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15 Frequently Asked Questions in ELISA Experiments

Posted on October 29, 2022 by Zeng YingELISA


ELISA experiment principle is simple, less steps, short process, the kit is relatively easy to operate, the experimental novice can easily get started. However, although the experiment is quick to start, it is not so easy to get reliable experimental results. In order to better assist the experiment, here will be concerned about the ELISA kits and samples frequently asked questions are organized as follows.

Catalogue: Equipment For Elisa Kit Manufacturing

ELISA kits

ELISA washer, ELISA coating machine

1. What species can ELISA kits detect?

The current ELISA kits are mainly for human, mouse and rat; a small number of indicators involve special species such as monkey, rabbit, pig and chicken.

If the ELISA kit is named with a specific point, it means that it is specifically for this species; if no species is specified, it means that the kit is universal within the species involved (e.g. human, mouse, rat, monkey, rabbit, etc.). For other specific species, please contact technical support to confirm the suitability of the test.

2. How many samples can be tested with the 96T ELISA kit? Do I need to do duplicate wells?

In order to ensure the accuracy of the experimental results, we recommend that the standards and samples are done in multiple wells, but does not force the ELISA experiment must be done in multiple wells / 3 multiple wells, customers can set their own needs according to the multiple well experiment.

Marking CurveSampleNumber of testable samples
No compound holeNo compound hole88
Do compound holeNo compound hole80
No compound holeDo compound hole44
Do compound holeDo compound hole40

3. Can the number of wells be reduced for a large number of samples to be tested?

To ensure the accuracy of the results, it is not recommended to reduce the number of wells.

4. The ELISA kit is to be used several times, but the samples are large, is it possible to make only one standard curve?

Although the operation steps of ELISA experiments are the same, the various influencing factors will change for each experiment. Therefore, the standard curve must be redrawn for each experiment to obtain more accurate and reliable detection results.

If you need to do multiple experiments, the highest concentration of the standard after dissolution can be stored in -20℃ condition in separate packs to avoid repeated freezing and thawing, and used effectively within half a month.

5. What is the difference between ELISA research kits and ELISA clinical kits ?

Clinical reagents are approved by authoritative agencies, such as FDA, CFDA, etc.. Research reagents are generally developed by manufacturers themselves and do not need to be verified by authoritative institutions.

Organizations dealing with clinical diagnostic products need to obtain the Medical Device Business Enterprise License. Research reagents do not need this document.

Clinical kits are generally only used to test a single type of secretory sample such as human serum/plasma. Research kits have a much wider range of sample types and tests than clinical kits. However, in general, the quality and stability of clinical kits are better than scientific kits.

6. Do ELISA kits contain toxic and harmful ingredients? How safe are the kits?

ELISA kits, except for the addition of BSA as a protective protein, do not contain any other ingredients of animal origin and meet the requirements of the Cartagena biosafety protocol. In addition, the preservative of the kit is proclin 300, which is less toxic and safe for the environment.

7. Can ELISA kits be used in parts? How do I store unused reagent components?

The ELISA kits can be used in different quantities. Our enzyme strips are detachable, so you can determine the number of strips you need for each experiment and the content of each reagent component according to your experimental needs.

The solubilized standards are recommended to be stored at the highest concentration/master batch, divided and stored at -20°C, avoiding repeated freezing and thawing, and used effectively within half a month.

Biotinylated antibody working solution and HRP enzyme conjugate working solution need to be prepared and used now. Unused concentrates and other components should be stored at 2~8℃/-20℃ respectively according to the instructions in a timely manner, and can be stored for 6 months.

ELISA samples


8. What type of vacuum blood collection tube should be used to collect serum plasma samples in ELISA?

Serum: For ELISA tests, non-anticoagulant/procoagulant blood collection tubes are required

(usually red, yellow, orange-capped blood collection tubes)

Plasma: for enzyme-linked immunosorbent assay, blood tubes with anticoagulant. (usually green, purple-capped tubes)

After ELISA sample collection, if assay is performed within 1 week, it can be stored at 2-8°C. If it cannot be assayed in time, please dispense it in single-use amount and freeze it at -20°C (assay within 1 month) or -80°C (assay within 3 months) to avoid repeated freezing and thawing.

Before testing, slowly thaw frozen samples and centrifuge to remove precipitates generated during the freeze-thaw process.

9. How can hemolysis be avoided as much as possible when collecting?

Hemolysis can be caused by a variety of physicochemical factors and toxins. In vitro, hemolysis can be caused by mechanical forceful shaking, cryogenic freezing and other triggers. To avoid the effect of hemolysis on test results, attention should be paid to.

