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ELISA & Multiplexing Focus ELISA & Multiplexing AH diagnostics: Your Science – Our Focus Dedicated to helping life science and diagnostics laborato- Representing more than 35 suppliers, our portfolio covers ries perform their best by providing innovative, high quality your entire workflow from A to Z. products and expert knowledge. With offices in Copenhagen, Aarhus, Oslo, Stockholm and We offer the ultimate cutting edge technologies for ELISA, Helsinki, we serve the Nordic region with our 30 years of flow cytometry, imaging, pathology, protein analysis, qPCR, experience in the biotech field. single-cell analysis and bio appliance. Let Us Find the Right Solution for You The selection of ELISA kits is vast, and new ones are added each year. It can be confusing to find the right ELISA kit for your project, but we are here to guide you. Whether your priority is accuracy, time use, budget or another requirement, we can help you find the best ELISA kits for your project. 5,700 From coat-it-yourself to ready-to-use ELISA kits ELISA kits Among our ELISA kits, you will find ready-to-use, coat-it-yourself, pre-coated, high sensitivity, phospho, and IVD/CE-marked ELISA kits. The kits are, depending on the target, based on different technologies in order to obtain the best possible sensitivity. If we don’t have the ELISA kit you are looking for, we are happy to source the product Build Coat it Pre- 1 wash for you your own yourself coated only Let ELISA instruments do the monotonous work We have a wide selection of washers and readers from trusted brands such as BioTek and Grifols. Save time and let the instruments do the 20 routine work. Our trained product specialists provide you with com- petent guidance for your specific instrument or kit needs, including in- formation and instructions on sensitivity, analytic range, species, price, species number of assays, etc. Kits for research and diagnostics • Allergy • Epigenetics • Apoptosis • Growth factors • Arthritis • Infection diseases • Autoimmunity • Inflammation • Bone metabolism • Matrix biology • Cancer Drugs • Metabolism • Cell based • Obesity • Chemokines • Respiratory diseases • Cytokines • Stress • Drug discovery • Vitamins • Endocrinology • And more 14 ELISA suppliers 3 10 Tips for Your ELISA Assay 1. Consistency is the key to any assay While consistency may seem like a given and an obvious goal, there are several assumptions that can innocently affect con- sistency between plates run in an assay. Often, one of these assumptions is that lot mixing is safe to do for conserving re- agent. Actually, you should never mix components between kit lots. Each assay is lot-specific and designed to give a de- fined level of performance when used before the assigned expiry date. If you mix lots, you may inadvertently introduce a reagent that may have already started to degrade which will result in a less than optimal assay or erroneous results. Lot-to-lot consistency between assay runs are ensured by con- sistently handling the assay reagents, following the protocol precautions each time, and adhering to the incubation times provided. In short, consistency should be introduced any- where and everywhere possible for the best and most useful More accurate data is, of course, vital to all researchers. They data. A break in consistency could mean less accurate results need to have absolute confidence in their data in order to for one plate to another and when dealing with samples from more assertively align their own data with other similar, pub- the same source/study, which can ultimately lead to conflict- lished findings. ing, hard-to-explain data. 4. Avoid contamination 2. Strive for reproducibility Having low coefficient of variability (%CV) between sample For ELISA users, reproducibility increases reliability. Reproduc- replicates is key to a successful and well-run assay. Keep %CV ibility can be affected by mishandling or inconsistently prepar- between duplicates low by using fresh pipet tips for each ad- ing the kit reagents and/or samples. It is important to make dition to the assay. New tips prevent cross-contamination be- sure that all kit reagents are at room temperature for approx- tween wells, which in turn keep the background and %CVs imately 30 minutes before every assay. Many of the reagents low and increase data reliability. Also, it is not recommended contain temperature-dependent components that may sepa- to pour reagent from a reservoir back into the original bottle rate from the solution when cold. Using the reagents at room as this can inadvertently cause contamination as well. Not ev- temperature ensures that you have consistent binding kinetics eryone takes the time to calculate or review the %CVs for an and consistent color development from one assay to another. assay, but it can be quite helpful in gauging consistency and Temperature changes can affect