Human thrombin-antithrombin complex,TAT ELISA Kit

Instructions
Code CSB-E08431h
Size 96T,5×96T,10×96T
Trial Size 24T ELISA kits trial application
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Product Details

Description

The Human thrombin-antithrombin complex (TAT) ELISA kit is a sandwich immunoassay specifically designed and validated for the quantitative detection of TAT in the serum, plasma, or tissue homogenates. It is not intended for diagnostic use. This assay kit was designed and optimized for cardiovascular research use in humans. The kit has undergone rigorous quality control in multiple parameters, including sensitivity, specificity, precision, linearity, recovery, and inter-batch difference. Refer to the product instructions for more details.

This assay employs the quantitative sandwich enzyme immunoassay technique, in which TAT in the samples or standards are sandwiched between pre-coated TAT antibody and Biotin-conjugated antibody specific for TAT. HRP-avidin is then added to the wells. Following a wash to remove any unbound reagent, the TMB substrate solution is added to the wells and color develops in proportion to the amount of TAT bound in the initial step. The color development is stopped upon adding the stop solution, and the intensity of the color is measured at 450 nm via a microplate reader. The levels of TAT in the samples can be determined by referring to the O.D. (optical density) of the samples to the standard curve.

TAT is a complex formed during antithrombin (AT)'s neutralization of thrombin. Thrombin loses irreversibly its enzymatic activity in the meantime. Elevated TAT is an indicator for an excess of thrombin generation. Increased thrombin levels hyper-activate the blood coagulation cascade. The relatively long half-life of TAT enables direct measurement. TAT Plasma TAT complex is valuable to diagnose and evaluate the risk of thromboembolic events in the clinical.

Target Name thrombin-antithrombin complex,TAT
Alternative Names N/A
Abbreviation TAT
Species Homo sapiens (Human)
Sample Types serum, plasma, tissue homogenates
Detection Range 62.5 pg/mL-4000 pg/mL
Sensitivity 15.6 pg/mL
Assay Time 1-5h
Sample Volume 50-100ul
Detection Wavelength 450 nm
Research Area Cardiovascular
Assay Principle quantitative
Measurement Sandwich
Precision
Intra-assay Precision (Precision within an assay): CV%<8%
Three samples of known concentration were tested twenty times on one plate to assess.
Inter-assay Precision (Precision between assays): CV%<10%
Three samples of known concentration were tested in twenty assays to assess.
Linearity
To assess the linearity of the assay, samples were spiked with high concentrations of human TAT in various matrices and diluted with the Sample Diluent to produce samples with values within the dynamic range of the assay.
 SampleSerum(n=4)
1:1Average %94
Range %90-99
1:2Average %99
Range %94-103
1:4Average %88
Range %82-93
1:8Average %95
Range %91-100
Recovery
The recovery of human TAT spiked to levels throughout the range of the assay in various matrices was evaluated. Samples were diluted prior to assay as directed in the Sample Preparation section.
Sample TypeAverage % RecoveryRange
Serum (n=5) 9288-96
EDTA plasma (n=4)9892-103
Typical Data
These standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed.
pg/mlOD1OD2AverageCorrected
40002.773 2.753 2.763 2.576
20002.134 2.178 2.156 1.969
10001.342 1.368 1.355 1.168
5000.795 0.756 0.776 0.589
2500.524 0.502 0.513 0.326
1250.378 0.398 0.388 0.201
62.50.317 0.327 0.322 0.135
00.186 0.188 0.187  
Materials provided
  • A micro ELISA plate --- The 96-well plate has been pre-coated with an anti-human TAT antibody. This dismountable microplate can be divided into 12 x 8 strip plates.
  • Two vials lyophilized standard ---Dilute a bottle of the standard at dilution series, read the OD values, and then draw a standard curve.
  • One vial Biotin-labeled TAT antibody (100 x concentrate) (120 μl/bottle) ---Act as the detection antibody.
  • One vial HRP-avidin (100 x concentrate) (120 μl/bottle) ---Bind to the detection antibody and react with the TMB substrate to make the solution chromogenic.
  • One vial Biotin-antibody Diluent (15 ml/bottle) ---Dilute the Biotin-antibody.
  • One vial HRP-avidin Diluent (15 ml/bottle) ---Dilute the HRP-avidin solution.
  • One vial Sample Diluent (50 ml/bottle)---Dilute the sample to an appropriate concentration.
  • One vial Wash Buffer (25 x concentrate) (20 ml/bottle) ---Wash away unbound or free substances.
  • One vial TMB Substrate (10 ml/bottle) ---Act as the chromogenic agent. TMB interacts with HRP, eliciting the solution turns blue.
  • One vial Stop Solution (10 ml/bottle) ---Stop the color reaction. The solution color immediately turns from blue to yellow.
  • Four Adhesive Strips (For 96 wells) --- Cover the microplate when incubation.
  • An instruction manual
Materials not provided
  • A microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.
  • An incubator can provide stable incubation conditions up to 37°C±5°C.
  • Centrifuge
  • Vortex
  • Squirt bottle, manifold dispenser, or automated microplate washer
  • Absorbent paper for blotting the microtiter plate
  • 50-300ul multi-channel micropipette
  • Pipette tips
  • Single-channel micropipette with different ranges
  • 100ml and 500ml graduated cylinders
  • Deionized or distilled water
  • Timer
  • Test tubes for dilution
Troubleshooting
and FAQs
ELISA kit FAQs
Storage Store at 2-8°C. Please refer to protocol.
Lead Time 3-5 working days

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