Traditional ELISA kits vs. Ready-To-Use ELISA kits

Our new ‘Ready-To-Use’ ELISA kits feature:

  • Shortened experimental time 
  • Pre-diluted detection reagents
  • Enhanced stability to allow for storage of the whole kit at 4oC

These kits still maintain our high level of quality and sensitivity, as well as our 12-month shelf life.

Our Ready-To-Use kits are easier and faster to use as compared to our Traditional ELISA kits.

Reagents and Materials Provided

Traditional​ 96T ELISA Kits

  • Pre-coated, ready to use 96-well strip plate: 1
  • Plate sealer for 96 wells: 2
  • Standard: 2
  • Standard Diluent: 1 x 20mL
  • Detection Reagent A: 1x120μL   (1x70μL  for competitive kits)
  • Detection Reagent B: 1x120μL
  • Assay Diluent A: 1 x 12mL
  • Assay Diluent B: 1 x 12mL
  • TMB Substrate: 1 x 9mL
  • Stop Solution: 1 x 6mL
  • Wash Buffer (30 x concentrate): 1x20mL
  • Instruction manual: 1

Ready-To-Use 96T ELISA kits

  • Pre-coated, ready to use 96-well strip plate: 1
  • Plate sealer for 96 wells: 2
  • Standard: 2
  • Standard Diluent: 1 x 20mL
  • Detection Solution A: 1x12mL
  • Detection Solution B: 1x12mL
  • TMB Substrate: 1 x 9mL
  • Stop Solution: 1 x 6mL
  • Wash Buffer (30 x concentrate): 1x20mL
  • Instruction manual: 1

 

 

Traditional 48T ELISA Kits

  • Pre-coated, ready to use 96-well strip plate: 1
  • Plate sealer for 96 wells: 1
  • Standard: 1
  • Standard Diluent: 1 x 10mL
  • Detection Reagent A: 1x60μL (1x35μL for competitive kits)
  • Detection Reagent B: 1x60μL
  • Assay Diluent A: 1 x 6mL
  • Assay Diluent B: 1 x 6mL
  • TMB Substrate: 1 x 4.5mL
  • Stop Solution: 1 x 3mL
  • Wash Buffer (30 x concentrate):1x10mL
  • Instruction manual: 1

 

Ready-To-Use 48T ELISA kits

  • Pre-coated, ready to use 96-well strip plate: 1
  • Plate sealer for 96 wells: 1
  • Standard: 1
  • Standard Diluent: 1 x 10mL
  • Detection Solution A: 1x6mL
  • Detection Solution B: 1x6mL
  • TMB Substrate: 1 x 4.5mL
  • Stop Solution: 1 x 3mL
  • Wash Buffer (30 x concentrate):1x10mL
  • Instruction manual: 1

 

 

 

Materials required but not supplied in kit
  • Microplate reader with 450± 10nm filter.
  • Precision single or multi-channel pipettes and disposable tips.
  • Microcentrifuge tubes for dilutions.
  • Deionized or distilled water.
  • Absorbent paper for blotting the microtiter plate.
  • Container for the diluted wash buffer.
Storage of the kits
For unopened kits

Traditional ELISA kits: All the reagents should be kept according to labels on the vials. The TMB Substrate, Wash Buffer (30X concentrate) and the Stop Solution should be stored at 4oC upon receipt while the others should be at -20oC.

Ready-To-Use ELISA kits: All the reagents should be stored at 4oC upon receipt.

For opened kits

Once the kit is opened, the remaining reagents still need to be stored according to the above storage conditions. In addition, return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.

Note:

For the expiration date of the kit, please refer to the label on the kit box. All components are stable until this date.

It is highly recommended to use the remaining reagents within 1 month of opening.

Sample Collection and Storage

Please note which sample types are recommended for the kit you are interested in. If you have any questions or would like to know if we can make a custom kit to fit your needs, please contact us.

