Agglutination Reactions

Agglutination Reactions

  • Agglutinins are the antibodies capable of clumping antigen-containing particles (microbial cells, erythrocytes, etc.) with the formation of visible agglomerates.
  • The addition of specific immune serum to a suspension of microbial cells leads to their agglomeration into the large visible complex, looking like flakes or granules. This phenomenon is determined as microbial agglutination.
  • Overall, the agglutination reaction results from the interaction of erythrocytes, microbial and other cells with the specific immune serum. The antigen in agglutination reaction must be presented in corpuscular (particle) form.
  • In 1896 F. Widal indicated that the serum of patients with enteric typhoid fever was able to cause the specific agglutination of salmonellas – the causative agents of this disease.
  • Further, it was found that in great sets of infectious diseases specific antibodies (or agglutinins) begin to rise, and their growth becomes the specific hallmark of the disease.
  • A specific antibody (agglutinin) and the corpuscular antigen (agglutinogen) are the immunochemical reagents for the agglutination reaction. Their specific binding occurs in saline-containing medium and requires the definite quantitative ratio of the reagents.
  • The mechanism and external manifestations of the agglutination test primarily depend on the corpuscular nature of an antigen with a large number of epitopes (multivalent structure).
  • On the other hand, agglutinating antibodies must be bivalent or better polyvalent with two or more active sites. In that way agglutination of at least two microbial cells is performed via the bridge of an antibody molecule, where two active sites of the same antibody are bound to different bacterial cells. Spatial spread of agglutinate lattice results in visible cell agglomeration.
  • Meanwhile, the mechanism of antigenic precipitation is also very similar. Both reactions are accompanied by the production of visible aggregates.
  • The agglutination reaction is characterized by high specificity. Nevertheless, the antigenic structure of bacteria for agglutination is extremely variable. The same bacterial cell can exhibit group, species, and type-specific antigens. Type-specific antigens are also known as serovar-specific. Group or species-specific antigens show cross-reactivity between many related bacteria.
  • The variation of antigens in the microbial cells is a regular process; it reflects intraspecies and interspecies variability or similarity of bacteria.
  • Thus, upon the immunization of an animal with the cells of one microbial species, agglutinins can appear not only to the species of immunization but also to some other related bacterial species that possess the common group-specific antigens.
  • That is why group agglutination is observed sometimes, resulting in non-specific clumping of closely related microbial cells bearing the same group antigens.
  • For isolation of specific agglutinins in sera of animals immunized by a complex of bacterial cell antigens, the method of adsorption of cross-reactive agglutinins on the related bacterial cells is employed. Agglutinating sera obtained by this technique (proposed by A. Castellani in 1902) is called adsorbed monospecific serum.
  • Monospecific antibody reagents make it possible to determine more precisely the species and serovar specificity of the causative agents of various diseases (e.g., salmonellosis, shigellosis, etc.)
  • Thus, agglutinating sera are used as non-adsorbed or adsorbed products that are group, species or serovar specific.
  • Motile bacterial cells carry somatic O- and flagellar H-antigens. Upon animal immunization, both O-agglutinins and H-agglutinins are produced. The bacteria, covered with capsular K- or Vi-antigen, poorly react with O-antisera, but easily agglutinate with anti-Vi-sera.
  • The manifestations of agglutination reaction depend on the ratio of antigen/antibody concentrations, the density of microbial suspension, pH and ionic strength of the medium, the temperature of incubation, the quality of reagent mixing, etc.
  • Microbial agglutination test has many practical applications being used for serological diagnosis of various infectious disorders.
  • In most of the clinical cases, the serological diagnosis means the laboratory diagnosis of the infectious diseases made by determination of specific antibodies directed against their causative agents.
  • In some other cases, the serological diagnosis of the disease is made by the detection of specific antigens in the patient’s serum.
  • Determination of specific antibodies is essential for the diagnosis of enteric typhoid fever and paratyphoidal diseases (Widal’s agglutination test), brucellosis (Wright’s reaction), as well as for leptospirosis, tularemia, and other diseases. In all these cases the specific antibodies or agglutinins are determined in patients’ sera by means of known microbial cells containing specific microbial antigens called diagnosticums.
  • In turn, the agglutination reaction is also used for the identification of unknown microbial cultures isolated from patients and sick animals. It is performed by means of agglutinating sera that contain antibodies of known specificity. This test determines microbial serological properties, resulting in serological identification of microbial species.
  • To get the fast preliminary results of serological reactions, rapid agglutination tests are commonly used.
  • They are performed as tentative slide agglutination tests with concentrated specific immune serum.
  • These reactions can be applied either for microbial identification (for instance, to identify cholera vibrios in patients with cholera) or for the determination of antimicrobial antibodies (Huddleson’s test in brucellosis, reactions for tularemia, etc.).
  • In case of positive results of slide agglutination test, the extended tube agglutination test with serial dilutions of immune serum is elaborated.

Passive Hemagglutination and Other Indirect Agglutination Techniques

  • Besides direct agglutination, indirect agglutination is employed for laboratory diagnosis of infectious diseases. For instance, indirect agglutination is used for detection of antibodies produced against the antigens devoid of corpuscular structure (e.g., soluble proteins).
  • The most effective is the indirect (or passive) hemagglutination test. For this reaction, the antigen is usually adsorbed on the surface of chemically fixed xenogenic (e.g., cattle) erythrocytes (erythrocyte antigenic diagnosticum).
  • Hemagglutination is performed with erythrocyte diagnosticum and patient’s sera. As an example, Vi-antibodies present in sera of carriers of enteric fever salmonellae is determined by hemagglutination with Vi-Ag erythrocyte diagnosticum.
  • Hemagglutination reaction is quite sensitive and allows the detection of antibodies in greater dilutions.
  • These tests are usually carried out in the wells of plastic agglutination plates, where the agglutination patterns of the cells on the bottom of the well are readily observed. This technique provides more sensitive detection than macroscopical clumping.
  • In medical practice, the reaction of iso hemagglutination is used for determination of human blood groups in case of blood transfusion.
  • Modern variations of indirect agglutination tests include latex-agglutination, acrylic beads agglutination, gold nanoparticles agglutination followed by the microscopical count of the reactions and some other tests. Quantification of initial levels of agglutination can be achieved by laser nephelometry.
  • These advanced reactions develop markedly higher sensitivity and specificity than conventional ones.

Agglutination Reactions