Serological Reactions: Precipitation Reactions

Serological Reactions: Precipitation Reactions

Precipitation Reactions (PRs)

• Reactions are similar to agglutination reactions (ARS) in terms of performance conditions.

Differences Between Precipitation and Agglutination Reactions

Types of Precipitation Reactions

In Liquid Phase

• Ring precipitation reaction
• PR with nephelometric account

In Solid Phase

• PR performed in gel (double immunodiffusion)
• Mancini radial immunodiffusion
• Immunoelectrophoresis

Ring Precipitation Reaction

I. Goals of the Reaction

• Seroindication:
  • Detection of the causative agent of Siberian ulcer (Ascoli's reaction).
  • Antigen: Somatic O-antigen of Bacillus anthracis.
• Forensic medicine (criminalistics):
  • Detection of the species of blood or other biological fluids at crime scenes.
  • Antigen: Protein molecules of blood plasma and other biological fluids.

II. Where the Reaction is Performed

• In narrow precipitation test tubes.

III. Components of the Reaction

• Precipitating serum (introduced into the test tube).
• Antigen solution (carefully layered on top of the serum along the test tube wall).
• Important: Components should not be mixed.

Antigen Types

• Thermal extract from animal fur (Ascoli's reaction):
  • Boil animal fur in HCl or H2SO4 for an hour (anthrax pathogens destroyed, thermostable somatic O-antigens remain).
  • Filtration to obtain thermal extract.
• Extract from bloodstain (criminalistics):
  • Blood stains extracted with sterile saline solution at +4…+6 °C for 18-72 hours.

IV. Accounting of the Reaction

• Visual detection of a turbidity ring (ring of precipitation).

V. Interpretation of Results

• Positive reaction: Presence of antigen in study material → precipitation ring visible at the border of precipitating serum and antigen solution.
• Negative reaction: Absence of antigen in study material → no precipitation ring visible.

Precipitation Reaction with Nephelometric Account

I. Goals of the Reaction

• Detection of the quantity of antigens in research material (proteins, enzymes, hormones, vitamins, oncomarkers, microbial antigens, etc.).
• Detection of the quantity of antibodies in biological fluids (quantitative detection of IgA, M, G, E in blood serum, autoaggressive antibodies, antibodies against microorganisms, etc.).

II. Where the Reaction is Performed

• In test tubes.

III. Components of the Reaction

• Diagnostic precipitating serum (known antibodies to detect unknown antigens).
• Solution of the unknown antigen.
• OR
• Diagnosticum (known antigen to detect unknown antibodies).
• Sample containing the unknown antibodies (e.g., blood plasma).

IV. Accounting of the Reaction

• Using a nephelometer (measures the degree of turbidity of the solution).
• The degree of turbidity is directly proportional to the amount of antigens or antibodies in the test sample.
• Greater amount of detected antigen/antibody → more turbid solution.
• Turbidity depends on the quantity of antigen-antibody complexes and is determined by the optical density of the solution.

V. Interpretation of the Results

• The amount of antigens or antibodies is determined using a calibration line.
• Calibration line: A graph of the dependence of the optical density of the obtained solution on the number of antigens or antibodies in the sample.
• The calibration line is constructed before the reaction using solutions containing known amounts of antigens or antibodies (standards).

Precipitation Reaction Performed in Gel (Double Immunodiffusion)

Goal of the Reaction

• Seroidentification (determination of the toxigenicity of pure culture of Corinebacterium diphtheriae).

II. Where the Reaction is Performed

• In Petri dishes.

III. Components of the Reaction

• Diagnostic precipitating antitoxic serum (known antibodies against exotoxins of Corinebacterium diphtheriae).
• Pure culture of Corinebacterium diphtheriae (unknown antigen).

Procedure

• A strip of filter paper soaked with anti-diphtheria diagnostic antitoxic serum is placed in the center of the Petri dish with the nutrient medium.
• Pure cultures of Corinebacterium are sown at the edges of the filter paper.

IV. Account of the Reaction

• Visual detection of turbid arcs of precipitation.

V. Interpretation of the Results

• Positive reaction: If a pure culture of corynebacteria is toxigenic (produces diphtheria exotoxin), precipitation arcs form between the filter paper and the bacterial culture.
• Negative reaction: If a pure culture of corynebacteria is non-toxigenic (non-pathogenic), there will be no arcs of precipitation.

Mancini Radial Immunodiffusion

I. Goal of the Reaction

• Detection of the quantity of IgA, IgM, IgG, and IgE in blood serum of a patient.

II. Where the Reaction is Performed

• On a glass plate.

III. Components of the Reaction

• A layer of molten agar mixed with diagnostic serum (containing antibodies against human immunoglobulins of the corresponding class) is applied to the surface of a glass plate.
• Wells of the same diameter are cut in agar.
• Standards (standard blood serum containing immunoglobulin solutions in known concentrations) are introduced into the first four wells cut out in agar.

Additional Information

• Agar-agar is a mixture of agarose and agaropectin polysaccharides obtained by extraction from red algae (Phyllophora, Gracilaria, Gelidium, Ceramium).

Procedure Continued

• The investigated blood serum of patients is added to the remaining wells.
• Incubation (studying immunoglobulins diffuse into the agar, forming precipitation rings in the zone of equivalent ratios with standard antibodies).
• The diameter of the precipitation ring is larger if more of the corresponding immunoglobulins are contained in the patient's blood serum.

Analysis

• According to the standards, a calibration line is made (a graph in which a specific level of immunoglobulin in the blood serum corresponds to a certain diameter of the precipitation ring).
• By measuring the diameters of the rings around the wells with the studied blood serum of patients, the levels of immunoglobulins of a certain class in the patient's blood serum are determined using the calibration line.

Immunoelectrophoresis

I. Goals of the Reaction

• Detection of the blood plasma protein composition (immunoelectrophoregram of the blood plasma).
  • Antigen: The specific protein of the patient's blood plasma.
• Detection of the presence of impurities in protein preparations (e.g., in diagnostic or therapeutic serums).
  • Antigen: Protein molecules contained in the preparation (including impurities).

II. Where the Reaction is Performed

• On a glass plate.

III. Components of the Reaction

• Molten agar is applied to a glass plate.
• Agar curdles.
• Wells are cut in the agar gel.
• Antigen is placed into the wells (patient's blood plasma or pharmacological preparation).
• Electrophoresis is performed → antigens are separated and ranged according to their molecular weight.

Procedure after Electrophoresis

• A groove is cut in agar parallel to the movement of the antigen.
• Diagnostic serum (containing antibodies against plasma proteins or proteins contained in pharmacological preparation) is added into the groove.
• Incubation.
• Antigens and antibodies diffuse towards each other, and arcuate lines of precipitation are formed.
• In the zones of correspondence between antigen and antibody, each antigen has its own precipitation arc.