ELISA Troubleshooting

Understanding The ELISA Troubleshooting

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First of all, to talk about this topic, you need to know what ELISA actually is. Its full name is enzyme-linked immunosorbent assay. It is a technique specially designed for quantifying and detecting proteins, peptides, hormones, and antibodies. While using ELISA, there is an antigen that should be immobilized to a solid surface. After that, it is structured with antibodies that are linked to an enzyme.

One way of detecting is accomplished by assessing the activity of an enzyme that is conjugated via incubation so it can lead to the production of a measurable product. Plus, the most essential thing during the process of detection is the interaction between antibodies and antigen.

ELISAs are usually performed in 96 or 384 well-made polystyrene plates, whose job is to passively bind proteins and antibodies. The process of binding and immobilizing of reagents is what is making ELISA simple to perform and design.

When the reactants of the ELISA are immobilized on the microplate surface, they are then starting the process of separating the bound from non-bound material, which is important during the assessment. These are the few reasons why ELISA is such a powerful tool.

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Types of ELISA

ELISAs are known for performing multiple modifications up to the basic procedure. There are four types of ELISA, and they are direct, indirect, competitive, and sandwich. The main ingredient, which is the immobilization of the antigen you want, is usually accomplished by the direct method. It is done by direct absorption, which goes on the assay plate. Another way it can be done is indirectly in ways such as capturing antibodies that are attached to the plate.

After some time, the antigen is detected directly or indirectly. Antibodies that are detected are then labeled with AP (alkaline phosphatase) or even HRP (horseradish peroxidase). A large quantity of substrates is now available to be performed with ELISA via AP or HRP.

When choosing which substrate, it mostly depends on the assay’s sensitivity and the instruments available for detecting signals, such as luminometer, fluorometer, or spectrophotometer.

Direct ELISA

In this detection method, antigens that are coated on multi-well plates are detected by an antibody that is conjugated directly to an enzyme. This method is a smart option if there are no commercial kits for your targeted protein.

There are advantages to this method, such as being quicker because fewer steps are being used, and only one antibody is required. One more thing which is also an advantage is cross-reactivity is eliminated for the secondary antibody.

There are also disadvantages to this method are cell smear, minimal signal amplification, no flexibility, time-consuming, expensive, immunoreactivity of primary antibody might be affected. If you want to read more, be sure to click this link.

Indirect ELISA

In this detection method, the antigen that is coated to a multi-well plate is usually detected in two to three stages of layers. First comes the unlabeled antibody, and then comes the enzyme-labeled antibody, which is then bound to the primary antibody.

There are advantages to this method, such as a wide variety of labeled antibodies that are available commercially, versatility, maximum immunoreactivity, more enormous sensitivity.

The disadvantages of this method are cell smear, more incubation steps are required, and sometimes cross-reactivity.

Sandwich ELISA

This particular method works in the way of pairing antibodies, where each antibody is meant for specific epitopes of the antigen molecule. There are advantages to this method, such as high specificity, flexibility, sensitivity, and it is suitable for very complex or impure samples.

Competitive ELISA

This method is based more on surface or plate kind of assays, where the plate is mainly coated with antibodies that are reactive to molecules.

Troubleshooting areas

Sometimes when testing out assays, errors can occur, and it is perfectly normal. This is why there are many tips, guides to optimize kits to perform better, and also when troubleshooting ELISA, it is good practice to know what to do in random kinds of scenarios. Furthermore, the following paragraph is going to be written about the areas in which researchers have problems.

These are one of the most occurring troubleshooting areas such as high signal, out of range, high variation, the background can be high, no signal at all, poor standard curve, etc. These are only some of the few which can be troubling for scientists.

But, there is always a solution for every problem, as anything can be solved if you pay enough attention to the details around the issue. Always make sure to study the guides properly and the scenarios which can occur during research.

Mistakes can cost you if you do not focus on the problems ahead of you, and that is why knowing all the scenarios which can happen will be of great benefit to you.

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