Alpco FAQs

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.

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.

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.

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

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

Inadequate sample mixing

Pipetting technique

Inadequate plate washing

Carry over from high and low samples

Wrong sample dilution buffer

Inadequate sample mixing

Pipetting technique

Inadequate plate washing

Carry over from high and low samples

Wrong sample dilution buffer

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

Inadequate plate washing

Contamination of the substrate with enzyme conjugate

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

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

Incorrect reagent handling

Poor standard curve

Error in reconstitution of standards and/or controls

Wrong curve fit used

Improperly prepared sample

Sample IDs mixed up

Error in sample dilution

Discrepancy in reported units

Error in experimental design

Interfering substances

Specificity

Delays due to reconstitution or dilution

Reagents not at temperature

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

Inter-operator variability

Differences in pipette calibration

Differences in instrumentation

Variation in environmental conditions

Differences in sample handling

Plate Shaking Techniques

Sample Calculator

Plate Layout

Plate Washing Technique Video

Pipetting Tips, Reverse, Forward Mode Video