Serum

Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximately 1000×g. Assay freshly prepared serum immediately or store samples in aliquots at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.

Plasma

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8℃ within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquots at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.

Tissue homogenates

The preparation of tissue homogenates will vary depending on tissue type. In general, tissues should be rinsed thoroughly in ice-cold PBS (0.02mol/L,pH 7.0-7.2) to remove excess blood and weighed before homogenization. Mince the tissues to small pieces and homogenize them in 5-10 mL of PBS with a glass homogenizer on ice (Micro Tissue Grinders work as well). The resulting suspension should be sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates should be centrifugated for 5 minutes at 5000×g. Remove the supernatant and assay immediately or aliquot and store at ≤-20 ℃.

Cell Lysates

Cells must be lysed before assaying according to the following directions:

  1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly).
  2. Wash cells three times in cold PBS.
  3. Resuspend cells in 1X PBS  and subject to ultrasonication 4 times (or Freeze cells at≤-20℃. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle 3 times.)
  4. Centrifuge at 1500×g for 10 minutes at 2 - 8℃ to remove cellular debris. Cell culture supernates and Other biological fluids - Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquot at -20℃ or -80℃. Avoid repeated freeze/thaw cycles.
Saliva

Collect saliva using a collection device or equivalent. Centrifuge samples for 15 minutes at 1,000×g at 2-8oC. Remove particulates and assay immediately or store samples in aliquot at ≤-20oC. Avoid repeated freeze/thaw cycles.

Urine

Aseptically collect the first urine of the day (mid-stream), collect directly into a sterile container. Centrifuge to remove particulate matter, assay immediately or aliquot and store at ≤-20oC. Avoid repeated freeze-thaw cycles.

Milk

Centrifuge samples for 15 minutes at 10,000×g at 2 - 8oC. Collect the aqueous fraction and centrifuge twice more for a total of 3 cycles. Assay immediately or or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles.

Tears, Cell culture supernatant and other biological fluids

Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquots at -20oC or -80oC. Avoid repeated freeze/thaw cycles.

Note:                                                
  1. Samples to be used within 5 days may be stored at 4oC, otherwise samples must be stored at -20oC (≤1 month) or -80oC (≤2 months) to avoid loss of bioactivity and/or contamination.
  2. Sample hemolysis will influence the results; hemolytic specimen used as samples will not be detected.
  3. When performing the assay, bring samples to room temperature.
Reagent Preparation
* Bring all kit components and samples to room temperature (18-25oC) before use.
Standard 

Reconstitute the Standard with Standard Diluent, keep for 10 minutes at room temperature, shake gently (not to foam). Please refer to the manual for the concentration of the standard in the stock solution. Prepare 7 tubes containing 0.5mL Standard Diluent and use the diluted standard to produce a double dilution series. Mix each tube thoroughly before the next transfer.

Detection Reagent A and Detection Reagent B

Traditional ELISA kits: Briefly spin or centrifuge the stock Detection A and Detection B solutions before use. Dilute to the working concentrations with Assay Diluent A and B, respectively (1:100).

Ready-To-Use ELISA kits: Detection Solutions A and B are already at the correct concentrations and do not need to be diluted further.

Wash Solution 

Dilute 20mL of Wash Solution concentrate (30×) with 580mL of deionized or distilled water to prepare 600mL of Wash Solution (1×).

TMB substrate 

Aspirate the needed dosage of the solution with sterilized tips. Do not dump the residual solution back into the vial.

