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General Information, valid for any Immunoassay

  • Do not mix components from different kit lots!
  • All reagents and specimen shall be at RT (18-25°C) before the test starts
  • Use a pipetting scheme to verify an appropriate plate layout.
  • Pipette in duplicate to be able to identify potential pipetting errors
  • No modification of test procedure and sample handling
  • Do not exceed incubation times, temperatures or shaking
  • No interruption of assay steps
  • All wells should be handled in the same order and time sequences.
  • Avoid contamination of reagents, pipettes and wells/tubes
  • Washing steps are crucial for good test results.
  • Standard curve appraisal: Use also OD Standard / OD highest Standard *100
  • Most of the IBL ELISAs use wavelength 450nm + Reference 620nm (some with 405nm).
  • In case no signal: conjugate or substrate forgotten?
  • TMB solution turns to blue, adding stop solution turns blue to yellow.
 

FAQ (Frequently Asked Questions)

Chapter notes for your instruction for use.

Please look up IFU chapter:
Intended use and/or Cross reactivity
Please look up IFU chapter:
Intended use - validated sample types are indicated
Please look up IFU chapter:
Cover sheet and Materials supplied.

  1. Check the number of determinations on "IFU cover sheet".
  2. Check how many wells are needed for standards (STD) and controls (CTR) (double determinations are recommended) in "Materials supplied".
  3. Start calculation.

Example: 6 STD and 2 CTR = 16 wells are needed for the calibration curve and controls.
96 wells – 16 wells = 80 wells for single samples left (40 wells for duplicates)

Please look up IFU chapter:
Storage and Stability
Please look up IFU chapter:
Specimen collection and storage
Please look up IFU chapter:
Materials required but not supplied
Please look up IFU chapter:
Warnings and precautions
Please look up IFU chapter:
Procedure notes
Please look up IFU chapter:
Calculation of Results

Please look up IFU chapter:
Calculation of Results
When obligatory required, the dilution factor is indicated respectively. In all other cases ONLY the additionally diluted samples have to be multiplied with the corresponding dilution factor.

Please look up IFU chapter:
Calculation of Results

Please look up IFU chapter:
Materials supplied

Please look up IFU chapter:
Performance

Other Questions

Yes you can, if you warm the solution up at 37°C until it is completely dissolved. Mix vigorously.

A 1:10 dilution for example means combining 1 unit volume of solute (the material to be diluted) with (approximately) 9 unit volumes of the solvent to give 10 units of total volume.

The test run is considered invalid, when the quality control criteria for the respective lot are not fulfilled. Standards and controls must be measured within the indicated acceptable range.
Example:

When creating the standard curve, the correct representation of the axes must be observed.
In many tests, the x-axis is logarithmic. However, since a logarithm of "0" cannot be calculated, the concentration of the zero standard needs to be entered e.g. as 10% of standard B.
 

Issue - Causes - Solutions

Causes

  • Poor-quality water was used to wash plates or to prepare wash solution
  • Insufficient washing or washer performance
  • Wash system contained an alternate wash formulation
  • Deteriorated substrate (colored)
  • Longer incubation times than recommended
  • Incorrect standard curve dilutions prepared
  • Reagents were intermixed, contaminated or prepared incorrectly

Solution

  • Use appropriate washing procedure and prepare wash solution as indicated in the IFU.
  • Make sure the washer is functioning properly and all wash buffers are removed before adding the substrate.
  • Use fresh TMB substrate solution which should be clear and colorless prior to addition to wells. Use a clean V bottom container prior to pipetting substrate solution into wells. Do not modify the test procedure! The indicated pipetting volumes, incubation times, temperatures and pretreatment steps have to be performed strictly according to the instructions.
  • Avoid contamination of reagents, pipettes and wells/tubes. Always make fresh buffers. Use new disposable plastic pipette tips for each component and specimen. Do not interchange caps. Always cap not used vials. Do not reuse wells/tubes or reagents.

Causes

  • TMB Substrate Solution was contaminated
  • Reaction not stopped (Colour will keep developing if the substrate reaction is not stopped.)
  • Plate left too long before reading on the plate reader
  • Incorrect dilutions prepared
  • Longer incubation times than recommended
  • Contamination of buffers
  • Substrate incubation carried out in the light (assay specific!)

Solution

  • Use fresh TMB substrate solution which should be clear and colorless prior to addition to wells. Use a clean V bottom container prior to pipetting substrate solution into wells.
  • Make sure that substrate reaction is stopped by using the stop solution of the kit. Do not let plate stand too long before reading on the plate reader! (Colour will keep developing, though at a slower rate if stop solution has been added) Do not modify the test procedure! The indicated pipetting volumes, incubation times, temperatures and pretreatment steps have to be performed strictly according to the instructions.
  • Avoid contamination of reagents, pipettes and wells/tubes. Always make fresh buffers. Use new disposable plastic pipette tips for each component and specimen. Do not interchange caps. Always cap not used vials. Do not reuse wells/tubes or reagents. Few Assays require that the substrate incubation should be carried out in the dark. Ensure substrate is not exposed to light—store in a dark place. Limit exposure to light while running assay.

