Lecture Presented by Dr.D.W.DeshkarDWD

COMPLEMENT FIXATION TESTCOMPLEMENT FIXATION TEST• Introduction:

The complement fixation test (CFT) was extensively used in syphilis serology after being introduced by Wasserman in 1909.

Complement is a protein (globulin) present in normal serum.

Whole complement system is made up of nine components: C1 to C9

Complement proteins are heat labile and are destroyed by heating at 56°C for 20 – 30 minutes.

Complement binds to Ag-Ab complex When the Ag is an RBC it causes lysis of RBC’s.

PrinciplePrinciple• Complement takes part in many of the immunological

reactions. It gets absorbed during the combination of antigens and antibody.

• This property of antigen–antibody complex to fix the complement is used in complement fixation test for the identification of specific antibodies.

• The hemolytic system containing sheep erythrocytes (RBC) and its corresponding antibody (amboceptor) is used as an indicator which shows the utilization or availability of the complement.

• If the complement is fixed then there will be no lysis of sheep erythrocytes, thus denoting a positive test.

• If the complement is available then there will be haemolysis which is a property of complement, denoting a negative test.

Components of CFTComponents of CFT• The test requires five reagents and is carried out in two steps.

Test System• Antigen: It may be soluble or particulate.

• Antibody: Human serum (May or may not contain Antibody towards specific Antigen)

• Complement: It is pooled serum obtained from 4 to 5 guinea pigs. It should be fresh or specially preserved as the complement activity is heat labile (stored at -30 °C in small fractions). The complement activity should be initially standardized before using in the test.

Indicator System (Haemolytic system)• Erythrocytes: Sheep RBC

• Amboceptor (Hemolysin): Rabbit antibody to sheep red cells prepared by inoculating sheep erythrocytes into rabbit under standard immunization protocol.

Positive TestPositive Test• Step 1:

At 37°CAntigen + Antibody + Complement Complement gets fixed

(from serum) 1 Hour

• Step 2: At 37°C

Fixed Complement complex + Haemolytic system No haemolysis 1 Hour (Test Positive)

Negative TestNegative Test

• Step 1: At 37°C

Antigen + Antibody absent + Complement Complement not fixed 1 Hour

• Step 2: At 37°C

Free Complement + Haemolytic system Haemolysis 1 Hour (Test Negative)

Results and Interpretations:Results and Interpretations:

• No haemolysis is considered as a positive test.

• Haemolysis of erythrocytes indicative of a negative test.

1 2 3 4

A

B

Microtiter plate showing Haemolysis (Well A3, A4 and B4) and No Haemolysis (Well A1,A2,B1 &B2)

Indirect complement fixation testIndirect complement fixation test

• Seras of duck, turkey, parrot, horse, cat unable to fix guinea pig complement.

• For such seras indirect complement test employed.• Test is set up in duplicate.

• After first step as in routine complement fixation test, the standard antiserum known to fix complement is added to one set.

• If test serum contained antibody ,the antigen being utilised in the first step.

• Standard antiserum thus unable to fix the complement.

• Haemolysis in indirect test Positive result.

Conglutinating complement Conglutinating complement absorption testabsorption test

• Alternative method for systems that are unable to fix guinea pig complement.

• Uses horse complement which being non hemolytic.

• Indicator system :- Sensitised sheep erythrocytes mixed with bovine serum.

• Bovine serum contains a beta globulin component called Conglutinin , which acts as Ab to complement.

• Conglutinin causes agglutination of sensitised sheep erythrocytes (Conglutination) if they have combined with complement.

• If the horse complement utilised by the Ag – Ab interaction during first step = No agglutination of sensitised cells.

Other complement dependent Other complement dependent serological testsserological tests

• 1. Immobilisation Test – When live motile Treponema pallidum is mixed with patient’s serum in presence of complement the organism is immobilised = Treponema pallidum immobilisation test.

• 2. Immune Adherence – The Ag – Ab complexes in some bacteria such as V.cholerae, T.pallidum adhere to particulate material like RBCs, platelates. This bacterial adherence to cell

Immune Adherence .

• Cytolytic or Citocidal Tests – Live V. cholerae + Its Antibody + Complement Bacterium lysed.

