Introduction
The Agglutination Test is a clinical assay that detects Antibodies or Antigens in different fluids of the body such as saliva, urine, cerebrospinal fluid, or blood.
During an agglutination test, antibodies react with antigens on cells, forming visible clumps or aggregates known as agglutinates. We can see the clumping with the naked eyes. Agglutinins are antibodies that are involved in the development of clumping in the Agglutination Test. The agglutination test is selective because the antigen will only mix with its homologous antibody to produce an antigen-antibody complex or clumps.
This complex is strong and cannot be reversed. The affinity of the reaction determines the strength of this reaction. Agglutination reactions occur more readily in IgM than in IgG. Singer and Plotz first described this test as the Rheumatoid Factor Test in 1965. The interaction of antibodies with antigens causes clearly visible clumping or aggregation, which is known as an agglutination reaction.
Prozone phenomenon
The prozone phenomenon occurs when an excessive amount of antigen and antibody inhibits the agglutination response. If the number of antibodies is greater than the number of antigens, the reactions of agglutination can be suppressed. This type of inhibition is referred to as the prozone phenomenon. When either an antibody or an antigen is present in excess, the prozone phenomenon occurs.
The prozone phenomenon also increases the number of antibody binding sites in comparison to antigen epitopes. As a result, antibodies attach to antigens only univalently rather than multivalently. Antibodies that have chosen univalent binding cannot cross-link one antigen to another.
Objective of the agglutination test
The agglutination test is used to detect the presence of antibodies produced against a certain antigen, virus, or bacteria.
Samples collection for agglutination test
Agglutination tests are used to identify antigens and antibodies in many types of body fluids, including saliva, blood, urine, and cerebrospinal fluid (also called as lumbar puncture). Following sample collection, they were combined with latex beads coated with a specific antibody or antigen. If the suspected antigen or antibody is present, clumps will form. This test takes between 15 minutes and an hour to complete.
Types of Agglutination Tests
Agglutination is categorized into three forms based on the type of response between antigen and antibody.
Direct agglutination test
Cells, insoluble particles, or antigens are agglutinated directly using antibodies, in this method.
The direct agglutination test is classified into two types:
Slide agglutination
To accomplish the agglutination, blood samples will be mixed with Anti-A, Anti-B, and Anti-D antibodies on a slide.
Procedure for slide agglutination
- The sample specimen is first placed on a clean microscopic slide in this process.
- On this sample drop, add antiserum.
- Then, using a sterilized loop, thoroughly mix them.
- Allow for agglutination. Examine the formation of agglutination.
Interpretation of results
In the slide agglutination reaction, a positive result can be detected by the development of a simple clump. Within a few minutes, clumping of test sample will occur in this test.
Applications of slide agglutination test
- This method is used for the identification of bacteria from clinical specimens.
- It is used for blood grouping.
Tube Agglutination
This technique is used to identify a specific antibody in a blood sample that contains a constant amount of antigen.
- In the following method, the serum to be examined is first serially diluted in test tubes.
- A standardized amount of known antigen is added to each Test tube and thoroughly mixed.
- The tubes are incubated at an ideal temperature.
- After a few hours of incubation, agglutination is complete, and tubes are checked for clumping.
Result Interpretation
Typically, a result is stated as tighter. The maximum dilution of test serum at which positive agglutination occurs.
Applications of test tube agglutination
- This test is used for Brucellosis testing.
- The above mentioned method is used in Widal test that is used to diagnose enteric fever.
- In serodiagnosis of typhus fevers, this method used for the Weil-Felix reaction.
- It can also be used in the Paul-Bunnel test.
Indirect Agglutination test
When a soluble antigen is being used in an agglutination reaction, it is frequently coated on a carrier particle, and agglutination occurs on the surface of the carrier molecule; this sort of reaction is known as an indirect agglutination reaction. In indirect agglutination tests, carrier molecules such as RBC, latex, or bentonite are utilized.
Reverse passive agglutination test
In this test, the antibody is coated on a carrier molecule, which then detects antigen in the patient’s serum.
Applications of agglutination tests
- Agglutination testing is used for blood grouping and cross-matching.
