Agglutination:Latex Agglutination

University of SulaimaniSchool of Science

Department of Biology

Practical Immunology4th Class

Lab 6: 19. 11. 2014

Hemin M. HusseinMSc Infection & Immunity

hemin.hmh@gmail.com

Lab. 6

• Explain latex agglutination (direct and indirect).

• Numerate some applications of latex agglutination technique.

• Perform latex agglutination technique efficiently.

Objectives

Lab. 6

Agglutination Reaction

Particulate

AntigenAntibody 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

Lab. 6

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.

Lab. 6

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

Lab. 6

HCG Latex Agglutination Test

Lab. 6

Latex Particle

Anti-HCG AntibodyHCG Hormone

(Urine)

Latex Agglutination Test for Pregnancy

Lab. 6

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

Lab. 6

HCG

Lab. 6

HCG Latex Agglutination Test:

• 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.

Procedure

Lab. 6

C-Reactive Protein (CRP)Latex Agglutination Test

Lab. 6

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.

Lab. 6

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.

An elevated CRP level is a sensitive although nonspecific, indicator of inflammation

Lab. 6

Latex Agglutination Test for C-Reactive Protein

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.

Principle

CRP

CRP

Lab. 6

1. Allow kit reagents and patient serum to come to room temperature.

2. Transfer one drop (50µL) of patient's serum to the test circle on the slide.

3. Shake the latex reagent, then using the dropper provided, add one drop of the suspension to the circle

4. Mix the drops a disposable stirrer ensuring coverage of the test circle with the mixture.

5. Gently and evenly rock and rotate the test slide for 2 minutes and under direct strong light source examine the slide for agglutination.

6. A positive result is indicated by the obvious agglutination of the latex in clear solution.

Qualitative slide test: Procedure

Latex Agglutination Test for C-Reactive Protein

Lab. 6

1. 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.

2. Begin with a 1:2 dilution of patient serum obtained by mixing equal parts specimen and glycine-saline buffer. Blend the tube contents thoroughly.

3. 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.

4. Perform a slide agglutination test on each dilution, and look for agglutination.

Semiquantitative slide test: Procedure

Latex Agglutination Test for C-Reactive Protein

Lab. 6

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.

Lab. 6

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

Lab. 6

References• Hay FC & Westwood OM. (2002). Practical Immunology. 4th ed. Blackwell

Science Ltd. Oxford.• Kindt TJ, Osborne BA & Goldsby RA. (2006). Kuby Immunology. 6 th 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.

• http://www.americanpregnancy.org/duringpregnancy/hcglevels.html• http://www.lab21.com/biotec/Products/LatexSerology/tabid/1428/language/en-

US/Default.aspx• http://labtestsonline.org/understanding/analytes/hcg/tab/faq• http://www.americanpregnancy.org/duringpregnancy/hcglevels.html• http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab17/lab17.html#pregan