Causes

  • Laboratory temperature was too low or reagents / plates were too cold Incubation periods were too short
  • Wrong conjugate was used, conjugate was prepared incorrectly or has deteriorated
  • Assay plate was read at wrong wavelength, or reader was malfunctioning
  • Washer system had microbial contamination or contained an alternate wash formulation.
  • Assay plates were compromised or previously used.

Solution

  • Make sure that all reagents and specimens have reached room temperature (18-25 °C) before starting the assay. Adjust laboratory temperature!
  • Incubation times affects results. All wells should be handled in the same order and time sequences. It is recommended to use an 8-channel Micropipette for pipetting of solutions in all wells.
  • Do not modify the test procedure! The indicated pipetting volumes, incubation times, temperatures and pretreatment steps have to be performed strictly according to the instructions.
  • Check microplate reader for possible disorders and/or if wavelength is set correctly. Check washer system for microbial contamination.
  • Only use the components of the kit box. Do not interchange reagents. Do not reuse wells/tubes or reagents.

Causes

  • Reagents were used in the wrong order or an assay step was omitted Conjugate or Substrate forgotten
  • Wrong conjugate was used, conjugate was prepared incorrectly or has deteriorated Pipettes properly calibrated

Solution

  • Repeat the test and make sure that ALL kit reagents and used (in the correct order).
  • Calibrate the pipettes
  • Do not modify the test procedure! The indicated pipetting volumes, incubation times, temperatures and pretreatment steps have to be performed strictly according to the instructions.
  • Follow instructions for use.
  • Store all kit components as indicated in the IFU and on the labels.
  • Check expiration dates of kit and reagents.

Causes

  • Excessive time was taken to add samples controls or reagents to the assay plate.
  • Multichannel pipette was not functioning properly.
  • There was inconsistent washing or washer system malfunctioning.
  • There was poor distribution of antibody in the sample.

Solution

  • Once the test has been started, all steps should be completed without interruption. Make sure that required reagents, materials and devices are prepared ready at the appropriate time.
  • Calibrate the pipettes
  • Use appropriate washing procedure.
  • Increase the number of washing steps (at least one more)
  • Firmly tap the plate after on absorbent paper

Causes

  • Washing is essential for good precision! Which wash solution has been used?
  • Poor-quality water was used to wash plates or to prepare wash solution.
  • Insufficient washing or poor washer performance.
  • Wash system contained an alternate wash formulation.
  • Obstruction of washer needle
  • Defective Washer

Solution

  • Increase the number of washing steps (at least one more)
  • Firmly tap the plate after on absorbent paper
  • Pipetting in duplicate important to identify pipetting errors
  • Carefully pipette the reagents in order to avoid splashing / contamination to other wells
  • Daily Maintenance of washer

Causes

  • Uneven temperature
  • Evaporation
  • Stacked plates

Solution

  • Seal the plate completely with a plate sealer during incubations. If 37°C incubation is indicated, make sure plate is in the center of incubator.
  • Avoid stacking plates during incubation.
  • Use duplicates or triplicates for all samples in order to note any large variations in the results for a given sample.

Causes

  • There was inconsistent washing or washer system malfunctioning.
  • Insufficient plate agitation
  • Edge effects
  • Pipette inconsistent / Multichannel pipette errors
  • Plate sealers not used or reused
  • Non-homogenous samples
  • Well allowed to dry out
  • Cross well contamination
  • Bottom of the plate is dirty / Well bottom scrapped
  • Bubbles in wells

Solution

  • Use appropriate washing procedure.
  • Calibrate the pipettes
  • Thoroughly mix samples before pipetting
  • Ensure the plate and all reagents are at room temperature
  • Avoid contact with the bottom of the well during pipetting. Aim the pipette tip to the side of the well to avoid disrupting the bottom.
  • Ensure no bubbles are present prior to reading the plate.

Causes

  • Incorrect standard concentrations used (check QC-Certificate of lot!)
  • Poor washing (standards do not differentiate properly)
  • Standard was incompletely reconstituted or was incorrectly stored
  • Incubations done incorrectly (temperature, time, shaking…)
  • Curve doesn’t fit scale
  • Wrong preparation of kit reagents

Solution

  • Check pipetting and washing technique - double-check calculations.
  • Try plotting use different scales (according to IFU e.g. log-log, 4 parameter logistic curve fit).
  • Reconstitute standard according to the protocol provided and follow storage instructions.
  • Follow protocol for storage, incubation and agitation.

Causes

  • Samples contain no or below detectable levels of analyte
  • Samples contain analyte concentrations higher than the highest standard point
  • Insufficient washing
  • Incorrect dilutions prepared

Solution

  • If samples are below detectable levels, it may be possible to use high sample volume.
  • Samples may require further dilution. Use appropriate washing procedure.
  • Prepare reagents freshly or as described in the IFU.
  • Check pipetting technique and double-check calculations.

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