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|Category:||Elisa Test Kit||Format:||96T/KIT|
TSH Streptavidin ELISA Kit 96wells/kit, high accuracy, Elisa Sandwich method, for quantitative measurement
Product Name: TSH Streptavidin ELISA Kit
TSH Streptavidin ELISA Kit is intended for the quantitative measurement of TSH in human serum.
Human TSH ELISA (Enzyme-Linked Immunosorbent Assay) Kit allows the quantitative measurement of human TSH levels in serum and plasma, urine, and cell culture supernatants. A 96-well plate (in a strip format with 12 strips and 8 wells per strip) is coated with an antibody specific for human TSH. TSH standards and experimental samples (prepared as described in “Reagent Preparation”) are added to the wells where the TSH is “captured” by the bound antibody. A series of washes is performed and the immobilized TSH in the standard and sample wells is then bound by biotinylated anti-human TSH antibody. The wash sequence is repeated to remove the unbound “detection” antibody, followed by addition of HRP-conjugated streptavidin that will lock on to the detection antibody. The washes are repeated and the specific color signal is generated with TMB substrate pipetted into the wells. Stop Solution will make the blue color turn to yellow, and the strip is then ready to read at 450 nm.
Principle of Test:
TSH is a solid phase sandwich ELISA method. The samples, and anti-TSHHRP/Biotin conjugate are added to the wells coated with Streptavidin. TSH in the patient’s sample forms a sandwich between two specific antibodies to TSH. Unbound protein and HRP conjugate are washed off by wash buffer. Upon the addition of the substrate, the intensity of color is proportional to the concentration of TSH in the samples. A standard curve is prepared relating color intensity to the concentration of the TSH.
|A. Microplate with||One 96-well (12 strips with 8 well/strip) microplate|
|immobilized “Capture”||pre-coated with antibody to human TSH|
|B. Wash Buffer||25 ml of wash buffer concentrate (20x)|
|C. Standards||Two vials of recombinant human TSH protein|
|D. Assay Diluent A||30 ml of diluent buffer with 0.09% sodium azide as|
|preservative (For standard and sample (serum/plasma)|
|E. Assay Diluent B||15 ml of 5x concentrated buffer (For standard and sample|
|(urine/cell culture medium) dilution)|
|F. “Detection” Antibody||Two vials of biotinylated human TSH antibody (each vial is|
|enough to assay one-half of the microplate)|
|G. HRP-Streptavidin||200 µl of 600x concentrated HRP-conjugated streptavidin|
|H. TMB Substrate||12 ml of 3,3’,5,5’-tetramethylbenzidine (TMB) in buffer|
|I. Stop Solution||8 ml of 0.2 M sulfuric acid‡|
1. Bring all reagents and samples to room temperature (18 - 25ºC) before use. It is recommended that all standards and samples be run at least in duplicate.
2. Label removable 8-well strips as appropriate for your experiment.
3. Add 100 µl of each standard (see Reagent Preparation step 3) and sample into appropriate wells. Cover wells and incubate for 2.5 hours at room temperature with gentle shaking.
4. Discard the solution and wash 4 times with 1X Wash Solution. Wash by filling each well with Wash Buffer (300 µl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5. Add 100 µl of 1X prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
6. Discard the solution. Repeat the wash as in step 4.
7. Add 100 µl of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
8. Discard the solution. Repeat the wash as in step 4.
9. Add 100 µl of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
10. Add 50 µl of Stop Solution (Item I) to each well. Read at 450 nm immediately.
Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
A. TYPICAL DATA
These standard curves are for demonstration only. A standard curve must be run with each assay.
The minimum detectable dose of Human TSH was determined to be 4 pg/ml.
Minimum detectable dose is defined as the analyte concentration resulting in an absorbance that is 2 standard deviations higher than that of the blank (diluent buffer).
C. SPIKING & RECOVERY
Recovery was determined by spiking various levels of Human TSH into the sample types listed below. Mean recoveries are as follows:
|ORIENT NEW LIFE MEDICAL CO., LTD.|
|Email:||Jerry @ newlifebiotest .com|
Contact Person: Jerry Meng