Serological methods

Serological methods are a set of screening techniques that are based on the reaction between antigen and antibody. One component of the reaction is always known, the other we investigate. Serology is used mainly in microbiology to diagnose pathogens. It is usually faster than cultivation and allows the capture of even poorly culturable agents (eg viruses ). According to the specific diagnostic procedure, serology can be classified as a direct and indirect method of detecting a pathogenic agent.

Guiding principle
Blood serum is most often used for serology, but it is also possible to use other biological materials (sputum, urine, saliva, cerebrospinal fluid ). There are three main principles:


 * 1) antigen detection,
 * 2) antibody detection,
 * 3) monitoring of cellular immunity.

Antigen detection
We detect antigens using a set of known antibodies. Both polyclonal antibodies, which bind to several epitopes of the putative antigen in our sample, and monoclonal antibodies , which allow a more accurate determination of the pathogen type, are used. The presence of antigenic molecules indicates the presence of an infectious agent.

Demonstration of antibodies
We detect antibodies with a known antigen. The response to the presence of antigenic molecules is usually a response of the immune system. Due to the different dynamics of antibody production, their detection does not necessarily mean an acute disease. In the early stages of infection, IgM antibodies (sometimes IgA ) appear, later a class of IgG that can persist after the disease. For testing, it is necessary to make more samples and evaluate them over time. Examination is problematic in immunodeficient patients.

Indirect agglutination
Indirect agglutination helps to determine the type of antigen isolated from the patient using known serum antibodies. In Escherichia coli, this reaction makes it possible to distinguish individual strains. Identification of serovar O157: H7 is crucial in severe variants of diarrheal diseases or hemolytic uraemic syndrome.

Direct agglutination
Direct agglutination is a method in which antibodies in a patient's serum are determined by adding a suspension of known bacteria. Antigens are found on the surface of bacteria (flagella, liposaccharides). Binding of the antibody to the O-antigen, the polysaccharide chain of the liposaccharide ( endotoxin ), causes a compact precipitate to form. Flagellar H-antigens form a clot resembling fluffy clouds or misty glass. Direct agglutination is used to detect Salmonella (so-called Widal reaction) in typhoid fever, Brucella bacteria (Wright reaction) or rickettsia (Weil-Felix reaction).

Direct hemagglutination
In direct hemagglutination, the surface molecules of the erythrocytes are antigenic molecules. The positive reaction, which manifests itself in the agglutination of red blood cells, is caused by the binding of antibodies produced by foreign organisms to these structures. An example is the Paul-Bunnel reaction to detect infectious mononucleosis.

HIT
HIT or hemagglutination inhibition is used in influenza viruses and other hemagglutinin -bearing diseases. Virus-bound antibodies inhibit hemagglutination.

Agglutination on carriers
Antigens or antibodies are placed on a support in this method. The carrier may be erythrocytes or latex media. In the so-called passive agglutination, the antigens ( endotoxin , Salmonella Vi factor ) are placed on the erythrocytes and aggregate after the addition of serum. We can also passively prove Trepone pallidum ( Dubos-Hiddlebrok reaction ). Latex tests used in the diagnosis of rheumatoid arthritis have gamma globulin as the antigen. Furthermore, latex carriers are used for direct detection of streptococci, meningococci , pneumococci.

Precipitation
Upon precipitation, an insoluble complex is formed by the reaction of the antibody with a soluble (colloidal) antigen. A suitable medium is an aqueous solution or an agar or agarose gel.

Precipitation in solution

 * Ring reaction: in a test tube, the antibody solution is overlaid with the antigen solution. The precipitate appears in the form of a ring at the layer interface.
 * Flocculation reaction: by mixing the antigen and antibody solution, the complex precipitates in the form of flakes . This response is the basis of VDRL syphilis screening.

Gel precipitation
Antigen and antibody solutions are added to the wells cut in the gel. The diffusion of solutions through the gel subsequently creates a certain concentration in the zones, the so-called precipitation line. The lines form at the antigen-antibody meeting site and are visible as precipitates. An example of this reaction is the detection of a toxin in C. diphtherieae.

Double arrangement according to Ouchterlony
A central well is cut into the agar, where the patient's serum is dripped. Several antigen wells are then cut in the area. Precipitation lines corresponding to the specificity of the antigen and antibodies are formed between the central well and the wells with antigens after diffusion:


 * the lines merge smoothly (antigens are identical),
 * the lines partially merge, one of them continues (antigens are related),
 * the lines intersect independently (unrelated antigens).

Possible results of gel precipitation

Complement fixation
Complement fixation reactions are based on complement properties. Free complement lyses erythrocytes and is capable of binding to the antigen-antibody (AP) complex. Once complement complements the AP complex, it can no longer disrupt erythrocytes. In a patient's blood serum sample, complement must be inactivated by temperature. Instead, guinea pig serum complement is supplied. The Bordet-Wasserman reaction is a less widely used method for diagnosing syphilis. The antigen is cardiolipin, which forms a complex upon contact with Treponema pallidum serum antibodies. With a positive reactionerythrocytes do not show lysis (complement is bound to the AP complex). Negative reactions are manifested by cell lysis (antibodies against T. pallidum are not present, cardiolipin has nothing to form a complex that could bind to complement). Today, for example, evidence of brucellosis, listeriosis , Mycoplasma pneumoniae and various viral infections work on this principle.

Neutralization
In neutralization reactions, the properties of antigens are inhibited. In the case of viral infections (so-called VNT = virus neutralization tests), the presence of antibodies is manifested, for example, by their inability to infect the experimental animal. The neutralization reactions therefore include the above-mentioned HIT. Another use is in the detection of antistreptolysin O, antibodies against streptolysin O in streptococci type A, C, G (eg Streptococcus pyogenes). Add a known concentration of streptolysin to the patient's serum. If antibodies are present, its effects are neutralized and no added erythrocytes are lysed. Concentrations of added streptolysin less than 200 IU are the norm, concentrations greater than 200 IU are after a recent infection or its consequences (eg glomerulonephritis ).

Methods of labeled antibodies
These methods use antibodies or antigens labeled with various indicators (eg fluorescent dyes, enzymes). We can include here:


 * ELISA ,
 * RIA ,
 * Western blotting ,
 * immunofluorescence.

related articles

 * Cultivation

Reference

 * 1) JULÁK, Jaroslav. Practical exercises and seminars in medical microbiology. 2nd edition. Prague: Karolinum, 2009. 113 pp. 37.  ISBN 978-80-246-1141-9.

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