated by comparing the measured value to the expected amount of Zonulin in samples.

Sample Type Recovery Range (%) Average (%)

serum (n=5)

91-103

95

EDTA plasma (n=5)

87-99

91

Cell Culture Media (n=5)

91-99

95

Linearity

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

Serum (n=5) EDTA Plasma (n=5) Cell Culture media (n=5)

1:2

Range(%)

Average(%)

85-101

93

98-109

105

94-105

101

1:4

Range (%)

Average(%)

84-96

90

100-109

105

91-100

95

1:8

Range (%)

Average (%)

94-103

100

91-103

97

88-99

93

1:16

Range (%)

Average (%)

96-110

104

93-102

96

92-104

100

Samples were spiked with high concentrations of Zonulin and diluted with Standard & Sample Diluent. The diluted samples were within the range of the assay.

Kit Components

Component Specifications Storage

ELISA Microplate (Dismountable)

8x12 strips

-20°C , 6 months

Reference Standard

2 vials

-

Concentrated Biotinylated Detection Ab (100×)

1 vial, 120 µL

-

Concentrated HRP Conjugate (100×)

1 vial, 120 µL

-20°C (shading light), 6 months

Reference Standard & Sample Diluent

1 vial, 20 µL

4°C, 6 months

Biotinylated Detection Ab Diluent

1 vial, 14 µL

-

HRP Conjugate Diluent

1 vial, 14 µL

-

Concentrated Wash Buffer (25×)

1 vial, 30 µL

-

Substrate Reagent

1 vial, 10 µL

4°C (shading light)

Stop Solution

1 vial, 10 µL

4°C

Plate Sealer

5

-

Product Description

1 copy

-

Certificate of Analysis

1 copy

-

Protocol

*Note: Protocols are specific to each batch/lot. For the exact instructions please follow the protocol included in your kit.

Step Procedure

1.

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.

2.

Aliquot 100µl of standard solutions into the standard wells.

3.

Add 100µl of Sample / Standard dilution buffer into the control (zero) well.

4.

Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.

5.

Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.

6.

Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.

7.

Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.

8.

Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.

9.

Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.

10.

Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.

11.

Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.

12.

Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Zonulin Background

Zonulin gene (HP gene)

The gene that codes for Zonulin is the Haptoglobin gene (HP). Primarily a gene that encodes Haptoglobin, Zonulin is the pre-protein form of Haptoglobin. This gene is located on chromosome 16q22.1 and consists of several exons and introns. The specific genetic sequence of the Zonulin gene determines the structure and function of the zonulin protein.

Hp exists in two allelic forms in the human population, so-called Hp1 and Hp2. One of the well-known variations in the Zonulin gene is the presence of different copy numbers of the HP2 allele. The HP2 allele has two repeated units, while the more common HP1 allele has a single repeated unit. Some studies have suggested a potential association between the HP2 allele and increased intestinal permeability or susceptibility to certain diseases.

Zonulin Protein

Zonulin is a protein that belongs to the family of proteins called pre-haptoglobins. It is composed of approximately 47 kilodaltons (kDa) and is comprised of 402 amino acids. The zonulin protein consists of a signal peptide, which helps in its secretion, followed by two functional domains: the N-terminal and C-terminal domains.

The N-terminal domain of zonulin contains a region known as the "occludin-binding region," which interacts with occludin, a tight junction protein. This interaction is believed to play a role in the regulation of tight junction permeability. The N-terminal domain also contains a sequence of amino acids that is similar to a domain found in certain bacterial proteins known as zonula occludens toxin (Zot). This similarity suggests that zonulin may have evolved from a bacterial precursor.

The C-terminal domain of zonulin contains a repeat region, known as the "zonulin-receptor-binding domain." This region is involved in binding to specific receptors on the surface of cells, triggering intracellular signaling pathways that affect tight junction permeability.

the structure of zonulin allows it to interact with occludin and potentially other proteins, as well as engage with specific receptors on cell surfaces. These interactions and signaling pathways modulate the tight junctions between epithelial cells, influencing the permeability of the intestinal barrier and potentially impacting overall gut health.

