Human CD8 alpha / CD8A ELISA Kit

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SKU:
HUFI00839
€599

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Human CD8 alpha / CD8A ELISA Kit - Information

The ELISA Genie CD8 alpha / CD8A ELISA Kit can assay for CD8 alpha / CD8A in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our CD8 alpha / CD8A 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 CD8 alpha / CD8A 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 CD8 alpha / CD8A ELISA Kit - Data

Description

Identifies cytotoxic/suppressor T-cells that interact with MHC class I bearing targets. CD8 is thought to play a role in the process of T-cell mediated killing. CD8 alpha chains binds to class I MHC molecules alpha-3 domains.

Post-Translational Modification

All of the five most C-terminal cysteines form inter-chain disulfide bonds in dimers and higher multimers, while the four N-terminal cysteines do not.

Uniprot ID P01732
Alias

CD8A/T-cell surface glycoprotein CD8 alpha chain/T-lymphocyte differentiation antigen T8/Leu-2

Detection method

Sandwich ELISA Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of CD8A concentrations in serum plasma and other biological fluids.

Size

96T

Range

2.81-180ng/ml

Sensitivity

< 1.688ng/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of CD8A and the recovery rates were calculated by comparing the measured value to the expected amount of CD8A in samples.

Matrix Recovery range(%) Average(%)
serum(n=5) 89-103 96
EDTA plasma(n=5) 85-102 92
UFH plasma(n=5) 85-103 95
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of CD8A 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) 85-104% 88-105% 86-105% 85-104%
EDTA plasma(n=5) 90-99% 83-85% 83-94% 86-98%
UFH plasma(n=5) 80-100% 89-98% 81-100% 81-99%
CV(%)

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

Note

For Research Use Only

Human CD8 alpha / CD8A ELISA Kit Protocol

The below protocol is a sample protocol for Human CD8 alpha / CD8A 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 CD8 alpha / CD8A 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 CD8 alpha / CD8A 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 CD8 alpha / CD8A 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 CD8 alpha / CD8A ELISA Kit Protein Information

UniProt Protein Function:Function: Identifies cytotoxic/suppressor T-cells that interact with MHC class I bearing targets. CD8 is thought to play a role in the process of T-cell mediated killing. CD8 alpha chains binds to class I MHC molecules alpha-3 domains.
UniProt Protein Details:

Subunit structure: In general heterodimer of an alpha and a beta chain linked by two disulfide bonds. Can also form homodimers. Shown to be expressed as heterodimer on thymocytes and as homodimer on peripheral blood T-lymphocytes. Interacts with the MHC class I HLA-A/B2M dimer. Interacts with LCK in a zinc-dependent manner. Ref.9

Subcellular location: Isoform 1: Cell membrane; Single-pass type I membrane protein. Isoform 2: Secreted.

Post-translational modification: All of the five most C-terminal cysteines form inter-chain disulfide bonds in dimers and higher multimers, while the four N-terminal cysteines do not

By similarity.

Involvement in Disease: CD8 deficiency, familial (CD8 deficiency) [MIM:608957]: An immunologic defect characterized by absence of CD8+ cells, leading to recurrent bacterial infections.Note: The disease is caused by mutations affecting the gene represented in this entry.

Sequence similarities: Contains 1 Ig-like V-type (immunoglobulin-like) domain.

NCBI Summary:The CD8 antigen is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates efficient cell-cell interactions within the immune system. The CD8 antigen acts as a coreceptor with the T-cell receptor on the T lymphocyte to recognize antigens displayed by an antigen presenting cell in the context of class I MHC molecules. The coreceptor functions as either a homodimer composed of two alpha chains or as a heterodimer composed of one alpha and one beta chain. Both alpha and beta chains share significant homology to immunoglobulin variable light chains. This gene encodes the CD8 alpha chain. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Nov 2011]
UniProt Code:P01732
NCBI GenInfo Identifier:116035
NCBI Gene ID:925
NCBI Accession:P01732.1
UniProt Secondary Accession:P01732,Q13970, Q4ZG17, D6W5M8,
UniProt Related Accession:P01732
Molecular Weight:
NCBI Full Name:T-cell surface glycoprotein CD8 alpha chain
NCBI Synonym Full Names:CD8a molecule
NCBI Official Symbol:CD8A Ãƒ‚ 
NCBI Official Synonym Symbols:CD8; MAL; p32; Leu2 Ãƒ‚ 
NCBI Protein Information:T-cell surface glycoprotein CD8 alpha chain; T8 T-cell antigen; T cell co-receptor; OKT8 T-cell antigen; T-cell antigen Leu2; Leu2 T-lymphocyte antigen; CD8 antigen, alpha polypeptide (p32); T-lymphocyte differentiation antigen T8/Leu-2
UniProt Protein Name:T-cell surface glycoprotein CD8 alpha chain
UniProt Synonym Protein Names:T-lymphocyte differentiation antigen T8/Leu-2
Protein Family:CD81 antigen
UniProt Gene Name:CD8A Ãƒ‚ 
UniProt Entry Name:CD8A_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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