- These are used to identify Cryptococcus neoformans antigen in cerebrospinal fluid or serum.
- The Agglutination Test is used to assess whether the quantity of antibodies or antibody titer against a certain infectious agent in a patient’s blood is increasing.
- This test is also used to detect Haemophilus influenza, Pneumococcus, and Meningococcus capsular antigens.
- The agglutination test is used to detect Clostridium difficile toxins A and B, rotavirus, and Escherichia coli 0157:H7 from suspicious E coli colonies.
- Anti-streptolysin O antibody was detected using this assay.
- This reaction test is frequently used to diagnose disease caused by a bacterium that is difficult to detect or grow in the clinical laboratory.
- The ezoic agglutination test is also used to diagnose beta-hemolytic Streptococcus.
- The Agglutination Test is used to detect antibodies to the three species of Brucella that produce brucellosis or undulant fever; Franciscella tularensis, which causes tularemia; and Epstein-Barr virus, which causes mononucleosis.
Advantages of agglutination tests
- These tests require less time.
- They are easy to perform.
- These require high degree of analytic sensitivity.
- They can detect an enormous variety of antibodies.
Disadvantages of Agglutination tests
- Their susceptibility to false-negative reactions caused by the prozone phenomenon.
- These reactions are semi-quantitative.
Examples of agglutination tests
The Widal test
This test is performed in a test tube. The widal test includes two forms of typhoid bacillus antigen: H, or flagellar antigen, antigen O, or somatic antigen.
Protocol of Widal test
This test is carried out in the following steps:
- This test involves adding a sample of dead typhoid bacterial cells to a test tubes series containing the patient’s serially diluted serum.
- For 30 minutes, the test tubes are incubated at 37 degrees Celsius.
- Ezoic centrifugation is used to determine the amount of agglutination that has occurred.
Result interpretation
The higher the titer in this test, the larger the individual’s antibody response to the disease.
Hemagglutination test
Hemagglutination is often referred to as haemagglutination. RBCs were typed using a hemagglutination assay. On a clean slide, the test blood sample is first mixed with antisera (Anti-A, B, D). If the antigen is present in the blood sample, it will form visible clumps on the slide, revealing the individuals’ blood type.
Applications of hemagglutination test
- Blood typing or blood grouping is done with this.
- In a haemagglutination assay, it is used to quantify virus dilutions.
Latex Agglutination test
This test involves attaching antibodies or antigens to the surface of latex beads, which are made of polystyrene. If antigen is present in the test sample, it will form cross-linked aggregates of latex beads and antigen by combining with the combining sites of the surface antibody of the latex beads. Latex beads range in size from 0.8m to 1um.
Latex agglutination has been shown to detect bacterial polysaccharides at levels as low as 1.0 ng/ml. Because the pH, osmolarity, and ionic concentration of the solution influence the amount of binding that occurs, the conditions under which latex agglutination procedures are carried out must be carefully standardized.
Furthermore, some bodily fluid elements, such as rheumatoid factor, have been examined to induce false-positive results in latex agglutination systems. To address this issue, before testing, all specimens should be boiled or treated with ethylene diamine tetra-acetic acid (EDTA).
Commercial testing is typically done on cardboard cards or glass slides. Reactions are evaluated on a scale of 1+ to 4+, with 2+ being the least amount of agglutination detected in a positive sample. Control latex is tested alongside the latex (coated with antibodies from the same animal species from which the specific antibody was produced). If both the test and control latex respond with the patient specimen or culture isolate, the test is termed nonspecific and so it is uninterpretable.
Applications of latex agglutination test
- Capsular antigens of Pneumococcus, Haemophilus influenza, and Meningococcus are detected.
- This test aids in the detection of Cryptococcus antigen.
Latex agglutination inhibition test
This test is based on antibody competition between a latex-drug conjugate and any drug present in the sample (usually urine). In the mixing well of a slide containing antibody reagent, buffer, and latex reagent, the urine sample is mixed.
- In the absence of the drug, the latex-drug conjugate binds to antibody, forming big particles that agglutinate. As a result, agglutination indicates the absence of drugs in the urine specimen.