Basis for measurement of anticholera Ab.

Neutralisation TestsNeutralisation Tests

• In Vivo Tests –

1. Toxigenicity Test – Done for detection of toxin of C.diphtheriae

2. Shick Test –

• In Vitro Tests –

1.Antistreptolysin ‘O’ (ASO) Test –

2. Virus neutralisation Tests –

3. Nagler Reaction -

OpsonisationOpsonisation

• A process by which opsonin ( complement) combines with an antigen and facilitates phagocytosis.

• Opsonin Index – Ratio of phagocytic activity of the patient’s blood for a given bacterium , to that of a normal individual.

• Phagocytic Index – Average number of phagocytosed bacteria per PMNs from stained blood films.

ImmunofluorescenceImmunofluorescence• Immunofluorescence is the labeling of antibodies or antigens

with fluorescent dyes. • This technique is sometimes used to make viral plaques more

readily visible to the human eye. • Immunofluorescent labeled tissue sections are studied using a

fluorescence microscope.

• Fluorescence is the property of certain dyes which absorb rays of one particular wavelength ( ultraviolet light)& emit rays with different wavelength ( visible light)

• Fluorescein is a dye which emits greenish fluorescence under UV light. It can be tagged to immunoglobulin molecules.

ImmunofluorescenceImmunofluorescence

• There are two ways of doing IF staining– Direct immunofluorescence– Indirect immunofluorescence

1. Direct immunofluorescence• Ag is fixed on the slide• Fluorescein labeled Ab’s are layered over it• Slide is washed to remove unattached Ab’s• Examined under UV light in an fluorescent microscope• The site where the Ab attaches to its specific Ag will

show apple green fluorescence• Use: Direct detection of Pathogens or their Ag’s in

tissues or in pathological samples

ImmunofluorescenceImmunofluorescence

• Direct Immunofluorescence

ImmunofluorescenceImmunofluorescence

2. Indirect immunofluorescence:

• Indirect test is a double-layer technique

• The unlabelled antibody is applied directly to the tissue substrate

• Treated with a fluorochrome-conjugated anti-immunoglobulin serum

ImmunofluorescenceImmunofluorescence

Indirect immunofluorescence of iron-regulated cell wall mannoprotein FIT1 of S. cerevisiae

 Immunofluorescence image of Cryptosporidium parvum oocysts

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

Fluorescence polarization Fluorescence polarization immunoassay (FPIA)immunoassay (FPIA)

• Principle• Add reagent antibody and fluorescent-tagged

antigen to patient serum • Positive test

– Antigen present in patient serum binds to reagent leaving most tagged antigen unbound

– Unbound labeled antigens rotate quickly reducing amount of polarized light produced

• Negative test– If no antigen present in patient serum, tagged

antigen binds to reagent antibody – Tagged antigen-antibody complexes rotate slowly

giving off increased polarized light

• Berson & Yallow (1959) first described RIA.

• RIA utilised for quantitation of hormones, drugs, hepatitis B surface antigen, IgE, & viral antigens.

• Ag or Abs are labeled with radioactive isotopes and traced.

• Can detect antigens up to picogram ( 10-12g).

• RIA is based on competition for fixed amounts of specific antibody between a known radiolabeled Ag & unknown unlabeled (test) Ag.

• After Ag – Ab reaction, the Ag is separated into ‘Free’ & ‘Bound’ fractions & their radioactivity measured.

RadioimmunoassayRadioimmunoassay( ( RIARIA))

Enzyme Linked Immunosorbent Assay Enzyme Linked Immunosorbent Assay (ELISA)(ELISA)

• ELISA – an immunological test, using an enzyme as a label to determine presence of target protein.

• The enzyme linkage or labeling allows you to follow your target protein and if present (qualify) and at what amounts (quantify).

• An enzyme conjugate is an enzyme bound or joined with an antibody which binds with your target protein.

• This enzyme labeling is a safe and effective way to track your antibody.

TYPES OF ELISATYPES OF ELISA

• There are three main methods that form the basis to all ELISAs.

