www.medicos11.

com 2011

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.gamspg.webs.com Page 1
 www.medicos11.com 2011

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 – Mothers serum (incomplete Ab) + RBCs : sensitization of RBC
Rh incmpatinbility) : (fetal blood) RBCs of erythroblastic occurs in vitro  washed  add coombs sera 
infants  washed free of unattached protein  coombs sera agglutination
 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 Polystyrene latex particles (0.8-1 m in dia) : adsorbs several
antisheep RBCs)  add serum  if RA is + : Agglutination 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

www.gamspg.webs.com Page 2