Antibodies Detection Tests for Disease Diagnosis

Antibodies are glycoproteins produced by our body in response to invading organisms or toxins (antigens).

When a pathogen (bacteria, virus, protozoa, or helminth) enters our body and anyhow manages to reach our bloodstream, antigen-presenting cells (such as macrophage, dendritic Cells or B Cells) circulating in the blood stream captures those pathogens and destroy, or present it to appropriate T Cells.  Receptors present in the Naïve B cells recognizes the surface proteins of the pathogen molecule. Antigens/other signals (such as MHC-II-peptide:TCR interaction, CD40-CD40L interaction) activates these naïve B cells. This results in the proliferation of antigen-specific cells, clonal expansion, and differentiation into effectors cells that actively secrete antibodies

All these things leads to activation of naïve B cells and convert it to antibody producing cells (plasma cells). After the plasma cells start production of antibodies, the concentration of the antibodies in the blood begin to raise slowly as initially formed Antibodies are consumed in antigen capture (formation of Ag-Ab complex) and neutralization.  As the time passes on or as the disease progress, the concentration of antigen in the blood stream starts to decline and reaches to negligible amount whereas the concentration of Antibodies increases and reaches to detectable level. Seroconversion is the period between the infection and appearance of detectable antibodies in the serum and the duration of which differs among infections. 

Presence of Antibodies in Blood ≠ Infection

In any systemic infections, the appearance of antigen (Ag) in the blood always precedes the appearance of antibodies so for the diagnosis of any infections initially antigen detection tests or culture (when available) are used and serological tests are used later. If the pathogen does not reach the bloodstream or fails to mount sufficient humoral response, antibody detection tests may not be applicable to detect such infections. For example, antibody detection tests are not used to detect local or superficial skin infections (e.g. dermatophytoses), cholera (caused electrolyte abnormalities due to cholera toxin), Giardiasis, etc.

Presence of the antibodies against a particular infection (or pathogen of interest) should not be interpreted as presence of infection at that point of time, because antibodies may be present in the serum of individuals due to any of the following conditions:

1. Vaccination
2. Previous infection or sub-clinical infection (which is already subsided)
3. Infection with another organism having antigenic similarity with the pathogen of interest
4. Passively acquired from mother: If the person is newborn, he/she might have got those antibodies from his/her mother through the placenta or breast milk (All newborns from mother who are antibody positive for that particular disease/pathogen will have passively transferred maternal IgG). In such cases, titer can be high but may not indicate infection in the baby.

Following approaches should be used during interpretation of the serological tests:

1. Four-fold rise in antibody titre between acute and convalescent-phase.
2. Detection of IgM antibody against the pathogen of interest (indicates recent infection).
3. Antibody titre should be more than the prevailing titer (cut-off titre) in the general population.

Currently, we have various tests to detect the presence of antibodies in the body fluids of the patient (serum, CSF), some of the commonly used test methods are agglutination test (latex, hemagglutination test), neutralization test, complement fixation test, immunochromatographic tests (ICT formats; Dipstick, cassette etc), Radio-Immuno Assay (RIA), Immunofluorescence Assay (DFA/IFA), ELISA (various types).

The choice of the test methods for a particular pathogen of interest differs based on the nature of the organism, cost of the test procedure and facility available in the diagnostic laboratories.

Following infections can be diagnosed serologically:

1. Dengue Fever

• Detection of anti-dengue antibodies (IgM/IgG or both) is one of the commonly used approach for the diagnosis of dengue virus infection. IgM antibodies are detectable in 99% of patients by day 10 after onset of illness.
  • IgM detection: Diagnosis of primary disease and in distinguishing dengue from other flavivirus infections.
  • IgG Detection is useful in diagnosing secondary disease.  This test is complicated by cross-reactivity of IgG antibodies to heterologous flavivirus antigens.
• A variety of methods are available for the detection of anti-dengue antibodies which are IgM or IgG ELISA, Hemagglutination inhibition test, and Rapid Diagnostic kits etc.

2.Hepatitis B Infection

The diagnosis of Hepatitis B infection depends on finding the presence of hepatitis B virus (HBV)-specific antigens (HBsAg, HBeAg) or antibodies (anti-HBc, anti-HBs, IgM anti-HBc). The presence/Absence of a particular or combination of “markers” will help to find out different phases of HBV infection and to determine whether a patient has acute or chronic HBV infection or is immune to HBV infection (due to prior infection or vaccination).

