More on Serology

The world of serology can be confusing. We broke down what serology is in our Basics of Serology blog post from 2019. At Galaxy Diagnostics, we go beyond to explain difficult concepts in comprehensible ways. In this post, we will take a deeper dive into a few serology testing methods we use in our labs.

An overview

Serology refers to the study of blood serum. Serum is the substance that is created when red and white blood cells are separated from the rest of the blood. It consists mainly of electrolytes, hormones, and an assortment of proteins.

At Galaxy Diagnostics, when we perform serology testing, we are focusing on specific proteins in the serum: antibodies. Antibodies, or immunoglobulins (Ig), are proteins created by the immune system as part of a complex, multi-pronged process that attacks other proteins the body has identified as threats. Antibodies are often Y-shaped and have binding locations specific to the target, known as an “antigen.” An antigen is a specific part of a protein the immune system has identified as a threat. Once an antigen is bound, it may be broken down immediately or other immune cells may come in to destroy it.

There are five classes of antibodies:

• IgG make up about 75% of the antibody population. They are known to bind bacteria and toxins.
• IgA make up about 15% of the antibody population. They are mainly responsible for protecting mucous membranes and the gastrointestinal tract.
• IgM make up about 10% of the antibody population. They are the largest antibody and serve as a preliminary defense to antigens
• IgD make up about 0.5% of the antibody population. They may play a role in the production of antibodies as well as the protection of the respiratory tract.
• IgE make up about 0.01% of the antibody population. They are responsible for protecting the body from allergens and part of a typical allergic reaction.

IgM and IgG are the most relevant antibodies for serum-based antibody testing for vector-borne diseases.

Our Assays

Immunofluorescence Assay (IFA)

IFA measures the reactivity of antibodies in a sample to antigens at different titrations (dilutions). This method uses fluorescently labeled antibodies that connect with antibodies in the sample if they are bound to the target antigen. The reaction is then observed under a fluorescent microscope to determine the sample’s reactivity.

There are 4 main components to performing an IFA:

1. The antigens – Typically immobilized on a slide. The antigens Galaxy Diagnostics uses are Bartonella henselae, B. quintana, B. vinsonii berkhoffii, and B. koehlerae within mammalian cells.
2. The sample – Our lab uses blood serum. We are focused on IgG antibodies that are specific to Bartonella spp
3. The titers – At Galaxy Diagnostics, serum samples are serially diluted in a sample buffer. This generation results in what are known as “titers”.
4. The secondary antibody – This antibody is marked with fluorescent dye. It will connect with the antibody from the sample if those antibodies are connected to the antigens. This special chain of connections causes a reaction that can be seen under a fluorescent microscope.

Going back to step three, a dilution, or titration, occurs when the ratio of sample to buffer decreases. This may look different across labs. A good way to think about it is to imagine four containers that each have an equal volume of water. You add two drops of orange food coloring to the first container and examine the water’s color. It will be a brilliant orange, because there is a lot of food coloring. You then take two drops of the orange water from the first container, add it to the second, and examine the color. It will now be a dull orange because there is less food coloring in this dilution. You would then repeat the process for the third and fourth container. Through this process, you’ve learned something about how many molecules of food coloring, something that you normally could not see, are in the water.

 

ELISA

ELISA stands for enzyme linked immunosorbent assay. Our assay is a plate-based testing method that captures antigens and measures the antibody response to said antigen by observing enzyme reactions.

It is best to think of this test in terms of “if…then” statements.

1. The antigens are immobilized on the plate.
2. The sample is added to the plate.
   1. If antibodies specific to the antigens are present, then they will bind to the antigens.
3. Next, a secondary enzyme-linked antibody is introduced to the environment. This secondary antibody is seeking to connect with the primary antibody that had bound the antigen.
   1. If the antigen was not originally bound, then the secondary antigen does not connect to anything.
4. An enzyme substrate is introduced to the plate, and it measures reactions using color.
   1. If the secondary antibody is connected to the primary antibody, then a reaction occurs that the enzyme substrate visualizes.

 

Western Blot

Western blot is an immunoassay that allows researchers to identify specific proteins among a wide range of proteins. Proteins are first separated by molecular weight and transferred to a paper-like membrane. The sample is introduced to the antigen proteins on the membrane.

A secondary antibody is introduced to the membrane and will connect to the primary antibody. A substrate is added to view the reaction that occurs when the secondary antibody connects to the primary antibody bound to the antigen.

Galaxy Diagnostics offers Bartonella IFA serology tests and Borrelia burgdorferi ELISA and Western blot tests

 

Conclusion

Serology can be a powerful supporting test method for a pathogen. The body’s immune system produces antibodies, signaling the presence of pathogens that can be detected in a laboratory. However, those signals themselves can be difficult to interpret as they are not always the same and can indicate different things about when – and if – a pathogen has been encountered by the body’s immune system.

 

References

Henrik’s Lab. (n.d.). ELISA (Enzyme-linked immunosorbent assay). YouTube. https://www.youtube.com/watch?v=ERk0hwqhyDw

BioBest. (n.d.). ELISA testing. https://biobest.co.uk/how-does-an-elisa-testing-work/

MBL International. (2016). Types of antibodies. https://www.mblintl.com/resources/scientific-resources/fundamentals-for-planning-research/types-of-antibodies/

Smith, Z., & Roman, C. (2022, April 16). Fluorescence. Chemistry LibreTexts. https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Spectroscopy/Electronic_Spectroscopy/Radiative_Decay/Fluorescence

ONI. (2021). Simple overview: Immunofluorescence. https://oni.bio/nanoimager/super-resolution-microscopy/immunofluorescence/#:~:text=Immunofluorescence%20(in%20short%2C%20IF),molecules%20under%20a%20light%20microscope.

Ryu, W-S. (2017). Diagnosis and methods. In Molecular virology of human pathogenic viruses (Ch. 4, pp. 47-62). Academic Press. https://www.sciencedirect.com/science/article/pii/B9780128008386000047 – this source lays out all three testing methods nicely

Animated Biology with Arpan. (n.d.). Western blot | Western blotting protocol | Application of Western blot | Limitations of Western blot. YouTube. https://www.youtube.com/watch?v=Yh69yHJMWPc

Mahmood, T., & Yang, P-C. (2012). Western blot: Technique, theory, and trouble shooting. North American Journal of Medical Sciences, 4(9), 429-434. https://doi.org/10.4103/1947-2714.100998 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/