Description
Mouse CD4 (Cluster Of Differentiation 4) ELISA Kit
The Mouse CD4 (Cluster of Differentiation 4) ELISA Kit is specially designed for the precise measurement of CD4 levels in mouse serum, plasma, and tissue lysates. This kit offers exceptional sensitivity and specificity, guaranteeing accurate and consistent results for various research purposes.CD4 is a key immune cell surface marker that plays a critical role in the immune response by helping to coordinate the activation of T-helper cells.
Monitoring CD4 levels is essential in studying immune function, autoimmune diseases, and infectious diseases. This ELISA kit provides a valuable tool for researchers seeking to understand the role of CD4 in immune regulation and disease progression.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Mouse |
| Detection Method: | Colormetric |
| Detection Range: | 0.39-25 ng/mL |
| Sensitivity: | 0.23 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Mouse CD4 in samples. No significant cross-reactivity or interference between Mouse CD4 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 Mouse CD4. 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 Mouse CD4 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 Mouse CD4, 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 Mouse CD4. The concentration of Mouse CD4 in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | CD4: Accessory protein for MHC class-II antigen/T-cell receptor interaction. May regulate T-cell activation. Induces the aggregation of lipid rafts. Associates with LCK. Binds to HIV-1 gp120 and to P4HB/PDI and upon HIV-1 binding to the cell membrane, is part of P4HB/PDI- CD4-CXCR4-gp120 complex. Interacts with HIV-1 Envelope polyprotein gp160 and protein Vpu. Interacts with Human Herpes virus 7 capsid proteins. Interacts with PTK2/FAK1; this interaction requires the presence of HIV-1 gp120. |
| UniProt Protein Details: | Protein type:Cell surface; Membrane protein, integral Chromosomal Location of Human Ortholog: 6 F2|6 59. 17 cM Cellular Component: cell surface; endoplasmic reticulum lumen; endoplasmic reticulum membrane; external side of plasma membrane; lipid raft; plasma membrane Molecular Function:enzyme binding; glycoprotein binding; immunoglobulin binding; MHC class II protein binding; protein homodimerization activity; protein kinase binding; zinc ion binding Biological Process: cell surface receptor linked signal transduction; cytokine production; defense response to Gram-negative bacterium; induction by virus of cell-cell fusion in host; maintenance of cellular protein localization; positive regulation of calcium-mediated signaling; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of protein kinase activity; positive regulation of T cell activation; positive regulation of T cell proliferation; regulation of T cell activation; T cell activation; T cell differentiation; T cell selection |
| UniProt Code: | P06332 |
| NCBI GenInfo Identifier: | 116015 |
| NCBI Gene ID: | 12504 |
| NCBI Accession: | P06332. 1 |
| UniProt Related Accession: | P06332 |
| Molecular Weight: | 24,473 Da |
| NCBI Full Name: | T-cell surface glycoprotein CD4 |
| NCBI Synonym Full Names: | CD4 antigen |
| NCBI Official Symbol: | Cd4 |
| NCBI Official Synonym Symbols: | L3T4; Ly-4 |
| NCBI Protein Information: | T-cell surface glycoprotein CD4 |
| UniProt Protein Name: | T-cell surface glycoprotein CD4 |
| UniProt Synonym Protein Names: | T-cell differentiation antigen L3T4; T-cell surface antigen T4/Leu-3; CD_antigen: CD4 |
| Protein Family: | CD40 ligand |
| UniProt Gene Name: | Cd4 |
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 |
| 25 | 2.396 2.438 | 2.417 | 2.358 |
| 12.5 | 1.601 1.621 | 1.611 | 1.552 |
| 6.25 | 0.974 0.94 | 0.957 | 0.898 |
| 3.13 | 0.445 0.459 | 0.452 | 0.393 |
| 1.57 | 0.239 0.225 | 0.232 | 0.173 |
| 0.79 | 0.173 0.149 | 0.161 | 0.102 |
| 0.39 | 0.104 0.118 | 0.111 | 0.052 |
| 0 | 0.052 0.066 | 0.059 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse CD4 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse CD4 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) | 1.28 | 2.92 | 9.81 | 1.40 | 2.82 | 8.99 |
| Standard deviation | 0.08 | 0.15 | 0.39 | 0.07 | 0.16 | 0.47 |
| C V (%) | 6.25 | 5.14 | 3.98 | 5.00 | 5.67 | 5.23 |
Recovery
The recovery of Mouse CD4 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) | 88-99 | 93 |
| EDTA plasma (n=5) | 90-106 | 98 |
| Cell culture media (n=5) | 93-107 | 101 |
Linearity
Samples were spiked with high concentrations of Mouse CD4 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 (%) | 86-97 | 85-99 | 85-97 |
| Average (%) | 92 | 91 | 91 | |
| 1:4 | Range (%) | 87-103 | 79-91 | 86-100 |
| Average (%) | 94 | 86 | 93 | |
| 1:8 | Range (%) | 94-109 | 83-98 | 86-102 |
| Average (%) | 100 | 89 | 93 | |
| 1:16 | Range (%) | 91-104 | 86-96 | 80-95 |
| Average (%) | 98 | 91 | 87 |
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.
