ELISA kit FAQs
Important Questions

Q: What products or services do you offer?

CUSABIO provides the highest quality ELISA kits for research use and custom ELISA service at reasonable costs allowing our customers to focus their precious time and resources on advancing their own research.

CUSABIO also offers food safety & drug residues ELISA kits in feed and food focusing mainly on chemical contaminants comprising on natural and artificial contaminants by antibiotics, mycotoxins and hormones testing solutions.

Q: Why do we choose your ELISA kits?

CUSABIO has a sound platform for the development of ELISA kit, mature antigen-antibody research and development system. We are proficient in a variety of ELISA technologies, and the quality of our ELISA kits is in the leading place worldwide. CUSABIO now offers a broad range of ELISA kits covering over 9,000 different assay targets. 100% Risk-free performance is guaranteed.

Q: Where can I find references from other researchers that have used your ELISA kits?

Our official website is updated to include the articles from researchers who have published their results after using CUSABIO ELISA kits. Please click here to see more: https://www.cusabio.com/m-248.html.

Application and Selection of Our Products

Q: How to choose a right ELISA kit?

It depends on the species and analyte you study and your purpose of analysis. You can make a better choice by referring to: https://www.cusabio.com/c-20299.html.

Q: What is the size of each ELISA Kit?

48T and 96T are available. If you order bulk quantity such as 5×96T and 10×96T, we could offer more discount. Now, you can also apply for a 24T trial size at low cost.

Q: What quality controls and performance evaluation do you do for every ELISA kit?

Linearity range, linearity, lower limit of detection, precision, recovery, stability, specificity and natural samples detection are carefully evaluated under strict QC standards to ensure good performance. Click here to know more about quality control.

Q: How sensitive are the ELISA kits?

The ELISA sensitivity is highlighted in the data sheet for each kit.

For example, Human Tumor necrosis factor α,TNF-α ELISA KIT, the sensitivity is 1.95 pg/mL.

Q: Do you offer a free sample for a trial?

We are now offering 24T trial size at low cost for all ELISA kits except food safety ELISA kits until the end of this year. It only costs $150/kit including shipping cost. Only one 24T trial size kit can be applied for each product for single customer. No quantity limit for products you can apply. For more information please kindly refer to: https://www.cusabio.com/24T_ELISA/.

Q: What components are provided in your ELISA kit?

Please refer to the "materials provided" section in the kit manual.

Q: Which preservatives do you frequently use?

We use Proclin 300 as preservatives.

Q: Do you offer standards or controls in every ELISA kit?

We offer positive and negative controls in qualitative ELISA kits, while we offer standards which can work as quality control in quantitative ELISA kits. Kit standards and the entire reagent system are matched. The customer can draw a good standard curve through the experiment, which proves that the whole system including the standards works well, so the standards can play a role of quality control. We suggest you run duplicates for the standards.

Q: Do you map the epitopes that the antibodies recognize in your kits? What is the clone ID for each antibody?

Unfortunately, the epitopes have not been mapped. But we have tested the specificity of the antibody and we fully guarantee the detection of the target with our ELISA kit. Sorry we do not have commercial clone ID.

Q: What kind of sample types have been validated with your ELISA kit? Can I use your ELISA kit for the sample type which is not mentioned in the manual?

Different ELISA kit has been validated in different sample types varying from serum, plasma, body fluid like urine, cell culture supernatant or cell/tissue lysate. Please refer to the kit protocol for sample types. Theoretically, if the concentration of the target is within the detection range, it can be detected out. We would like to suggest you do a pretest with 24T trial kit first if you want to test the sample type which is not mentioned in the manual.

Q: What kind of species is your ELISA kit specific to? Can I use your ELISA kit for the species which is not mentioned in the manual?

Different ELISA kit is designed for different species varying from human, rat, horse, rabbit, mouse, sheep, pig, bovine, dog, chicken, fish, monkey, plant, etc. Please refer to each protocol for species instruction. For different species, the antibody's specificity is different, and there is matrix interference. We can recommend the right kit if you have difficulty to find the kit of species you need.

Q: How many times do I have to run the ELISA standards and samples in each test?

It is recommended to run duplicated wells both for the standards and samples.

Q: How many samples could be detected with each ELISA Kit?

For a 96T sandwich ELISA kit (double-antibody), we suggest you run duplicates for the standard. In this way you can test 80 samples. We also suggest you run duplicates for the sample, and in this way you can test 40 samples. You can decide how many kits you need according to the number of your samples. If you run a pre-test, the number of samples you can detect in one kit will be less. Click here to see how to set up your plate.

Q: How much sample volume is needed?

