Elisa Kits

ELISA troubleshooting

 

1.Can I mix reagents from different ELISA Sets, or use them for other applications?

This is not recommended. The ELISA reagents are optimized for a particular set lot, and they are not recommended for applications other than ELISA.

2.What is the shelf life of Fn-test ELISA products?

FineTest Kits are guaranteed for 6 months from the date of receipt. For lot-specific expiration date, refer to the box label on each Kit or Set.

3.Can I use different components from different companies for my ELISA?

Antibodies used are different in different kits. The specificity of the antibodies partially dictate how much signal is being detected.
Recombinant standards used are different.  First of all, different kits may use recombinant proteins expressed and purified using different method. Second, recombinant proteins expressed from E. coli from the same source can show greater than 10 fold differences in term of immuno reactivity from lot to lot, primarily due to refolding inconsistency. Third, different kit standards can be produced and calibrated against different references. So far there is no universally accepted standardization for immuno reactivities.
Each FineTest ELISA product was developed and validated with reagent concentrations and protocols optimized for best analytical robustness.  Any changes to the reagents (standards, antibodies, matching matrices) and protocols etc all affect the final assay performance.

4.How can I obtain better signal and sensitivity for my ELISA assay?

Increase incubation times (1st incubation, detection, avidin-HRP or TMB substrate)
Shake plates during incubation steps
Make sure that the standard is completely reconstituted before use.
Increased washing and soaking in between washings to further decrease background.
If possible read the plate at 450 nm for background subtraction.
Improve duplicate CV% by controlling pipetting error, washing with bigger volume of washing buffer, etc

5.Should I use serum or plasma samples for my ELISA experiment?

This is dependent on the targets being detected and the biological processes. Customers are advised to study the difference between serum and plasma for the targets of interest and decide on the sample type to be used for quantification. Depending on targets, there may be a difference in concentrations of the targets between serum and plasma. The most important factor in preparing plasma or serum samples is consistency in preparation to ensure precise measurements. In general, plasma/serum samples should be free of particulate matters, contain no excess lipids, and have no hemolysis.
These types of contaminants will contribute to background, and adversely affect the precision of the assay. The key is to prepare the sample the same way each time. That is, centrifuging samples at the same speed, for the same time, removing the serum or plasma immediately after centrifugation and aliquoting and freezing the samples in the same time. Avoid repeated freezing and thawing cycles.

6.Why are my sample concentrations different on theFN-testkits vs other Elisa kits?

Currently, it is very difficult to obtain exactly the same sample concentrations at pg/ml levels when comparing different kits from different vendors, partly because of following reasons:
• As mentioned in the question above, the immuno reactivity for the reagents vary from different suppliers.
• In the area of sample quantification particularly at pg/mL ranges, the general consensus is that it is more important to have matching biological trend instead of matching absolute concentrations.  This is to say that the same sample measured by different kits from different vendors should show the same pattern of biological changes predicted for the relevant biological treatments.
•  For some samples, it is not uncommon that the cytokine concentrations vary a few-fold between kits for the reasons mentioned above.
• It is critical that kits produced by the same vendor to keep its assay products consistent from lot-to- lot over time.
We tested ELISA standards from competitor’s kits. Results showed clear discrepancies in the relative potency of the standards in term their immuno reactivities.

7.Can I use your ELISA kits for my tissue samples?

Almost all of fn-test ELISA products can be used for tissue/ cell extract/homogenate samples as long as the samples are prepared in such a way that they are compatible with immune-reactivity:

  • Tissues/cells should be lysed or homogenized in a neutral pH buffer that contains no denaturing chemicals (such as urea, thiourea, SDS).
  • No or minimal levels of detergent (SDS, Triton X-100 etc).
  • No excessive ionic strength (salt concentration greater than physiological ionic strength).
  • The buffers should contain sufficient protease inhibitor cocktails to preserve the target proteins from proteolytic degradation by enzymes released from cells.

8.What antibody clones are used in your ELISA Kits?

That information is proprietary. However the clonality (polyclonal or monoclonal) and host species details may be provided upon request.

9.For some of your ELISA kits, why do my serum samples require dilution with assay buffer?

Dilution with assay buffer is required to minimize the matrix difference between the samples and the standards to achieve better accuracy.

10.What is the sensitivity of your ELISA kit?

the sensitivity values are mentioned in the manual for each kit.

11.What is the expected concentration of a particular analyte in biological samples (e.g. in serum samples of naïve animals or normal humans)?

