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    ELISA Tips and Tricks to Optimize Results

    ELISA Kit Tips and Tricks

    ELISA (Enzyme Linked Immunosorbent Assay) kits identify and quantify specific protein sequences and other research targets within a sample. Good ELISA kits support and validate your research by producing accurate, quantitative results. Samples suitable for use with ELISA kits include: plasma, serum, saliva, cell lysates and cell culture supernatants. Choose the right type of kit for your research, usually: direct, indirect, sandwich or competitive ELISA. A good quality kit will be carefully designed to help you achieve the best results and will be able to withstand a reasonable amount of variance in technique. Our team is happy to advise you.

    Your time is valuable, to get the best out of your ELISA kit, we help you avoid the pitfalls and provide helpful tips and tricks. Our informed insights are based on years of experience, understanding and listening.

    Biorbyt continue to support the scientific community to achieve the best results possible. Our tips and tricks save time and effort and support you to produce reliable and accurate results enabling you to research and publish with confidence.

    1. Kit Compatibility

      Before choosing your kit, ensure it is compatible with your target and the nature of your sample. Ideally, the kit you select should have been characterized in the same matrix as the sample you are testing, i.e. plasma, serum, urine, tissue culture, saliva etc. Check the assay detection ranges and sensitivity of the kit are suitable and that it is able to detect the target in the species in which you’re working.

    2. Have a Complete Understanding of the Assay Before You Begin Testing

      Each kit is created for detection of specific targets and with specific capabilities, it is not ‘one size fits all’. Knowing the sensitivity and specificity of your ELISA kit, and how to augment each step with precision, will generate an accurate and reliable standard curve displaying quantification of your target. Each kit will contain target specific reagents and buffers. You need to know and understand each set of parameters at the start, e.g. type of antibody, incubation times, temperatures and reporter system. Familiarizing yourself with this in advance will save a lot of time and frustration.

    3. Sample Preparation

      Before performing your main experiments, establish the correct dilution range by using a small sample. Your samples must be compatible with the microtiter plate assay format. The amount of biological marker being tested will vary. Use the guidelines within the kit as a reference point, you are aiming for data that fits within your sample standard curve. Be aware, samples which contain interference factors such as Bilirubin will produce inaccurate results.

    4. Antibodies

      If you are developing your own ELISA test, your choice of antibody will depend on your requirements for specificity and affinity. Monoclonal antibodies, by definition are likely to give more specific binding leading to decreased background signal. Monoclonals can be used on their own or in combination with polyclonals. Polyclonals will generate a higher signal but there will be a higher level of non-specific binding. You will need to carry out thorough testing each time as polyclonals will show more batch-to-batch variation. ‘Matched pairs’ refers to a combination of monoclonal, polyclonal or both which are used within an assay for the detection of a single antigen and have been validated to work together, binding to different epitopes and working as a good ‘capture’ and ‘detection’ pair.

    5. Maximize Your Samples

      To get the most out of your kit, Biorbyt would advise that you carry out a couple of test assays using control samples at a range of dilutions to obtain standard curves. Conserve your most valuable samples until you have a clear idea of the right dilutions to use. Once you know the samples and dilutions to use you can plan the best layout of your plate. Use all wells, following the kit’s guidelines. Add more detection reagent if necessary according to the information provided.

    6. Reproducibility

      Ultimately, you are aiming for accuracy and reproducibility to create usable and valuable data. To achieve consistency, optimized performance and accurate results, be fully cognizant of the protocol throughout. Remain consistent and be systematic in your approach.

      • Before running the assay, allow about 30 minutes for the kit reagents to reach room temperature or the temperature stated in the information provided. Frozen samples should also be allowed to thaw completely before being used and repeated freeze-thaw cycles should be minimized to less than three times.

      • Keep environmental conditions, e.g. temperature and humidity constant throughout the assay and between assays.

      • Ensure all equipment is calibrated including pipettes, plate washers and readers.

      • Use sufficient quantities of antibody.

      • Substrate solutions should be freshly prepared, not stored for hours ahead of use.

      • Handle samples consistently throughout testing and follow the same procedures each time.

      • Throughout the process, visually inspect the tips and wells to check aspiration, addition of reagents and withdrawal. Levels should be equal.

      • Don’t be tempted to mix lots between kits to get more out of your reagent. ELISA kits are each prepared so that the contents work together. Mixing lots between assays could adversely affect performance.

      • Once a reagent has left the bottle, it should not be returned.

      • Don’t allow the wells to dry out once testing has begun, keep the tray sealed to prevent drying.