3.HIV/AIDS

Most people will develop detectable antibodies within 2 to 8 weeks (the average is 25 days) of HIV infection. Some individuals take longer to develop detectable antibodies. A person should consider a follow-up test more than three months after their last potential exposure to HIV.
Note: serological testing is not sufficient to identify Neonatal HIV infection (as they might have passively acquired antibodies from mother). p24 Antigen testing or virological testing should be done on such cases.

• Tests:
  • RDTs (e.g.Tri-Dot Assay for HIV and HIV-determine Assay),
  • ELISA and
  • Western blot

4. Post Streptococcal Infection with Streptococcus Pyogenes

• Test Name: Antistreptolysin O (ASO) titer. ASO titre is requested for a patient has symptoms of post-streptococcal complications such as rheumatic fever and glomerulonephritis.
• Antigen used: polystyrene latex particles coated with Streptolysin O
• Antibody detected: Qualitative and semi-quantitative determination of anti-streptolysin-O antibodies (ASO) in serum. ASO titre greater than 200 IU/ml is indicative of disease by epidemiological and clinical studies

5. Syphilis

• Presumptive diagnosis of syphilis is made by testing blood samples (CSF in case of neurosyphilis) for the presence of antibodies against Treponema pallidum or lipoidal materials.
• Test:
  • Nontreponemal test (i.e., Venereal Disease Research Laboratory [VDRL] or Rapid Plasma Reagin [RPR])
  • Treponemal test (i.e., fluorescent treponemal antibody absorbed [FTA-ABS] tests, Treponema pallidum passive particle agglutination [TP-PA] assay, or Treponema pallidum hemagglutination assay (TPHA).
• Antigen used: Based on the tests method in use (Treponemal or Non-treponemal test), the antigen used will vary. E.g. RPR antigen contains cardiolipin, lecithin, cholesterol, 10% choline chloride, EDTA, charcoal etc in buffer whereas TPHA test antigen is erythrocytes (chicken or avian) sensitized with sonicated T. pallidum, Nichols strain.

6. Typhoid Fever

• Test Name: Widal test: It is widely used diagnostic test for the diagnosis of Typhoid fever in developing countries. Currently it is being criticized/abandoned due to lack of specificity). Widal test is positive after the tenth day of the disease and may be false positive if an individual previously received a TAB vaccine.
• Antigen used:  killed colored suspension of S. enterica serotype Typhi O antigen, S. entericaserotype Typhi H antigen and S. enterica serotype Paratyphi AH antigen and S. enterica serotype Paratyphi BH antigen
• Antibodies detected: Widal test measures agglutinating antibody levels against O, H, AH and BH antigens.

7. Visceral Leishmaniasis (Kala-azar)

• Test Name: rK39 test
• Antigen used: k-39 (K39 represents 39 amino acid repeats encoded by a kinesin-like gene)
• Antibody detected:  Detection of the presence of anti-rK-39 antibody (IgM and IgG) in human

8. Hepatitis C infections

The hepatitis C antibodies are detected serologically for presumptive disease diagnosis. HCV antibody testing detects the antibody after an individual is infected with the Hepatitis C virus. This does not mean that the person is recently infected because antibodies against HCV remain in a person for a lifetime, even when there is no active HCV. So, a confirmatory RNA test is required if the serological result shows reactive.

9. Infectious Mononucleosis

It is the clinical manifestation of primary Epstein-Barr virus (EBV) infection in young children. Two serological tests, heterophile antibody testing, and specific antibody testing, are performed to detect EBV serologically.
Various agglutination card (monospot) tests help to measure heterophile antibodies. 50% of patients under five years of age and 80-90% of adolescents and adults with infectious mononucleosis only carry the heterophile antibodies. Likewise, the false positive and negative results rate is high, so CDC does not recommend it.
The antibodies are present only for three months of infection, so IgM to the EBV viral capsid (VCA) means primary infection. IgG against VCA develops during early infection and persists for life. IgG against EBV nuclear antigen develops later and persists for life. So, the presence of IgG against EBV must be correlated with other diagnosis similar in symptoms.

References

1. Delforge M. L. (2011). De l’utilité ou non de la sérologie infectieuse: morceaux choisis [On the usefulness of serology testing in infectious diseases: selected topics]. Revue medicale de Bruxelles, 32(4), 285–288.
2. Feng, Z., Li, Z., Sui, B., Xu, G., & Xia, T. (2005). Serological diagnosis of infectious mononucleosis by chemiluminescent immunoassay using capsid antigen p18 of Epstein-Barr virus. Clinica chimica acta; international journal of clinical chemistry, 354(1-2), 77–82. https://doi.org/10.1016/j.cccn.2004.11.010