Human CD3e / CD3 epsilon ELISA Kit

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SKU:
HUFI01659
¥77,817.89

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Human CD3e / CD3 epsilon ELISA Kit - Information

The ELISA Genie CD3e / CD3 epsilon ELISA Kit can assay for CD3e / CD3 epsilon in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our CD3e / CD3 epsilon ELISA Kits Work?

The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound CD3e / CD3 epsilon is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Human CD3e / CD3 epsilon ELISA Kit - Data

Description

The CD3 complex mediates signal transduction, resulting in T-cell activation and proliferation. Required for normal immune responses (PubMed:15546002, PubMed:8490660).

Post-Translational Modification

Uniprot ID P07766
Alias

CD3E(T-cell surface glycoprotein CD3 epsilon chain)/CD3e/CD3e antigen/CD3e antigen epsilon polypeptide(TiT3 complex)/CD3e molecule epsilon(CD3-TCR complex)/CD3-epsilon/T3E/T-cell antigen receptor complex epsilon subunit of T3/T-cell surface antigen T3Leu-4 epsilon chain/TCRE

Detection method

Sandwich ELISA Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of CD3E concentrations in serum plasma and other biological fluids.

Size

96T

Range

0.625-40ng/ml

Sensitivity

< 0.375ng/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of CD3E and the recovery rates were calculated by comparing the measured value to the expected amount of CD3E in samples.

Matrix Recovery range(%) Average(%)
serum(n=5) 85-96 91
EDTA plasma(n=5) 95-103 99
UFH plasma(n=5) 85-102 94
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of CD3E and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 87-100% 94-99% 92-101% 93-103%
EDTA plasma(n=5) 82-99% 83-99% 88-101% 86-99%
UFH plasma(n=5) 82-98% 83-95% 94-100% 82-99%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Human CD3e / CD3 epsilon ELISA Kit Protocol

The below protocol is a sample protocol for Human CD3e / CD3 epsilon ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human CD3e / CD3 epsilon present in their sample.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Sandwich Protocol

Sandwich ELISA Protocol

Kit Protocol:

1.Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3.Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6.Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

Human CD3e / CD3 epsilon ELISA Kit components

96 Assays

Storage

ELISA Microplate(Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The ELISA Genie Human CD3e / CD3 epsilon ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

Sample Preparation

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample TypeProtocol

Serum

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

Plasma

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.

Urine & Cerebrospinal Fluid

Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.

Cell culture supernatant

Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.

Cell lysates

Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Tissue homogenates

The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.

Tissue lysates

Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Breast Milk

Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Human CD3e / CD3 epsilon ELISA Kit Protein Information

UniProt Protein Function:CD3E: a T cell surface glycoprotein that is a component of the T cell antigen receptor. The recruitment of Nck by CD3 epsilon reveals a ligand-induced conformational change essential for T cell receptor signaling and synapse formation. Contains 1 immunoglobulin-like domain and 1 ITAM domain.
UniProt Protein Details:

Protein type:Receptor, misc.; Membrane protein, integral

Chromosomal Location of Human Ortholog: 11q23

Cellular Component: T cell receptor complex; integral to plasma membrane; plasma membrane; immunological synapse; alpha-beta T cell receptor complex; intercellular junction; external side of plasma membrane

Molecular Function:protein binding; transmembrane receptor activity; receptor signaling complex scaffold activity; protein heterodimerization activity; receptor signaling protein activity; T cell receptor binding; SH3 domain binding; protein kinase binding

Biological Process: regulation of immune response; T cell activation; positive regulation of interleukin-2 biosynthetic process; signal complex assembly; positive regulation of calcium-mediated signaling; negative thymic T cell selection; T cell receptor signaling pathway; positive regulation of interleukin-4 production; regulation of apoptosis; G-protein coupled receptor protein signaling pathway; positive regulation of interferon-gamma production; positive regulation of peptidyl-tyrosine phosphorylation; cell surface receptor linked signal transduction; negative regulation of smoothened signaling pathway; T cell costimulation; protein complex assembly; positive regulation of alpha-beta T cell proliferation; positive regulation of T cell proliferation; positive regulation of T cell anergy; transmembrane receptor protein tyrosine kinase signaling pathway; response to nutrient

Disease: Immunodeficiency 18

NCBI Summary:The protein encoded by this gene is the CD3-epsilon polypeptide, which together with CD3-gamma, -delta and -zeta, and the T-cell receptor alpha/beta and gamma/delta heterodimers, forms the T-cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. The genes encoding the epsilon, gamma and delta polypeptides are located in the same cluster on chromosome 11. The epsilon polypeptide plays an essential role in T-cell development. Defects in this gene cause immunodeficiency. This gene has also been linked to a susceptibility to type I diabetes in women. [provided by RefSeq, Jul 2008]
UniProt Code:P07766
NCBI GenInfo Identifier:1345708
NCBI Gene ID:916
NCBI Accession:P07766.2
UniProt Related Accession:P07766
Molecular Weight:
NCBI Full Name:T-cell surface glycoprotein CD3 epsilon chain
NCBI Synonym Full Names:CD3e molecule
NCBI Official Symbol:CD3E Ãƒ‚ 
NCBI Official Synonym Symbols:T3E; TCRE; IMD18 Ãƒ‚ 
NCBI Protein Information:T-cell surface glycoprotein CD3 epsilon chain
UniProt Protein Name:T-cell surface glycoprotein CD3 epsilon chain
UniProt Synonym Protein Names:T-cell surface antigen T3/Leu-4 epsilon chain; CD_antigen: CD3e
UniProt Gene Name:CD3E Ãƒ‚ 
UniProt Entry Name:CD3E_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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