Alpco FAQs

If your questions are not listed below, please contact technical support:

Q. What kind of sample type can be used with the kit I purchased?

A. Each type of kit has been validated for the sample types indicated in its protocol. All other sample types would have to be validated internally.

Q. Is this kit cross reactive with another species?

A. Each type of kit has been validated for the species indicated in the protocol. Although cross reactivity may exist, validation of other species would need to be performed internally.

Q. How many standards and controls do I run?

A. Each kit will include at least one set of standards (or one vial of standard to be diluted) and in most cases one or two controls. Standards and controls (if included) must be run each time the assay is performed and it is highly recommended to run all standards/controls/and samples in duplicate.

Q. Can I use reagents from other kits or lots?

A. No. Do not substitute components from other kits or lots.

Q. Can I modify incubation times or temperatures?

A. No. Please do not deviate from the protocol.

Q. Can I use an expired kit?

A. No. The kit should not be used if it is past the expiration date specified on the kit label.

Q. Can I use a kit that has been left at room temperature?

A. Please be sure that the kit and its components are stored as indicated in the kit insert. For additional information about kit stability at room temperature please contact the Stratech Technical team.

Q. Can I skip running the control(s) on my assay?

A. No. If a control(s) is provided always be sure to run the control(s) with each assay. The concentrations of the controls should fall within the range specified on the certificate of analysis.

Q. Can I wash my plate with a multichannel pipette?

A. We do not recommend the use of a multichannel pipette. Wash buffer must be dispensed with adequate and equal force to properly wash the wells. We recommend the use of a Statmatic wash nozzle or an automated plate washer. Please refer to the Learning Center tab of the Resources section of the website for a video displaying proper washing technique or click here.

Q. Is it necessary to make a layout of my samples?

A. It is extremely important to know where your standards, controls and samples are located on the plate so that results are generated appropriately. Click here for a downloadable platemap. It also helps to label the strips on the plate (use small tabs at each end) in the event that strips become loose while decanting.

Q. Why is the washing step so important?

A. Correct washing of the plate is a very critical and important aspect of running any successful ELISA. Consistency in washing the plate is essential. Washing the plate too rapidly or too slowly, incomplete washing or aspirating, and allowing the wells to sit dry are all factors that will affect the precision of the assay. We do not recommend the use of a multichannel pipette for this purpose. Multichannel pipettes do not apply enough pressure to thoroughly remove the unbound material and this will result in higher background noise. A video displaying proper washing technique is available in the Learning Center section of the website or click here.

Q. How many times can I freeze and thaw my samples?

A. Recommended freeze/thaw cycles vary based on kit. We recommend to avoid repeated freeze and thaw cycles unless otherwise indicated in the manual. If the collected sample amount allows for it, prepare and freeze aliquots to eliminate or reduce freeze/thaw cycles.

Q. Can I use my own "homemade" buffers?

A. Each kit contains diluents and buffers that are formulated to closely match specific sample types. The manufacturer will not guarantee the performance of the kit if components that were not provided with the kit are used to run the assay.

Q. What should I do if I run out of diluent?

A. Enough reagents are provided to run the entire kit when the protocol is followed. If your type of study requires a greater dilution, calculate the amount of diluent needed prior to setting up the assay and additional diluent may be available for purchase.

Q. My controls are out of range. Is my data invalid?

A. The concentration of the controls must be within the range specified in the certificate of analysis. If the controls are out of range the assay may be invalid. Also, make sure the curve fit recommended in the protocol has been used. If more than one recommendation is provided use the regression method that best fits the standard data points.

Q. I didn't get good values from my standard curve. Can I use the example values from the protocol?

A. No. Never use example values to calculate your assay results. Sample concentrations should be generated from the standard/calibrator values obtained with each assay.

Q. My samples generated OD values out of the standard range. Can I still calculate concentrations?

A. Only sample values that fall within the range of the standard curve can be used. Values outside of this range are generally not linear and can lead to incorrectly extrapolated results.

Q. My standard curve is fine but I didn’t get a signal with my samples. Why?

A. The sample may contain the analyte but it is undetectable based on sensitivity of the kit. The matrix of the sample may also be masking the detection. Ensure that the dilutions have been made as stated in protocol and review the procedure to ensure all reagents were added with the correct volume and in the correct order.

Q. How many samples can I run?

A. ALPCO now offers a tool to help with calculating the number of samples that can be run in a kit. Once you review the protocol to determine the number of standards, controls, and blanks that may be required for the specific kit you plan to run click here to go to the Sample Calculator.

Q. What is reference wavelength?

A. A reference wavelength is a secondary wavelength used for correction (normalization) of the principal wavelength OD values. It helps to account for imperfections in the plate.

Q. Why is 450nm the most common measure wavelength?

A. The measurement wavelength is determined by the substrate used in each kit. The most common one is TMB. It produces a blue color measurable at a wavelength of 650nm. It can be used in end point assays by stopping the reaction with either 1M phosphoric acid or 1M sulfuric acid. A yellow reaction product is formed upon acidification that is measurable at 450 nm.

Q. What is the difference between measurement wavelength and reference wavelength?

A. The main difference between measure wavelength and reference is that the first one has been chosen to read the absorbance produced by the substrate and the second one (reference) to detect imperfections in the plate.

Q. Can I read the plate without having a reference wavelength?

A. Yes, you can. However, the use of a reference wavelength may improve the sensitivity of the assay.

ELISA Troubleshooting Guide

Inadequate plate washing

For automated washers: Check nozzles for clogs, verify dispense volume, and perform regular cleaning to prevent and remove any buildup. Refer to owner’s manual for additional maintenance information.

