Antigen-Antibody reactions: Agglutination and types

 General feature of antigen antibody reactions
  1. The reaction is specific; an antigen combines only with its homologous antibody and vice versa. The specificity however is not absolute and cross reactions may occur due to antigenic similarity or relatedness.
  2. Entire molecules react and not fragment.
  3. There is no denaturation of the antigen or the antibody during the reaction.
  4. The combination occurs at the surface, therefore it is the surface antigens that are immunologically relevant.
  5. The combination is firm and irreversible. The firmness of the union is influenced by the affinity and avidity of the reaction.
    1. Affinity refers to the intensity of attraction between the antigen and antibody molecules. It is a function of the closeness of fit between an epitope and the antigen combining region of its antibody.
    2. Avidity is the strength of the bond after the formation of the antigen antibody complexes. It reflects the overall combining property of the various antibody molecules in an antiserum, possessing different affinity constants with the multiple epitopes of the antigen.
  6. Antigens and antibodies can combine in varying proportions, unlike chemicals with fixed valencies. Both antigens and antibody are multivalent, antibodies are generally bivalent, though IgM molecules may have five or ten combining sites. Antigens may have valencies up to hundreds.
Prozone phenomenon

At high antibody concentrations, the number of antibody binding sites may greatly exceed the number of epitopes present in the antigens.

Prozone phenomenon

As a result, most antibodies bind antigen only univalently instead of multivalently. Antibodies that bind univalently can not crosslink one antigen to another. Prozone effects are readily diagnosed by performing the assay at a variety of antibody ( or antigen) concentration. As one dilutes to an optimum antibody concentration, one sees higher levels of agglutination. When using polyclonal antibodies incomplete antibodies also causes prozone effect.

Agglutination reactions
The interaction between antibody and a particulate antigen results  in visible clumping called agglutination. Antibodies that produce such reactions are called agglutinins. Better agglutination takes place with IgM antibody than with IgG antibodies. Excess of an antibody also inhibits agglutination reaction; this inhibition is called prozone phenomenon.
  1. Agglutination is more sensitive than precipitation for the detection of antibodies.
  2. Agglutination occurs optimally when antigens and antibodies react in equivalent proportions.
The prozone phenomenon may be seen when either an antibody or an antigen is in excess. Incomplete or monovalent antibodies do not cause agglutination, though they combine with the antigen. They may act as blocking antibodies, inhibiting agglutination by the complete antibody added subsequently.

Types of agglutination

  1. Slide agglutination: Serotyping.
  2. Tube agglutination: e.g. Widal test.
  3.  Indirect (passive agglutination): where soluble antigens are coated on vehicle particle e.g. latex particle, RBCs.

Slide agglutination.

Widal Test: Sample showing H positive in screening test
Widal Test: Sample showing H positive in screening test
  • When a drop of the appropriate antiserum is added to a smooth, uniform suspension of a particulate antigen in a drop of saline on a slide or a tile, agglutination takes place.
  • A positive result is indicated by the clumping together of the particles and the clearing of the drop. Depending up on the titre of the serum, agglutination may occur instantly or with in seconds.
  • Clumping occurring after a minute may be due to drying of the fluid and should be disregarded.
  • It is essential to have on the same slide a control consisting of the antigen suspension in saline, without the antiserum, to ensure that the antigen is not autoagglutinable.
  • Slide agglutination is a routine procedure for the identification of many bacterial isolates from clinical specimens. It is also the method used for blood grouping and cross matching.

Fig: How to perform slide agglutination
Tube agglutination

 This is the standard quantitative method for the measurement of antibodies.

Fig. (a).Tube agglutination test for determining antibody titer.
  • When a fixed volume of a particulate antigen suspension is added to an equal volume of serial dilutions of an antiserum in test tubes, the agglutination titre of the serum can be estimated.
  • Tube agglutination is routinely employed for the serological diagnosis of typhoid, brucellosis and typhus fever ( weil- felix reaction).

Widal test

The procedure involves adding a suspension of dead typhoid bacterial cells to a series of tubes containing the patient’s serum, which has been diluted out to various concentrations. After the tubes have been incubated for 30 minutes at 37° C, they are centrifuged and examined to note the amount of agglutination that has occurred. The reciprocal of the highest dilution at which agglutination is seen is designated as the antibody titer of the patient’s serum. For example, if the highest dilution at which agglutination occurs is 1:320, the titer is 320 antibody units per milliliter of serum. Naturally, the higher the titer, the greater is the antibody response of the individual to the disease.

