Human zonulin ELISA kit

Code CSB-EQ027649HU
Size 96T,5×96T,10×96T How to order?
Trial Size 24T ELISA kits trial application
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Product Details

Description

This Human zonulin ELISA Kit was designed for the quantitative measurement of Human zonulin protein in serum, plasma, tissue homogenates. It is a Sandwich ELISA kit, its detection range is 0.625 ng/mL-40 ng/mL and the sensitivity is 0.156 ng/mL .

Target Name zonulin
Abbreviation zonulin
Species Homo sapiens (Human)
Sample Types serum, plasma, tissue homogenates
Detection Range 0.625 ng/mL-40 ng/mL
Sensitivity 0.156 ng/mL
Assay Time 1-5h
Sample Volume 50-100ul
Detection Wavelength 450 nm
Research Area Others
Assay Principle quantitative
Measurement Sandwich
Precision
Intra-assay Precision (Precision within an assay): CV%<8%      
Three samples of known concentration were tested twenty times on one plate to assess.  
Inter-assay Precision (Precision between assays): CV%<10%      
Three samples of known concentration were tested in twenty assays to assess.    
             
Linearity
To assess the linearity of the assay, samples were spiked with high concentrations of human zonulin in various matrices and diluted with the Sample Diluent to produce samples with values within the dynamic range of the assay.
  Sample Serum(n=4)  
1:1 Average % 93  
Range % 86-97  
1:2 Average % 94  
Range % 89-98  
1:4 Average % 96  
Range % 93-105  
1:8 Average % 94  
Range % 86-98  
Recovery
The recovery of human zonulin spiked to levels throughout the range of the assay in various matrices was evaluated. Samples were diluted prior to assay as directed in the Sample Preparation section.
Sample Type Average % Recovery Range  
Serum (n=5) 95 90-101  
EDTA plasma (n=4) 97 90-104  
             
             
Typical Data
These standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed.
ng/ml OD1 OD2 Average Corrected  
40 2.148 2.066 2.107 2.008  
20 1.455 1.353 1.404 1.305  
10 0.867 0.895 0.881 0.782  
5 0.557 0.571 0.564 0.465  
2.5 0.393 0.375 0.384 0.285  
1.25 0.264 0.276 0.270 0.171  
0.625 0.195 0.199 0.197 0.098  
0 0.098 0.099 0.099    
Materials provided
  • A micro ELISA plate --- The 96-well plate has been pre-coated with an anti-human zonulin antibody. This dismountable microplate can be divided into 12 x 8 strip plates.
  • Two vials lyophilized standard --- Dilute a bottle of the standard at dilution series, read the OD values, and then draw a standard curve.
  • Biotin-labeled zonulin antibody (100 x concentrate) 1 x 120 μl ---Act as the detection antibody.
  • HRP-avidin (100 x concentrate) 1 x 120 μl --- Bind to the detection antibody and react with the TMB substrate to make the solution chromogenic.
  • Biotin-antibody Diluent 1 x 15 ml ---Dilute the high concentration Biotin-antibody to an appropriate working solution.
  • HRP-avidin Diluent 1 x 15 ml ---Dilute the high concentration HRP-avidin solution to an appropriate solution.
  • Sample Diluent 1 x 50 ml---Dilute the sample to an appropriate concentration.
  • Wash Buffer (25 x concentrate) 1 x 20 ml --- Wash away unbound or free substances.
  • TMB Substrate 1 x 10 ml --- Act as the chromogenic agent. TMB interacts with HRP, eliciting the solution turns blue.
  • Stop Solution 1 x 10 ml --- Stop the color reaction. The solution color immediately turns from blue to yellow.
  • Four Adhesive Strips (For 96 wells) --- Cover the microtiter plate when incubation.
  • An instruction manual
Materials not provided
  • A microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.
  • An incubator can provide stable incubation conditions up to 37°C±5°C.
  • Centrifuge
  • Vortex
  • Squirt bottle, manifold dispenser, or automated microplate washer
  • Absorbent paper for blotting the microtiter plate
  • 50-300ul multi-channel micropipette
  • Pipette tips
  • Single-channel micropipette with different ranges
  • 100ml and 500ml graduated cylinders
  • Deionized or distilled water
  • Timer
  • Test tubes for dilution
ELISA Data Analysis Watch ELISA data processing video & download Curve Expert if needed
Troubleshooting
and FAQs
ELISA kit FAQs
Storage Store at 2-8°C. Please refer to protocol.
Lead Time 3-5 working days

