ELISA Technical Manual and Troubleshooting Guide v.032017 2 Contents: Page Kit

ELISA Technical Manual and Troubleshooting Guide v.032017 2 Contents: Page Kit Storage Guidelines 3 Materials and Equipment Required 4 Performing the ELISA Assay 5 - 8 Sample Handling and Storage 9 Troubleshooting Guide 10 - 17 3 BioChek ELISA Test Kit Storage Guidelines • General • Storage of a complete kit • Using the kit • Handling of partially used plates General The kit is composed of coated microtiter plates, sample diluent, substrate buffer, stop solution and positive and negative controls (aqueous solutions containing salts), conjugate reagent (aqueous solution with antibodies and enzymes), washbuffer and freeze dried substrate tablets. The most critical components of the kit are the conjugate and microtiter plates. These are coated at precise concentrations with antigens that are immobilized to the surface. The coated microtiter plates are sensitive to moisture and temperature. Moisture will affect the stability of the binding of the antigen to the microtiter plates, therefore it is important to keep the kit box away from water, vapour or ice. High temperatures (> 45°C, 113°F) or low temperatures (< 2 °C, 35.6°F) may have a similar effect. The other components are (due to the packing materials) only sensitive to temperature. The complete kit is most stable at a temperature between 2 – 8 °C (35.6- 46.4°F). Storage of a complete kit The kit should be stored at 2 – 8 °C (35.6- 46.4°F). Often there is condensation on the walls of the refrigerator, therefore make sure the kit doesn’t come in contact with the walls of the refrigerator. Also, prior to storing the kit or components of a kit in the refrigerator, make sure there is no condensation on the kit components. Using the kit When planning to run an assay, remove from the refrigerator only the number of microtiter plate(s) that will be needed along with all the kit components and controls at least 2 hours prior to starting the assay. Handling of partially used plates In some instances, only a ‘partial plate’ may be required. When working with partial plates, cover the unused wells of the plate with adhesive tape (i.e. scotch) prior to and during the assay. After the assay, dry, mark, and cover the used wells with adhesive tape. Then REMOVE the tape from the unused wells to avoid condensation and place the used plate with desiccant pouch in a re- sealable bag. Specify the date and number of remaining wells. Put the plate back in the original kit box and in the refrigerator. 4 Materials and Equipment Required for Running a BioChek ELISA Test • Room with temperature between 22 and 27 °C (72-80°F) • Refrigerator to store kits • Freezer to store samples • Personal computer (IBM compatible with minimum 8GB RAM) with BioChek software. • Precision Pipette and disposable tips which are able to pipette 5 μl (i.e. single channel adjustable 1-10µl) • 8 or 12 channel pipettes/repeater pipettes with disposable tips able to pipette volumes of 50, 90, 100, and 245μl (i.e. 5-50µl and 30-300µl) • 96 well flat bottom dilution plates (min vol/well - 350µl) • Distilled or de-ionized water: -/+ 300 ml is required for the full washing procedure of 1 test plate. • 4- Clean Reagent reservoirs (Dedicate 1 for each specific reagent) • Tissues or other absorbing paper • 4- 10 ml Volumetric pipettes (Dedicate each for a certain reagent) • Graduated cylinder – 1L • Timer (portable and with alarm for 1 hour, 30 and 15 minutes) • Microtiter plate reader with 405 nm filter • Microtiter plate washer: manual or (semi)- automatic • Vortex shaker • Sealable tubes for sample collection 5 Performing the ELISA Assay Before you start Make sure that all required equipment, plastic ware, etc. is ready to use. Critical factors for running an ELISA test are: • Temperature of reagents (need to get to room temperature 22-27°C, 72-80°F) • Temperature of laboratory (22-27°C, 72- 80°F) • Time for the various incubation steps Other factors are: • ELISA plates on an insulated surface • No direct sunlight on ELISA plates/reagents • Plates on a clean surface • Use good quality and well maintained equipment Standard Operating Procedure for BioChek ELISA test kits • Preparation Running the assay Preparation: • Take samples out of freezer/refrigerator to allow them to reach room temperature. If samples were frozen, allow them to thaw. Prior to use, shake samples vigorously, either manually or with a vortex shaker. • Have all equipment ready to use on the bench (see previous