- If a drug is present in the urine sample, it competes for the limited amount of accessible antibody with the latex conjugate. A significant amount of the drug prevents particle formation and agglutination, and a positive urine sample has no effect on the appearance of the mixture of test.
The Weil-Felix reaction
The Weil-Felix reaction for screening of typhus fever is a heterophile agglutination test based on the presence of a common antigen shared by typhus rickettsiae and some proteus bacilli strains. The streptococcus MG agglutination test is another example of a heterophile agglutination test used to diagnose primary atypical pneumonia. The Paul Bunnel test and the cold agglutination test are two examples of agglutination tests that use red blood cells as antigens. In primary atypical pneumonia, the cold agglutination test is positive. At 4°C, the patient’s sera agglutinates human O group erythrocytes, and at 37°C, the agglutination is irreversible.
Coagglutination test
Coagglutination is the same as latex agglutination. The antibodies are linked to a particle to increase the visibility of the agglutination reaction created between antigen and antibody. In this procedure, initially killed particles are used with Staphylococcus aureus. Protein A can also be used (which is an antibody-binding protein found in the cell walls of Staphylococcus aureus). Staphylococci bind just the base of the heavy chain component (Fc) of the antibody, leaving both (Fab) antigen-binding ends free to form complexes with specific antigen.
Applications of Coagglutination test
- Antigen detection in serum, urine, and CSF.
- Several commercial providers are preparing coagglutination reagents for the identification of streptococci, including Lancefield groups A, B, C, D, F, G, and N; Streptococcus pneumoniae; Neisseria meningitides; N gonorrhea; and Haemophilus influenza types A to F cultivated in culture.
Coombs test
Direct Coombs test
It involves the detection of partial antibodies on RBCs from patients. Agglutination is caused by antibodies linked to the surface of RBCs (patient RBCs) and antihuman globulin.
Indirect Coombs test
Antibodies in patients’ serum are detected using the indirect Coombs test. Rhesus positive RBC + Patient serum (if incomplete circulating Abs cover the surface of the RBC) + Antihuman globulin that forms the bridge, will result in the agglutination
Risks of agglutination tests
The level of risk varies according to the type of test.
Saliva and Urine Tests
The urine or saliva tests pose no danger.
Blood test
The size of veins and arteries varies from person to person and from one side of the body to the other. It may be more difficult to obtain a blood sample from some persons than from others. Other minor dangers associated with having blood collected include:
- Continuous bleeding
- Fainting or feeling dizzy
- Hematoma (blood clot under the skin)
- Infection (a small danger whenever the skin is punctured)
Cerebrospinal Fluid test
Lumbar puncture has the following risks:
- Subdural hematomas are caused by bleeding into the spinal canal or around the brain.
- Discomfort during the examination.
- Headache that lasts for several hours or days after the test. If your headaches linger for more than a few days (particularly when sitting, standing, or walking), you may have a “CSF-leak.” If this happens, you should consult your doctor.
- Anesthetic hypersensitivity (allergic response).
- Infection is caused by the needle puncturing the skin.
References
- Aoyagi K, Ashihara Y, Kasahara Y. Immunoassays and immunochemistry. In: McPherson RA, Pincus MR, eds. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 24th ed. Philadelphia, PA: Elsevier; 2022:chap 45.
- Esmail S, Knauer MJ, Abdoh H, Voss C, Chin-Yee B, Stogios P, Seitova A, Hutchinson A, Yusifov F, Skarina TJCrm. 2021. Rapid and accurate agglutination-based testing for SARS-CoV-2 antibodies. 1(2).
- Gillet P. 2011. Malaria Rapid Diagnostic Tests: Laboratory aspects in the diagnostic setting.
- Yoshida T, Mandour AS, Sato M, Hirose M, Kikuchi R, Komiyama N, Hendawy HA, Hamabe L, Tanaka R, Matsuura KJFiVS. 2022. Pulmonary thromboembolism due to immune-mediated hemolytic anemia in a cat: A serial study of hematology and echocardiographic findings. 9: 930210.
- https://www.sciencedirect.com/topics/medicine-and-dentistry/agglutination-test
