A N T I G L O B U L I N T E S T.docx

ANTIGLOBULINTEST

o Contains only one antibody specificity, either anti-IgG or antibody to specific complement components such as C3b or C3d

PRINCIPLE Antihuman globulins (AHGs) obtained from immunized nonhuman species bind to human globulins such as IgG or complement, either free in serum or attached to antigens on red blood cells (RBCs). HISTORY 1908 – Moreschi described the principle of the test. He used rabbit antigoat serum to agglutinate rabbit RBCs that were sensitized with low nonagglutinating doses of goat antirabbit RBC serum 1945 – Coombs and associates described the use of the antiglobulin test for the detection of weak and nonagglutinating Rh antibodies in serum 1946 – Coombs and coworkers described the use of AHG to detect in vivo sensitization of the RBCs of babies suffering from hemolytic disease of the newborn 1947 – Coombs and Mourant demonstrated that the antibody activity that detected Rh antibodies was associated with the anti-gamma globulin fraction in the reagent 1951 – Dacie presented the first indication that there might be another antibody activity present that influenced the final reaction. 1957 – Dacie and coworkers published data showing that the reactivity of AHG to cells sensitized with warm antibodies resulted from anti-gamma globulin activity, whereas anti-nongamma globulin activity was responsible for the activity of cells sensitized by cold antibodies. TYPES OF ANTIHUMAN GLOBULIN REAGENTS Polyspecific AHG o Contains antibody to human IgG and to the C3d component of human complement. Other anticomplement antibodies, such as anti-C3b, anti-C4b, and anti-C4d, may also be present Monospecific AHG

PREPARATION OF AHG The classic method of AHG production involves injecting human serum or purified globulin into laboratory animals such as rabbits. May be of polyclonal or monoclonal response o Polyclonal Antibodies Mixture of antibodies from different plasma cell clones o Monoclonal Antibodies Derived from one clone of plasma cells Polyclonal AHG Production o Usually prepared in rabbit. Sheep or goats for large volumes o Different colonies are immunized with IgG and C3 antigens o Uses serum from many donors o Both colonies are hyperimmunized o The animals are bled if the antibody potency and specificity meets the predetermined specifications

o Absorption with A1, B, and O cells o Block titrations are performed Monoclonal AHG Production o Devised by Kohler and Milstein o Immunization of mice with purified human globulin o Mouse spleen cells are fused with myeloma cells o Hybridomas are screened for antibodies with required specificity and affinity o Propagated in tissue culture or by inoculation into mice The reagent also contains buffers, stabilizers, and bacteriostatic agents and may be dyed green for identification

Reaction Medium o Albumin TWO (2) TYPES OF COOMBS TEST: DIRECT ANTIGLOBULIN TEST o A one-stage technique used to detect in vivo sensitization

o Low Ionic Strength Solution (LISS) Enhances antibody uptake and allows incubation time to be decreased

o Clinical applications: Hemolytic Disease of the Newborn

Introduced by Low and Messeter o Polyethylene Glycol (PEG) A water-soluble linear polymer

Hemolytic Transfusion Reaction Autoimmune and drug-induced hemolytic anemia o Can detect 100 to 500 IgG molecules per RBC and 400 to 1,100 C3d molecules per RBC INDIRECT ANTIGLOBULIN TEST o A two-stage technique used to determine in vitro sensitization of RBCs o Clinical Applications: Compatibility testing Antibody Screen & Identification RBC Phenotyping Titration of incomplete antibodies o A positive reaction is obtained when there are 100 to 200 IgG or C3 molecules on the cell o Tasks and its purposes: Incubate RBCs with antisera Allows time for antibody molecule attachment to RBC antigen Perform a minimum of three saline washes Removes free globulin molecules Add antiglobulin reagent Forms RBC agglutinates Centrifuge Accelerates agglutination by bringing cells closer together Examine for agglutination Interpret test as positive or negative Grade agglutination reactions Determines the strength of reaction Add Check Cells Check for neutralization of antisera by free globulin molecules FACTORS AFFECTING THE ANTIGLOBULIN TEST Ratio of serum to cells o 40:1 (2 drops of serum and 1 drop of 5% RBC suspension)

Effective on concentrating the antibody Anti-IgG is the reagent of choice Temperature o Optimal at 37 degrees Celsius Incubation Time o Saline – 30 to 120 minutes o LISS or PEG – 10 to 15 minutes Washing of RBCs o Must be saline-washed for a minimum of three times before adding the AHG rgt Saline for Washing o Should be fresh or buffered to a ph of 7.2-7.4 Addition of AHG o AHG should be added to the cells immediately after washing Centrifugation for Reading o Centrifugation and resuspending the cells are crucial step in the technique o CBER-recommended: 1000RCF for 20 seconds o 500 RCF for 15 to 20 sec

o Reverse ABO typing Specific Gel A specific reagent is incorporated into the gel Antigen determination Gel Low Ionic Antiglobulin Test (GLIAT) AHG reagent is incorporated into the gel Provides a safe, reliable, and easy-to-read AHG test 910 rpm for 10 minutes

MODIFIED AND AUTOMATED ANTIGLOBULIN TEST TECHNIQUES Low Ionic Polybrene Technique o Relies on low ionic conditions to rapidly sensitize cells with antibody o Polybrene is a potent rouleaux-forming reagent o Monospecific anti-IgG reagent must be used Enzyme-Linked Antiglobulin Test o The amount of color produced is measured spectrophotometrically and is proportional to the amount of antibody present o OD is measured at 405 nm Solid Phase Technology Gel Test o Detects RBC antigen-antibody reactions by means of using a chamber filled with polyacrylamide gel o Three (3) types of gel tests: Neutral Gel Does not contain any specific reagent and acts only by its property of trapping agglutinates Applications: o Antibody screening and ID with enzyme-treated or untreated RBCs