Optimization of ELISA is often required even when matched sets of antibody reagents are bought. Where a poor signal is seen at the end of the ELISA four parameters may be varied to make improvement. Each one should be varied independently and an optimum dilution factor or value determined for routine use.
1. Vary the pH of carbonate coating buffer between pH 7.0 and 10.0 in 0.5 pH unit steps. Also, try PBS at pH 7.4. Some antibodies, particularly monoclonals will bind better at one specific pH value.
2. Make a dilution of the capture antibody in coating buffer from 0.5 to 10 |g/mL and use to coat ELISA wells. The binding capacity of ELISA wells may be reduced if the concentration of antibody is not optimal. Overdilution, and paradoxically underdilution may lead to poor well binding.
3. Make a dilution of biotinylated detection antibody from 0.05 to 1.0 Mg/mL and use to determine the optimum for the reporter antibody.
4. Make dilutions of streptavidin-HRP conjugate between 1:1000 and 1:40,000 and use to determine the optimum concentration for this reagent.
1. The quality of the antibodies used is perhaps the most important aspect in setting up a good ELISA. Antibodies need to have a high affinity for the sample to be measured, low crossreactivity to other substances and work well together as a pair. Mismatched antibody pairs can lead to poor signals from test substances and high nonspecific background.
2. The pH of the coating buffer affects the amount of antibody, which will bind to the plate. Basically, a higher pH will result in more antibody binding, but may have a detrimental effect upon its immuno-activity. Thus a pH must be found that is suitable for the antibody in question, and this can vary dramatically. When beginning optimization of the assay, test a range of carbonate buffers from pH 7.0 to 10.0 as well as PBS pH 7.4. We usually find a carbonate buffer pH of 9.5 gives good results. In some cases we have found commercially available coating antibodies recommended by the manufacturer to be adsorbed onto the plate at pH 7.4, to be far more effectively adsorbed at higher pH's. This improves the lower detection limit of sensitivity by up to 1000%, and thus allowing the coating concentration to be vastly reduced and creating an extremely cost-effective assay.
3. There can be a significant difference between the protein binding capability of different makes, and even batches of microtiter plate. The only real way to choose a suitable plate is by trial and error or by using a recommended make known to bind the antibody that you intend to coat with.
4. Although the HRP/TMB system is usually a good, reliable, and sensitive combination, HRP has a number of alternative substrates that can be used, such as o-phenylene diamine. There are also numbers of options for the enzyme used other than HRP, such as alkaline phos-
phatase, which can be used in combination with the substrate ^-nitro-phenol phosphate. It is important to note that if alkaline phosphate is used, the wash buffer must not contain phosphate. Usually in this case a tris-buffered rather than phosphate buffered wash buffer is used. The choice of enzyme-substrate system depends upon a number of factors including price, sensitivity and whether a spectrophotometer filter is available for the substrate specific wavelength to be measured.
5. Occasionally, an ELISA can give rise to unexpected results in wells close to the edge of the microplate. The phenomenon is often referred to as the "edge effect" whereby unexpectedly low or high OD readings are observed. In some cases this is a result of light or heat sources. For example, if the plate is being incubated near a strong light source such as a window any reaction that is photosensitive (such as the substrate reaction) may give rise to elevated OD levels in those wells closest to the light. More commonly, temperature difference causes the edge effect. This is particularly common when plates are stacked one upon the other or where a solution is taken straight from the fridge and the incubation step is performed at 37°C for a short period of time. In such cases, the outside wells are the first to heat up and thus the reaction rate is greater there, most likely because of the faster rate of movement of molecules at this higher temperature. To avoid these complications, plates should ideally be separated from one another during incubation periods and liquids should be adjusted to the temperature that the incubation step is to be performed at. Overnight incubation steps at 4°C tend to give better results than 37°C with respect to lowering the edge effect. It is also advisable to perform incubation in the dark or in subdued light. Sealing the plates or using a 100% humidified environment can also help.
6. If the major aim of the ELISA is to obtain quantification of substances present in extremely low concentrations there are a number of adaptations to the technique that can be used. For example, Alkaline Phosphatase enzyme systems can be used to lock the colorimet-ric reaction into a circular redox cycle producing an end product such as red formazan. This results in a hugely amplified signal in comparison to standard amplification methods (3). Chemiluminescent-amplified ELISA principles have also been used to give very high sensitivity (4). In fact, this modification of the ELISA development stage can be optimized to measure as little as 1 zeptomole (approx 350 molecules!) of alkaline phosphatase (5). Although extremely sensitive, such techniques are extremely time consuming to set up and optimize, and are far more expensive than the simple colorimet-ric ELISAs described in this chapter.
Was this article helpful?