Immunoassays: Spotlight on the ELISpot Technique

An immunoassay is a bioanalytical technique used to determine the presence or quantity of a substance, based on the reaction of an analyte and an antibody.¹ Immunoassays play a critical role in diagnosing diseases and monitoring therapeutic drugs, as well as in clinical studies in the drug discovery and pharmaceutical sectors.¹ In this post, we’ll look at the different types of immunoassays, and focus on the unique ELISpot technique.

“Immunoassays combine the principles of chemistry and immunology, enabling scientific tests for detection of the analytes of interest. This technique is used for the detection and quantification of an analyte at low concentration present in a complex mixture of assay chemicals or biological fluids. The technique depends on the specificity and high affinity of antibodies for their complementary antigens.”  – A. Sharma, 2014, Bhabha Atomic Research Centre, Mumbai, India²

The Importance of Immunoassays³

Immunoassays (IAs) are essential tools with widespread importance in various settings. In the clinical setting, they are crucial for diagnosing diseases like influenza and hepatitis, as well as monitoring similar diseases and managing their treatments. They are also becoming vital in the home and workplace through point-of-care testing. IAs are also used to detect pollutants in the environment and to test food for allergens and foodborne diseases like salmonella and E. coli. Overall, immunoassays play a pivotal role in various fields, ensuring the health and safety of individuals and the environment.

ELISA Immunoassays

There are many different types of IAs and are typically classified according to the indicator molecule that's used to label the antigen or antibody.² These indicator molecules range from radioisotopes, fluorophores, or luminol and derivatives.² But the most common immunoassay is the ELISA.⁵ ELISA stands for Enzyme-Linked-Immunosorbent Assay and as the name suggests, utilizes an enzymatic reaction for detection.⁵

Common uses for ELISA include:⁷

• Detecting and measuring antibodies against bacterial, viral or fungal infections
• Detecting and measuring autoantibodies (e.g. in Type 1 diabetes)
• Detecting and estimating hormone levels, like human chorionic gonadotropin (HCG) and testosterone
• Tracking disease outbreaks in communities
• Screening blood donations for possible viral infections
• Screening for the presence of ‘recreational’ (non-medical) drugs like amphetamines or cocaine 
• Detecting the presence and estimating the levels of tumor markers
• Detecting allergies 
• Determining whether a patient has received a specific vaccine

The ELISpot Technique

ELISA vs ELISpot:

ELISA and ELISpot are closely related immunosorbent assays designed to detect specific protein analytes.⁸ However, ELISA is used to determine the total concentration of secreted signaling proteins, while ELISpot is used to detect individual cytokine-secreting cells.⁸

How the ELISpot technique works:⁴

During an ELISpot assay, the cells that secrete the analyte to be studied, like a cytokine or antibody, are cultured along with coating antibodies in the solid phase, e.g. in plate wells. The cells are stimulated in order to measure their response in terms of analyte secretion. In the presence of the target analyte, the coating antibodies will detect and capture the target from the media. 

If a cell produces the analyte of interest, a positive signal or ‘spot’, will form in the immediate region. These spots can then be counted. So, while ELISA would measure the total output of a group of cells, ELISpot can go further by differentiating between the total number of producing and non-producing cells.

With proper adjustments of assay conditions, the ELISpot technique can be adapted to study almost any protein-secreting cells. It can be used for screening cell lines to find the best antibody pairs for an immunoassay platform, or it can enable multiple fluorescence tags to monitor several analytes at once.

How is ELISpot used?