For a 96T double antibody ELISA kit, the sample volume needed for testing one sample is 100μL, but considering the loss during the assay, you can prepare 120 μL. We suggest you run duplicated wells, so in this way it takes about 250 μL for testing one sample.

For a 96T competitive ELISA kit, the sample volume needed for testing one sample is 50μL, but considering the loss during the assay, you can prepare 70μL. We suggest you run duplicated wells, so in this way it takes about 150μL for testing one sample.

Q: Is it possible to mixedly use the reagents of two different ELISA kits? Can I only purchase the standard/antibody in the kit?

No, the reagents in the ELISA kits with different lot numbers cannot be mixed and we do not sell the components in the kit separately. Hope you could understand.

Q: What's your recommended dilution for human serum?

Please refer to the kit manual. For different ELISA kit, the dilution ratio is different. We would like to suggest that you do a pretest first and confirm the proper dilution ratio.

Q: Do you add BSA and preservative in your kit? What is your stop solution?

Some components in the ELISA kits contain BSA. We use commercial Proclin 300 as our preservative and 4N sulfuric acid as stop solution in most of ELISA kits.

Q: Which kind of antibody do you use in ELISA kits?

Different kits have different antibodies. If you need this information, please send inquiry to our tech team.

Q: Can I incubate at room temperature?

Incubation is performed at 37℃ for all ELISA kits except for food safety & drug residues ELISA kits as it is the best temperature for combination of antigen and antibody. We do not recommend the room temperature since it is not stable.

Q: What is TMB? Why should I use dual-spectrum to test my sample using TMB system?

TMB(3,3',5,5'-Tetramethylbenzidine) is chromogenic substrate used in ELISA. TMB can act as a hydrogen donor for the reduction of hydrogen peroxide to water by peroxidase enzymes such as horseradish peroxidase. The reaction will be terminated and the color developed in the wells will turn from blue to yellow upon addition of the stop solution. The absorbance value should be read at 450 nm. We recommend you use dual-spectrum to test your sample to avoid the error caused by interference and scratch in the bottom.

Q: How should I wash the plate?

Please ensure the same volume of wash buffer per well and you'd better use multi-channel pipette. If you use the ELISA automatic washer, please make sure it’s clean enough without contamination. No rinse but gently wash. Hold the plate vertically and wash it thoroughly to reduce the edge effect.

General Troubleshooting Guide for ELISA

Q: What is ELISA? What is the common procedure of ELISA?

ELISA is the abbreviation of the enzyme-linked immunosorbent assay used to identify the presence of specific proteins and to determine their concentrations. There are three kinds of ELISA including ”sandwich”, competitive and indirect ELISA. The common procedures are Storage, Reagents preparation, ELISA plate, Samples or reagents adding, Incubation, Washing and Reading. Please refer to our manual for details.

Q: Why is yellow or light yellow color developed in the wells of the whole plate when stop solution is added?

a. Incorrect reagents added. You should check the components and Lot No. of reagents to ensure correct reagents of the kit.

b. Insufficient plate washing. You should be sure all wells are filled with buffer with the same volume during every wash step. After final wash, blot plate forcefully on paper towel to remove residual buffer.

c. Contamination of the pipette tips and chromogenic agent container with the enzyme conjugate or contamination of blank wells with positive control. You should change pipette tips between reagents and use separate reservoirs for each reagent. Use pipette during operation.

d. Substrate exposed to light or contaminated prior to use. You should keep substrate in the dark until ready to dispense into wells.

e. Concentration of detection antibody or avidin-HRP is too high. You should check calculations or try again after further dilution.

f. Incubation time or color developing time is too long. You should follow the kit protocol strictly.

g. Wrong wavelength used when taking readings. Wavelength should be 450nm with a 650nm wavelength correction for TMB.

Q: Why is there no color developed in the wells of the whole plate including the positive control when color development is finished?

a. Reagents were wrongly used. You should check the label carefully when preparing or using.

b. Chromogenic agent inactive with the contamination of enzyme conjugate during wash or sample addition steps. You should ensure no enzyme inhibitor in the enzyme conjugate container. Check and ensure the washing buffer container clean.

c. A reagent or a step of the procedure taken by mistake. You should read the kit protocol carefully and follow each step strictly.

