Tests for the Antibodies and Process of Isolating and Purifying Antibodies

Amelie Nothomb^*
*Correspondence: Amelie Nothomb, Department of Medicinal Chemistry, University of Illinois, Chicago, USA, Email:

Description

Antibody purification is a process of isolating and purifying antibodies, also known as immunoglobulins (Ig), from a complex mixture such as serum, ascites fluid, or cell culture supernatant. Antibodies are proteins produced by the immune system in response to the presence of foreign substances, such as pathogens or antigens and are used in various applications, including research, diagnostics and therapeutics. There are several methods used for antibody purification, depending on the source material and the desired level of purity. Some commonly used methods for antibody purification include protein A/G/L affinity chromatography: This method takes advantage of the specific binding of antibodies to proteins A, G, or L, which are bacterial proteins that bind to the Fc region of antibodies. The antibody-containing sample is passed through a column containing immobilized protein A, G, or L and the antibodies selectively bind to the column. After washing to remove impurities, the antibodies can be eluted from the column, resulting in highly purified antibodies [1].

Similar to protein A/G, this method utilizes the binding of antibodies to proteins L or G, which are derived from bacterial cells. Protein L has broader specificity and can bind to a wider range of antibodies, including those that do not bind to protein A or G. Protein L/G chromatography can be useful for purifying antibodies from species such as rabbit or camelids, which may not bind well to protein A or G protein G sepharose or Protein A sepharose beads. Sepharose beads are commonly used as a solid support for protein A or protein G and the beads are packed into a column. The antibody-containing sample is then applied to the column and the antibodies selectively bind to the protein A or protein G beads. After washing to remove impurities, the antibodies can be eluted from the column, resulting in purified antibodies [2].

This method utilizes the differences in charge of proteins, including antibodies, to separate them based on their isoelectric point (pI). Antibodies can be purified using either cation exchange chromatography (for antibodies with a lower pI) or anion exchange chromatography. The sample is loaded onto a column containing ion exchange resin and the antibodies can be eluted by adjusting the pH or salt concentration of the elution buffer. This method separates proteins based on their size, with larger proteins eluting earlier from the column than smaller proteins. Size exclusion chromatography can be used as a complementary step after other purification methods to further purify and remove remaining impurities, or as a standalone method for purifying antibodies based on their size. Protein A/G/L or protein L/G magnetic beads: Similar to the column-based methods, magnetic beads coated with protein A, protein G, protein L, or protein L/G can be used to capture antibodies from a sample. The beads are mixed with the sample and the antibodies selectively bind to the beads. The beads are then separated using a magnetic field and the antibodies can be eluted from the beads, resulting in purified antibodies [3].

Antibodies can also be precipitated from a sample using protein A or protein G, which selectively bind to the Fc region of antibodies. After precipitation, the mixture is centrifuged to pellet the precipitated antibodies, which can then be resuspended and further purified if needed. These are some common methods used for antibody purification. The choice of method depends on factors such as the source of the antibodies, the desired level of purity and the specific requirements of the downstream application. It is important to carefully consider the characteristics. Tests for antibodies, also known as serological tests or antibody assays, are used to detect the presence of specific antibodies in a person's blood or other body fluids. These tests can provide valuable information about a person's immune response to an infection or vaccination. Here are some common types of tests for antibodies.

ELISA is a widely used serological test that detects antibodies by using enzymes to produce a colour change. It can be used to detect different types of antibodies, such as IgM, IgG, or IgA, depending on the specific variant of ELISA used. ELISA tests are commonly used for screening and diagnosis of various infections, including viral infections such as HIV, hepatitis and COVID-19, as well as bacterial and parasitic infections. Rapid diagnostic tests are point-of-care tests that provide quick results, usually within minutes, without the need for complex laboratory equipment. These tests typically use a lateral flow or a similar format and can detect antibodies against specific pathogens. They are commonly used in resource-limited settings or for rapid screening purposes. Immunofluorescence assay (IFA): IFA is a technique that uses fluorescent labels to detect antibodies [4].

It involves exposing a person's blood sample to a known antigen and then adding fluorescent-labelled antibodies that bind to any antibodies present in the sample. The fluorescence can be visualized under a microscope, indicating the presence of specific antibodies. Neutralization assays are used to determine the functional ability of antibodies to neutralize the infectivity of a virus or toxin. These assays involve exposing the antibodies to the virus or toxin in the presence of cells and then measuring the reduction or inhibition of viral or toxin activity. Neutralization assays are commonly used in research or in the evaluation of vaccine efficacy. Western blot is a technique that can detect specific antibodies against multiple antigens simultaneously. It involves separating proteins from a person's blood sample by size using gel electrophoresis, transferring them onto a membrane and then using labelled antibodies to detect the presence of specific antibodies [5].

Conclusion

It's important to note that interpretation of antibody test results should be done in consultation with a qualified healthcare professional, as false positives or negatives can occur and results should be interpreted in the context of a person's clinical history, symptoms and other relevant factors.

Acknowledgement

None

Conflict of Interest

The author shows no conflict of interest towards this article.

References

1. Anderson, Annette C., Daniel Jonas, Ingrid Huber and Lamprini Karygianni, et al. "Enterococcus faecalis from food, clinical specimens and oral sites: Prevalence of virulence factors in association with biofilm formation."Front Microbiol 6 (2016): 1534.

Google Scholar, Crossref, Indexed at

2. Aas, Jørn A., Bruce J. Paster, Lauren N. Stokes and Ingar Olsen, et al. "Defining the normal bacterial flora of the oral cavity."J Clin Microbiol 43 (2005): 5721-5732.

Google Scholar, Crossref, Indexed at

3. Kouidhi, Bochra, Tarek Zmantar, Kacem Mahdouani and Hajer Hentati, et al. "Antibiotic resistance and adhesion properties of oral Enterococci associated to dental caries."BMC Microbiol 11 (2011): 1-7.

Google Scholar, Crossref, Indexed at

4. Dahlen, Gunnar, Susanne Blomqvist, Annica Almstahl and Anette Carlen. "Virulence factors and antibiotic susceptibility in enterococci isolated from oral mucosal and deep infections."J Oral Microbiol 4 (2012): 10855.

Google Scholar, Crossref, Indexed at

5. Siqueira Jr, J. F. and I. N. Rocas. "Diversity of endodontic microbiota revisited."J Dent Res88 (2009): 969-981.

Google Scholar, Crossref, Indexed at