Description
Rat OCLN (Occludin) ELISA kit
The Rat OCLN (Occludin) ELISA Kit is specifically designed for the precise measurement of occludin levels in rat serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit delivers accurate and consistent results, making it suitable for various research purposes.Occludin is a key protein involved in maintaining the integrity of tight junctions in epithelial and endothelial cells, essential for regulating paracellular permeability.
Dysregulation of occludin has been linked to several conditions, including inflammatory bowel disease, multiple sclerosis, and various autoimmune disorders. Therefore, this ELISA kit serves as a valuable tool for studying these diseases and potentially identifying new therapeutic targets.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Rat |
| Detection Method: | Colormetric |
| Detection Range: | 0.16-10 ng/mL |
| Sensitivity: | 0.10 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Rat OCLN in samples. No significant cross-reactivity or interference between Rat OCLN and analogues was observed. |
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 Rat OCLN. 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 Rat OCLN 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 Rat OCLN, 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 Rat OCLN. The concentration of Rat OCLN in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | occludin: May play a role in the formation and regulation of the tight junction (TJ) paracellular permeability barrier. It is able to induce adhesion when expressed in cells lacking tight junctions. Interacts with TJP1/ZO1 and with VAPA. Localized at tight junctions of both epithelial and endothelial cells. Highly expressed in kidney. Not detected in testis. Belongs to the ELL/occludin family. 7 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Membrane protein, integral; Cell adhesion; Motility/polarity/chemotaxis; Membrane protein, multi-pass Cellular Component: apical plasma membrane; apicolateral plasma membrane; cell junction; cytoplasmic vesicle; cytosol; endocytic vesicle; integral to membrane; intercellular junction; plasma membrane; tight junction Molecular Function:protein domain specific binding; S-adenosylmethionine-dependent methyltransferase activity; thiopurine S-methyltransferase activity Biological Process: intercellular junction assembly and maintenance; methylation; S-adenosylhomocysteine metabolic process; S-adenosylmethionine metabolic process |
| UniProt Code: | Q6P6T5 |
| NCBI GenInfo Identifier: | 40254754 |
| NCBI Gene ID: | 83497 |
| NCBI Accession: | NP_112619. 2 |
| UniProt Secondary Accession: | Q6P6T5,Q9Z303, |
| UniProt Related Accession: | Q6P6T5 |
| Molecular Weight: | 59,186 Da |
| NCBI Full Name: | occludin |
| NCBI Synonym Full Names: | occludin |
| NCBI Official Symbol: | Ocln |
| NCBI Protein Information: | occludin |
| UniProt Protein Name: | Occludin |
| Protein Family: | Occludin |
| UniProt Gene Name: | Ocln |
| UniProt Entry Name: | OCLN_RAT |
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 |
| 10 | 2.414 2.444 | 2.429 | 2.348 |
| 5 | 1.613 1.619 | 1.616 | 1.535 |
| 2.5 | 0.996 0.986 | 0.991 | 0.91 |
| 1.25 | 0.462 0.474 | 0.468 | 0.387 |
| 0.63 | 0.307 0.279 | 0.293 | 0.212 |
| 0.32 | 0.197 0.179 | 0.188 | 0.107 |
| 0.16 | 0.133 0.137 | 0.135 | 0.054 |
| 0 | 0.075 0.087 | 0.081 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat OCLN were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat OCLN 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) | 0.49 | 1.39 | 3.52 | 0.49 | 1.53 | 3.78 |
| Standard deviation | 0.03 | 0.06 | 0.19 | 0.03 | 0.08 | 0.13 |
| C V (%) | 6.12 | 4.32 | 5.40 | 6.12 | 5.23 | 3.44 |
Recovery
The recovery of Rat OCLN 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) | 93-108 | 100 |
| EDTA plasma (n=5) | 93-107 | 100 |
| Cell culture media (n=5) | 85-97 | 91 |
Linearity
Samples were spiked with high concentrations of Rat OCLN 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) | ||
| 1:2 | Range (%) | 95-111 | 88-100 | 86-97 |
| Average (%) | 103 | 95 | 91 | |
| 1:4 | Range (%) | 91-105 | 82-92 | 83-94 |
| Average (%) | 99 | 87 | 88 | |
| 1:8 | Range (%) | 93-107 | 81-95 | 83-95 |
| Average (%) | 99 | 87 | 89 | |
| 1:16 | Range (%) | 93-106 | 87-99 | 87-102 |
| Average (%) | 100 | 92 | 94 |
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 ×12 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 mL | 4°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 | 4°C(shading light) |
| Stop Solution | 1 vial, 10 mL | 4°C |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
- 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.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 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.
- 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.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 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.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
