Vitamin E ELISA Kit

Details for Product No. ABIN1884657, Supplier: Log in to see
Antigen
Reactivity
Chemical
Alternatives
Kits with alternative reactivity to:
9
3
2
1
1
1
1
1
1
1
1
1
Method Type
Competition ELISA
Detection Range
1.563-100 μg/mL
Minimum Detection Limit
1.563 μg/mL
Application
ELISA
Options
Supplier
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Supplier Product No.
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'Independent Validation' Badge
Antigen Vitamin E
Lot Number AK0017FEB08040 and AK0017AUG30057
Method validated ELISA
Positive Control

ABIN1884657 AK0017FEB08040: 40 human plasma samples in duplicates.

ABIN1884657 AK0017AUG30057: 3 plasma samples were assayed 20 times. Run spiked samples 1, 2, and 3 triplicates. Assay plasma and serum of sample 1 in triplicates and plasma and serum of sample 2 in duplicates.

Negative Control

Sample diluent

Notes

ABIN1884657 generates satisfactory results. The kit has high sensitivity and is able to detect vitamin E even at low concentrations. The recovery of vitamin E is good.

Protocol
  • Reagent preparation and the assay are performed according to the manufacturer’s instructions.
  • Add 50μL of the standard or the sample to each well.
  • Immediately add 50μL of the biotinylated detection antibody to each well and incubate for 45min at 37°C.
  • Aspirate the solution in the wells and wash wells 3x with 350µl wash buffer.
  • Add 100μL of the HRP conjugate to each well and incubate for 30min at 37°C.
  • Aspirate the solution in the wells and wash wells 5x with 350µl wash buffer.
  • Add 90µl of the substrate reagent in dim light and incubate plate for 15min at 37°C protected from light.
  • Add 50µl of the stop solution to each well in the same order the substrate reagent was added.
  • Read OD450 after 20min using a Biotek synergy multimode reader.
Experimental Notes
  • The intra assay CV for blank and standards ranged from 0.2% to 10.9 % for ABIN1884657 from both tested lots (A and C).

  • The average intra assay % CV was 6.9% for 40 samples that were assayed in duplicates (lot AK0017FEB08040) Table 2.

  • The intra assay % CV for the samples 1, 2 and 3 was around 10.5%, 11.9% and 10.6%, respectively and the intra-assay % CV for spiked samples 1, 2, 3, were 4.29%, 2.68%, 11.05%. The recovery of vitamin E from the spiked samples ranged between 83% and 98% with ABIN1884657 lot AK0017AUG30057 (Table 4).

  • We found that two samples that were repeated from the first plate did not replicate. It is unclear if it is due to the sample quality.

  • We have observed that our OD450 values for all standard and blanks were lower than the OD450 normally observed and the reason is not known at this point.

