ROLE OF SEROLOGY IN INFECTIOUS DISEASES
Dr. Nigam Prakash Narain M.D., D.C.H., Ph.D., M.R.C.P. (UK), F.I.A.P*
Associate Professor of Pediatrics, Patna Medical College *
Role of serology in diagnosis of Infectious Diseases has been a landmark in the history of Modern Medicine and its importance will remain the same in spite of all the advancements as they are simple, feasible, economical and available even in remote places.
General features of Antigen-antibody reactions
The antigen antibody reactions have following features:
  1. The reaction is mostly specific as an antigen combines with homologous antibody though it is not absolute and there may be cross reactions.
  2. The combination occurs at surface and therefore surface antigens are immunologically relevant.
  3. The combination is firm but reversible. The firmness of the union is dependent on affinity and avidity of the reaction. Affinity refers to the intensity of the attraction between antibody and antigen whereas avidity is the strength of the bond of complexes.
  4. Antigen and antibody can combine in different proportions.

Two important parameters of serological tests are sensitivity and specificity. Sensitivity refers to the ability of the test to detect even very minute quantities of the antigen or antibody whereas specificity refers to the ability of the test to detect the homologous antigen and antibody.
Serological Reactions in Clinical Practice
Precipitation Reaction

When a soluble antigen combines with its homologous antibody in the presence of electrolytes (NaCl) at a suitable temperature and pH, antigen-antibody complexes form an insoluble precipitate which when sedimented is called precipitation and once not sedimented is called flocculation. The reaction can occur in liquid media or in gels as Agar, Agaron and Polyacrylamide. Best precipitation occurs in optimal concentrations of antigen and antibody. There may be prozone phenomenon in presence of antibody access and the best precipitation occurs in the middle tubes which is known as Zone of Equivalence. Zoning is important to strike a balance.

Mechanism of Precipitation

Lattice Hypothesis describes a multivalent antigen combining with bivalent antibody in varying proportions. Lattice is formed when alternate molecules of antigen and antibody are combined.

Application of Precipitation reaction

Precipitation test may be used either as Quantitative or Qualitative test. It is very sensitive as even 1 Ig of protein is detected by precipitation and so is being used successfully for identification of blood and animal stains or food adulterations.
The following types of precipitation reactions are being used:

Ring test
Ring test is done by layering of antigen on antibody in a narrow tube and is used for Streptococcal grouping.

Slide test
It is done by a drop each of antigen and antibody mixed by shaking which is seen as flocculation as in VDRL test for Syphilis.

Tube test
Serial dilutions of toxins/toxoids are added to tubes containing fixed amount of antitoxin as in Kahn test for Syphilis.
Immunodiffusion
It is done by allowing precipitation in a gel. There are various types of this reaction:
  • Single diffusion in one dimension
  • Double diffusion in one dimension
  • Single diffusion in two dimensions
  • Double diffusion in two dimensions
  • Immunoelectrophoresis
Immunoelectrodiffusion
Development of precipitation can be speeded up by electrically driving the antigen and antibody types:
  • One dimension Double electrophoresis (Counter immune electrophoresis)
  • One dimension single electrophoresis (Rocket Electrophoresis)

Agglutination reaction
When a particulate antigen is mixed with its antibody in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. In this also Zone phenomenon holds true.

Applications
Slide agglutination-When a drop of antigen is added to a smooth uniform suspension of a particulate antigen in a drop of saline on a slide visible to naked eye it is slide agglutination as in blood grouping and cross matching.

Tube agglutination
This is a standard method for measurement of antibody since by adding a fixed volume of particulate antigen to same volume of serial dilution of antibody as in Widal test, test for Brucellosis, Weil-Felix reaction for Typhus, Paul Bunnell test.
  • Anti Globulin test (Coomb's Direct and Indirect test)
  • Passive agglutination test

This is done by precipitation by agglutination by attaching soluble antigen to surface of carrier particles as RBC (Human or sheep), Latex or Bentonite examples being Rose-Waaler test. They are very sensitive but less specific.
Complement Fixation Test
In this reaction, complement is absorbed during antigen-antibody reaction. In the presence of appropriate antibody complement either lyses RBC or kills, immobilizes or phagocytosis bacteria. Examples are Wassermann reaction, Immunoadherence for Vibrio cholerae, Treponema pallidum Immobilisation, etc.
Neutralization
Examples are:
  • Virus Neutralization in animal, egg or tissue cultures
  • Toxin Neutralization as in Schick test for Diphtheria, ASO titre estimation
Opsonisation
Opsonins are labile substances present in fresh normal serum which facilitate phagocytosis. Similarly heat stable factors are called Bacteriotropins.
Immunofluorescence
Fluorescence is the property of an absorbing light rays of a particular wavelength and emitting rays with a different wavelength. Fluorescent dyes are shown up brightly as they convert ultra violet to visible light-fluorescent dyes (Fluorescein Isetheocyanate and lissamine rhodamine can be conjugated with antibodies, antigens or complements). Examples are - Rabies Virus detection in mouse brain, Treponema detection in Syphilis.
Radioimmunoassay
This was a great advancement in serology as antigen or antibody were labeled to either radioisotopes or enzymes and they could quantify the complexes appropriately.

Examples are:
  • Hormone assays
  • Drug assays
  • Tumour markers
  • IgE/Viral antigens
  • Complement binding assays
Enzyme immunoassay
Of late, this has been most widely used serological tests as this is sensitive, simple, safe, versatile and economical out of all other tests.

Types:
  1. Homogenous
  2. Heterogenous (ELISA) - 2 steps - Separation of Bound and Free fractions either by Centrifugation or absorption to solid surface. It is so named because of use of absorbent material specific o antigen or antibody. It uses wells which are colour guided.
Types of ELISA
  • Non-competitive or Sandwich ELISA as done for Anti-HIV detection
  • Capture ELISA
  • Immunometric ELISA
  • Cylinder or Cassette ELISA (for HIV-1 and HIV-2)
Immunoelectroblot techniques
It combines the sensitivity of ELISA with better specificity.
Steps:
  • Separation of Ligand-antigen complexes by gel electrophoresis
  • Blotting of electrophoresed ligand fraction on nitrocellulose membranes
  • Enzyme or Radioimmunoassay
  • Example-western Blot test for HIV
Immunochromatographic tests
They are quite popular as they are simple, economical and reliable as for example:
  • HBsAg detection

Other newer tests..
  • Immunoelectronmicroscopy
  • Immunoferritins
  • Immunoenzyme tests
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