How to understand antibody application

Table 1: main applications of antibodies

The antibody is a soluble form of B lymphocyte antigen receptor, which is specifically produced by mature B lymphocytes.

The antibody immunoglobulin molecules consist of two heavy chains and two light chains, which can be divided into five subtypes according to their structure.

The molecules of the antibody are linked by a disulfide bond, which gives the whole molecule some flexibility.

There is no light chain in the molecule called a variable or Fc segment (the crystalline part).

This region is determined by a set of genes, all of which are consistent with the subtype of antibody in the same genus.

The parts of the molecule that have both heavy chains and light chains are called constant or Fab segments (antigen binding regions).

The extremes in this region contain high variable sites that identify and combine target antigen epitopes.

Fab is also determined by a set of genes, but requires further somatic mutation to produce unique and highly specific variable sites (figure 1).

Figure 1. Molecular structure of antibodies

• Protein detection and purification

  Immunoprecipitation

  The immunological precipitation (IP), also known as the "pull down" experiment, is that the antibody is labeled and precipitated (" pull down ") in the cell lysis fluid, in biological fluids, or in other solutions, to isolate the corresponding target antigen.

  By antibodies Fc period of combination of different categories of beads (such as magnetic beads, agarose, agarose gel), reoccupy centrifugal or other mechanical methods to separate from the total protein antibody antigen complex (figure 2).

  
  

2. The schematic diagram of standard immune precipitation method.

IP detection is very popular in many cell and molecular biology studies.

According to its basic principle, IP is often used to purify target antigen for follow-up study.

IP is also commonly used to study the interaction between homeostasis cells or the various proteins in the cells that are processed.

In the studies of protein interactions via IP, one of the known protein antibodies will be used for precipitation steps, then through such as western blot method (see below) to determine the combined with the known proteins and common precipitation separation of another kind of protein molecules.

Immunosorption experiment

Enzyme-linked immunosorbent assay (ELISA) used for a specific soluble protein molecules of qualitative and quantitative analysis, such as serum, or liquid samples, such as biological humoral and cell culture supernatant of specific antibodies or proteins in common concentration.

This method is used to detect the ability of the protein or antibody and the ability of antibody specific binding target antigen.

In general, this method can be through the testing experiment samples under a certain wavelength absorbance, coupled with a known antigen or antibody concentration standard curve, calculated the antigen or antibody concentration in the samples.

There are three commonly used ELISA detection methods.

Direct ELISA method

Direct enzyme-linked trials use different quantified monoclonal antibodies to determine the concentration of a particular antigen in a solution.

For example, direct ELISA can determine the level of cytokines in the cells by measuring the cytokines in the cell culture to determine the level of cytokines in this group of cells (via stimulation or inhibition).

The direct ELISA method is often carried out in the "sandwich" method, which is that the two monoclonal antibodies bind to the same target antigen with different antigen epitopes, and the target antigen is sandwiched in the middle.

First, the "capture" antibody package is on the test board.

After washing and sealing, the solution is added to the determination hole, and the "capture" antibody is used to capture the target antigen in the solution.

The rest of the protein molecules in the sample were washed to the test hole to be added to the biotin "detection" antibody.

The antibiotin protein compound enzyme was added to bind the antibody of biotin.

Finally, the substrate of the enzyme is added to the determination hole.

The relative quantification can be carried out according to the determination of specific color changes in the hole: the more the color change, the higher the antigen concentration in the sample.

It is possible to make the standard curve of the hole by the sample of the target antigen concentration.

The detection process does not require "capture antibodies";

In other words, the antigen can be directly packaged into the detection hole for subsequent detection.

However, the specificity of target antigen was detected by double-antibody sandwich method (figure. 3).

Figure 3. Flow diagram of the standard direct sandwich ELISA method.

Indirect ELISA method

Indirect enzyme-linked tests are used to detect antigen-specific antibodies in a solution.

In this test, a specific antigen will be directly packaged into a sample hole in the test plate.

Samples that may contain such antigen-specific antibodies (such as serum, heteronoma cell culture medium) are added to the detection hole and the specific antibody antigen binds to each other.

Next, the second antibody, which can be incorporated into the antibody Fc segment, is added to the detection hole to bind to the antibody already binding to the specific antigen.

Finally, the substrate was then added to the colorimetric substrate, and the substrate was uncoupled with the enzyme enzyme of the second antibody, which was reflected in the determination of the absorbance to determine the content of the antibody.

Competitive ELISA method

A competitive enzyme test is usually used to detect small antigen molecules.

In such tests, the unknown concentration of target antigen samples were added to the detection hole of the same target antigen that had been given the known concentration.

Then the marker antibody that detects target antigen is added.

The soluble antigen antibody complexes were then washed, and the antibody levels in the detection pore were detected.

The final detection of the signal intensity of the competitive ELISA method is generally the opposite of that of other ELISA methods.

In particular, the higher the free antigen concentration in the sample, will be combined with the more antibodies, therefore to test plate fixed antigen antibody will reduce, so the last detected signal will become weak (figure 4).

Figure 4. Flow diagram of competitive ELISA method.

Enzyme-linked immunospeckle method

Enzyme-linked spots like sandwich ELISA method to detect cytokines and other soluble factors, by combining with monoclonal antibody on the test board to capture the antigen, and can be used for the colorimetric determination of enzyme-linked second monoclonal antibody testing to capture the target antigen.

But the difference is that in the enzyme-linked immune spot detection, cytokine production in the cells can be directly to cultivate the bag had been captured by the antibody and closed in advance of the test plate, this test can be captured by a single cell from a spot the secretion of cytokines.

After developing a specific segment, discard the cells in the test board.

The rest of the subsequent detection steps are very similar to the common ELISA method.

The final step is not to analyze the absorbance at a certain wavelength, but to visually observe or use a specialized reader-board in the microscope.

The ELISPOT method of successful detection of cytokine cytokine cells in each inspection hole has many different shades of color, and each spot corresponds to a single cell.

ELISPOT method is particularly suited to detect cell a very small number of cells, such as detection of immune antigen specific T cells in mice, such a very small number of cells with other methods generally difficult to detect (figure 5).

Figure. 5. Enzyme-linked immunostain method.

A. Flow diagram.

B. Representative results of the enzyme linked immunostain detection.

Immunoadsorption technology can be used in combination with microarray technology to realize high-throughput proteomics measurement.

In this type of test, a pre-made glass or polystyrene slide is coated with a captured antibody or sample (such as a cell lysis product).

The former (packet capture antibody) similar to the classic sandwich ELISA, ELISPOT, because the target antigen between fixed antibodies and free, the latter (direct) package is sample similar to the direct ELISA method, because the target antigen directly on the glass slide.

The detection of antibodies in these two methods is aimed at the fluorescent labeling antibody of various known antigens.

The technology can quickly screen out proteins that express or activate changes.

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