Validation Images
ELISA validation image for Vitamin E ELISA Kit (ABIN1884657) A OD450 measurements, standard deviation, intra assay precision, and calcu...
Purpose The kit is a competitive enzyme immunoassay for in vitro quantitative measurement of VE in serum, plasma and other biological fluids
Sample Type Plasma, Serum, Biological Fluids
Analytical Method Quantitative
Detection Method Colorimetric
Specificity This kit recognizes natural and recombinant VE. No signif icant cross-reactivity or interference between VE and analogues was observed. Note: Limited by existing techniques, cross reaction may still exist, as it is impossible for us to complete the cross-reactivity detection between VE and all the analogues.
Sensitivity 0.938 μg/mL
Components Micro ELISA Plate(Dismountable)
Reference Standard
Concentrated Biotinylated Detection Ab (100×)
Concentrated HRP Conjugate (100×)
Reference Standard & Sample Diluent
Biotinylated Detection Ab Diluent
HRP Conjugate Diluent
Concentrated Wash Buffer (25×)
Substrate Reagent
Stop Solution
Plate Sealer
Product Description
Certificate of Analysis
Material not included Microplate reader with 450nm wavelength filter
High-precision transferpettor, EP tubes and disposable pipette tips
37℃ Incubator
Deionized or distilled water
Absorbent paper
Loading slot for Wash Buffer
Research Area Signaling, Metabolism
Application Notes ELISA Plate: The just opened ELISA Plate may appear water-like substance, which is normal and will not have any impact on the experiment results.
Add Sample: The interval of sample adding between the first well and the last well should not be too long, otherwise will cause different pre-incubation time, which will significantly affect the experiment’s accuracy and repeatability. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. Parallel measure ment is recommended.
Incubation: To prevent evaporation and ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Do not let the strips dry at any time during the assay. Strict compliance with the given incubation time and temperature.
Washing: The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. Residual liquid in the reaction wells should be pat dry against absorbent paper in the washing process. But don’t put absorbent paper into reaction wells directly. Note that clear the residual liquid and fingerprint in the bottom before measurement, so as not to affect the micro-titer plate reader.
Reagent Preparation: As the volume of Detection Ab and HRP Conjugate is very small, liquid may adhere to the tube wall or tube cap when being transported. You better hand-throw it or centrifugal it for 1 minute at 1000rpm. Please pipette the solution for 4-5 times before pippeting. Please carefully reconstitute Standards, working solutions of Detection Ab and HRP Conjugate according to the instructions. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10μL for once pipetting. Do not reuse standard solution, working solution of Detection Ab and HRP Conjugate, which have been diluted. If you need to use standard repeatedly, you can divide the standard into small pack according to the amount of each assay, keep them at -20°C to -80°C and avoid repeated freezing and thawing.
Reaction Time Control: Please control reaction time strictly following this product description!
Substrate: Substrate Solution is easily contaminated. Please protect it from light.Stop Solution: As it is an acid solution, please pay attention to the protection of your eyes, hands, face and clothes when using this solution.
Mixing: You’d better use micro-oscillator at the lowest frequency, as sufficient and gentle mixing is particularly important to reaction result. If there is no micro-oscillator available, you can knock the ELISA plate frame gently with your finger before reaction.
Security: Please wear lab coats and latex gloves for protection. Especially detecting samples of blood or other body fluid, please perform following the national security columns of biological laboratories.
Do not use component from different batches of kit(washing buffer and stop solution can be an exception)
To avoid cross-contamination, change pipette tips between adding of each standard level, between sample adding, and between reagent adding. Also, use separate reservoirs for each reagent. Otherwise, the results will be inaccurate!
Comment

Information on standard material:
The formulation of the standard is 0.01 M PBS. The standard contains additives (1 % BSA).

Information on reagents:
Reagents include 1 M SO2. Azide, thimerosal, 2-mercaptoethanol (2-ME) or any other poisonous materials are not used.

Information on antibodies:
The provided antibodies and their host vary in different kits. All antibodies are affinity purified

The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.

Sample Volume 50 µL
Plate Pre-coated
Protocol This ELISA kit uses Competitive-ELISA as the method. The microtiter plate provided in this kit has been pre-coated with VE. During the reaction, VE in the sample or standard competes with a fixed amount of VE on the solid phase supporter for sites on the Biotinylated Detection Ab specific to VE. Excess conjugate and unbound sample or standard are washed from the plate, and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of VE in the samples is then determined by comparing the OD of the samples to the standard curve. 6th Edition, revised in June, 2015 www.elabscience.com 4
Reagent Preparation

Bring all reagents to room temperature (18-25°C) before use.
Wash Buffer: Dilute 30 mL of Concentrated Wash Buffer into 750 mL of Wash Buffer with deionized or distilled water. Put unused solution back at 4°C. If crystals have formed in the concentrate, you can warm it with 40°C water bath (Heating temperature should not exceed 50°C) and mix it gently until the crystals have completely dissolved. The solution should be cooled to room temperature before use.
Standard: Prepare standard within 15 minutes before use. Reconstitute the Standard with 1.0 mL of Sample Diluent, let it stand for 10 minutes until it dissolved fully. This reconstitution produces a stock solution. Then make serial dilutions as needed (Making serial dilution in the wells directly is not permitted). The Sample Diluent serves as the zero (0).
Biotinylated Detection Ab: Calculate the required amount before experiment (100µL /well). In actual preparation, you should prepare 100~200µL more. Centrifuge the stock tube before use, dilute the concentrated Biotinylated Detection Ab to the working concentration using Diluent for Biotinylated Detection Ab (1:100).
Concentrated HRP Conjugate: Calculate the required amount before experiment (100µL /well). In actual preparation you should prepare 100~200µL more. Dilute the Concentrated HRP Conjugate to the working concentration using Diluent for Concentrated HRP Conjugate (1:100).
Substrate Reagent: As it is sensitive to light and contaminants, so you shouldn't open the vial until you need it! The needed dosage of the reagent can be aspirated with sterilized tips and the unused residual reagent shouldn't be dumped back into the vial again.
Note: please don't prepare the reagent directly in the Diluent vials provided in the kit. Contaminated water or container for reagent preparation will influence the result.

