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Notes #09 Compliment Fixation Test (CFT) _538 - Google Docs

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Notes #09 Compliment Fixation Test (CFT) _538 - Google Docs

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lablife618
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© © All Rights Reserved
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‭www.chandanmlt.

com‬‭(RanKplus)‬
‭Hand written Notes‬
‭ ntigen and antibody reaction part -III‬
A
‭Introduction :‬
‭ . CFT(compliment fixation test) :‬
C
‭Complement :‬‭the complement system also known as complement‬‭cascade, Is a part of the immune‬
‭system that enhance the ability of antibodies and phagocytic cells to clear microbes and damaged cells‬
‭from an organism , promote inflammation and attack the pathogens cells membrane .‬

‭3 Different complement pathways are :‬


‭A) Classical complement pathway‬
‭B) The alternative complement pathways‬
‭C) Mannose binding lectin pathway.‬
‭Principle of CFT :‬
‭The antigen-antibody complexes have ability to 'fix ' complement. This reaction has no visible effect.‬
‭To detect the fixation of complement , an indicator system consisting of sheep erythrocytes coated‬
‭with‬‭amboceptor‬‭(rabbit antibody to sheep erythrocytes)‬‭is used. Complement can lyse these‬
‭erythrocytes coated with antibodies.‬
‭If complement is fixed and utilized in the antigen-antibody reaction , there is no free complement to‬
‭act on the indicator system and hence no lysis of erythrocytes, which indicates the positive‬
‭complement fixation test.‬
‭Lysis of erythrocytes indicates that complement was not fixed in the first step and therefore, the‬
‭serum is negative for antibodies (negative CFT).‬

‭ rocedure :‬
P
‭The serum (to be tested) should be inactivated by heating at 56°C for 30 minutes to destroy any‬
‭complement activity the serum may have and to remove some non-specific inhibitors of complement.‬
‭The antigen may be used as soluble or particulate. Fresh guinea pig serum is used as source of‬
‭complement. Complement activity is heat labile so it is used as fresh.‬

‭ . A known antigen (cardiolipin/Viral Ag/sheep RBC) is mixed with inactivated patient’s serum‬
1
‭2. Add a measured amount of complement ( Guineapig serum) in the test system‬‭3. The test‬
‭system is incubated at 37°C for about 1 hour.‬
‭4. After 1 hour an indicator system (sensitized RBC) is added to the test system and again‬
‭incubated at 37°C for 30 minutes‬
‭ . If Ag and Ab matches, they form Ag-Ab complex and utilizes complement.‬
5
‭6. Observe the result.‬
‭Observation :‬
‭▪‬‭If hemolysis is observed‬‭: it indicates the absence‬‭of specific antibody in patient serum, so that‬
‭complement has not been used which lysed the sensitized RBC giving hemolysis.‬‭▪‬‭If no‬
‭ emolysis is observed:‬‭it indicates that the patient‬‭serum contains antibody which reacts with Ag‬
h
‭to form Ag-Ab complex and then fix complement .so that no complement is available to‬
‭hemolyse sensitized RBC.‬
‭Result interpretation:‬
‭1.‬‭Positive CFT‬‭: if no hemolysis is observed, it indicates‬‭positive complement fixation test.‬
‭Antigen- antibody reaction and complement fixation occurs, so NO free complement is‬
‭available to lyse the RBC.‬
‭2.‬‭Negative CFT‬‭: if hemolysis of RBC observed, it‬‭indicates negative complement test. NO‬
‭complement fixation occurs, so the complement remain free and it hemolyse the RBC.‬
‭Applications of CFT:‬
‭▪‬‭CFT is used to detect and quantify antibody that‬‭does not agglutinate or precipitate with its‬
‭antigen.‬
‭▪‬‭CFT can detect antibody at level less than 1 microgram‬‭per millilitre.‬
‭▪‬‭CFT is also used to detect antigen‬
‭▪‬‭CFT can be used to‬‭screen large numbers of viral or bacterial infection.‬

‭………End…….‬

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