Citations

Customer Reviews and Q&A

 Customer Reviews
Average Rating:
5.0 - 1 reviews

Submit a Review here

Sample type: Serum

Sample species: Human

Review: The assay is straightforward and easy to run. Low detection in serum (0.3-12ng/ml) but differences between samples clearly apparent. Freeze-thawing of the same serum samples causes a significant decrease in zonulin levels (average decrease of 0.7ng/ml after one further freeze-thaw).

By Anonymous

 Q&A
Q:

please provide the following information regarding Human zonulin ELISA Kit (CSB-EQ027649HU)
1) Does this kit detect pre-haptoglobin-2?
2) Also will it not react with mature haptoglobin-2? mature haptoglobin-1? and pre-haptoglobin-1?
3) Is the capture antibody a monoclonal or polyclonal?
4) What is the host of the capture antibody?
5) What’s the immunogen or immunogen range of the capture antibody?
6) What’s is the species of the immunogen of the capture antibody, human or something else?
7) Is the detection antibody a monoclonal or polyclonal?
8) What is the host of the detection antibody?
9) What is the immunogen or immunogen range of the detection antibody?
10) What is the species of the immunogen of the detection antibody? Human or something else?
11) What’s is that standard? A recombinant protein or something else? If a recombinant protein, what is the host?
12) If recombinant protein, is it full length or partial?
13) What is the species of the standard– human or something else?
14) What sample type was tested with this kit (serum? plasma? Something else?) and what were the reference values.

A:
Thanks for your inquiry!
CSB-EQ027649HU Human zonulin ELISA kit
Human zonulin is the precursor for haptoglobin-2 (pre-HP2).
This kit is designed against full length protein. There are 2 strains of antibodies in the kit.
Capture antibody is mouse monoclonal antibody and immunogen is 293 cell-derived recombinant protein.
Detection antibody is rat monoclonal antibody and immunogen is 293 cell-derived recombinant protein.
Sorry we didn't do epitope mapping before, so we are not sure if it can detect properdin, pre-haptoglobin-1, mature haptoglobin-1 and -2 or not.
Standard is 293 cell-derived recombinant human protein. Sorry we can not disclose the sequence of the standard and immunogen.
We have this kit available if you need it:
CSB-E08584h Human Haptoglobin,Hpt/HP ELISA KIT
We tested undiluted serum and plasma before and the test value is 0.625--2.5ng/ml.
Pls let me know if you have any further questions. Thank you.
Q:

Thank you for your response. Is there any way you can provide the precise immunogen of pre-HP2 was that you used for antibody generation? If you cannot provide the precise immunogens of the 2 MoAbs, can you provide the exact total protein sequence (# ?, from which protein database?) you have used to generate the 293-derived recombinant zonulin?

A:
Thanks for your inquiry!
The immunogen is 293 cell-derived recombinant protein, but sorry that we can not disclose the sequence.
This kit is designed according to: https://www.uniprot.org/uniprot/P00738 .
Pls let me know if you have any further questions. Thank you.
Q:

Does your kit quantify the whole human haptoglobin or specific subgroup?
"Recently, Fasano’s group identified the molecular identity of zonulin as prehaptoglobin-2 (pre-HP2).47"
Reference 47 is
47. Tripathi A, Lammers KM, Goldblum S, Shea-Donohue T, Netzel-Arnett S, Buzza MS, Antalis TM, Vogel SN, Zhao A, Yang S, et al. Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2. Proc Natl Acad Sci U S A 2009; 106:16799-804http://dx.doi.org/10.1073/pnas.0906773106; PMID:19805376
Can you help us to quantify only prehaptoglobin-2 only? If we quantify all haptoglobin, the other haptoglobins will dilute actual zonulin level.
If this kit isn’t suitable for the needs, perhaps do you have another one, which can detect only prehaptoglobin-2? Please advise.