For manual washing: Ensure that all wells are being forcefully overfilled with wash buffer. Refrain from using a multichannel pipette for washing. Consider switching from a wash bottle to a hand-held manifold or automated plate washer, particularly when running chemiluminescence assays. A video displaying proper washing technique is available here.

Do not skip wash or soak steps.

Problem with multichannel pipette during the addition of the conjugate or substrate
Check multichannel pipette calibration. Ensure all tips are affixed securely and drawing the same volume of liquid each time tips are changed. Watch for bubbles and consider a reverse pipetting technique to ensure air bubbles do not interfere with pipetting accuracy. See video on forward and reverse pipetting.

Pipetting technique

Check single pipette calibration. Watch for bubbles and consider a reverse pipetting technique to ensure air bubbles do not interfere with pipetting accuracy. See video on forward and reverse pipetting.

Bubbles in wells

Be sure to forcefully tap plates dry after each wash step to minimize residual wash buffer in the wells. Bubbles remaining in the wells after addition of assay reagents, particularly substrate, should be removed prior to plate incubation and plate reading. A clean pipette tip can be used to remove bubbles.

Poor precision between replicates for samples only (high CV values)

Inadequate sample mixing

Pipetting technique

Inadequate plate washing

Carry over from high and low samples

Wrong sample dilution buffer

Poor precision between replicates for samples only (high CV values)

Inadequate sample mixing

Pipetting technique

Inadequate plate washing

Carry over from high and low samples

Wrong sample dilution buffer

Poor standard curve (does not match previous results or Certificate of Analysis)

Inadequate plate washing

Pipetting technique

Inadequate mixing of reagents

Insufficient shaking of plate

Wrong volume of reagents added to the well

Storing of prepared reagents

Low or inconsistent ambient temperature

Differences in chemiluminescence instrumentation

All wells turned bright blue for colorimetric assay

Inadequate plate washing

Contamination of the substrate with enzyme conjugate

High background (blank or 0 standard values are too high)

Inadequate plate washing

Old or contaminated wash buffer

Contamination of the substrate with enzyme conjugate

Wrong conjugate dilution

Wrong filter used in the plate reader

No color development or very low OD readings for standards and samples

Wrong filter used in the plate reader

Error in reagent preparation

Inadequate mixing of reagents

Insufficient shaking of plate

Mix-up in reagents

Reagents contaminated or added in the wrong order

Alternate component lot or expired component used

Assay incubation times not followed correctly

Delayed reading of plate

Control out of range

Incorrect reagent handling

Poor standard curve

Error in reconstitution of standards and/or controls

Wrong curve fit used

Unexpected results for samples

Improperly prepared sample

Sample IDs mixed up

Error in sample dilution

Discrepancy in reported units

Error in experimental design

Interfering substances


Assay drift: difference in values from beginning to end of plate

Delays due to reconstitution or dilution

Reagents not at temperature

Extended time between addition of reagent to first well and last well

Inter-assay variability (poor precision for controls or samples tested on different plates)

Inter-operator variability

Differences in pipette calibration

Differences in instrumentation

Variation in environmental conditions

Differences in sample handling


Plate Shaking Techniques

Instructions for proper plate shaking with an ELISA assay

Sample Calculator

“How many samples can I test on my 96 well ELISA plate?


Plate Washing Technique Video

Recommendations for hand washing and drying an ELISA plate

Pipetting Tips, Reverse, Forward Mode Video

Pipetting Tips, Reverse, Forward Mode Video

Q. What are the basic components of an HPLC system?

A. A functioning HPLC system includes a sampler, pump, column, detector and data processor. A degasser and column oven may also be used.

Q. How many types of detectors are available for HPLC?

A. There are UV, fluorescence, electrochemical, conductivity, refractive index, evaporative light scattering, chiral, radioactive and mass spectrometry detectors.

Q. I am not sure which column to use?

A. Check the assay protocol to instructions on the appropriate column. The most commonly used column is C18 which covers a wide range of applications.

Q. How do I make a column last longer?

A. Filter samples to remove particulate and make sure the pH of the mobile phase is within specifications for the column. If the column will be stored for an extended period of time, flush with methanol or acetonitrile.

No peaks

Check injectors, they may be congested.

Double peaks

Potential dead volume in fittings and/or column. Renew fittings and/or column.

Contaminating peaks

  • Injector may be dirty. Clean injector.
  • There may be contamination at the head of the column. Change direction of the column and rinse for 30 minutes at a low flow rate (0.2ml/min) with mobile phase.
  • There may be air in the system. Degas pump.
  • Vials may be contaminated. Use new vials or clean with methanol.

Variable retention times

  • There may be a drift in temperature. Use a column oven.
  • Imprecise pump delivery. Check pumps and degas system if needed.
  • System is not in steady state yet. Rinse system mobile phase for 15 minutes.

Baseline is drifting

  • Detector lamp may not have reached working temperature. Wait until appropriate temperature is reached.
  • Detector lamp may be too old and needs to be replaced.
  • System is not in steady state yet. Rinse system mobile phase for 15 minutes.
  • Imprecise pump delivery. Check pumps and degas system if needed.

Baseline is not smooth

  • Imprecise pump delivery. Check pumps and degas system if needed.
  • Detector flow cell is dirty. Try cleaning flow cell.

Chemiluminescent Plate Reader Settings

Reader Settings


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