How to understand antibody application（4）
Now we're going to go on to the next one and continue to learn about the application of cytology outside the body of the antibody
Application of cytological methods outside the body
Flow cytometry - cell group analysis
This is the most common application of antibodies in biomedical research and in clinical practice. Antibodies can be directly tagged on the cell surface and directly detected by flow cytometry for various cell groups.
The fluorescence antibody staining was very fast in single cell suspension.
The staining method is not only specific, but also sensitive.
In addition, most flow cytometry can detect three or more fluorescent antibodies simultaneously, so it can detect the differentiated cell subgroups and the surface proteins associated with differentiation, activation and apoptosis.
Antibody must go through strict selection, ensure the antibodies against a target on the cell surface antigen epitope expression, as well as the blocking agent (total source host immunoglobulin IgG) should be to avoid certain cells, such as various types of lymphocytes Fc receptor on nonspecific combination (figure 9).
Figure 9. Flow diagram of cell surface molecular markers in magnetic assisted cell separation/flow cytometry.
The representative flow cytometry results map showed CD44 and CD107a of CD8 + T cell surface markers with magnetic secondary cells.
Cell separation and removal
Fluorescence assisted cell separation (FACS) is an antibody that USES fluorescence labeling to bind specific cells and then select single cells based on specific fluorescence parameters.
This method is quick and specific.
But specific flow cytometry is needed.
An antibody that has a magnetic bead can also be used to separate or separate cells, a process known as a magnetic assisted cell separation (MACS).
In the process of MACS, cells are marked with antibodies tagged with cell surface tags.
The cells are then incubated with small magnetic beads that bind to the label of the antibody.
Finally, through a powerful magnet, it is very easy to isolate the cells that have been marked with magnetic microbeads (figure 9).
Internal study application
To study the function of a particular cell group, antibodies can be given to remove such cells.
In immunological studies, for example, antibodies can be used to remove specific lymphocyte subgroups in mice, so that the immune response to a specific antigen can be observed.
Similarly in vitro studies, antibodies can bind cells' surface receptors or neutralize the soluble factors in the body.
Applied to this kind of method of antibody, are generally by hybridoma mass production, to avoid reactions to heterologous antigen, and need to be purified to remove cell culture reagents and other possible pollution.
Mass spectrometry
Mass spectrometry (MS) is an analytical technique that measures the mass of the product ions (M/Z) to detect, identify and quantify molecules in simple and complex matrices.
Today MS is an irreplaceable tool in a wide range of areas such as proteomics, drug development, environmental analysis, and biomedical research.
In particular, the protein analysis, because of the use of MS instruments, has experienced very rapid development, so it can be more accurate and deeply qualitative similar molecules.
However, MS still has some problems to be solved, such as the huge amount of data generated, and the presence of high abundance proteins to cover up the protein that is of interest.
The latter point has been partially overcome by MS targeted methods, such as multiple/select response monitoring (MRM/SRM).
In other words, MS sensitivity has some problems, but it also produces highly accurate and specific data.
Under this framework, combined with a method based on the affinity of antibodies, MS target molecules with the method based on immune first capture, then use MS analysis, the strategy at the same time increase the specificity and sensitivity.
The combination of immunoassay and MS has developed several quantitative and qualitative methods.
Stable isotope standard and the use of anti-peptide antibody capture
MS technology allows for the absolute quantitative determination of specific proteins in the plasma.
This can be achieved by using a mixture of synthetic peptides and heavy isotopes (AQUA).
In 2004, Anderson et al. developed stable isotope standards and the method of capture (SISCAPA) by anti-peptide antibody, which can be enriched by using anti-peptide antibodies that are fixed in the nanoparticles.
The experiment consisted of four steps: I) using trypsin digestion protein samples;
2) add the heavy isotope standard peptide such as AQUA;
The specific anti-peptide antibody immunoenrichment iv was used to quantify the peptide by electrospray ionization mass spectrometry (ESI - MS).
SISCAPA has several applications, such as the determination and quantification of the biomarkers of serum transferrin receptor (sTfR) for breast cancer patients.
However, the improvement of target MS determination results in a combination of SISCAPA and MRM in immune-ms research, resulting in the so-called immune MRM.
The immune - MRM
Multi-response monitoring mass spectrometry (MRM - MS) is a targeted quantitative MS method with high specificity and precision.
In order to increase the sensitivity of the measurement, it is possible to immunize MRM through the mixture of immuno-affinity and enrichment target peptide.
The technique is repeatable, multiple, and highly sensitive and specific.
The main problem with widespread use of immune MRM is the lack of proven antibodies to the technology.
Antibodies are usually produced for the classical immune market (such as enzyme-linked immunosorbent, protein imprinting), whereas the antibodies to MRM should correspond to short, straight-linked peptides.
Several studies have been completed to investigate the use of monoclonal antibodies in immunomrm.
Clearly, these antibodies are superior to polyclonal antibodies.
Unfortunately, monoclonal antibodies are expensive and they are produced by hybrid systems for a long time.
Recently, the feasibility of immunomrm monoclonal antibody against trypsin peptide antigen by using recombinant B cell cloning has been proved.
The immune - MALDI
Immune - specifically (iMALDI) is similar to SISCAPA methods: even in this case, the use of peptide antibody capture peptide, but then the enrichment of the sample point to a specific goal and by matrix assisted laser desorption ionization (MALDI) MS analysis.
This approach has been used to diagnose different diseases, such as high blood pressure.
However, one of the main limitations of iMALDI is the inability to multimeasure.
The immune - MALDI
Immune - specifically (iMALDI) is similar to SISCAPA methods: even in this case, the use of peptide antibody capture peptide, but then the enrichment of the sample point to a specific goal and by matrix assisted laser desorption ionization (MALDI) MS analysis.
This approach has been used to diagnose different diseases, such as high blood pressure.
However, one of the main limitations of iMALDI is the inability to multimeasure.
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