Is it important to store at the recommended temperature?

• ELISA and Western blot kits have to be kept at 2-8 °C at all times.
• A kit which has been frozen cannot be used.
• When storing kits avoid using devices with automatic defrost which causes fluctuations in emperature.

Are components in kits interchangeable?

• Use components exclusively from one package unless stated otherwise in the manual.
• Do not mix reagents from different lots or from different manufacturers.
• After a test is completed, put all the components back in the original box. This helps to avoid mixing of components from different batches and manufacturers.

Might the laboratory environment affect the test results?

• A kit needs to be brought up to room temperature prior to use. In case of insufficient or too sudden warming a significant shift in measured values will occur and the results might not be valid.
• If possible, perform the test in areas with normal laboratory temperature (20-25 °C). Higher or lower temperature affects the test results.
• Do not test in the presence of reactive vapors (acids, alkalis, aldehydes) or in dusty environments. Such contaminates have the potential to affect enzymatic activity of the conjugate.

How should my samples be prepared for testing?

• It is necessary to bring all of the specimens to room temperature and mix well prior to use. Concentration differs greatly at the bottom and the surface of the solutions due to low temperatures during storing. Inhomogeneous samples can cause false positive or negative reactions.
• Samples containing visible particles must be centrifuged slowly prior to testing.
• Always use freshly diluted samples. When rerunning samples, a fresh dilution of the sample should be made.

Might the nature of the sample affect the test result?

• Samples which are bacterially contaminated, hemolytic, chylous or containing  anticoagulants in plasma (except citrate) might affect the test results. Serum (plasma) heat inactivation might cause non-specific results.
• Well-sealed samples can be stored for 1 week at 2-8 °C. When storing for prolonged period of time samples must be kept at -20 °C in tightly closed containers (e.g. Eppendorf). We do not recommend repeated thawing, which can lead to non-specific results.

What are the critical points when working with individual components of the kit?

Tools

• For the preparation and use of reagents Always use clean, preferably disposable devices. Any contamination can lead to bad results.
• Never use the same container (tray) for preparing or pipetting reagents simultaneously. Do not use containers with metal lids for reagents.
• For preparing the working dilutions, use only distilled water from high quality stills with no contamination by metal ions from the electrodes.
• To prepare the working solutions it is necessary to use clean containers that have been rinsed with deionized (distilled) water or use disposables.

Microplate

• Before opening the bag with plate, bring up to room temperature in order to avoid condensation from humid air. The remaining unused strips should always be returned to the bag from the original package along with a desiccant and hermetically closed.  Protect from moisture.
• Antigen-coated microplate strips can be used only once.
• Follow the marks for each set of strips to avoid errors in their confusion.
• When washing and pipetting do not touch the inner surface of microplate wells as this can damage the bound antigen.

Rules for working with solutions

• All reagents must be mixed well before use. Due to low temperatures during storage the concentration at the bottom and at the top of the solution differs greatly. Tap bottoms of vials against a hard surface prior to use in order to remove remains of the solution on the lid.
• Close all reagent bottles immediately after use to prevent evaporation and contamination of the content
• Do not confuse lids from reagents. Avoid cross-contamination of reagents. May lead to their mutual degradation.
• Do not return residues of the reagents into vials since this might cause degradation of the remaining content.

Solutions

• The reagents which are sensitive to light (TMB - Complete) need to be kept away from direct sunlight.
• The TMB solution should be colorless (or possibly slightly bluish). If it is deep blue, this indicates degradation and this solution should not be used for the assay.
• If a test contains controls in working dilution, do not dilute it further.
• Controls must be included in every run of a test. This is the only way to determine whether the test performed properly or not.

What to check before pipetting?

• Pipetting requires using only calibrated and validated tools that ensure good reproducibility of results. Poor volumes of reagents in test lead to erroneous and abnormal results.
• Before performing the test, check the volume settings on pipettes. Always dispense only the specified volume of reagents.
• Make sure that strips adhere well to the frame before pipetting into microplate. There is a risk of capsizing resulting in damage to washer or photometer.
• Check the label on the vial prior to pipetting to avoid unnecessary mistakes when confusing components.