chapter, pg. 4 for required equipment) • Label reservoirs and graduated pipettes/cylinders • Dedicate reagent reservoirs and graduated pipettes/cylinders to the same solution (DILUENT, CONJUGATE, SUBSTRATE AND STOP SOLUTION) if planning to reuse. Write on each item using a marker pen. Clean after use with distilled water. • Prepare required conjugate volume (enough for each well) Measure the required amount of conjugate into reservoir and place the remaining conjugate back in the refrigerator immediately. Note: 100µl of conjugate will be needed per well for all samples and controls being tested. For example, for a full plate, 9.6 ml will be needed. Therefore, it is recommended to dispense 11 ml into the reagent reservoir with a graduated pipette to easily cover require volume. • Let kit components warm to room temperature 2 hours prior to starting the test Remove the required number of microtiter plates, all kit components (bottles), and the reference control from the box in the refrigerator in order to allow reagents to reach room temperature (22-27°C, 72-80°F). A minimum of 2 hours should pass before starting with the test. (see page 6 for preparation of wash buffer and substrate reagent). • Prepare dilutions of samples Samples may be diluted during the 2 hour warm up time (see Making Sample Dilutions section). • Make a layout for sample location Make a layout of where samples will be dispensed on the microtiter plate. This can be done using the BioChek software (see software manual for instructions). 6 ATTENTION: When using only part of a plate, cover the unused wells of the plate with Plate Sealant tape, prior and during the assay. Do not use ordinary adhesive tape, as this may affect the chemistry of the plate. The covered wells must remain dry while running the assay. Making sample dilutions In dilution microtiter plate or empty plate: Preparation Have layout for the samples ready. For example, to prepare a 1:50 diluton, always first dispense 5 µl of sample in the bottom of the wells of an uncoated microtiter plate according to the plate layout. After all the samples have been added do a visual check to see that a drop of serum is present in each well. Add 245 µl of sample diluent to each well by reverse pipetting. This will give a 1:50 dilution. NOTE: Whenever making a pipetting mistake while dispensing sample, NEVER try to remove wrongly added sample out of a well or tube and try to replace it with the correct sample! Just mark the well on the layout in order to skip it when entering the plate layout in the BioChek Software (see software manual). COVERED DILUTED SAMPLES CAN BE STORED FOR 7 DAYS IN THE REFRIGERATOR. Now everything is ready to start the assay. From now on, it will take about 2 hours to complete the test. However, some kits require more time to run. Don’t start if time doesn’t allow you to finish the test. Preparing the wash buffer Dissolve one sachet of wash buffer in 1 litre of distilled water. Pour the powder in a 1 litre bottle or flask and rinse the residual salt in the sachet once with a small volume of distilled water. Shake the bottle or flask very vigorously to be sure that all the salts have dissolved. Prepared wash buffer can be stored for at least 4 weeks when stored in an air-tight container/bottle. Only use wash buffer that as is clear as crystal. Preparing substrate reagent Make the substrate reagent by calculating the number of tablets required (2 per plate) and the volume of substrate reagent (11 ml per plate). First add the tablets, then the reagent. Never touch the tablets with your hands. It is recommended to prepare fresh substrate reagent during sample incubation. Mix substrate prior to use. Tablets must be fully dissolved. Running the Indirect ELISA Assay 1. Add Samples and Pre-diluted Controls a. Fill the wells on the test plate, except the wells which will contain the pre- diluted controls, with either 90 µl (for assays requiring a 1:500 final dilution) or 50 µl (for assays requiring a 1:100 final dilution) of sample diluent. Warning: when adding diluted samples or pre-diluted controls, use clean tips for new samples. 7 b. Add 100 µl of pre-diluted controls (including reference controls) to the appropriate wells (see diagram A). c. Use the multichannel pipette and dispense either 10 µl (for a final dilution of 1:500) or 50 µl (for a final dilution of 1:100) of the 1:50 diluted samples uploads/Ingenierie_Lourd/ elisa-technical-manual-and-troubleshooting-guide.pdf

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