Note:
  1. Do not perform a serial dilution directly in the wells.
  2. Prepare standard within 15 minutes of performing the assay. Do not dissolve the reagents at 37oC directly.
  3. Detection Reagent A and B are sticky solutions, therefore slowly pipette them to reduce the volume errors.
  4. Traditional ELISA kits: Carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, avoid foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to pipette more than 10μL at a time to ensure accuracy.                 
  5. Ready-To-Use ELISA kits: The reconstituted Standards can be used only once.
  6. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
  7. If crystals have formed in the Wash Solution concentrate (30×), warm to room temperature and mix gently until the crystals are completely dissolved.
  8. Any contaminated water or container used during reagent preparation will influence the detection result.
Sample Preparation
  • Reddot Biotech is only responsible for the kit itself, not for the samples consumed during the assay. The experimenter should always calculate the total amount of samples needed prior to beginning the experiment.
  • The experimenter should also predict the approximate concentration before beginning. If the expected values are not within the range of the standard curve, it is recommended to determine the optimal dilution for your sample.
  • If the sample type you are using is not indicated in the manual, you should conduct a preliminary experiment to ensure the effectiveness of the kit before beginning the actual experiment.
  • Using a chemical lysis buffer to prepare tissue or cell extraction samples is not recommended as it may cause unexpected reactions and results due to the contents of the lysis buffer.
  • Due to the possibility of mismatching between antigens of other origins and the antibodies used in our kits, it is NOT recommended to use native or recombinant proteins from other manufacturers as they may not be recognized by our kit.
  • Samples from cell culture supernatant may not be detected by our kit as they can easily be influenced by factors such as cell viability, cell number and/or sampling time.
  • Fresh samples that have not been stored for long periods are recommended for use with our kits. If samples are stored unfrozen for long periods of time, protein degradation and denaturation can occur, resulting in contradictory or incorrect results.
Assay Procedure
For Sandwich ELISA Kits
  1. Determine wells for diluted standard, blank and sample. Prepare 7 wells for the standards, and 1 well for the blank.  Add 100μL each of dilutions of standard (read Reagent Preparation), blank, and samples into the appropriate wells. Cover with the Plate sealer. Incubate for 2 hours at 37oC.
  2. Remove the liquid from each well, do not wash.
  3. Add 100μL of Detection Reagent A working solution (Detection Solution A in Ready-To-Use Kits) to each well. Incubate for 1 hour at 37oC after covering it with the Plate sealer.
  4. Aspirate the solution and wash with 350μL of 1× Wash Solution in each well using a squirt bottle, multi-channel pipette, manifold dispenser or auto-washer, and let it sit for 1~2 minutes. Remove the remaining liquid from all wells completely by tapping the plate onto absorbent paper. Wash thoroughly 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper.
  5. Add 100μL of Detection Reagent B working solution (Detection Solution B in Ready-To-Use Kits) to each well. Incubate for 1 hour at 37oC after covering it with the Plate sealer.
  6. Repeat the aspiration/wash process for a total of 5 times as conducted in step 4.
  7. Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15-25 minutes at 37oC (Do not exceed 30 minutes). Protect from light. The liquid will turn blue with the addition of the Substrate Solution.
  8. Add 50μL of Stop Solution to each well. The liquid will turn yellow with the addition of the Stop solution. Mix the liquid by tapping the side of the plate. If the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
  9. Remove any drops of water and fingerprints on the bottom of the plate and confirm there are no bubbles on the surface of the liquid. Run the microplate reader and take measurements at 450nm immediately.
For Competitive ELISA Kits
  1. Determine wells for diluted standard, blank and sample. Prepare 7 wells for the standards, and 1 well for the blank. Add 50μL each of dilutions of standard (read Reagent Preparation), blank and samples into the appropriate wells, respectively. And then add 50µL of Detection Reagent A (Detection Solution A in Ready-To-Use Kits) to each well immediately. Shake the plate gently (using a microplate shaker is recommended). Cover with a Plate sealer. Incubate for 1 hour at 37oC. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
  1. Aspirate the solution and wash with 350μL of 1× Wash Solution to each well using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1-2 minutes. Remove the remaining liquid from all wells completely by tapping the plate onto absorbent paper. Wash thoroughly 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper.
  2. Add 100μL of Detection Reagent B working solution (Detection Solution B in Ready-To-Use Kits) to each well. Incubate for 1 hour at 37oC after covering it with the Plate sealer.
  3. Repeat the aspiration/wash process for total 5 times as conducted in step 2.
  4. Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15-25 minutes at 37oC (Do not exceed 30 minutes). Protect from light. The liquid will turn blue with the addition of Substrate Solution.
  5. Add 50μL of Stop Solution to each well. The liquid will turn yellow with the addition of Stop solution. Mix the liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
  6. Remove any drops of water and fingerprints on the bottom of the plate and confirm there are no bubbles on the surface of the liquid. Run the microplate reader and conduct measurement at 450nm immediately.
Note:
  1. Assay preparation: Keep appropriate numbers of wells for each experiment and remove extra wells from the microplate. Remaining wells should be resealed and stored at -20oC for Traditional kits, 4oC for Ready-To-Use Kits.  
  2. Samples or reagents addition: Please use freshly prepared Standard. Carefully add samples to wells and mix gently to avoid foaming. Do not touch the well walls. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and specimens, although not required, is recommended. To avoid cross-contamination, change pipette tips between additions of standards, samples, and reagents. In addition, use separated reservoirs for each reagent.
  3. Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods in between incubation steps. Once reagents are added to the well strips, DO NOT let the strips dry at any time during the assay. Incubation time and temperature must be controlled.
  4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential for good performance of the kit. After the last wash, remove any remaining Wash Solution by aspirating or decanting, and remove any drops of water or fingerprints on the bottom of the plate. Insufficient washing will result in poor precision and false elevated absorbance reading.
  5. Controlling of reaction time: Observe the change of color after adding TMB Substrate (e.g. observation once every 10 minutes), if the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in an inaccurate absorbance reading.
  6. TMB Substrate can easily be contaminated. Protect it from light as well as physical contaminants. 
  7. The environmental humidity may have an effect on the results obtained from the kit. If the humidity in your facility is less than 60%, using a humidifier is recommended.

