Frequently asked questions ELISA

ELISA - general

ELISA - specific

ELISA - troubleshooting

ELISA - general

What is a sandwich ELISA?
A sandwich enzyme-linked immunosorbent assay (ELISA) is a sensitive assay for the determination of protein/peptide levels in biological fluids such as cell culture supernatant, plasma or serum. The assay uses an immobilized coating antibody specific for the analyte of interest. After the analyte is bound to the immobilized antibody, a second biotinylated antibody specific for the analyte is used for detection. The analyte is now 'sandwiched' between the two antibodies. By using HRP (horseradish peroxidase)-labeled streptavidin, the analyte can be quantified by use of an HRP-specific substrate (e.g. TMB).

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ELISA - specific

Which types of ELISA products does U-CyTech offer?
We offer 5-plate format ELISA kits and matched ELISA antibody pairs for 10-plate and 20-plate format.
The ELISA kits contain all reagents necessary to perform 480 ELISA determinations, including optimized coating and detection antibodies, standards, conjugate (streptavidin-HRP), substrate (TMB), stop solution and reagents to prepare the blocking, dilution and wash buffer. In addition, the kit provides you with eight 96-well ELISA plates and ten adhesive cover slips.
The ELISA antibody pairs contain optimized coating and detection antibodies, standards and a streptavidin-HRP conjugate for 960 or 1920 ELISA determinations. These reagents have been validated for assay performance with the same secondary reagents and materials as included in the U-CyTech ELISA kits.
Hundreds of peer-reviewed publications describe the successful use of U-CyTech’s ELISA tests and/ or apply our high affinity antibodies in other immunoassays. Please find them in our Reference database.

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Do U-CyTech ELISA kits contain pre-coated plates?
No, our kits do not contain pre-coated plates. We recommend to coat the ELISA plate(s) one day before you perform the assay.

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Do I need to optimize the coating and detection antibody concentrations?
No, U-CyTech ELISA products contain optimized concentrations of coating and detection antibodies. The ELISA manual (for kits) and Technical data sheet (for antibody pairs) should be followed to ensure optimal assay performance. You can find the Manual and Technical data sheet in our Manual database.

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How should I store U-CyTech ELISA products upon arrival?
Store the kits and antibody pairs at 4 °C upon arrival. We recommend to store the lyophilized SPP conjugate vial at -20 °C in the dark to maintain long-term stability.
The optimal storage conditions of each reagent can be found in the ELISA manual (for kits) and Technical data sheet (for antibody pairs). You can find the Manual and Technical data sheet in our Manual database.

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For which type of samples are the ELISA kits validated?
Most of our ELISA kits are validated for cell culture supernatant, serum and plasma. The validated samples types are mentioned in the Typical data sheet of each ELISA kit. You can find the Typical data sheets in our Manual database.

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Can the ELISA kits be used for non-validated sample types (such as tissue homogenates)?
Unfortunately, U-CyTech has not routinely validated other sample types than the ones mentioned in the Typical data sheet of the ELISA kits. This does not mean that the ELISA kit cannot be used for other sample types. We suggest to perform spike and recovery experiments to determine if a non-validated sample type can be analyzed with a particular kit.

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What is the best method to store my biological samples for analysis?
It is recommended to aliquot the samples and freeze them at ≤ -20 °C to prevent cytokine degradation. If samples are run within 24 hours, they may be stored at 4 °C. Avoid repeated freeze-thaw cycles. Do not heat serum or plasma samples. Prior to assay, frozen samples should be completely thawed and mixed well.

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How many sample and standard replicates should I test?
It is recommended to test standards and samples at least in duplicate.

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How many samples can be analyzed with one 5-plate ELISA kit?
When analyzing all samples in duplicate, you can run in total 200 samples (one dilution per sample, and including 7 standard dilutions and one blank for each plate).

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What can I do when my samples are small in size?
In our ELISA kit we recommend to add 100 µl sample per well (96-well plate). This sample should be diluted at least 1:1 in Dilution buffer. In case of working with small animals, like marmosets, mice or rats the amount of sample available is not always sufficient for adding 100 µl diluted sample per well. As an alternative we can recommend the use of 384-well plates from Greiner bio-one (cat. no. 781061) instead of the 96-well plates that come with our ELISA kits. These 384-well plates require only 20 µl of diluted sample per well.
When using a 384-well plate, we recommend to use a volume of 20 µl per well for the coating antibody solution, standard, blank, diluted samples, detector antibody solution, SPP conjugate, TMB substrate solution and Stop solution. For blocking, we recommend a volume of 50 µl per well. Please check whether your plate reader is able to read such plates.
Please do not hesitate to contact us when you have any further questions on the use of 384-well plate in our ELISAs.

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Why is there a sample dilution necessary in the U-CyTech ELISA kits?
There are primarily two different reasons for dilution of your sample. Most analytes are present in high quantities in biological fluids exceeding the concentrations in the standard curve. Another reason for dilution is to limit interfering factors present in complex matrices.

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How sensitive are the U-CyTech ELISA kits?
The assay sensitivity of the ELISA kit is mentioned on the Typical data sheet of each particular kit. You can find the Typical data sheets in our Manual database.

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Once the ELISA plates have been coated, how can they be stored and for how long?
Antibody coated plates can be prepared up to one week in advance. Seal the plate against evaporation and store the plate at 4 °C. Be aware that for long-term storage, the plate and reagents should be kept sterile. Moreover, it is recommended that after coating, the wells are washed with sterile wash buffer and blocked with sterile blocking buffer. The blocking buffer can be left in the wells until use.

