Human CLDN1 (Claudin 1) ELISA Kit (HUES01890)

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HUES01890
€539

Description

ELISA Kit Technical ManualMSDS

Human CLDN1 (Claudin 1) ELISA Kit

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 CLDN1 . 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 CLDN1 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 CLDN1, 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 CLDN1. The concentration of Human CLDN1 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range0.16-10 ng/mL
Sensitivity0.10 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human CLDN1 in samples. No significant cross-reactivity or interference between Human CLDN1 and analogues was observed.

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
102.487
2.521
2.5042.43
51.758
1.818
1.7881.714
2.51.025
1.011
1.0180.944
1.250.485
0.503
0.4940.42
0.630.284
0.268
0.2760.202
0.320.191
0.179
0.1850.111
0.160.123
0.137
0.130.056
00.069
0.079
0.074--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CLDN1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CLDN1 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.501.144.110.531.253.96
Standard deviation0.030.050.130.040.050.20
C V (%)6.004.393.167.554.005.05

Recovery

The recovery of Human CLDN1 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=5)88-10395
EDTA plasma (n=5)95-108102
Cell culture media (n=5)88-10294

Linearity

Samples were spiked with high concentrations of Human CLDN1 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)92-10798-11590-104
Average (%)9910598
1:4Range (%)89-10483-9486-99
Average (%)968993
1:8Range (%)94-10884-9687-101
Average (%)1008992
1:16Range (%)92-10782-9584-96
Average (%)988889

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human CLDN1 (Claudin 1) ELISA Kit (HUES01890) Assay 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.

Human CLDN1 (Claudin 1) ELISA Kit (HUES01890) Protein Information

UniProt Protein Function:Claudin-1: Plays a major role in tight junction-specific obliteration of the intercellular space, through calcium- independent cell-adhesion activity. Acts as a co- receptor for HCV entry into hepatic cells. Can form homo- and heteropolymers with other CLDN. Homopolymers interact with CLDN3, but not CLDN2, homopolymers. Directly interacts with TJP1/ZO-1, TJP2/ZO-2 and TJP3/ZO-3. Interacts with MPDZ and INADL. May interact with HCV E1 and E2 proteins. Strongly expressed in liver and kidney. Expressed in heart, brain, spleen, lung and testis. Belongs to the claudin family.
UniProt Protein Details:

Protein type:Membrane protein, integral; Cytoskeletal; Membrane protein, multi-pass

Chromosomal Location of Human Ortholog: 3q28-q29

Cellular Component: integral to membrane; integral to plasma membrane; tight junction

Molecular Function:identical protein binding; protein binding

Biological Process: calcium-independent cell-cell adhesion; intercellular junction assembly and maintenance; protein heterooligomerization; protein homooligomerization

Disease: Ichthyosis, Leukocyte Vacuoles, Alopecia, And Sclerosing Cholangitis

NCBI Summary:Tight junctions represent one mode of cell-to-cell adhesion in epithelial or endothelial cell sheets, forming continuous seals around cells and serving as a physical barrier to prevent solutes and water from passing freely through the paracellular space. These junctions are comprised of sets of continuous networking strands in the outwardly facing cytoplasmic leaflet, with complementary grooves in the inwardly facing extracytoplasmic leaflet. The protein encoded by this gene, a member of the claudin family, is an integral membrane protein and a component of tight junction strands. Loss of function mutations result in neonatal ichthyosis-sclerosing cholangitis syndrome. [provided by RefSeq, Jul 2008]
UniProt Code:O95832
NCBI GenInfo Identifier:6685283
NCBI Gene ID:9076
NCBI Accession:O95832.1
UniProt Related Accession:O95832
Molecular Weight:22,744 Da
NCBI Full Name:Claudin-1
NCBI Synonym Full Names:claudin 1
NCBI Official Symbol:CLDN1  
NCBI Official Synonym Symbols:CLD1; SEMP1; ILVASC  
NCBI Protein Information:claudin-1
UniProt Protein Name:Claudin-1
UniProt Synonym Protein Names:Senescence-associated epithelial membrane protein
Protein Family:Claudin
UniProt Gene Name:CLDN1  
UniProt Entry Name:CLD1_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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