The ELISA test detects numerous diseases, which is why it has been widely used in medicine and more. However, the ELISA test is best known for its diagnosis of Lyme disease. What is the ELISA test? How to interpret the results? What is the price of the test?

Contents:

  1. ELISA test - what does it detect? Use of the ELISA
  2. ELISA test - what is the test?
  3. ELISA test and Lyme disease - when to perform?
  4. ELISA test - price. How much does an ELISA test cost?
  5. ELISA test - results. Can they be false positives?
  6. ELISA test - results. Can they be false negative?

ELISA test , i.e. the enzyme-linked immunosorbent assay, is a commonly used laboratory method that serves, among others, for the quantitative detection of antibodies in the blood.

Due to thisELISA testfoundapplicationin the diagnosis of bacterial diseases, such asLyme diseasewith Lyme.

The advantage of the ELISA method is its simplicity, quick execution and high sensitivity. The ELISA method is performed using special plastic plates with wells filled with e.g. Borrelia proteins (antigens) and specific monoclonal antibodies, which are designed to detect antibodies in the patient's sample.

ELISA test - what does it detect? Use of the ELISA

The high sensitivity and simplicity of the ELISA method made it applicable in the diagnosis of many diseases, but it is best known as the diagnostic method of Lyme disease.

In the diagnosis of Lyme borreliosis, the so-called indirect ELISA, which allows to detect specific IgM or IgG in the tested material. In the same way, antibodies can be detected for diagnostic purposes:

  • viral infections: herpes viruses (herpes, herpes zoster, Epstein-Barr, cytomegalovirus), HIV, hepatitis viruses (HBV and HCV)
  • protozoan infections: toxoplasmosis, giardiasis
  • autoimmune diseases: celiac disease (anti-tTG or anti-DPG antibodies), Hashimoto's disease (anti-TG and anti-TPO antibodies), inflammatory bowel diseases (anti-ASCA and anti-ANCA antibodies)
  • parasitic diseases and allergies,in which specific IgE antibodies are detected. Their increased level may indicate the presence of allergies, e.g. to pollen and food, or parasitic invasions (armed tapeworm, human roundworm)

There are also modifications to the ELISA method (so-called sandwich ELISA), which allow the detection of a tested sample of proteins other than antibodies. Then the aim of the research is to detect in the biological material:

  • fragments of viruses or bacteria, most often these are antigens specific to a given pathogen
  • serum hormones, e.g. estrogen, prolactin, TSH, fT4, cortisol
  • enzymes e.g. calprotectin, pancreatic elastase, alkaline phosphatase
  • tumor markers, e.g. CA-125
  • presence of drugs in the serum

This method is also used in the food industry, where trace amounts of allergens (e.g. milk, peanuts), GMOs or toxins in food can be detected.

ELISA test - what is this test?

The diagnosis of e.g. Lyme borreliosis using the ELISA method consists in detecting immune proteins in biological material - antibodies that arise during the response against Borrelia antigens.

Antibodies are tested in two classes, IgM and IgG. IgMs are the first antibodies to appear in the body upon contact with bacteria and to decline over time.

Their place is taken by more persistent IgG antibodies that can stay in the body for several dozen years.

In this way, the presence of bacteria in the body is indirectly tested by detecting antibodies specifically directed against Borrelia spirochetes.

Antibodies at various stages of infection may be directed against a variety of Borrelia proteins. For example, at an early stage it is the p41 protein.

In the later stages, more and more proteins start to appear, such as p21, p30, p39, p43. This is why the diagnosis of Lyme disease is extremely difficult.

The selection of bacterial proteins is extremely important for the test to have a high diagnostic value. Therefore, routinely in the diagnosis of Lyme disease, II-generation or III-generation ELISA tests should be used, in which the sets of proteins are selected so as to obtain the most reliable result.

ELISA test and Lyme disease. When to perform?

Lyme borreliosis is a bacterial disease caused by the bacteria Borrelia burgdorferi and its many varieties.

Disease diagnosis is not easy due to the complexity of the immune response against Borrelia and the imperfections of laboratory methods.

The most widely used laboratory method is the highly sensitive ELISA method which detects specific antibodies raised against bacterial proteins.

In the diagnosis of Lyme borreliosis, an indirect ELISA method is used in which the following stages are distinguished:

  • incubation of the wells on the plate with material collected from the patient, e.g. serum. If there are antibodies against Borrelia in the tested material, they bind to proteins at the bottom of the reaction well
  • then the excess serum is rinsed out with a special washing fluid (buffer) and the so-called conjugate, i.e. an antibody linked to an enzyme that binds to previously linked antibodies in the reaction wells
  • Substrate for the conjugate is added later and there is no (positive) or no color reaction (negative)
  • the last step is the reading by the reader (spectrophotometer) of the concentration of antibodies against Borrelia in the patient's serum

The ELISA method for Lyme disease is performed with blood, and in the case of neurological symptoms (neuroborreliosis), also with the cerebrospinal fluid.

