Anti-Endothelin-Receptor-B IgG Antibody ELISA Kit

Catalog No. ABIN1068572

Product Details

Target: Anti-Endothelin-Receptor-B IgG Antibody

Reactivity: Human

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Purpose: The anti-Endothelin Receptor B Antibody-EIA is designed for the determination of antibodies (IgG) against the Endothelin receptor subtype B in serum and plasma.

Sample Type: Serum, Plasma, Cell Culture Supernatant

Analytical Method: Quantitative

Characteristics: The CellTrend anti-Endothelin Receptor B- Antibody-EIA is an antibody screening test.

Components: MTP Microplate strips, Endothelin Receptor B coated: 12 x 8
Wash buffer, 10fold conc. : 50 ml
Diluent sample, ready to use : 50 ml
Diluent conjugate, ready to use : 14 ml
Standards, ready to use [2.5 - 5 - 10 - 20 - 40 U/ml] : 1 ml
Positive control, ready to use : 1 ml
Negative control, ready to use : 1 ml
anti-human-IgG, HRP conjugate, 100fold conc.: 0,2 ml
TMB substrate, ready to use: 12 ml
Stop solution, ready to use (0.5 M sulphuric acid): 12 ml

Material not included: Deionized or distilled water, Graduated cylinder, Micropipettes, multipipette, Microplate reader

Application Details

Plate: Pre-coated

Protocol: Endothelin- receptor B has been pre-coated onto a microtiter plate. During the first incubation the anti-Endothelin receptor B- Antibodies of the samples are immobilised on the plate. The autoantibodies are detected with a POD labeled anti-human IgG antibody. In the following enzymatic substrate reaction the intensity of the colour correlates with the concentration and/ or avidity of anti-Endothelin receptor B- antibody.

Reagent Preparation:

Bring all reagents to room temperature before use. If crystals have formed, mix gently until the crystals have completely dissolved.
The microplate strips MTP are ready to use. Remove excess strips (breakable) from the frame, reseal in the bag with the desiccant and store at 2-8 °C.
Dilute the wash buffer BUF│WASH│10x with deionized or distilled water 1:10 (e. g. 50 ml + 450 ml water). The diluted solution is stable for 30 days at 2-8 °C.
Dilute the HRP conjugate CONJ│ENZ 100x with diluent DIL│Conj 1:100 (e. g. 50 µl + 4950 µl diluent DIL│Conj). The required amount of conjugate solution should be prepared freshly.
Dilute the human serum or plasma samples with diluent DIL│SPE 1:100 (e. g. 5 µl + 495 µl diluent). Store undiluted samples at
20 °C.
Standards CAL│1-5, positive control CONTROL│+, negative control CONTROL│-, the diluent sample DIL│SPE and the diluent conjugate DIL│Conj, are ready to use. Assay procedure It is recommended that all samples and standards be assayed in duplicate. 1. Prepare all reagents and samples as directed in the previous section. 2. Pipette 100 µl of diluted samples, standards, controls or diluent DIL│SPE (as blank) into the wells. 3. Seal wells with adhesive strip and incubate for 2 hour at 4°C temperature. 4. Aspirate fluid from wells and wash three times with 300 µl wash buffer. After the last wash, invert the plate and tap on a clean paper towel. 5. Dispense 100 µl of diluted HRP conjugate into each well 6. Seal wells with adhesive strip and incubate for 1 hour (with shaking) at room temperature. 7. Repeat the wash as in step 4. 8. Dispense 100 µl of TMB substrate SUBS│TMB solution into each well. 9. Incubate for 20 minutes at room temperature in the dark. 10. Add 100 µl of stop solution SOLN│STOP to each well. 11. Determine the absorbance within 30 minutes at 450 nm. A reference wavelength of 620 nm/690 nm is recommended.

Assay Procedure:

Assay procedure summary:
A. Preparation:
1. Bring all reagents to room temperature
2. Dilute wash buffer 1:10
3. Dilute samples with diluent sample 1:100
4. Dilute freshly HRP conjugate 1:100 with diluent conjugate
B. Performance:
1. Pipette 100 µL of samples, standards, controls into the wells
2. Incubate for 2 hours at 4°C temperature
3. Wash three times with 300 µL of wash buffer
4. Dispense 100 µL of HRP conjugate solution

5. Incubate for 1 hour (with shaking) at room temperature
6. Wash three times with 300 µL of wash buffer
7. Dispense 100 µL of TMB substrate solution
8. Incubate for 20 minutes at room temperature in the dark
9. Add 100 µL of stop solution
10. Measure absorption at 450 nm

Calculation of Results:

Create a standard curve using computer software capable of generating a curve fit (four parameter fit, x-axis: linear, anti-ETBR-Ab standard points (2.5 U/ml, 5 U/ml, 10 U/ml, 20 U/ml, 40 U/ml), y-axis: linear, absorbance). The sample concentrations can be calculated from the standard curve. A run is considered valid if the positive control is in the expected range (see label) and the negative control is less than the 10 U/ml. Samples over the standard curve can be assyed again using a higher dilution factor (e.g. 1:500). In this case the concentration read from the standard curve must be multiplied by the additional dilution factor (e.g. 5 for 1:500 dilution).

Assay Precision: Intra-assay precision (CV) (n=8) Probe 1 (21.2 U/ml): 4.4% - Inter-assay precision (CV) (n=8) 20.1 U/ml: 3.7%

Restrictions: For Research Use only

Handling

Handling Advice: The assay procedure should be carried out only by qualified and well trained employees. Lipaemic, icteric, haemolysed or microbially contaminated specimen may cause interference. Do not mix reagents from different lots. Some components of the kit contain human blood derivatives. No known test method can offer complete assurance that products derived from human blood will not transmit infectious agents. Therefore, all blood derivatives should be considered potentially infectious. It is recommended that these reagents and human specimens be handled using established good laboratory working practices. Some components of this kit contain Thimerosal, a mercury containing compound. Follow routine precautions for handling hazardous chemicals. Avoid contact with skin and mucous membranes when handling reagents, which contain preservatives (see materials provided). Wash thoroughly with water in case of contact and possibly look up a doctor. The stop solution contains 0.5 M sulphuric acid. Wash thoroughly with water in case of contact with skin. In case of contact with eyes rinse with much water and look up a doctor. Do not allow the wells to become dry once the assay has begun.

Storage: 4 °C

Target Details

Target: Anti-Endothelin-Receptor-B IgG Antibody

Target Type: Antibody

Background: Endothelins (ET) are 21-amino acid vasocon- stricting peptides produced primarily in the endothelium having a key role in vascular homeostasis. It mediates the effects through G-Protein-coupled receptors, the Endothelin receptors. There are two key receptor types, ETA and ETB. ETB is primarily located on the endothelial cells that line the interior of the blood vessels. When endothelin binds to ETB receptors, this leads to the release of nitric oxide, diuresis and mechanisms that lower blood pressure.