A:
Thanks for your inquiry!
Zonulin is different from Haptoglobin.
CSB-EQ027649HU detects zonulin (zonulin is prehaptoglobin-2);
CSB-E08584h detects haptoglobin;
We have validated these kits before and there is no cross reactivity between them. You can choose the proper kit you need.
Pls let me know if you have any further questions. Thank you.
Q:

Thank you for your reply,
Could you please provide us with, as much as you can, detailed information about these two kits:
1) Capture antibody or capture antigen – immunogen type, sequence (including species source and accession no. if available), expression host, clonality and host species.
2) Detection antibody (if applicable) - immunogen type, sequence (including species source and accession no. if available), expression host, clonality and host species.
3) Standard – if recombinant protein please specify sequence (including species source and accession no. if available) and expression host.
4) Please send the manuals
5)“We have validated these kits before and there is no cross reactivity between them.”
Debbie, how exactly were the kits validated? Please share with us your validation data. What did you test by validation process?
6) The customer also has an additional question. Do you have the clone from Pascale Galea (Bio Rad), mentioned in the attached articale. If yes, are you able to develop an ELISA kit with the antibody per customer request? Perhaps you use a similar clone (please refer to attachment)?
The customer has a concern regarding validation of kits, they need to exclude any possibility of cross reactivity with other haptoglobins.

A:
Thanks for your further inquiry.
CSB-EQ027649HU
Capture antibody is mouse monoclonal antibody and detection antibody is rat monoclonal antibody.
Immunogen is 293 cell-derived recombinant protein.
Standard is 293 cell-derived recombinant protein.
Pls find attached manual.
CSB-E08584h
Capture antibody is goat polyclonal antibody and detection antibody is goat polyclonal antibody.
Immunogen is purified crude extract of natural samples.
Standard is purified crude extract of natural samples.
Pls find attached manual.
The antibodies in these 2 kits are specific to its target. We tested the cross reactivity before and found the antibody used in
CSB-EQ027649HU can not detect Haptoglobin. The raw materials used in the kits are for our internal traceability, so we are sorry that it is not convenient for us to disclose this information.. Hope you could understandard.
Pls let me know if you have any further questions. Thank you.
Q:

I would like to use this kit for fecal samples. Please confirm that fecal samples can be used for this assay and if possible suggest a dilution and recommended preparation protocol for the samples.

A:
Thanks for your inquiry!
We will send you manual soon by email.
Sorry we didn't test fecal samples before, We suggest you do a pretest first if you want to test it.
Q:

I wanna know whats being measured? there are two analytes that can be measured for this assay, and I wonder which is being measured by this kits.This kit measures the concentration of human zonulin in human serum, plasma and other samples. How's that measured?
By measuring the conc of one of these proteins: properdin or haptoglobin2. Which of these does your kit measure?

A:
Thanks for your inquiry!
CSB-EQ027649HU
This kit is for quantitative determinantion of zonulin rather than properdin or haptoglobin2.
The specificity of raw materials for this kit is validated. As this is our core information related to R&D, we can not disclose more information. Hope you could understand.
Q:

We are looking for ELISA kits to measure zonulin in an HIV-positive population to monitor intestinal permeability (since zonulin is a tight junction protein). However, we are finding that most suppliers that advertise kits for zonulin are actually kits for haptoglobin, a related but different protein involved in hemoglobin modulation. We have found dozens of commercial kits that have this problem. Can you confirm that this kit is for zonulin and not haptoglobin? Many thanks.

A:
Thanks for your inquiry!
Zonulin is different from Haptoglobin, and we have both kits.
CSB-EQ027649HU It detects zonulin.
CSB-E08584h It detects haptoglobin.
We tested the cross reactivity between them before and there is no cross reactivitivitie.
The antibody in CSB-EQ027649HU can not detect haptoglobin.
Pls let me know if you have any further questions. Thank you.
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Address
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