Since every sample is unique, it is difficult to predict as this may depend on the sample preparation and the nature of the analyte. For fn-test kits refer to the respective ELISA manual for more information.

12.How many samples can I run with your kit?

It depends on how your samples are analyzed whether in duplicate or triplicate. For example you can run 80 samples with no replicates or 40 samples in duplicates and so on.

13.Can samples such as serum be reused?

It is recommended that for accurate results samples be stored aliquoted for one-time use only. However it is empirical to find out if reusing samples work for a particular analyte as it will depend on sample stability.

14.Can I add an extra standard at the lower end/at the higher end of the standard curve to enhance the sensitivity of my assay?

We don’t recommend this. Sensitivity of a kit depends on the individual components and their collective validation as a kit and it will not change by adding extra points to the standard curve.

15.Can I use your matched ELISA pair antibodies with protein standard from another company?

we don’t recommend it and we also can’t guarantee the performance of the kit as indicated in the product manual. Antibodies may not recognize or show poor binding towards the protein standard if the immunogen protein used to generate these antibodies is different in terms of structure or sequence. It is therefore empirical to find out if it works. It is best to acquire the ELISA kit components from one source.

16.Can I use the recombinant standard provided with the kit for bioassay?

No. It is not recommended because the ELISA protein standards are not sterile, may contain other carrier proteins, and not tested for bioactivity. Therefore this material is not bioassay grade.

17.Can I use the capture and detection antibody provided as part of an ELISA kit for other applications such as flow cytometry staining?

Since the antibodies are validated in house for ELISA, it is empirical to find out if the antibodies work for applications such as flow cytometry. We recommend you use flow cytometry validated antibodies for this purpose.

18.Does phenol red in the medium interfere with an ELISA assay?

No.

19.My sample may have very low levels of analyte. What methods can I use to improve detection?

For our kit we recommend following the prescribed protocol. We can’t guarantee kit performance once fixed protocols are changed. Below are just general suggestions for our ELISA Standard format.

a) Control background noise: For example, increase number of washings and soaking times in between washes.
b) Control assay precision: For example, be more careful and more consistent in your pipetting, use fresh paper towels for tapping plates to avoid contamination of avidin-HRP from dirty paper towels and increase washing volumes.
c) Increase incubation times: However this usually also increases background so the assay sensitivity may not necessarily increase.
d) Concentrate your sample if possible.
e) Use a five parameter logistic curve fitting method, which can accurately calculate sample concentration at the lower end of the standard curve. In many cases, samples indeed contain very low to non-detectable levels. No matter how you manipulate the assay you may still not be able to obtain detectable concentrations.

20.I am using the biotin and purified formats of the same antibody clone to try to set up my own ELISA, but I’m having no success.

If you are using the same clone for both the detection and capture antibodies, the epitope may already be occupied by one of the antibodies and prevent binding of the other. You should always choose different clones for your capture and detection antibodies.

21.In your ELISA Kits, there is a step that calls for a washing of the plates before even adding any sample to it. What is the purpose of this step?

We typically use a stabilizer for pre-coated plates. The washings were designed to remove these components before you start the assay. If you do not do the washings, the effect on assay performance is negligible.

22.We ran out of capture/detection antibody in our ELISA kit. Can we use a standalone/single antibody to replace it?

No, we don’t recommend it.

23.I have multiplefn-testELISA kits that I want to run simultaneously. Can I use the same wash buffer for all the kits?

The Wash buffer is the same for all the current FineTest kits. All the part numbers on the Wash Buffer bottles in these kits should be the same. For ELISA Standard sets, we provide a recipe for the wash buffer on each kit’s technical data sheet. This recipe is the same for all ELISA sets.

24.The OD value of those diluted sample is higher than that of samples without dilution.

There are too many proteins in the serum, which might blocks target antibody-antigen combining, making a proper dilution could reduce the block. If the OD value of the sample in the range of our ELISA kit, we suggest you to calculate based on the diluted OD value.

25.Is there any rewords to publish papers using FineTest ELISA kit?

If you published paper using our ELISA kit, you will obtain US$150-US$550 coupon, please contact the sales for details.

26.What is the stability of your products?

All of our products have been passed strict stability test, we conduct quality test for each single product before shipping out.

27.How long it takes for ELISA experiment?

3.5-4 hours for Sandwich method, 2-2.5 hours for competitive method.