When collecting blood intravenously, the pumping pressure should not be too high.

The amount of blood collected at one time should not be too small.

The whole blood collected should not be shaken vigorously.

Centrifugation speed should not be too high.

The collected whole blood should be processed into serum/plasma as soon as possible and the whole blood should not be frozen or thawed.

If anesthesia is required for blood collection, anesthetics without hemolytic effect should be used.

10. How should the process of "repeated freeze-thawing" mentioned in tissue/cell sample collection be performed?

Tissue samples: after tissue grinding, place them at -80°C for 1h/liquid nitrogen for 0.5h, then place them in a 30°C water bath with slight shaking to make them thaw rapidly, and repeat the operation 1-2 times.

Cell samples: freeze-thaw 2-3 times repeatedly according to the above operation steps. In case of membrane proteins, sonication can be done appropriately, but the temperature and frequency of sonication need to be controlled.

It is recommended to add protease inhibitors to the sample in advance. PMSF is recommended in routine cases.

11. How can I determine if an ELISA sample needs to be diluted or otherwise processed?

The detection range of the kit is not the same as the concentration range of the analyte in the sample. It is recommended to estimate the concentration of the analyte in the sample through relevant literature before the experiment and determine the actual concentration of the sample through pre-experiment.

In case of routine sample types, ELISA technical support can be consulted to obtain the recommended dilution of the sample and arrange for pre-experimental testing.

If the concentration of the analyte to be measured in the sample is too high or too low, make an appropriate dilution or concentration of the sample.

If the concentration of the analyte in the sample is much lower than the minimum detection limit of the kit, it is recommended to select a more sensitive kit for the assay.

12. What to do with high-fat samples in ELSIA experiments?

High-fat samples contain fat, not homogeneous solution, if directly on the sample, it will affect the binding of antigen antibody, resulting in inaccurate measurement values. It is recommended to centrifuge at high speed (5000×g) first, let it stand for 10 min, take the middle layer of more clarified liquid, then centrifuge once more at high speed (≥5000×g), take the middle and lower layer of clear liquid and use it for testing.

13. What is the ELISA sample dilution protocol?

The ELISA kit detection range is not equivalent to the concentration range of the analyte to be tested in the sample. Before the experiment, it is recommended to estimate the concentration of the analyte in the sample from the relevant literature and determine the actual concentration of the sample through pre-experiments.

If your ELISA assay sample requires dilution, the reference dilution protocol is as follows.

Dilution timesDilution program
Dilution 100-foldOne-step dilution. Take 5μL of sample into 495μL of standard & sample diluent and make a 100-fold dilution.
Dilution 1000 timesTwo-step dilution. Take 5 μL of sample into 95 μL of standard & sample diluent and dilute 20 times, then take 5 μL of 20 times diluted sample into 245 μL of standard & sample diluent and dilute 50 times, total dilution 1000 times.
Dilution 100000 timesThree steps dilution. Take 5μL of sample into 195μL of standard & sample diluent and dilute 40 times, then take 5μL of 40 times diluted sample into 245μL of standard & sample diluent and dilute 50 times, and finally take 5μL of 2000 times diluted sample into 245μL of standard & sample diluent and dilute 50 times, for a total dilution of 100000 times.

For each dilution step, the volume of liquid taken should not be less than 3 μL and the dilution should not exceed 100 times. Each dilution step should be mixed well.

ELISA data processing


14. What fitting function is used for the ELISA standard curve? Can I use Excel to make a standard curve? Why is the standard curve I made not a straight line?

It is recommended to use Origin software or other software containing a 4-PL (four-parameter logistic) model to fit the standard curve. 4-PL fitting method can more accurately reflect the linear relationship between concentration and absorbance, and thus more accurately obtain the concentration of the substance to be measured in the sample. These two fitting methods are not recommended for ELISA standards.


15. How to use Origin to plot standard curves & calculate ELISA results?

The result calculation of ELISA experiment includes standard curve fitting and sample concentration calculation. The average OD values of the standard and sample replicate wells were calculated, and the OD values of the blank wells were subtracted as the correction values. With the concentration as the horizontal coordinate and OD value as the vertical coordinate, a four-parameter logistic function standard curve was fitted on double logarithmic coordinates, and then the sample OD value was substituted into the standard curve to obtain the sample concentration value. If the sample was diluted before measurement, the actual concentration of the sample should be multiplied by the dilution factor.

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