Frequently asked questions ELISA

ELISA - general

ELISA - specific

 
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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 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 described the successful use of U-CyTech’s ELISA tests and/ or apply our high affinity antibodies in other immunoassays. See 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 coating 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 storing 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 sample 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 samples types (such as tissue homogenates)?
Unfortunately, U-CyTech has not routinely validated other sample types than those 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 performing spike and recovery experiments to determine if a non-validated sample type can be analyzed.

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What is the best method to store my biological samples for analysis?
It is recommended to aliquot samples and store 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.

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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 have less volume?

In our ELISA kit we recommend adding 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 volume of sample available is not always sufficient for adding 100 µl diluted sample per well. As alternative we can recommend the use of 384-well plates from Greiner bio-one (cat. no. 781061) instead of the 96-well plates included in our ELISA kits. These 384-well plates require only 20 µl of diluted sample per well.
When using a 384-well plate, we recommend using 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 measure these plates.

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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:

  1. Most analytes are present in high quantities in biological fluids exceeding the concentrations of the standard curve. 
  2. 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 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 it 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 stay 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 mention 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.

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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. Incubate for 10-20 sec followed by emptying 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 determining 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 standard curves 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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My question is not mentioned here, how can I contact U-CyTech?

The researchers of U-CyTech build on >20 years experience on the development and performance of the ELISAs for human, 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 our customer service:
E-mail: cs @ ucytech.com

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