 Two types of antigens are used, the H or the flagellar antigen and the O or the somatic antigen of the typhoid bacillus.
Round bottomed felix tubes are used for agglutination. Agglutinated bacilli spread out in a disc like pattern at the bottom of the tubes. The tube agglutination test for brucellosis may be complicated by the prozone phenomenon and the presence of blocking antibodies. Several dilution of the serum should be tested to prevent false negative results due to prozone.
The weil- felix reaction for serodiagnosis of typhus fever is a heterophile agglutination test and is based on the sharing of a common antigen between typhus rickettsiae and some strains of proteus bacilli. Another example of the heterophile agglutination test is the streptococcus MG agglutination test for the diagnosis of primary atypical pneumonia.
Examples of agglutination tests using red blood cells as antigens are the Paul Bunnel test and the cold agglutination test. The cold agglutination test is positive in primary atypical pneumonia. The patient’s sera agglutinate human O group erythrocytes at 4°C the agglutination being irreversible at 37°C.
Hemagglutination is used in blood typing
Agglutination reactions are routinely performed to type red blood cells. In typing for the ABO antigens, RBCs are mixed on a slide with antisera to the A or B blood group antigens. If the antigen is present on the cells, they agglutinate, forming a visible clump on the slide. Determination of which antigens are present on donor and recipient blood is the basis for matching blood types for transfusions.
Particle agglutination
Numerous procedures have been developed to detect antigen via the agglutination (clumping) of an artificial carrier particle such as a latex bead with antibody bound to its surface.
Latex agglutination
Antibody molecules can be bound in random alignment to the surface of latex (polystyrene) beads. Antigen present in a specimen being tested binds to the combining sites of the antibody exposed on the surfaces of the latex beads, forming cross- linked aggregates of latex beads and antigen.
  • The size of the latex bead (0.8µm or larger) enhances the ease with which the agglutination reaction is recognized.
  • Levels of bacterial polysaccharides detected by latex agglutination have been shown to be as low as 1.0 ng /ml because the pH, osmolarity and ionic concentration of the solution influence the amount of binding that occurs, conditions under which latex agglutination procedures are carried out must be carefully standardized.
  • Additionally, some constituents of body fluids such as rheumatoid factor, have been found to cause false- positive reactions in the latex agglutination systems available. To counteract this problem. It is recommended that all specimens be treated by boiling or with ethylenediaminetetraacetic acid (EDTA) before testing.
  • Commercial test systems are usually performed on cardboard cards or glass slides; manufacturers recommendations should be followed precisely to ensure accurate results.
  • Reactions are graded on a 1+ to 4+ scale, with 2+ usually the minimum amount of agglutination seen in a positive sample.
  • Control latex (coated with antibody form the same animal species from which the specific antibody was made) is tested alongside the latex. If the patient specimen or the culture isolate reacts with both the test and control latex, the test is considered non specific and therefore uninterpretable.
  • Latex tests are very popular in clinical laboratories to detect antigen to Cryptococcus neoformans in CSF or serum and to confirm the presence of beta- hemolytic streptococcus form the culture plates. Latex tests are also available to detect Streptococcus agalactiae, Clostridium difficile toxins A and B and rotavirus.
In this case the particles are killed and treated Staphylococcus aureus  ( cowan I strain), which contain a large amount of an antibody- binding protein, protein A, in their cell walls. In contrast to latex particles, these staphylococci bind only the base of the heavy chain portion (Fc) of the antibody, leaving both (Fab)antigen- binding ends free to form complexes with specific antigen.
 Several commercial suppliers have prepared coagglutination reagents for identification of streptococci, including Lancefield groups  A, B , C, D, F , G and N; Streptococcus pneumoniae; Neisseria meningitides; N gonorrhoeae; and Haemophilus influenzae types A to F grown in culture. The coagglutination reaction is highly specific but may not be as sensitive for detecting small quantities of antigen as latex agglutination. Thus, it is not usually used for direct antigen detection.
Latex agglutination inhibition test
Latex agglutination inhibition test a) negative b) positive
The latex agglutination inhibition test relies on competition for the antibody between a latex- drug conjugate and any drug that may be present in the sample (mostly urine). A urine sample is placed in the mixing well of a slide containing antibody reagent, buffer and latex reagent.
a) If the drug is absent, the latex- drug conjugate binds to the antibody and forms large particles that agglutinate. Therefore agglutination is evidence for the absence of drugs in the urine specimen
b) If a drug is present in the urine sample, it competes with the latex conjugate for the small amount of available antibody. A sufficient quantity of the drug will prevent the formation of particles and agglutination and a positive urine sample does not change the appearance of the test mixture.
Coombs test
  1. Direct coombs test: Detection of incomplete antibodies on patients RBCs. Antibodies attached on the surface of the RBCs ( patient RBCs)  + Antihuman globulin = agglutination.
  2. Indirect coombs test:Detection of antibodies in patients sera. Rhesus positive RBC + Patient serum ( if contains incomplete circulating Abs coats the surface of the RBC)+ Antihuman globulin which makes the bridge = agglutination

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