How does the pipetting technique affect the test results?

• When pipetting samples always make sure to swap tips to prevent the cross-contamination and test degradation
• Pipette into the wells in the same rhythm and sequence for all consecutive reagents, especially if a larger number of samples is used, in order to achieve more standardized results.
• Two consecutive samples of a single patient (paired sera) must always be examined side by side in a single assay.  This is the only way to achieve dependable results.
• Avoid cross-contamination of other wells when pipetting reagents into the wells of a microplate.
• Antigen-coated wells must not be allowed to dry up during the test.
• Pipette the reagents right after washing. There is a risk of degrading the results.
• After pipetting into the last well, cover the microplate with a lid, put the microplate into a thermostat and set the alarm clock. Strict adherence to the incubation period is very important.

Is it important to follow the number of washing cycles?

• Follow the specified number of washing cycles and the way they are supposed to be done (complete filling and extraction from wells). Proper washing has a major influence on the test result.
• When using the washer, make sure to wash it thoroughly after the previous test. Another solution in the device can affect your results. It is crucial to remove the remaining washing solution from the wells by tapping onto an absorbent material after the washing. Ignoring this step would lead to dilution of the following reagent and interference with the result.
• Use the proper method of washing. If the wells are not extracted properly there is a risk of confusing the samples and reaching false positivity during the microplate shake out after incubation
• Wash the microplate right after the incubation is finished. Delaying the washing step prolongs the incubation time which leads to nonstandard results.

What is the advantage of incubation at 37 °C compared to incubation at room temperature?

• Standardized temperature in the incubator step eliminates the negative environmental factors - such as fluctuations in ambient temperature in different seasons. Higher or lower temperature affects the measured values. Incubation at 37 °C allows for shorter incubation time, thereby reducing the overall time of the test.
• Check the correct temperature setting on the thermostat. Temperature does not always match the value on the LCD screen device inside the thermostat. Do not put plates on the heated surface of the device.
• Remove all the bubbles from the wells by gently tapping the microplate frame before incubation in thermostat. Air bubbles change the standard volume in the wells.
• Always cover the microplate with a lid prior to incubation to prevent evaporation of reagents in the incubator and potential contamination of the exposed wells.

What to check before measuring the absorbance?

• Check the settings of the wavelength.
• Check if the underside of the wells is dry and clean. Use a suitable material leaving no fibers for wiping. A soiled bottom of the well can cause erroneous results.
• Measure the color intensity of the solutions in the wells within 30 minutes after stopping the reaction.

How to properly handle the WB strips?

• The strips are relatively fragile and require cautious handling. After opening the bag and tabs with strips use tweezers to carefully  grasp the strips at the label area.
• Immediately return any unused strips in the bag with desiccant and reseal it with the enclosed sealing strip to prevent contact with moisture and thereby degradation of strips. Avoid any contact with the membrane.
• Put strips immediately into trays. Strips must be in the tray always turned face up during the detection( ie the side, where the starting line is designated). These strips must be wetted completely.

What are the possibilities of the Westernblot test validation method?

• WB strips contain a so-called cut-off line which is designed to control the functionality and sensitivity of the kit and also facilitates the evaluation of the test. If the cut-off line is absent, the sensitivity required for correct performance of the test had not been reached.
• For additional validation, use positive and negative controls (included in the kit and are in the final dilution).
• Validity is met if the final appearance of the strip after using positive control appears the same as the validation strip shown on the protocol inside the kit. (Positive control contained in the kit may not contain all the specific lines). After the reaction is complete, immediately transfer the strips from trays on filter paper and dry to avoid increasing the background of strips.
• Do not evaluate the strips before they are in completely dry condition - wet and thoroughly dried strip vary in the intensity of the color lines and background.