ELISA Kit for the Quantitative Analysis of Human CTLA-4

 

 

 

The human CTLA-4 ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human CTLA-4 in cell culture supernatants,human serum and plasma.THE ELISA KIT IS FOR RESEARCH USE ONLY.Please read this instruction manual carefully and check out the material provided before use, and you can contact with our company if any questions. You can enter our website or call us for other aim.

Introduction

Cytotoxic T-Lymphocyte-Associated molecule-4 (CTLA-4), also known as cluster of differentiation 152 , is a type I trans-membrane T cell inhibitory molecule that is a member of the Ig superfamily. Within the ECD, human CTLA4 shares 68%, 71% and 83~86% aa sequence identity with mouse, rat and porcine/bovine/rabbit/feline/canine CTLA4, respectively. A 174 aa form that lacks trans-membrane and cytoplasmic sequences (sCTLA4) is possibly secreted. In mouse, an isoform lacking the Ig-like domain has ligand independent inhibitory activity and is termed liCTLA4.

CTLA-4 plays a role in immune self-tolerance, which is important for avoiding autoimmune diseases. Unfortunately, this mechanism can be co-opted in order to suppress immune responses in the context of cancer.CD28, which is structurally related to CTLA4, is constitutively expressed on naïve T cells and promotes T cell activation when engaged by B72 on antigen presenting cells (APC) within the immunological synapse (IS). In contrast, CTLA4 is recruited from intracellular vesicles to the IS beginning 1, 2 days after T cell activation. It forms a linear lattice with B71 on APC, inducing negative regulatory signals and ending T cell activation.

Principles of the Test

The kit is a solid sandwich enzyme-linked immunosorbent assay for detection of human CTLA-4. An anti-human CTLA-4 monoclonal antibody has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into wells. The human CTLA-4 in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The human CTLA-4 biotin-conjugated antibody were added and binds to human CTLA-4 captured by the first antibody, which formed a sandwich. Streptavidin-HRP would be added and binds to the biotin conjugated antibody, then free Streptavidin-HRP would be removed during a wash step. After this, substrate solution would be added and catalyzed by the HRP, and a colored product is formed. The intensity of the colored product is used to calculate in proportion to the amount of human CTLA-4 in the original specimen.

Materials provided with the kit:

Reagent	96/48Test Kit
Assay Buffer	5ml/3 ml
Human CTLA-4 Antibody-Coated Wells	12 strips/6 strips
Standard Diluent	10ml/5ml
Human CTLA-4 Standard	2/1vial(s)
Human CTLA-4 Detection Antibody	10ml/5ml
Streptavidin-HRP	10ml/5ml
Wash Buffer Concentrate 20×	30ml/15ml
TMB	10ml/5ml
Stop Solution	5ml/3 ml
Plate Covers	3/2
Complete Instruction Manual	1

Specimen Collection

1. Collecting specimen as following:

A. The particulate of the cell culture supernatants should be removed before use.

B. Serum was obtained from clot at room temperature.

C. Please collect plasma with EDTA.

D. Assay immediately or store samples at -20℃. Avoid free-thaw cycles.

2. Antiseptic and anticoagulant should not appear in Serum

samples.

3. Any particulate should be removed from samples before use.

4. Do not use grossly hemolyzed or lipemic samples.

Note: Strongly recommend that the serum and plasma samples should be diluent as doubling dilution before use.

Precautions for use:

1.Please storage the Kit at 2～8℃。

2. Washing buffer concentrate may have crystal in low temperature, and you can melt its in water-bath before use.

3. Please discard the remains after use of the dissolved standard.

4. Avoid contact of substrate solution with oxidizing agents and metal.

5. Usage of disposable pipette tips avoid microbial contamination or cross-contamination of reagents or specimens.

6. Do not mix or substitute reagents with those from other lots or other sources.

7. To ensure the adequate mixture of added reagents, please tap gently the plate after the wells were filled with liquid.

8. Incubation temperature should be 25～28℃.

9. Wash step was crucial for whole assay process.

10. Duplicate wells of the same sample were recommended in

assay process.

11. Avoid the foam while pour the liquid into wells.

12．For serum or plasma samples ,the biotin-conjugated antibody should be incubate for at least 90 minutes.

Reagent Preparation

1.The reagents should be warmed up to room temperature before use.The remanent reagents must reseal and put into refrigeratory again as soon as possible.

2.Dilute 1.5ml of wash buffer Concentrate into 19ml deionized or distilled water to work.

3. If you have a 5x standard diluent, please dilute it with double steaming water or deionized water.

4. Add standard diluent to the bottle according to the volume of the label and wait15 minutes for complete dissolution. Incubation temperature should be 25～28℃。And in turn add the half concentration diluent by standard diluent

Wash step:

Automated microplate washer or operating by pipette: Each well should be pour into300ul wash buffer and soak 15 or 30 seconds,then be aspirated, five times process were repeated. After the last wash, remove remaining wash buffer by aspirating. Invert the plate and blot it against clean paper towels.

Materials Required But Not Provided

1. pipettes and pipette tips

2. Microwell strip reader capable of reading at 450 nm (540 nm as optional reference wave length)

3. automated microplate washer

4.Glass-distilled or deionized water

Assay procedure

1.The needed strips were putted into the frame, the remains were returned into foil pouch and resealed.

2.Blank well were recommended, which only color reagent and stop solution be added. It is suggested that each testing with gradient density of standard for standard curve.

3.Add 100ul of standard or sample.Cover with the Plate Covers provided.Incubate for 2 hours at room temperature If assay the serum sample,you should add 50μl assay buffer with 50μl sample into the wells,if the protein concentration is higher than the range of the Kit, add the same quantitys assay buffer with the sample, the deficiency should be complemented with sample diluent to 100μl per well.

4.Five times wash process were repeated.

5.Add 100ul of detection antibody. Cover with the Plate Covers provided.Incubate for 1 hour at room temperature.

6.Five times wash process were repeated.

7.Add 100ul of Streptavidin-HRP. Cover with the Plate Covers provided. Lucifugal incubation for 20 minutes at room temperature.

8.Five times wash process were repeated.

9.Add 100ul of TMB，Lucifugal incubation for 20 minutes at room temperature.

10.Add 50ul of stop solution to each well, determine the optical

density of each well within 10 minutes.

Calculation of Results

1.Duplicates should be within 20 percent of the mean. Average absorbance values for each set of duplicate samples were used as detection results.

2.The blank absorbance values of subtract should be deducted.

3.Drawing a best fit curve through the points of graph. Draw the standard curve by plotting assayed OD value (on the Y axis) vs. concentration (on the X axis). The sample concentration was obtained based on its OD value ​​founding in the standard concentration curve.

4.If the values obtained are not within the expected range of the standard, Samples should be dilute and assay again

Typical Data and Standard Curve

concentration (pg/ml)	Typical data 1	Typical data 2	Average
0	0.151	0.132	0.1415
62.5	0.314	0.269	0.2915
125	0.438	0.384	0.411
250	0.724	0.648	0.686
500	1.126	0.974	1.05
1000	1.853	1.735	1.794
2000	2.82	2.884	2.852

Human CTLA-4 Standard Curve

 

 

Sensitivity,Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 28.2pg/ml.

Specificity : No significant cross-reactivity or interference with human B7-2，CD28，IL-17A  and mouse  CTLA-4.

Repeatability: The coefficient of variation between wells or plates is less than 10 percent.