    ELISA Kit Tips and Tricks
    1. Washing

      Careful washing is necessary to reduce background signal caused by unbound, conjugated antibody. It will therefore increase the assay’s signal-to-noise ratio. Washing at each step will help to maintain purely the specific binding events. Ensure wash volume is high enough to remove all traces of antigen or antibody from the wells and hence, reduce unwanted background signal. Maintain the recommended distance between the bottom of the well and the wash tips to reduce residual antibody/antigen containing fluid being created that will affect your signal. Floating heads are more flexible and easier to obtain a complete wash. Repeat wash cycles enough to ensure unwanted antigen and antibody are removed but the bound antibody remains in place. Follow the guidelines but Biorbyt recommends three washes after each incubation. This will need to be altered depending on whether your plate is manufacturer coated or if you have coated it. You may need to increase the number of wash cycles if you have coated your own plate.

    2. Buffers

      Use the buffers and diluents provided within the kit or those that are specified in the protocol. Buffers can be single or multi-function. They are used throughout the assay for coating, blocking, washing and for dilution. Coating is the process of adding a buffer to stabilize the antigen or antibody then incubating overnight to cause adsorption of the diluted antigen or antibody to the surface of the well. Biorbyt also provide multi-purpose, universal ELISA buffers that could potentially save time and energy, speeding the process up without compromising function.

    3. Blocking

      There are many blocking buffers available, some offering additional benefits such as increased stability, enhanced blocking, reduced cross-reactivity and reduced non-specific binding. A blocking buffer containing an unrelated protein can be used to prevent non-specific binding of the detection antibodies to the plate. Blocking buffers usually contain BSA or milk proteins dissolved in PBS. Biorbyt’s range of Blocking buffers will include the perfect solutions for your assay.

    4. Cross Contamination

      Forgive us for stating the obvious, but working in a clean and organised manner is the first step. Before beginning the assay, establish the amount of reagents you will need to use. Prepare the right amount of reagent and discard any excess. Avoid cross contamination of samples or reagents by changing pipette tips between each sample, standard or reagent addition. Be extra careful during liquid removal and washing. For each transfer, use new, disposable reagent reservoirs.

    5. Accuracy

      Accuracy throughout the procedure will increase confidence in your data. You are aiming to generate more than one standard curve with a high degree of accuracy. You will need to test your samples in duplicate at minimum, and preferably in triplicate. You need the smallest possible coefficient of variability (%CV) between each replicate. Outliers will be more obvious so can be investigated before being included in calculations and affecting your results. One possible cause of outliers is ‘Edge Effect’ caused by issues with production of multiwell plates or with assay processes that affect the outer wells. Things that will affect your CV include: incorrect storage and/or preparation of samples, bubbles in wells, incomplete plate washing, poor mixing of reagent, temperatures not controlled and poor pipetting technique. Samples with optical densities (OD) above or below the linear range of the standard curve will result in target concentrations being underestimated or overestimated, respectively.

    6. Obtaining Reliable Standard Curves

      It is crucial to follow the manufacturer\s recommendations for storage, handling and pipetting. Always use the recommended diluents, reagents and detergents, and keep to the correct conditions of pH, temperature and strengths for your kit. Check vials don’t contain any undissolved residue after spinning. Assays will not be identical, but there is a range of variation that is acceptable. A standard curve should be produced for each set of samples assayed. Generate each standard curve within the manufacturer’s recommended dilution range and ensure that you have sufficient data points. There are no benefits to trying to extend the curve, the antibodies used for binding will only produce data within a set range. If you notice the ‘Hook Effect’ in your results the probable cause is not enough analyte to bind with the quantity of antigen in your sample. This issue is also resolved by testing first at a range of dilutions. If you are finding sensitivity is low, it is worth checking:

      • That the kit was stored correctly

      • Your detection reagent is fully functioning

      • Your sample type and buffers are compatible

      • You had an adequate concentration/amount of target protein and of substrate

      • The adsorbance wavelength settings of your plate reader are correct

      • Incubation times are long enough

      • Your ELISA kit had the right level of sensitivity for your assay.

    7. Color Development

      After your careful preparation, optimize color development to display your results in the clearest way possible. Follow the guidelines within the kit for incubation times. Substrates used in ELISA kits for color detection are usually photometric. If you are using a colorimetric assay, all color development must take place in total darkness. If your protein is not being detected you could try increasing its concentration by diluting it less. If you detect a high background signal there are a number of things to investigate: contamination, ineffective washing, timings not adhered to, too much detection reagent, antibody concentration needs changing, wrong blocking buffer used, non-specific antibody binding or failure to follow any stage of the manufacturer’s guidelines.

    8. Data Analysis

      To maximise the potential of the data you have obtained, it’s worth the time and effort to get this stage right. Your assay instructions will suggest software for data analysis. Follow the suggested guidelines for best wavelength to read the plate. It is possible to apply factors to your calculations to correct for background, this should be included in the guide. The manual will also outline how to plot the data and generate a standard curve. Use a template spreadsheet to generate your curve automatically.

    Biorbyt’s team can provide advice for any stage of the process from purchase to completion. Our range of solutions and equipment are specifically designed to support your research.