www.medicos11.com 2011

w w w . g a m s p g . w e b s . c o m Page 1

Complement fixation test (CFT) Principle : Ag-Ab complexes fix complement Sensitivity : can detect 0.04 mg of Ab mg of Ag

5 separately standardized reagents : Ag

- (soluble/particulate) Ab (antiserum) :

- heated(inactivated) at 56 C for ½ hour (to destroy complement activity of serum as well as remove nonspecific inhibitors of complement)

Complement (guinea pig serum) : - freshly drawn/preserved in lyophilized/frozen / special preservatives (Richardson’s method) - titrated with diluents (physiological saline with calcium and magnesium ions) for complement activity : - 1 unit = MHD = Minimum hemolytic dose of complement = highest dilution of guinea pig serum –that lyses 1

unit volume of sheep RBCs in presence of excess hemolysin (amboceptor) – within a fixed time of 30-60 min – at a fixed temperature of 37 C

Sheep RBCs Amboceptor = rabbit Ab to sheep RBCs

Classic example : Wassermann Reaction (for syphilis) : 2 steps 1. Inactivated test serum + Wassermann Ag + 2 units of guinea pig complement incubation at 37 C for 1 hour (is

serum has Ab it will bind to Ag – ag-ab complex fix complement) 2. Add sensitized cells (Sheep RBCs coated with 4 MHD hemolysin) [no complement available] incubate at 37C for

30 min Results :

o Hemolysis (RBC lysis) = complement not fixed in step 1 & available to cause hemolysis in step2 = serum does not have antibodies = negative CFT

o No Hemolysis (no RBC lysis) = complement was used up in step 1 = serum has Ab = positive CFT Controls used :

o Ag & serum controls : to ensure they are not anticomplementary o Complement control : to ensure desired amount of complement added o Cell control : to ensure that sheep RBCs not undergo lysis in absence of complement

Indirect CFT : Certain avian (duck/turkey/parrot)& mammalian (horse , cat)sera don’t fix complement Step 1 is same : Inactivated test serum (avian/mammalian) + Wassermann Ag + 2 units of guinea pig complement incubation at 37 C for 1 hour(is serum has Ab it will bind to Ag – ag-ab complex cannot fix complement) In step 2 : test is set up in duplicate One set: add standardized antiserum known to fix complement (if test serum has Ab all Ag is used up in step1 & standardized serum cannot bind to Ag – cannot fix complement) Second set : not added standardized antiserum known to fix complement In both the set Add sensitized cells (Sheep RBCs coated with 4 MHD hemolysin) (complement is freely available to cause hemolysis in set1) incubate at 37C for 30 min Result : Hemolysis = positive Indirect CFT No hemolysis = negative indirect CFT Conglutinating complement absorption test : Used for system which cannot fix Guinea pig complement Test serum + nonhemolytic Horse complement + Indicator (sensitized sheep RBCs mixed with bovine serum) Bovine serum has – beta globulin = conglutinin = Ab to complement If no Ab in test serum : horse complement is available Agglutination of sheep RBCs if they have combined with complement =conglutination : negative test

www.medicos11.com 2011

w w w . g a m s p g . w e b s . c o m Page 2

If horse serum has been used up by Ag –Ab complex : no agglutination : positive test Coomb’s test : Antiglobulin test (1945 – Coombs , Mourant & Race) Use : detection of anti-Rh Ab Principle : serum (incomplete antiRh Ab) + Rh positive RBCs Ab coats RBC surface (though they are not agglutinated) washed free of all unattached protein add Rabbit Antisera against Human gammaglobulin = antiglobulin/coomb’s globulin agglutination of cells Direct coombs test (DCT) Indirect Coombs test (ICT) RBC sensitization with incomplete Ab occurs in vivo (HDN –Rh incmpatinbility) : (fetal blood) RBCs of erythroblastic infants washed free of unattached protein coombs sera agglutination

Mothers serum (incomplete Ab) + RBCs : sensitization of RBC occurs in vitro washed add coombs sera agglutination

Negative : HDN d/t ABO incompatibility Positive : Rh incompatibility Positive : HDN d/t Rh incompatibility Detects incomplete Ab on fetal RBCs Detects incomplete Ab in maternal serum Also detects incomplete=nonagglutinating ab : Brucellosis Passive Agglutination tests : detect Ab Only difference b/w precipitation & agglutination : physical nature of Ag (soluble in ppt & particulate in agglutination) Principle : attach soluble Ag – on surface of Carier particles make it particulate = convert precipitation agglutination = more convenient & more sensitive for Ab detection Carrier particles used :

1. RBCs (human/sheep) 2. Latex 3. Bentonite

Method of adsorption of soluble Ag on carrier particles: For polysaccharide Ag : simple mixing with cells (RBCs) For Protein Ag : Tanned RBCs Rose Waaler Test Latex agglutination fixation test For RA ASO / CRP / RA /HCG RA factor = IgM Ab Against Fc-of IgG = Ab to gamma globulin : able to agglutinate RBCs coated with globulins

Sheep RBCs + subagglutinating dose of Amboceptor (Rabbit antisheep RBCs) add serum if RA is + : Agglutination

Polystyrene latex particles (0.8-1 m in dia) : adsorbs several Ag

Very sensitive test Adv : yield high titres Disadv : more false Positives Reverse passive agglutination tests : detect Ag Instead of Ag , Antibody is adsorbed to carrier particles