Q: Why is weak color developed in all the wells of standards and samples?

a. Reagents expired or improperly stored. You should keep the kit well stored according to the kit protocol and use it before expiration date. Avoid contamination.

b. Reagents and samples were not brought to room temperature prior to use. You should put all the reagents and samples under room temperature for 30 minutes.

c. Pipette suction of fluids insufficient, pipette emissions too fast, fluids on the tip wall too much or tip wall unclean. You should use calibrated pipettes to ensure the pipette tips firmly matched and pipette slowly. Single use is recommended.

d. Insufficient incubation time. You should set the timer accurate.

e. Insufficient color development time. You should develop color in 15-30 minutes. 20 minutes is the best unless otherwise instructed.

f. Color development reagent added in incorrect order. You should follow the kit protocol strictly.

g. Too much washing or improper dilution of concentrated wash buffer. You should reduce washing impact force: dilute the concentrated wash buffer, control the washing time, and record the washing times and dosage according to the manual.

h. Unqualified distilled water. You should check and ensure the pH is neutral when preparation of wash buffer is finished.

i. Chromogenic agent inactive with the contamination of enzyme conjugate during wash or sample addition steps. You should ensure no enzyme inhibitor in the enzyme conjugate container. Check and ensure the washing buffer container clean. Ensure the purified water no contamination.

Q: Why does the standard curve look fine but weak color is developed in the wells of samples?

a. NaN3 preservatives from the samples inhibit the reaction of enzyme. You should avoid use this perservative in samples.

b. No strong positive samples on the detected ones, the result is normal. You should repeat the assay if you have any doubt.

Q: Why does it read low with normal results by eyes?

Wrong filter is used when taking readings. Wavelength should be 450nm with a 650nm wavelength correction for TMB.

Q: Why is repeatability poor?

a. Improper storage of the kit or poor storage environment. You should store all components as recommended on data sheet rather than room temperature for excess time.

b. Incorrect preparation of standard. You should reconstitute standard strictly with the recommended diluent according to the kit protocol. You should prepare reagents in 10 minutes prior to use and add them to wells promptly.

c. Insufficient mixing after adding samples. You should fully mix reagents in the vortex mixer when adding several reagents at the same time. Be careful when holding reagents to avoid splashing.

d. Poor repeatability of the plate readings. You should calibrate the plate reader.

e. Inconsistent incubation time, washing condition, color development condition and operators. You should repeat the assay of standard. Ensure consistent reactivity condition and operators.

f. Improper washing. You should add 200μL of wash buffer or fully fill into every well with pipette but no overflows are allowed. Check that all ports of the plate washer are unobstructed to ensure sufficient washing.

g. Uneven temperature. You should keep constant temperature during incubation to avoid temperature fluctuations.

h. Too much residual on the wall of the wells when adding or the bottom of wells scratched with pipette tip. You should lower the pipette tips along the wall of wells when adding slowly and carefully. Do not touch the bottom of wells.

i. Reused materials. You should change pipette tips between samples and reservoirs between reagents.

j. Occasional positive and negative values close to the cut off value. You should set 3 duplicates for the same sample and the same result over 2 samples.

k. Cross contamination when adding samples. You should avoid cross contamination when adding samples.

Q: Why is abnormal color developed?

a. Cross contamination during manual washing. You should reduce the cross contamination by promptly removing the liquid in wells after 3 times of filling washing buffer during manual washing and then setting the soak time the next times.

b. Cross contamination when patting the plate. You should use proper paper towels when patting the plate. Do not bring unrelated materials into the plate. Do not pat at the same place to avoid cross contamination.

c. Contamination due to long storage of samples. You should keep samples fresh or store them under low temperate to avoid contamination.

d. Abnormal developed color due to insufficient filling or too much residual when the plate washer is obstructed. You should fully fill into every well with pipette but no overflows are allowed. Check that all ports of the plate washer are unobstructed to ensure sufficient washing.

e. Coagulationor interference of precipitates or residual cell caused by incomplete centrifugation of samples. You should complete centrifugation of serum and plasma.

f. Wrong preparation of wash buffer or misuse of concentrated wash buffer. You should prepare wash buffer as manual required.

Shipping and Storage

Q: How long can I get the products after I place an order?

All our ELISA kits are made to order. The production time is about 3-5 working days, and it takes another 3-5 days for FEDEX or DHL delivery.

Q: How should I store the products?

Please store unopened kit at 2-8℃ and use the kit before expiration date. For opened kit, which is vulnerable to germ contamination, it’s better to use it as soon as possible. Do never store the ELISA plate at -20℃ and the substrate solution should be kept in the dark both during storage and usage.

Q: Are the plates pre-coated? How can I store the remaining wells or plates if I can not use in one time?

Yes. The strips are movable and it’s recommended to separate those wells needed for assay and store the rest at 2-8℃ in the dark without germ contamination.

Q: What is the shelf life of the products?

The expiration date is shown on the label of each kit. Generally, CUSABIO ELISA kit has a shelf life of 6 months from the date of production if it is unopened and stored at 2-8℃. For opened kit, please refer to the protocol for component-specific storage instructions.

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