ELISpot is commonly used:

• To monitor the immune response of patients
• To determine the efficacy of immunotherapies in clinical studies 
• As a reference method for identifying the best vaccine candidates 
• To diagnose tuberculosis by detecting interferon-gamma release from T cells
• To diagnose Lyme disease

“The easy-to-perform, high sensitivity of ELISpot technology can be utilized to answer many complex questions in the journey to making scientific breakthroughs.”  – A. Austin, Global Marketing Manager, Medix Biochemica⁴

Click on the links below to read studies which offer some fascinating insight into the benefits of the ELISpot technique:

• ELISpot assays with pp65 peptides or whole HCMV antigen are reliable predictors of immune control of HCMV infection in seropositive kidney transplant recipients⁹
• ELISpot for measuring human immune responses to vaccines¹⁰
• Unique strengths of ELISPOT for T cell diagnostics¹¹

Where to source ELISpot kits, sets and pairs

Medix Biochemica is an independent, international, and industry-leading partner to the in vitro diagnostic (IVD) Industry, offering a number of ELISpot kits, sets and pairs to enable our customers to get the results they need.¹²

Medix Biochemica is a market-leading IVD raw material supplier.¹³ We produce and supply high-quality antibodies, antigens and other critical IVD raw materials that allow our customers to manufacture diagnostic tests and supporting materials.¹³ Our expertise covers IAs, clinical chemistry, molecular diagnostics and more.¹³ 

View our comprehensive raw materials portfolio or contact us to find out how we can build a partnership to consistently meet your IVD needs.

References: 

1. Darwish IA. Immunoassay methods and their applications in pharmaceutical analysis: basic methodology and recent advances. Int J Biomed Sci. 2006;2(3):217-235. 
2. Sharma A, Gautam S, Bandyopadhyay N. Enzyme immunoassays: overview. In: Batt CA, Tortorello ML, eds. Encyclopedia of Food Microbiology (Second Edition). Academic Press; 2014:680-687. doi:10.1016/B978-0-12-384730-0.00099-9.
3. Vashist SK, Luong JHT. Chapter 1 - Immunoassays: an overview. In: Handbook of Immunoassay Technologies. Academic Press; 2018:1-18. doi:10.1016/B978-0-12-811762-0.00001-3.
   3a. Dhillon RS, Kelly JD, Srikrishna D, et al. Overlooking the importance of immunoassays. Lancet Infect Dis. 2016;16(10):1109-1110. doi:10.1016/S1473-3099(16)30338-3.
4. Expert opinion. Anthony Austin, Global Marketing Manager, Medix Biochemica. 2023.
5. Alhajj M, Zubair M, Farhana A. Enzyme linked immunosorbent assay. In: StatPearls. StatPearls Publishing; 2023. Accessed August 28, 2023. http://www.ncbi.nlm.nih.gov/books/NBK555922/.
6. Difference between competitive and noncompetitive elisa. Compare the Difference Between Similar Terms. Accessed September 8, 2023. https://www.differencebetween.com/difference-between-competitive-and-noncompetitive-elisa/.
7. Elisa technique. Cleveland Clinic. Accessed August 28, 2023. https://my.clevelandclinic.org/health/articles/24990-elisa.
8. What is the difference between Elisa and Elispot. Compare the difference between similar terms. Accessed August 28, 2023. https://www.differencebetween.com/what-is-the-difference-between-elisa-and-elispot/.
9. Zavaglio F, Rivela F, Cassaniti I, et al. ELISPOT assays with pp65 peptides or whole HCMV antigen are reliable predictors of immune control of HCMV infection in seropositive kidney transplant recipients. J Med Virol. 2023;95(2):e28507. doi:10.1002/jmv.28507.
10. Slota M, Lim JB, Dang Y, et al. ELISpot for measuring human immune responses to vaccines. Expert Rev Vaccines. 2011;10(3):299-306. doi:10.1586/erv.10.169.
11. Lehmann PV, Zhang W. Unique strengths of ELISPOT for T cell diagnostics. In: Kalyuzhny AE, ed. Handbook of ELISPOT: Methods and protocols. Methods in Molecular Biology. Humana Press; 2012:3-23. doi:10.1007/978-1-61779-325-7_1.
12. Medix Biochemica. Accessed August 28, 2023. https://www.medixbiochemica.com/.
13. About us. Medix Biochemica. Accessed August 30, 2023. https://about.medixbiochemica.com/.