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How critical are the incubation times mentioned in the ELISA manual?
To ensure optimal assay performance and reproducibility of the assay follow the incubation times as stated in the manual. However, the blocking buffer, detection antibody and conjugate can be incubated for 1 h at 37°C, 2 h at room temperature or overnight at 4°C without any problem.

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Is it possible to stack ELISA plates in the incubator?
It is critical that the plates are equally heated during incubation. Stacking of plates will lead to variation in temperature of the individual wells and consequently to higher CV values of the OD readings.

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What about washing?
Incomplete washing of the wells will adversely affect the assay.
Washing (manually or automated) can be performed as follows:
Completely empty the wells (do not touch the bottom). Then fill the wells with at least 250 µl wash buffer. Let it soak for 10-20 seconds and then empty the wells. Repeat these steps at least six times. After washing, the plate is inverted and tapped dry on absorbent paper.

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Why is the solution in the wells green after adding the stop solution?
This happens when the substrate in the well does not completely mix with the stop solution. After addition of the stop solution, tap the plate gently until the reagents are properly mixed and the solution turns yellow.

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Can I extend the standard curve (in either direction)?
Each ELISA kit is optimized and validated for the standard curve range provided on the Typical data sheet. U-CyTech always recommends to determine the concentration of the analyte in the linear portion of the curve. However, for some ELISA assays the standard curve may be extended, but this should be determined empirically by the end-user.

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What is the precision of U-CyTech ELISA kits?
The intra-assay CV of our ELISA kits is ≤ 5% and the inter-assay CV is ≤ 10%.

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How do I plot the standard curve?
Calculate the mean OD for each standard concentration. Next, calculate the mean OD for the blank and subtract this value from the mean OD of each standard concentration.
The standard curve can now be plotted as the standard concentration (x-axis) versus the corresponding OD (y-axis). Most laboratories have (plate reader) software that allows various methods of curve fitting. Since ELISA data are essentially sigmoid rather than linear, we recommend using the 4- or 5-parameter logistic fit for quantitative analysis of the samples. Alternatively, a linear regression curve may be acceptable for the linear portion of the curve consisting of at least 3 concentrations.
For samples, calculate the mean OD for each sample. Subtract the mean OD blank. The concentration of the analyte of interest can now be interpolated from the standard curve.

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ELISA - troubleshooting

The OD of my sample falls outside the standard curve range. What should I do?
Samples showing an OD below the lowest concentration of the standard curve should be re-analyzed at a lower dilution. Please keep in mind that the sample can also contain an analyte concentration below the detection limit of the assay. Samples showing an OD that exceeds the highest concentration of the standard curve should be re-analyzed at a higher sample dilution.

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The variability between my standard or sample replicates is too high. What could have happened?
There could be several reasons for high variability in the ELISA assay:
  1. Inaccurate pipetting: ensure accurate pipetting of volume (check pipettes) and avoid air bubbles.
  2. nadequate mixing of reagents: mix reagents adequately.
  3. Inadequate washing: increase the stringency of washes (particularly after the 'detection antibody' incubation step). Check the operation of the automated plate washer.
  4. Evaporation of solutions: ensure accurate sealing of the ELISA plate during each incubation step.
  5. Non-homogenous samples or samples with high particulate matter: mix samples thoroughly and remove particulates by centrifugation.
  6. Temperature differences between wells: do not stack the ELISA plates during reagent incubation.
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I am getting very low or no color development with the standards. What went wrong?
There could be several reasons for no or low OD values for the standards:
  1. Improper storage of reconstituted SPP conjugate: avoid storage at room temperature and prolonged exposure to light and heat.
  2. Incorrect incubation times and/or temperatures: ensure sufficient incubation times/temperatures and allow reagent solutions to reach room temperature before use.
  3. Improper quality or pH of used water: use distilled water (no tap water) and check the quality and pH of the distilled water.
  4. Improper antibody, conjugate or standard dilution: ensure correct dilution of antibodies, conjugate and standard.
  5. Activity loss of antibodies or conjugate: follow recommended storage conditions.
  6. Overly high washing/aspiration pressure from automated plate washer: check function of washing system or utilize manual washing.
  7. Working solutions contain sodium azide: avoid adding sodium azide in solutions, as this is a 'peroxidase activity' inhibitor.
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I am getting high background values (OD blank > 0.2). What could have happened?
There could be several reasons for high background values:
  1. Working solutions were contaminated: solutions should be clear and colorless. Use a clean container before addition into wells.
  2. Detection antibody or conjugate dilution was too concentrated or left to long on the plate: ensure proper dilution of detection antibody or conjugate and incubation time.
  3. Incubation time of TMB substrate is too long: shorten the incubation time of TMB substrate.
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The linearity and/or dynamic range of the standard curve are poor. What went wrong?
There could be two mean reasons for poor linearity and/or dynamic range of the standard curve:
  1. Improper standard dilutions: ensure proper dilution of standards (two-fold dilutions for the standard curve is recommended).
  2. Inaccurate pipetting: ensure accurate pipetting of volume (check pipettes) and avoid air bubbles.
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My question is not mentioned here, how can I contact U-CyTech?
The researchers of U-CyTech have over 20 years experience with the development and performance of the ELISAs. Not only for human but also for monkey, mouse and rat species. They are more than happy to provide you with additional advice and to share their experiences with you.

Please contract us:
E-mail: click here (info at uytech.com)

U-CyTech biosciences

Yalelaan 48

3584 CM Utrecht

The Netherlands


 
Phone: +31.30.253 5960

Fax: +31.30.253 9344


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