Zonulin Function and Pathology

Zonulin is a protein involved in regulating intestinal permeability and the integrity of tight junctions. Its normal function is to maintain a tight barrier between cells in the intestinal lining, controlling the passage of substances. However, dysregulation of zonulin can lead to increased intestinal permeability, known as leaky gut, which has been associated with various diseases. Zonulin dysregulation may contribute to autoimmune diseases, inflammatory bowel disease, metabolic disorders, neurological disorders, allergies, and asthma. Understanding the function and pathology of zonulin provides insights into the mechanisms underlying these conditions and potential avenues for therapeutic interventions.

Elevated zonulin levels may suggest increased intestinal permeability and dysfunction of the intestinal barrier. A zonulin test is a diagnostic test used to measure the levels of zonulin in the body. It is primarily performed to assess intestinal permeability and determine if there is increased permeability or "leaky gut" present. The zonulin test is not widely available and is still being researched as a diagnostic tool, so its clinical utility and accuracy are not yet fully established.

Citations

Le et al. Development of an Inflammation-Triggered In Vitro “Leaky Gut” Model Using Caco-2/HT29-MTX-E12 Combined with Macrophage-like THP-1 Cells or Primary Human-Derived Macrophages International Journal of Molecular Sciences (2023) Pub Med: 37108590

Citation Data

The significant increased permeability highly corresponded to a significant production of zonulin, a proposed regulator of the permeability of the intestinal barrier [51], in the “leaky gut” model after 6 h (Figure 5E) and 24 h (Figure 5F). As seen in Figure 5E, within 6 h, the mean concentration of zonulin in both compartments of the “leaky gut” model (2043 ± 464 pg/mL) was nearly 25-fold higher than in the control model (83 ± 143 pg/mL). After 24 h, the mean concentration of zonulin in both compartments of the “leaky gut” model (1982 ± 260 pg/mL) was around 1.3-fold higher than the control model (1664 ± 29 pg/mL) (Figure 5F).

 As shown in Figure 5E,F, while there was no change in Tight Junction Protein-1 (TJP1) between the “leaky gut” and control models after 6 h (24 ± 7 pg/mL and 22 ± 8 pg/mL, respectively) and 24 h (141 ± 12 pg/mL and 147 ± 6 pg/mL, respectively), the amount of released occludin in the “leaky gut” model increased from 66 ± 13 pg/mL at 6 h to 104 ± 49 pg/mL at 24 h. On the other hand, the amount of occludin in the control model decreased from 54 ± 15 pg/mL (6 h) to 51 ± 60 pg/mL (24 h).

Zonulin ELISA FAQs

Q: What assurances are there that this ELISA kit specifically detects zonulin?

Our ELISA kit is specifically designed to detect natural Zonulin. It can also theoretically detect recombinant Zonulin, but a pre-test is recommended to confirm this. The antigen used for generating the anti-Zonulin antibodies is full-length recombinant pre-HP2, expressed in HEK 293 cells.

Q: Does the Zonulin ELISA Kit recognize complement C3?

No, our Zonulin ELISA kit is designed to specifically recognize Zonulin and does not cross-react with complement C3. If you are interested in testing for complement C3, we recommend our specific Human C3 ELISA Kit.

Q: What information is available about the variability and coefficients of variation of the Zonulin assay?

All of our kits, including those for zonulin and occludin, are subject to strict quality control. Both the intra-assay and inter-assay coefficients of variation for our kits are less than 10%. For specific coefficient of variation information for each kit, please refer to the accompanying manuals.

Q: Can this kit be used for studies on intestinal permeability and how accurate is it in detecting pre-haptoglobin-2?

Yes, this ELISA kit is designed to detect the Precursor of haptoglobin 2. The capture and detection antibodies were generated against the sequence of human haptoglobin 19-406aa (#P00738). Sensitivity details will be shared via email upon request.

Q: How does the kit differentiate between zonulin/pre-HP2 and haptoglobin?

The kit employs anti-ZOT antibodies, which are known analogues of zonulin. These antibodies specifically recognize human zonulin (pre-HP2) and not mature haptoglobin. We have also verified through testing that our kit does not show significant cross-reactivity with mature haptoglobin proteins.

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Citations

Le et al. Development of an Inflammation-Triggered In Vitro “Leaky Gut” Model Using Caco-2/HT29-MTX-E12 Combined with Macrophage-like THP-1 Cells or Primary Human-Derived Macrophages International Journal of Molecular Sciences (2023) Pub Med: 37108590