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One of the most important aspects of any assay is consistency and standardization of conditions, as this will affect the reproducibility and accuracy of your results. In the initial stages of assay development, it is important to test a range of parameters, usually by completing a checkerboard dilution series to test various conditions in systematic manner. In addition, buffers, temperature, and humidity must be kept constant between and within experiments in order to produce standardized results.
In a typical ELISA, multiwell plates, multichannel pipets, and plate washers provide for more consistent and faster results, as well as higher throughput. It is very important to make sure that all pipettors used in ELISAs are properly calibrated on a regular basis, to prevent significant variation in the results. Furthermore, it is good technique to observe the level of the liquid in the pipet tip and the wells while following the procedure, to make sure no sample is far out of line with the others. This is particularly important when multi-channel pipets are used, as sometimes the tips in the end rows do not always attach fully to the pipettor.
Using the assumption that a matched pair of antibodies is available to the analyte and that the aim is to establish a sandwich ELISA, the initial task is to determine working concentrations for the antibodies.
Prepare capture antibody dilutions in coating buffer at 0.5, 1, 2 and 5 μg/ml. Then follow standard procedure for a sandwich ELISA which can be found on our ELISA protocols page, distributing the capture antibody as shown in Figure 7.
At the stage where sample addition would occur, add a high and low concentration of your analyte that reflects the expected working range. Finally include a blank, again following the layout in Figure 7 and standard sandwich ELISA protocol until the detection stage.
At the detection stage, prepare detection antibody dilutions at 1:200, 1:1,000, 1:5,000 and 1:25,000 in buffer, adding it as detailed in Figure 7. Including a high and low concentration of the analyte helps to determine the dynamic range. The low concentration analyte indicates the sensitivity of the assay. The blank will indicate non-specific binding.
Fig. 7. Antibody concentration optimization plate layout.
Find the set with the maximum signal-to-noise ratio/largest difference between low and high analyte concentrations; these are the antibody concentrations for further optimization.
Should the blank sample show excessive readings, above 0.2 absorbance units, these key components need to be checked for optimization: ELISA plate type and the blocking and washing buffers. If the background readings are appropriate but the sensitivity is not high enough further experimentation with matrix conditions, buffers, and incubation timings should be carried out. However, if no improvement is possible different antibody combinations need to be generated.
Matrix effects occur most often in plasma and serum samples where a series of components can cause interference. These can be cross-reactive or non-specific interactions to substances in the matrix or breakdown products that develop during the sample handling process. It is possible to reduce matrix effects by dilution of the sample; this should be verified by analyte recovery from spiked samples. Specialized buffers for sample dilution, coating, blocking and washing can ameliorate matrix effects and provide constant performance.
Specialist coating buffers are available which have been optimized for ELISA, such as BUF030 which has been developed to stabilize the adsorbed protein, preserving the antigenic regions and allowing greater binding reactivity in order to enhance the specific signal.
AbGuard® Plate Stabilizer (BUF063) simultaneously stabilizes and preserves microwell plates coated with proteins or other biomolecules and blocks any free binding sites. It preserves the biological activity of bound molecules and prevents degradation, denaturation and leaching. It also blocks free binding sites without creating any interference.
A wide range of sophisticated commercial blocking buffers are available, some of which are BSA-based (BUF032), but also contain preservatives to create a stable long-term environment. Another option is to use a high performance commercial blocking buffer, such as ELISA Ultrablock (BUF033), which is an optimized formulation that includes small protein fragments which thoroughly block less accessible surfaces of the multiwell dish. Ultrablock is recommended for use on mammalian samples, particularly, human, pig, and cow, as there is less chance of cross-reactivity. When maximal blocking strength is required, or for most sandwich ELISAs, preparations such as ELISA SynBlock (BUF034), an inert, synthetic blocking formula that reduces non-specific interference.
For greatest consistency, specialized plate washers are used to add and remove the wash liquid. Specialized wash buffers are also available, such as BUF031, which contains an optimized formulation of pH stabilizers, salts, and detergents that reduce background noise and enhance specific signal.
One type of specialty sample buffer used for sample dilutions is HISPEC assay diluent, BUF049A. This buffer is used to dilute a range of sample types or for the dilution of detection antibodies. It reduces cross-reactivity, non-specific binding, and matrix effects.
BUF037A is a specialized buffer that is recommended for use in sandwich ELISA assays with samples containing plasma, serum, or cell culture supernatant. This buffer contains goat serum proteins which reduce the difference between the sample matrix and the diluent used to generate the standard curve. It also contains a proprietary chelating agent that blocks interference from complement and thrombin in plasma and serum. It is easy-to-use and applied by pipetting 50-100 μl directly onto the plate before adding the sample. Since this buffer contains proteins derived from goat, it is not recommended for ELISAs that use anti-goat secondary antibodies for detection, as this will result in non-specific signal.
Some specialist buffers are multifunctional, allowing for simultaneous coating, stabilization, and blocking. One such example is AbGuard Plate Stabilizer (BUF063), which preserves the biological activity of adsorbed molecules and prevents degradation, denaturation, and leaching of the coating material. As a result, this buffer can shorten the length of time required for the assay and will increase efficiency by extending the life of coated plates.
Another option is Block Ace (BUF029), a buffer that can be used for blocking, sample dilution, and washing for ELISAs and western blots. It also improves ELISA results by reducing background and producing sharper standard curves.