28.Why there are some ELISA kits using competitive method?

The small molecule antigens or haptens can’t be tested by double antibody Sandwich method, because there are less than 2 positions, which is suitable for competitive method. The principle is the antigen of the sample competes the combine the position of the biotinylated antibody with the antigen on the solid support, the more content of the antigen in the sample, the less biotinylated antibody combines with solid support, then the coloration is lighter, means a lighter color indicates a higher concentration of the antigen in the sample. Those small molecule hormones use this method usually.

29.What if getting a bad performance using Fn-test ELISA kit?

Please contact customer service or tech support in Fn-test, we would like to help you analyze the experiment result. If we can’t figure out the reason based on experiment result, you could ship the products back to us for further testing. If it proved to be our quality which leads to bad performance, we would like to send replacement, if it proved to be operation wrongly, our tech support will offer improvement suggestions.

30.The difference of EDTA and EDTA.2Na?

EDTA included 4X -COOH( not soluble in water, dissolved PH=4.0) acid

EDTA

 

Top1: High background. The color reaction is too fast after adding TMB and all the wells turn to blue. The blank OD value is more than 0.5, sometimes is higher than 1.0.

A example

      

Re:

A.Non-exhaustive washing plate

1)Automatic Washing: Make sure the tips are not been blocked by any obstacle, like crystal. Fill each well completely with 350ul wash buffer.

2)Manual Washing: please comply with the washing process strictly which is stated in the manual. The absorbent paper must be oxidizer free and the tips need to be sterilized and clear. Add 350ul wash buffer.

3)After washing: clap the plate on absorbent papers or other absorbent material to absorb up the liquid of wells before the next step.

4)Attention: the washing before adding TMB is very important. The residual HRP enzyme will directly make the TMB color turn to blue.

 

B. Pollution

1)TMB has been polluted. TMB should be transparent, if it looks blue, then turns out to be polluted.

2)Check if the water used for formulating wash buffer is polluted or not. Because common water contain metal ion such as Fe3+, Cu3+, Co3+ and HClO which will cause TMB oxidation. We require using the double distilled water, three steamed water or deionized water to formulate wash buffer.

3)Check if the Elisa plate is damp or not. Check if any component reagents are expired.

4)Check if the experiment environment is polluted or not. There might be dust or substance in the air causing color.

 

Top2: The standard OD values we get are not as good as it stated on the manual.

Re:

1)Check if all the reagents have been stored under the manual requirement or not. Especially for the detection antibodies and SABC, if stored at -20℃, melting during the experiment will affect their activity severely.

2)Make sure the pipette is accurate. You can calibrate the pipette by testing the deionized water. (1000ul=1.0g 100ul=0.1g 10ul=10mg)

3)Equilibrate the TMB for 30 minutes at 37℃ before their incubation.

4)Do all the operations strictly comply with the shipment manual.

5)Make sure the kit is within expiry date.

 

Top3: The duplicate OD value varies a lot. The CV is high.

Re:

1)The deviation will be low if the duplicate OD values are between 0.5 and 2.5. When OD values are less than 0.5 or higher than 2.5, the CV will tend to high because of the operation deviation. In order to get an accurate CV, we suggest the duplicate well quantity is between 5 and 10.

2)Pay attention to your adding sample operation, Eg: Don’t let the tips touch the bottom and internal of the well. It just needs to let the tips touch the liquid and make the residual flow into the sample well.

3)Adding samples as soon and accurate as you can, don’t let any bubbles occur.

4)The pre-experiment is a must before testing sample. Test sample at different concentration to find out the best dilution rate. The inappropriate dilution rate will increase the deviation of duplicate OD value.

 

Top4: The color reaction is too fast and the blank OD value is less than 0.2. The kit works well but the sample OD value is negative.

Re:

1)Make sure the samples are under right storage. Proteins are easy to degrade. You had better store it at -80℃or test the fresh samples directly.

2)The matrix in the sample will affect the specific binding of antibody and antigen, and then cause the negative result. Based on this, you may need to dilute the sample at 1/5, 1/10, 1/100, 1/1000 to find out the best dilution rate. Diluting sample will reduce the matrix effect to help to get a positive result.

3)Some kits demand incubating samples and standard at 4overnight. If you still don’t get any positive OD values after sample dilution, we suggest you to incubate samples and standard at 4for 16h (Execute other operations comply with the shipping manual).

4)If you need to test special sample, please contact us for the tech support.

 

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