 

Test Principle
For Sandwich ELISA Kits

The microtiter plate provided in this kit has been pre-coated with an antibody specific to the target antigen. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to the target antigen. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only the wells that contain the target antigen, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of the target antigen in the samples is then determined by comparing the O.D. of the samples to the standard curve.

For Competitive ELISA Kits

This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to the target antigen has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled target antigen and unlabeled target antigen (Standards or samples) with the pre-coated antibody specific to the target antigen. After incubation, the unbound conjugate is washed off. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of the target antigen in the sample. After addition of the substrate solution, the intensity of color developed is proportional to the concentration of the target antigen in the sample.

Calculation of Results
For Sandwich ELISA Kits

Average the duplicate readings for each standard, control and sample, then subtract the average zero standard optical density. Construct a standard curve by plotting the mean O.D. and concentration for each standard and draw a best fit curve through the points on the graph or create a standard curve on log-log graph paper with the target antigen concentration on the y-axis and absorbance on the x-axis. Using plotting software, (for instance, curve expert 1.30), is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

For Competitive ELISA Kits

This assay employs the competitive inhibition enzyme immunoassay technique, so there is an inverse correlation between the target antigen concentration in the sample and the assay signal intensity. Average the duplicate readings for each standard, control, and samples. Create a standard curve on log-log or semi-log graph paper, with the log of the target antigen concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line through the standard points, or it can be determined by regression analysis. Using plotting software, (for instance, curve expert 1.30), is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Sensitivity

The sensitivity of the 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.

Typical Data for Sandwich ELISA Kits

In order to make the calculation easier, we plot the O.D. value of the standard (X-axis) against the known concentration of the standard (Y-axis), although concentration is the independent variable and O.D. value is the dependent variable. However, the O.D. values of the standard curve may vary according to the conditions of assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), plotting the log of the data to establish a standard curve for each test is recommended. Please refer to the manual included in your kit for the typical standard curve.

Precision

The general method to determine precision for all our kits is described below. Specifications for individuals lots of kits are available by request.

  • Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level the index were tested 20 times on one plate, respectively.
  • Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level the index were tested on 3 different plates, 8 replicates in each plate.
  • CV(%) = SD/mean(X)100
    • Intra-Assay: CV<10%
    • Inter-Assay: CV<12%
Stability

The stability of our ELISA kits are determined by the rate of the loss of activity. The loss rate of our kits is generally less than 5% within the expiration date under appropriate storage conditions.

To minimize any outside influences on the performance of our kits, operation procedures and lab conditions should be controlled at all times. Factors to pay specific attention to include:  room temperature, air humidity, and incubator temperature. It is also strongly suggested that the whole assay is performed by the same experimenter from the beginning to the end.

Assay Procedure Summary
For Sandwich ELISA Kits
  1. Prepare all reagents, samples and standards;
  2. Add 100µL standard or sample to each well. Incubate 2 hours at 37oC;
  3. Aspirate and add 100µL prepared Detection Reagent A (Detection Solution A in Ready-To-Use Kits). Incubate 1 hour at 37oC;
  4. Aspirate and wash 3 times;
  5. Add 100µL prepared Detection Reagent B (Detection Solution B in Ready-To-Use Kits). Incubate 1hour at 37oC;
  6. Aspirate and wash 5 times;
  7. Add 90µL Substrate Solution. Incubate 15-25 minutes at 37oC;
  8. Add 50µL Stop Solution. Read at 450nm immediately.
For Competitive ELISA Kits
  1. Prepare all reagents, samples and standards;
  2. Add 50µL standard or sample to each well. And then add 50µL prepared Detection Reagent A (Detection Solution A in Ready-To-Use Kits) immediately. Shake and mix. Incubate 1 hour at 37oC;
  3. Aspirate and wash 3 times;
  4. Add 100μL prepared Detection Reagent B (Detection Solution B in Ready-To-Use Kits). Incubate 1 hour at 37oC;
  5. Aspirate and wash 5 times;
  6. Add 90μL Substrate Solution. Incubate 15-25 minutes at 37oC;
  7. Add 50μL Stop Solution. Read at 450nm immediately.
Important Note
  1. Limited by the current conditions and scientific technology, it is impossible to conduct comprehensive identification and analysis tests on the raw materials provided by suppliers. As a result, it is possible there are some qualitative and/or technical risks.
  2. The final experimental results will be closely related to the validity of the products, operation skills of the end users and the experimental environments. Please make sure that sufficient samples are available to obtain accurate results.
  3. Kits from different batches may be a little different in detection range, sensitivity and color developing time. Please perform the experiment exactly according to the instruction manual included in your kit. Electronic ones on our website are for reference only.
  4. Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
  5. Protect all reagents from strong light during storage and incubation. All bottle caps of reagents should be closed tightly to prevent evaporation of liquids and contamination by microorganisms.
  6. There may be a foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microtiter plate from the storage bag until needed.
  7. Incorrect procedures during reagent preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 O.D. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment.
  8. Even the same experimenter may get different results from two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before the general assay for each batch is recommended.
  9. Each kit has undergone several rigorous quality control tests. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipment. Intra-assay variance among kits from different batches could arise from the above factors as well.
  10. Kits from different manufacturers with the same item might produce different results, since we have not compared our products with other manufacturers.
  11. The standard in this kit, as well as the antigens used in antibody preparation are typically recombinant proteins. Differently expressed sequences, expression systems, and/or purification methods can be used in the preparation of recombinant proteins. There is also the possibility of differences in the screening technique of antibodies and antibody pairs in our kits. As a result, we cannot guarantee that our kit will be able to detect recombinant proteins produced by other companies. We do NOT recommend using Reddot Biotech ELISA kits for the detection of other recombinant proteins.
  12. Validity period: 12 months.
  13. The instruction manual also works with the 48T kit, but all reagents in the 48T kit are reduced by half.
Precaution

The Stop Solution provided for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this reagent.

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