Learning Objectives Describe how agglutination reactions can be used to assess the presence of antibodies in a specimen. Key Points Agglutination reactions produce visible aggregates of antibody — antigen complexes when antibodies or antigens are conjugated to a carrier. Carriers used in agglutination methods could be artificial e.
There are various methods of agglutination reactions that follow the same principle, but they differ in the elements they employ based on the desired endpoint and the main purpose of the test. Key Terms avidity : The measure of the synergism of the strength of individual interactions between proteins.
The desorbed antibody also causes agglutinated lumps of red blood cells to form. These lumps cannot pass through the pores of the filter paper.
The immobilization and filtration of agglutinated red cells give reproducible identification of positive haemagglutination reaction. The presence or absence of visual agglutination enables a quick convenient method of determining the ABO and Rhesus status of the individual. Agglutination of red blood cells is used in the Coombs test. In cross-matching, agglutination occurring when donor and recipient's blood are incubated together indicates that the donor blood is incompatible for that particular recipient.
Leukoagglutination is when the particles involved are white blood cells. Agglutination is commonly used as a method of identifying specific bacterial antigens, and in turn, the identity of such bacteria.
Because the clumping reaction occurs quickly and is easy to produce, agglutination is an important technique in diagnosis. Two bacteriologists, Herbert Edward Durham and Max von Gruber , discovered specific agglutination in The clumping became known as Gruber-Durham reaction. Gruber introduced the term agglutinin from the Latin for any substance that caused agglutination of cells.
French physician Fernand Widal put Gruber and Durham's discovery to practical use later in , using the reaction as the basis for a test for typhoid fever. Widal found that blood serum from a typhoid carrier caused a culture of typhoid bacteria to clump, whereas serum from a typhoid-free person did not. This Widal test was the first example of serum diagnosis. Agglutination assays are usually quick and easy to perform on a glass slide or microtiter plate Figure 1. Microtiter plates have an array of wells to hold small volumes of reagents and to observe reactions e.
The wells come in many different sizes for assays involving different volumes of reagents. Figure 1. Microtiter plates are used for conducting numerous reactions simultaneously in an array of wells. The use of agglutination tests to identify streptococcal bacteria was developed in the s by Rebecca Lancefield working with her colleagues A.
Dochez and Oswald Avery. Production of antibodies against M protein is crucial in mounting a protective response against the bacteria. Lancefield used antisera to show that different strains of the same species of streptococci express different versions of M protein, which explains why children can come down with strep throat repeatedly.
Lancefield classified beta-hemolytic streptococci into many groups based on antigenic differences in group-specific polysaccharides located in the bacterial cell wall. The strains are called serovars because they are differentiated using antisera. Identifying the serovars present in a disease outbreak is important because some serovars may cause more severe disease than others. Figure 2. Antibodies against six different serovars of Group A strep were attached to latex beads.
Each of the six antibody preparations was mixed with bacteria isolated from a patient. The tiny clumps seen in well 4 are indicative of agglutination, which is absent from all other wells. This indicates that the serovar associated with well 4 is present in the patient sample. The method developed by Lancefield is a direct agglutination assay , since the bacterial cells themselves agglutinate.
A similar strategy is more commonly used today when identifying serovars of bacteria and viruses; however, to improve visualization of the agglutination, the antibodies may be attached to inert latex beads. This technique is called an indirect agglutination assay or latex fixation assay , because the agglutination of the beads is a marker for antibody binding to some other antigen Figure 2.
Indirect assays can be used to detect the presence of either antibodies or specific antigens. When mixed with patient serum, the antibodies will bind the antigen, cross-linking the latex beads and causing the beads to agglutinate indirectly; this indicates the presence of the antibody Figure 3. This technique is most often used when looking for IgM antibodies, because their structure provides maximum cross-linking.
One widely used example of this assay is a test for rheumatoid factor RF to confirm a diagnosis of rheumatoid arthritis. RF will agglutinate IgG-coated latex beads. Figure 3. Agglutination tests are widely used in underdeveloped countries that may lack appropriate facilities for culturing bacteria. For example, the Widal test , used for the diagnosis of typhoid fever , looks for agglutination of Salmonella enterica subspecies typhi in patient sera. The Widal test is rapid, inexpensive, and useful for monitoring the extent of an outbreak; however, it is not as accurate as tests that involve culturing of the bacteria.
The Widal test frequently produces false positives in patients with previous infections with other subspecies of Salmonella , as well as false negatives in patients with hyperproteinemia or immune deficiencies.
In addition, agglutination tests are limited by the fact that patients generally do not produce detectable levels of antibody during the first week or longer of an infection. A patient is said to have undergone seroconversion when antibody levels reach the threshold for detection. Typically, seroconversion coincides with the onset of signs and symptoms of disease.
However, in an HIV infection, for example, it generally takes 3 weeks for seroconversion to take place, and in some instances, it may take much longer. Since antibody levels change over time in both primary and secondary immune responses, by checking samples over time, changes in antibody titer can be detected.
For example, a comparison of the titer during the acute phase of an infection versus the titer from the convalescent phase will distinguish whether an infection is current or has occurred in the past. Agglutination of red blood cells is called hemagglutination. The test can also detect complement attached to red blood cells. These antibodies would activate complement, leading to red blood cell lysis and the subsequent jaundice.
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