1. Agglutination: Latex Agglutination
Practical Immunology
4th Class
Lab 6:
University of Sulaimani School of Science Department of Biology
Agglutination:
Latex Agglutination
Hemin M. Hussein
MSc Infection & Immunity

2. Objectives Explain latex agglutination (direct and indirect).
Numerate some applications of latex agglutination technique.
Perform latex agglutination technique efficiently.
Occurs in two stages:
Sensitization – cannot be seen
Lattice formation – visible
Antigen or antibody can be coated onto or an integral part of a carrier particle:
Latex particles
Red blood cells--HEMAGGLUTINATION
Charcoal
Bacteria
Agglutination indicates presence of substance being tested for.

3. Agglutination Reaction
Particulate
Antigen
Antibody
Agglutination
The interaction between antibody and a particulate antigen results in visible clumping called agglutination, due to the cross-linking between the antigens.
Types of particles:
Erythrocytes
Inert carriers such as (Latex bead)
Bacterial cells

4. Latex Agglutination Tests
The specificity of antigen-antibody interactions has led to the development of a variety of immunologic assays.
These assays can be used to detect the presence of either antibody or antigen. In all immunoassays, one of them (Ab or Ag) is unknown.
They have played vital roles in diagnosing diseases, monitoring the level of the humoral immune response, and identifying molecules of biological or medical interest.
The agglutination reaction has principally been exploited using erythrocytes as the particles and has been exploited in blood group serological techniques.
The simplest form of this test involves the agglutination of erythrocytes (as antigens).

5. Latex Agglutination Tests
Sheep red blood cells (SRBS) have been used in many immunological diagnostics.
Till now its in use, for example for the diagnosis of infectious mononucleosis (Epstein-Barr virus).
Over the past several years, there has been a shift away from red blood cells to synthetic particles, such as latex beads, as matrices for agglutination reactions.
The use of synthetic beads offers the advantage of stability.
Furthermore, agglutination reactions employing synthetic beads can be read rapidly, often within 3 to 5 minutes of mixing the beads with the test sample.
Agglutination reactions are simple to perform, do not require expensive equipment, and can detect small amounts of antibody
Till now its in use, for example for the diagnosis of infectious mononucleosis (Epstein-Barr virus).
Polystyrene is one of the most widely used plastics, the scale of its production being several billion kilograms per year. It is a colorless solid that is used, for example, in disposable cutlery, plastic models, CD and DVD cases, and smoke detector housings.

6. HCG Latex Agglutination Test

7. Latex Agglutination Test for Pregnancy
Anti-HCG Antibody
HCG Hormone
(Urine)
Latex Particle

8. Latex Agglutination Test for Pregnancy
Latex agglutination test for pregnancy comprises the use of suspensions of polystyrene latex particles sensitized with anti-serum to human chorionic gonadotropin (hCG). When mixed with urine or serum containing HCG, agglutination indicating a positive test for pregnancy.
First introduced in 1976
Measure Urinary or serum human Chorionic gonadotropin (hCG) levels.
This glycoprotein hormone is released by placenta.
The HCG becomes detectable 6-12 days post conception.
Reason for use
Detects pregnancy at very early stage
Earlier prenatal care
Several commercial latex agglutination tests are available;
Conception: the process of becoming pregnant involving fertilization or implantation
Early pregnancy detection allows the commencement of prenatal care, potential medication changes, lifestyle changes to promote a healthy pregnancy (appropriate diet; avoidance of alcohol, tobacco, and certain medications), early pregnancy termination if so desired .
Pregnancy can be diagnosed by 3 approaches.
Physical examination, Laboratory evaluation, Ultrasonography

9. HCG
hCG is a dimer consisting of a 145 amino acid beta-subunit that is unique to hCG and a 92 amino acid alpha-subunit. The alpha-subunit is identical to that for luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). The alpha and beta-subunits have separate genes on separate chromosomes (chromosomes 6 and 19, respectively). After synthesis, the alpha and beta-subunits are bonded with a noncovalent bond before being released into the circulation.

10. HCG Latex Agglutination Test:
Procedure
Allow kit reagents and patient sample to come to room temperature.
Place one drop of urine sample (50µL) on to the reaction area of the slide using a disposable pipette.
Shake the latex reagent, then add one drop and mix using a stirrer.
Gently and evenly rock and rotate the test slide for 2 minutes and under direct strong light source examine the slide for agglutination.
A positive result is indicated by the obvious agglutination of the latex in clear solution.