The synovial fluid should not be tested as there is a high probability of false-positive results.

The basis for the diagnosis of the disease are clinical symptoms indicating it, confirmed by the results of laboratory tests.

The addition of a laboratory test result without the presence of symptoms does not authorize the diagnosis of Lyme disease, as it has been shown that antibodies to Borrelia spirochetes are also detected in he althy people.

Serological diagnosis of Lyme borreliosis begins with a semi-quantitative ELISA screening test. If the result is negative, it means that no Borrelia spirochetes have been detected or the result is false-negative.

If the result is positive or weakly positive (equivocal), confirmatory Western blot testing, which is highly specific.

This is to confirm the "truthfulness" of the results obtained with the ELISA method. During diagnostics, you cannot skip any of the above steps and, for example, perform diagnostics using the Western blot method or make a diagnosis based on a positive result only using the ELISA method.

In Poland, the diagnosis of Lyme disease very often ends at the first screening stage, which causes a misinterpretation of positive or weakly positive results.

Worth knowing

ELISA test - price. How much does an ELISA test cost?

The test costs around 60zlotys and is made right away. However, you can wait about three months for the results of the referral.

ELISA test - results. Can they be false positives?

False positive results result from the cross-reactivity of IgM antibodies with other proteins. Antibodies produced by the body may "incorrectly" recognize an antigen of other microorganisms as the Borrelia antigen.

The most common "mistakes" occur in people infected with treponema pallidum, herpes viruses and in people with systemic lupus erythematosus and other rheumatic diseases.

ELISA test - results. Can they be false negative?

False negative results may appear in the early stage of infection, then the test should be repeated after about 3-4 weeks. This is called serological window, i.e. the initial period of infection in which the body has not yet "managed" to produce specific antibodies.

Similarly, in immunocompromised people who do not make enough antibodies.

A big problem, especially in very intense infections, is the appearance of the so-called immune complexes that consist of Borrelia spirochetes antigens and specific antibodies for them.

This makes the antibodies "invisible" to commonly used methods such as ELISA, and although they are present in the tested material, they cannot be detected.

In such cases, the laboratory can use breaking down immune complexes. This treatment is designed to release the antibodies. However, this method is not widely used due to the lack of standardization in laboratories.

Worth knowing

Why isn't Lyme disease diagnosis easy?

This is due to the complexity of the immune response against Borrelia and the limitations of laboratory methods. The diversity of varieties and the emergence of new infections causing infections, especially on the European continent, does not facilitate the development of effective diagnostic methods.

More varieties have recently appeared in Europe, such as Borrelia bissetii, Borrelia valaisiana and Borrelia spielmanii. In addition, in the body, Borrelia spirochetes can turn into cysts or stay in places of the body inaccessible to the cells of the immune system.

Being thus "invisible" to the immune system. In addition, it has been shown that the tissues may contain fragments of bacteria that have strongly pro-inflammatory properties. They are believed to be responsible for the chronic symptoms of Lyme disease, despite negative results in the studylaboratory.

References

1. Witecka-Knysz E. et al. Lyme disease: why is diagnosis so difficult? Laboratory Diagnostician April 2007.2. Moore A. et al. Current Guidelines, Common Clinical Pitfalls, and Future Directions for Laboratory Diagnosis of Lyme Disease, United States. Emerg Infect Dis. 2016 Jul; 22 (7) .3. Laboratory diagnosis of tick-borne diseases. Recommendations of the Working Group: National Chamber of Laboratory Diagnosticians, National Institute of Public He alth-National Institute of Hygiene, National Consultant in the field of infectious diseases, Department of Infectious Diseases and Neuroinfections, Medical University of Białystok, Polish Society of Virology, Warsaw 2014. Hosseini S. i co: Enzyme-linked Immunosorbent Assay (ELISA) From A to Z. Springer 2022.

About the authorKarolina Karabin, MD, PhD, molecular biologist, laboratory diagnostician, Cambridge Diagnostics Polska A biologist by profession, specializing in microbiology, and a laboratory diagnostician with over 10 years of experience in laboratory work. A graduate of the College of Molecular Medicine and a member of the Polish Society of Human Genetics. Head of research grants at the Laboratory of Molecular Diagnostics at the Department of Hematology, Oncology and Internal Diseases of the Medical University of Warsaw. She defended the title of doctor of medical sciences in the field of medical biology at the 1st Faculty of Medicine of the Medical University of Warsaw. Author of many scientific and popular science works in the field of laboratory diagnostics, molecular biology and nutrition. On a daily basis, as a specialist in the field of laboratory diagnostics, he runs the content department at Cambridge Diagnostics Polska and cooperates with a team of nutritionists at the CD Dietary Clinic. He shares his practical knowledge on diagnostics and diet therapy of diseases with specialists at conferences, training sessions, and in magazines and websites. She is particularly interested in the influence of modern lifestyle on molecular processes in the body.

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Research