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Research Applications

Applications in Immunoassays

Antibodies are used as the most critical reagent in most immunoassays. A signal is always needed to quantify the binding of the antibody to a specific target. The most widely used types of the signal label are a fluorescent or chemiluminescent group of compounds, and enzymes, such as alkaline phosphatase and horseradish peroxidase (HRP). The use of antibodies is particularly valuable in immunoassays, such as enzyme-linked immunosorbent assay (ELISA), western blotting (WB), immunoprecipitation (IP), flow cytometry (FCM), lateral flow assays (LFIAs), immunocytochemistry (ICC), immunohistochemistry (IHC) and others.

  • WB. Sample proteins are separated by electrophoresis and transferred onto a supporting matrix. The first antibodies bind against a certain protein of interest, followed by binding by enzyme-linked second antibodies to produce a signal that can be detected. Generally, WB can be used to detect the expression level, the size of protein, and the posttranslational modification status, such as phosphorylation and acetylation.
  • ELISA. Normally, the basic ELISA steps consist of plate coating, blocking, washing, signal generation, and plate reading sequentially. ELISA has four frequently used iterations: indirect, direct (immunocapture), sandwich (immunometric), and competitive. ELISA can be utilized to identify the presence and concentration of an antigen in a liquid sample.
  • FCM. FCM is widely utilized in immunology-associated researches and assays analyzing particles. FCM can detect multiple parameters in one sample at the same time. Parameter measurements of FCM include the size of cells and the expression level of markers. Therefore, FCM can be used in sorting different subpopulations of cells, liquid biopsy, detection of intracellular antigens and markers on the cell surface.
  • IP and Co-IP. IP is another widely used technique that can be used for the purification and concentration of the target protein from a liquid sample, followed by subsequent detection via western blotting and other assay techniques. Co-IP is an extension of IP to analyze protein-protein interaction.
  • IHC & ICC. IHC can show the tissue-specific and subcellular location of the antigen in the sample. This visualization is achieved by a fluorescent or chemiluminescent group of compounds. ICC is similar to IHC but is used to investigate the subcellular distribution of proteins. Compared with IHC, ICC provides a higher spatial resolution.

Applications in immunoassays. Fig.1 Applications in immunoassays.

Production of Monoclonal Antibody (mAb)

  • Hybridoma technique. The splenocytes from the immunization of mice are fused with myeloma cells to generate hybridoma cells that can produce antibodies. But this technology has drawbacks like a limited number of candidates, possible instability of the aneuploid cell lines, inability to provide antibodies against highly conserved antigens, and most of all its limited application to generate human antibodies.
  • Antibody phage display. The phage display technique involves an approach for the rapid identification of specific binders, such as single-chain fragment variable or Fab. Depending on the analysis of DNA sequence, human immunoglobulin G (IgGs) are constructed. Antibody phage display allows the generation of recombinant antibodies from different species.
  • Single B cell monoclonal antibody technologies. From infected or vaccinated donors, suitable B cells are isolated from peripheral blood mononuclear cells by FCM, following cloning the variable heavy and light chain of each identified B cell to inform the generation of mAbs.
  • Transgenic mouse. The human Ig genes were inserted into the mouse genome to replacing the Ig genes of the mouse. These mice can produce complete human antibodies after immunization. However, antibody generation is limited by the immune system.

Approaches for the development of therapeutic antibodies. Fig.2 Approaches for the development of therapeutic antibodies. (Lu, 2020)

Drawing on accumulated experience over years, Creative Biolabs shows excellent performance in the ViroAntibody field. We provide a series of services and products to support the development of advanced research and therapeutics. Please feel free to contact us for more information.

Reference

  1. Lu, R.M.; et al. Development of therapeutic antibodies for the treatment of diseases. Journal of Biomedical Science. 2020, 27:1.

All products and services are intended for Research Use Only, and NOT to be used in diagnostic or therapeutic procedures.

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