assay behavior, which in turn reliability. can lead to a lack of reproducibility in data. Striving for repro- ducibility is also vital when it comes to handling samples. Sam- ples should always be managed and handled by researchers in a very consistent manner. This can be done by introducing 5. Produce-reliable standard curves consistent sample collection protocols, minimizing freeze- For the most reproducible standard curves, be sure to serially thaw cycles, and optimizing dilution for each sample group dilute the curve using the range indicated in the assay manu- in the assay ahead of time, so that you can be confident that al. Adding points to either end of the curve will not increase a majority of the samples will fall in the most linear portion of sensitivity and will also not allow the kit to detect higher con- the standard curve. centrations. The upper and lower asymptotes of the curve are limited by the unique antibody characteristics which allow the antibodies to bind to the plate in the assay; expanding the curve will not change these limitations. Also, keep in mind that 3. Ensure assay accuracy it is important to make fresh standard curves for every assay/ Increase accuracy by making sure you have duplicates for all plate. Most assays indicate a suggested time limit in which points in the assay including background, standard and sam- freshly prepared standard curves should be used. It is vital to ple wells. It is simply good lab practice to run replicates for all follow all recommendations and precautions associated with points; this will allow you to immediately spot any obvious out- standard curve preparation. liers that might affect the average. For example, if you have an inadvertent pipetting issue with the n=1 in a sample well in the assay, this will definitely affect the OD, which in turn will affect the calculated average leading to an inaccurate interpolation from the curve for that particular sample. 4 6. Guarantee accurate sample analysis prevent everything from achieving equilibrium before mov- ing to the next step in the assay to large temperature changes For the most accurate sample analysis, it is very important for which also affect binding kinetics. If shaking is suggested, it is each end-user to optimize the dilution for their unique sam- always best to follow that guideline as well because in some ples because the levels of the biological marker being detect- assays, it does make a big difference in the resulting ODs. Ac- ed can vary as can potential matrix interference effects. It is, curacy and consistency of handling during plate incubation therefore, important to not only make sure the data lie on the greatly enhance assay performance. standard curve of the sample population, but it is also import- ant to make sure there is dilutional linearity in the sample dilu- tion. Even if there is a recommended dilution ratio for a sam- ple matrix, it is still crucial for each researcher to optimize their 10. Obtain optimal detection and data dilution ratio for their specific samples using the kit protocol recommendations only as a guide. At times, extraction, addi- analysis tional dilution, and others may be required for some samples. For optimal detection, read the plate at the wavelength indi- Optimization of the dilution is the only way to pre-determine cated in the assay manual and use the suggested data analysis this potential requirement. Some researchers underestimate software. We suggest using a 4Parameter (4P) Logistic Curve the importance of this optimization process and, in some in- Fit for data analysis in order to obtain the most accurate sam- stances, end up with sample data that is not linear, falls off the ple data. Most plate readers have this 4P software available curve, or is too variable, thereby rendering the data essentially already. If you are unsure of whether or not your plate reader unusable. has 4P software, we encourage you to contact the plate read- er manufacturer to confirm. If you have this software, they can walk you through opening it and setting up your plate tem- plate for plate read and the data analysis which occurs imme- 7. Be sure to include all background and diately following the read. control wells suggested in the protocol It is important to include all of the suggested background wells Source: Enzo Life Sciences in your assay to ensure the assay works well and to allow for efficient troubleshooting if issues arise. Some assays call for multiple control wells or background wells which all serve a slightly different purpose. If you are unclear about what cer- tain background wells and control wells are used for in an as- say, be sure ask technical support for clarification. 8. Use the provided/recommended diluents and buffers For best results, you will want to use the recommended di- luents only for your standard curve and sample preparation.
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