What is the latex agglutination test?
This type of agglutination test is performed by coating antibody or antigen on the surface of an artificial carrier particle, which is called latex bead (polystyrene)
If any antigen present in the test sample it will bind with at the combining site of antibody which is coated on the surface of the latex bead, by forming a cross-linked aggregates of latex beads and antigen. The size of latex beads varies from 0.8µm to 1um.
Latex agglutination test is also known as the latex fixation test. Clinically this test is used for the identification and typing of many important microorganisms.
In 1965, Singer and Plotz first introduced this test and was described as the Rheumatoid Factor Test, which is based on the latex agglutination.
Types of Latex Agglutination
Latex agglutination test is divided into two classes based on the processes of detection;
A. Latex Agglutination Test (LAT) for Antibody Detection
It is a passive agglutination test, in this method antigen is coated on the surface of latex beads to detect the antibody in the test sample.
B. Latex Agglutination Test (LAT) for Antigen Detection
It is a Reverse Passive Agglutination Test. In this method, antibody is coated on the surface of latex beads to detect the antigen in the test sample.
Principle of Latex Agglutination
Antibody or antigen molecules can be bound in random alignment to the surface of latex (polystyrene) beads. The number of antibody or antigen molecules bound to each latex particle is large, resulting in a high number of exposed potential binding sites. Antigen or antibody present in a specimen binds to the combining sites of the corresponding antigen/antibody exposed on the surfaces of the latex beads, forming cross-linked aggregates of latex beads and antigen/antibody. A large particle size of latex facilitates the visualization of the antigen-antibody reaction.
Materials Required
- 1.5 ml Vials.
- Microcentrifuge.
- Pipette.
- Microtips.
- Laboratory refrigerator.
- Glycine saline buffer.
- Blocking buffer.
- Antigen for coating.
- Latex beads.
- Test antiserum.
- Glass slides.
- Beaker.
- Toothpick.
Procedure
Coating of Latex
- To 20 μl of latex beads taken in a 1.5 ml vial add 40 μl of glycine-saline buffer.
- Add 60 μl of antigen to the latex and incubate at 37oC for 2 hours.
- Spin down at 5000 rpm for 10 minutes and carefully aspirate the supernatant.
- Resuspend the pellet in 1 ml of blocking buffer and spin down at 5000 rpm for 10 minutes.
- Repeat the washing once more.
- Add 90 μl of blocking buffer to the pellet, mix well.
- Incubate at 4oC, overnight.
Agglutination Test
- To 200 μl of glycine-saline buffer taken in a vial,add 4 μl of test antisera. ( 50 times diluted ).
- Add 50 μl of antigen to 50 μl of diluted antiserum in a 1.5 ml vial, mix well and incubate at room temperature for 10 minutes.
- Pipette 10 μl of coated latex onto a glass slides.
- Add 10 μl of diluted test antiserum to slide A.
- Add 10 μl of antiserum mixed with antigen (from step 8) to B.
- Add 10 μl of glycine-saline buffer to C.
- Take a toothpick and mix the content in each slide. Discard the toothpick after using in one slide (take a new one for the next slide ).
- After mixing, wait for 2 minutes to observe the result.
Result of Latex Agglutination
Positive Agglutination Result: The presence of White clumps indicates a positive result i.e suspected particle is present.
Negative Agglutination Result: The absence of white clumps indicates a negative result i.e Suspected particle is not present.
Use of Latex Agglutination
- n the clinical laboratory, it is used for the detection of antigen to Cryptococcus neoformans in CSF or serum.
- Used for the confirmation test of beta-hemolytic streptococcus from the culture plates.
- It also used for the detection of Streptococcus agalactiae, Clostridium difficile toxins A and B and rotavirus.
Latex agglutination inhibition test
In Inhibition test for latex agglutination involve a competition for antibody between a latex- drug conjugate and any drug that may be present in the sample (mostly urine). This test is performed in this following procedure;
- Prepared a mixer of antibody reagent, buffer, and latex reagent in a mixing well of a slide.
- Add a urine sample in this mixing well-containing mixer of antibody reagent, buffer, and latex reagent.
- 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
- 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.
Limitation
- Rheumatoid factor of body fluids has been found to cause false-positive reactions in the latex agglutination systems available, to prevent this problem it is recommended to boil the specimen with ethylenediaminetetraacetic acid (EDTA) before testing.
- Commercially latex agglutination is performed on cardboard cards or glass slides, which can create inaccurate results.
- 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.
Reference
- https://en.wikipedia.org/wiki/Latex_fixation_test
- https://www.ucsfhealth.org/medical-tests/003334
- https://microbeonline.com/immunology-note/
- https://vlab.amrita.edu/?sub=3&brch=69&sim=195&cnt=2
- https://www.ucsfbenioffchildrens.org/tests/003334.html
- https://www.lornelabs.com/news-events/blog/everything-you-should-know-about-latex-agglutination-tests
- https://medlineplus.gov/ency/article/003334.htm
- https://microbenotes.com/latex-agglutination-test/#applications-of-latex-agglutination-test
