ELISA FAQ

Why isn't my ELISA producing any color?
  • You may not have added the enzyme conjugate.
  • You may have used the capture antibody instead of the enzyme conjugate.
  • The positive control may be inactive.
  • You may not have added the PNP substrate tablet to the PNP substrate buffer.

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Can I coat plates and then store them?

All Agdia plates can be stored for longer periods of time if postcoated with the appropriate postcoat buffer. Please contact Agdia directly for more information.

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Why did color appear in all my testwells?
  • •  You may not have washed the plate properly.
  • • The substrate solution may have been contaminated. PNP tablets can be contaminated by touching them.
  • • The substrate solution or plate may have been exposed to bright light.
  • • You may have added positive samples to all wells.
  • • You may have used an old buffer that has become contaminated.
  • • (Reagent sets only) You may have coated the plate with enzyme conjugate, or with coating antibody contaminated with enzyme conjugate. You must use only new, sterile pipette tips.

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Why did color develop in all wells along the edge of the plate?
  • You may not have washed the edge wells properly.
  • Lower quality plates may cause an edge effect. This is not a problem with high-quality new plates such as those supplied by Agdia.

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Why did unexpected color develop in wells near positive samples or positive controls?

You may have contaminated surrounding testwells duing the wash step.  Please see the plate washing video in the how to video section.

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Why did I get what appears to be false positive results with my samples?
  • The plant sample is really positive.
  • Some carryover may have occurred on your grinding equipment.

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Why are my positive results weak?
  • Your kit components may have expired.
  • The pathogen titer or analyte titer may be low.
  • You may have used an inappropriate sample extraction buffer.
  • (Alkaline phosphatase tests) You may have prepared the PNP buffer using PBST wash buffer instead of distilled water. Phosphate in PBST can reduce the amount of color produced.
  • You may have over diluted the enzyme conjugate or substrate.

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Why does one of my test components have an odor of decay?

The test component may be contaminated and should not be used.

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How long does it take to perform an ELISA test?

Agdia leads the way with ELISA protocols that are flexible and reliable.   Our standard PathoScreen can be completed within 5 hours (sample 2hrs or overnight, conjugate 2 hours, substrate 1 hour).  Reagents can be completed in 9 hours (coat 4 hours or overnight, sample 2 hrs or overnight.

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What are the ideal temperature ranges for storing and performing the test?

Agdia products have been developed to be highly sensitive and specific while also being robust enough to withstand temperature fluctuations and unrefrigerated storage during shipping. The storage and incubation temperatures listed on our products and user guides reflect the average conditions observed in our laboratories. To ensure that your test is performing optimally, we recommend storing the refrigerated components at 2 - 8 °C (36 to 46 °F). When performing the test or storing components at room temperature, we recommend a range of 21 +/- 3 °C (65 to 75 °F). In many situations, temperature fluctuations beyond those listed above may not significantly affect the outcome of a qualitative test , however, Agdia can only guarantee test performance within the recommended temperature ranges. We understand that temperatures at your location may be different from those in our test validation laboratory, so if you have concerns about your local conditions, please feel free to contact us at 1-800-622-4342, or info@agdia.com or infolatina@agdia.com.

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