11. C-Reactive Protein (CRP)
Latex Agglutination Test

12. C-Reactive Protein (CRP)
Belong to a group of proteins known as acute phase reactants (APRs)
Whose plasma concentration changes in response to a variety of inflammation states including:
Infection
Surgery
Trauma
Some cardiac diseases
Malignancy
Tissue necrosis.
CRP was first identified in 1930 in the serum of a patient with pneumonia and was the first recognized APR.
After that, it was found that CRP is elevated in a variety of other acute inflammatory diseases.
It rises rapidly (24-48 hours) after inflammation and its early return to normal levels after successful therapy make CRP the best APR, suited for diagnostic purposes.

13. C-Reactive Protein (CRP)
CRP functions include:
Initiation of opsonization and phagocytosis, activation of complement by classical pathway, neutrophil and monocyte-macrophage.
So that, has an important role in recognition of microbial organisms and immunomodulation.
Because infection with bacteria lead to higher CRP levels than viral diseases, CRP levels may be helpful to distinguish bacterial infection from viral infection. !
An elevated CRP level is a sensitive although nonspecific, indicator of inflammation

14. Latex Agglutination Test for C-Reactive Protein
Principle
Latex beads are coated with antibodies to human CRP. When the latex suspension mixed with serum containing elevated CRP level on a slide, clear agglutination is seen within 2 minutes.
CRP
CRP

15. Latex Agglutination Test for C-Reactive Protein
Qualitative slide test: Procedure
Allow kit reagents and patient serum to come to room temperature.
Transfer one drop (50µL) of patient's serum to the test circle on the slide.
Shake the latex reagent, then using the dropper provided, add one drop of the suspension to the circle
Mix the drops a disposable stirrer ensuring coverage of the test circle with the mixture.
Gently and evenly rock and rotate the test slide for 2 minutes and under direct strong light source examine the slide for agglutination.
A positive result is indicated by the obvious agglutination of the latex in clear solution.

16. Latex Agglutination Test for C-Reactive Protein
Semiquantitative slide test: Procedure
If a positive reaction is obtained from qualitative slide test, the specimen may be serially diluted with a glycine-saline buffer in order to obtain a semiquantitative estimate of the CRP level.
Begin with a 1:2 dilution of patient serum obtained by mixing equal parts specimen and glycine-saline buffer. Blend the tube contents thoroughly.
Add 0.1 mL of buffer to the desired number of test tubes. Add 0.1 mL of 1:2 dilution to the first tube; mix and transfer 0.1 mL to the next additional tube, and continue according to the used kit.
Perform a slide agglutination test on each dilution, and look for agglutination.

17. Results
A positive reaction is reported when the specimen shows agglutination, indicating the presence of CRP in the serum at a level equal to or greater than 0.6 mg/dL.
The titer is represented by the last dilution that shows a positive reaction.
A negative reaction is characterized by a lack of visible agglutination in the undiluted specimen.

18. Recommended Cleaning Procedure of Used Cards
Used cards must be immediately immersed in disinfectant solutions.
The reaction circles must be physically rubbed with non-abrasive material to ensure removal of possible adhering particles.
Thoroughly rinse in purified water
Allow reaction card to dry
Spray cards with a 70% alcohol solution
Allow the alcohol to evaporate prior to re-use

19. References
Hay FC & Westwood OM. (2002). Practical Immunology. 4th ed. Blackwell Science Ltd. Oxford.
Kindt TJ, Osborne BA & Goldsby RA. (2006). Kuby Immunology. 6th ed. W. H. Freeman & Company.
Kartz A, Lee-Lewandrowski E & Lewandrowski KB. (2002). The Plasma Proteins. In: McClatchey KD. Clinical Laboratory Medicine. 2nd ed. Lippincott Williams & Willkins, A Wolters Kluwer Company, Philadelphia.
Wu AH. (2002). Diagnostic Enzymology & other Biochemical Markers of Organ Damage. In: McClatchey KD. Clinical Laboratory Medicine. 2nd ed. Lippincott Williams & Willkins, A Wolters Kluwer Company, Philadelphia.