Sample Collection Samples should be clear and transparent and be centrifuged to remove suspended solids.
Serum: Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation for 15 minutes at 1000×g. Collect the supernatant and carry out the assay immediately. Blood collection tubes should be disposable, non-pyrogenic, and non-endotoxin.
Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8°C within 30 minutes of collection. Collect the supernatant and carry out the assay immediately. Hemolysis samples are not suitable for ELISA assay!
Other biological fluids: Centrifuge samples for 20 minutes at 1000×g at 2 - 8°C. Collect the supernatant and carry out the assay immediately.
Tissue homogenates: You'd better get detailed references from other literatures before assay aiming at different tissue types. For general information, hemolysis blood may affect the result, so you should mince the tissues to small pieces and rinse them in ice-cold PBS (0.02M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then homogenized in PBS (the volume depends on the weight of the tissue) with a glass homogenizer on ice. To further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifugated for 5minutes at 5000×g to get the supernate.
Note:
Samples should be used within 7 days when stored at 2-8°C, otherwise samples must be divided and stored at -20°C (≤ 1month) or -80°C (≤ 6months) to avoid the loss of bioactivity and contamination. Avoid repeated freeze-thaw cycles.
Please take the samples to room temperature (18-25°C) without extra heating before performing the assay.
Please predict the concentration before assaying. If the sample concentration is not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
Assay Procedure

Bring all reagents and samples to room temperature before use. Centrifuge the sample again after thawing before the assay. All the reagents should be mixed thoroughly by gently swirling before pepitting. Avoid foaming. It's recommended that all samples and standards be assayed in duplicate.
1. Add Sample and Biotinylated Detection Ab: Add 50µl of Standard, Blank, or Sample per well. The blank well is added with sample diluent. Immediately add 50 µl of Biotinylated Detection Ab working solution to each well. Cover with the Plate sealer we provided. Gently tap the plate to ensure thorough mixing. Incubate for 45minutes at 37°C. (Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible.)
2. Wash: Aspirate each well and wash, repeating the process three times Wash by filling each well with Wash Buffer (approximately 350µl) using a squirt bottle, multi-channel pipette, manifold dispenser or automated washer. 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 pat it against thick clean absorbent paper.
3. HRP Conjugate: Add 100µl of HRP Conjugate working solution to each well. Cover with a new Plate sealer. Incubate for 30 minutes at 37°C.
4. Wash: Repeat the aspiration/wash process for five times as conducted in step 4.
5. Substrate: Add 90µl of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for about 15 minutes at 37°C. Protect from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30 minutes. When apparent gradient appeared in standard wells, you can terminate the reaction.
6. Stop: Add 50µl of Stop Solution to each well. Color turn to yellow immediately. The adding order of stop solution should be as the same as the substrate solution.
7. OD Measurement: Determine the optical density (OD value) of each well at once, using a microplate reader set to 450 nm. You should open the microplate reader ahead, preheat the instrument, and set the testing parameters.
8. After experiment, put all the unused reagents back into the refrigerator according to the specified storage temperature respectively until their expiry.

Calculation of Results

Average the duplicate readings for each standard and samples. Create a standard curve by plotting the mean OD value for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. It is recommended to use some professional software to do this calculation, such as curve expert 1.3 or 1.4. In the software interface, a best fitting equation of standard curve will be calculated using OD values and concentrations of standard sample. The software will calculate the concentration of samples after entering the OD value of samples. If samples have been diluted, the concentration calculated from the standard curve must be multiplied by the dilution factor. If the OD of the sample surpasses the upper limit of the standard curve, you should re-test it after appropriate dilution. The actual concentration is the calculated concentration multiplied dilution factor.

Restrictions For Research Use only
Handling Advice All the reagents in the kit should be stored according to the labels on vials. Unused wells should be returned to the foil pouch with the desiccant pack and resealed along entire edge of zip-seal. Substrate Reagent shouldn't be kept at -20 °C (Check!). Exposure of reagents to strong light should be avoided in the process of incubation and storage. All the taps of reagents should be tightened to prevent evaporation and microbial contamination. If not to store reagents according to above suggestions, erroneous results may occur.
Storage 4 °C/-20 °C
Storage Comment The unopened kit can be stored at 4℃ for 1 month. If the kit is not used within 1 month, store the items separately according to the conditions since the kit is received.
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