Components
Microtiter plate (96 wells stripwell) - 1
Enzyme conjugate - 1 vial
Standard A - 1 vial
Standard B - 1 vial
Standard C - 1 vial
Standard D - 1 vial
Standard E - 1 vial
Standard F - 1 vial
Substrate A - 1 vial
Substrate B - 1 vial
Stop solution - 1 vial
Wash solution - 1 vial
Balance solution - 1 vial
Instruction manual - 1
Material not included
Precision pipettors and disposable tips to deliver 10-1000 μL. A multi-channel pipette is desirable for large assays.
100 mL and 1 L graduated cylinders.
Distilled or deionized water.
Tubes to prepare sample dilutions.
Absorbent paper.
Microplate reader capable of measuring absorbance at 450 nm.
Centrifuge capable of 3000 x g.
Microplate washer or washing bottle.
Incubator (37 °C).
Data analysis and graphing software.
Reagent Preparation
Samples - Please predict the concentration before assaying. If concentrations are unknown or not within the detection range, a preliminary experiment is recommended to determine the optimal dilution.
Wash solution - Dilute 10mL of wash solution concentrate (100×) with 990mL of deionized or distilled water to prepare 1000mL of wash solution (1×).
Assay Procedure
1.Secure the desired number of coated wells in the holder then add 50 µL of Standards or Samples to the appropriate well of the antibody pre-coated Microtiter Plate.
2.Add 100 µL of Conjugate to each well. Cover and incubate for 1 hour at 37 °C.
3.Wash the Microtiter Plate using one of the specified methods.
4.Add 50 µL Substrate A to each well.
5.Add 50 µL Substrate B to each well. Cover and incubate for 15 minutes at 20-25 °C. (avoid sunlight)
6.Add 50 µL of Stop Solution to each well. Mix well.
7.Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader immediately.
Calculation of Results
The standard curve is used to determine the amount of samples.
First, All O.D. values are subtracted by the mean value of blank control well.
Construct a standard curve by plotting the average O.D. for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis, and draw a best fit curve using graph paper or statistical software to generate a linear regression, four parameter logistic (4-PL) curve-fit, or curvilinear regression of second degree.
Calculate the concentration of samples corresponding to the mean absorbance from the standard curve.
Precaution of Use
TMB is non-carcinogenic but it is hazardous in case of skin contact, eye contact, ingestion and inhalation. In case of contact, rinse affected area with plenty of water.
The stop solution provided with this kit is an acid solution. Wear protective gloves, clothing, and face protection.
Avoid generation of aerosols.
All blood components and biological materials should be handled as potentially hazardous. Decontaminate and dispose specimens and all potentially contaminated materials as they could contain infectious agents. The preferred method of decontamination is autoclaving for a minimum of 1 hour a 121.5°C.
Handling Advice
Influenced by the factors including cell viability, cell number and also sampling time, samples from cell culture supernatant may not be detected by the kit.
Fresh samples without long time storage are recommended for the test.
The coefficient of determination of the standard curve should be higher or equal 0.95 and the highest O.D. should be more than 1.0.
Samples should be collected in pyrogen/endotoxin-free tubes.
Read absorbance immediately after adding the stop solution.
Because TMB is light sensitive, avoid prolonged exposure to light. Also avoid contact between TMB and metal, otherwise color may develop.
Restrictions
For Research Use only
Datasheet