1. DIRECT ELISA

2. INDIRECT ELISA

3. SANDWICH ELISA

• All these systems (1-3) can be used in assays called:

4. COMPETITION OR INHIBITION ELISA

DIRECT ELISADIRECT ELISA

INDIRECT ELISAINDIRECT ELISA

SANDWICH DIRECTSANDWICH DIRECT

SANDWICH INDIRECTSANDWICH INDIRECT

ELISA ANIMATIONELISA ANIMATION

Uses of ELISAUses of ELISA

• Screening donated blood for evidence of viral contamination by – HIV-1 and HIV-2 (presence of anti-HIV antibodies) – hepatitis C (presence of antibodies) – hepatitis B (testing for both antibodies and a viral

antigen)

• Measuring hormone levels – HCG (as a test for pregnancy) – LH (determining the time of ovulation) – TSH, T3 and T4 (for thyroid function)

• Detecting infections – sexually-transmitted agents like HIV, syphilis and

Chlamydia – hepatitis B and C – Toxoplasma gondii

• Detecting allergens in food and house dust • Measuring "rheumatoid factors" and other

autoantibody in autoimmune diseases like lupus erythematosus

• Measuring toxins in contaminated food• • Detecting illicit drugs, e.g.,

– cocaine – opiates

Uses of ELISAUses of ELISA

ChemiluminescenceChemiluminescence• Chemiluminescence

Is the emission of light with limited emission of heat (luminescence), as the result of a chemical reaction.

Chemiluminescence compounds are used in this method to provide the signal (i.e. light) during the Ag- Ab reaction.

• Application –

- Drug sensitivity testing of M.tuberculosis.

Immunoelectroblot TechniquesImmunoelectroblot Techniques

• It combines the sensitivity of enzyme immunoassay with greater specificity.

• This technique – Combination of three separate procedures

1) Separation of ligand – Ag components by polyacrylamide gel electrophoresis.

2) Blotting of electrophoresed ligand fraction on nitrocellulose membrane strips.

3) Enzyme immunoassay or ( radioimmunoassay) to - detect Ab in test sera against various ligand fraction bands

- probe with known antisera against specific Ag bands. - Western blot test - serodiagnosis of HIV.

Immunochromatographic TestsImmunochromatographic Tests• The test system is a small cassette containing a membrane

impregnated with anti – HBsAg Ab colloidal dye conjugate.

• The membrane is exposed at three windows on the cassette.

• The test serum is dropped into first window .

• As the serum travels upstream by capillary action, a coloured band appears at the second window (test site) if the serum contains HBsAg , due to the formation of HBsAg antibody conjugate complex. = Positive reaction

• Absence of coloured band at test site = Negative reaction.

• Simultaneously the coloured band appears at the third window, which forms the inbuilt control, in the absence of which the test is invalid.

Immunoelectronmicroscopic TestsImmunoelectronmicroscopic Tests

• 1. Immunoferritin Test – Ferritin (electron dense subs.) conjugated Ab used to react with an Ag &

Ag-Ab reaction visualised under E.M.

• 2. Immunoelectronmicroscopy – Viral particles mixed with specific antisera & observed under E.M. which are seen as clumps. Hepatitis A virus, Virus causing diarrhoea.

• 3. Immunoenzyme Test – Some enzymes such as peroxidase can be conjugated with Ab. Tissue sections are treated with peroxidase labelled antisera to detect corresponding Ag. The peroxidase bound to Ag visualised under E.M.

To summeriseTo summerise• Agglutination Reactions –

1. Widal Test2. Weil Felix Test3. Brucellosis Test 4. Cold agglutination Test5. Paul Bunnel Test6. Rose Waller Test for RA7.Latex agglutination Test for

RA8. Coagglutination Test9. Coomb’s Test10. Blood Grouping & Cross

matching.

• Precipitation Reactions –1. V.D.R.L.Test2. R.P.R. Test3. Kahn Test4. C.F.T.5. Immunoelectrophoresis6. Counter

Immunoelectrophoresis7. Antitoxin neutralisation Test8. Antistreptolysin O Test9. Reverse Passive

Haemagglutination Test10. ELISA11. Western Blot Test12. Polyacrylamide Gel

Electrophoresis