SEROLOGY TESTS Flashcards by astrum glen

PRESERVING SERUM

Physical- refrigerate for 72 hrs at 4-6C.

Chemical- add 0.001g Merthiolate powder per mL of serum or 5% phenol or
tricresol at 0.1ml/ml of serum

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inactivation of serum

Physical- heat serum at 56C for 30 mins
or heat at 60-62C for 3-4 mins

Chemical- Add choline chloride

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–Frequency of positive result obtained in the testing of a
population who are truly positive

Sensitivity

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–Proportion of negative test results obtained on the population
who lack the antibody being detected

Specificity

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–Reaction between a single antigenic determinant (epitope)
and single antibody

Affinity

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–Strength of binding between an Antigen with many
determinants and multivalent Antibody.

Avidity

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immunologic reactions

primary, secondary and tertiary AG-AB rxn -

PRIMARY AG-AB REACTION
- MOST SENSITIVE
–No visible reaction, first union of Ag-Ab
–Farr, Equilibrium, Dialysis, ELISA, RIA , IFA
SECONDARY AG-AB REACTION - LESS
SENSITIVE
–Visible reaction
–Precipitation, Agglutination, Neutralization, complement fixation
TERTIARY AG-AB REACTION
–In vivo Ag-Ab reaction
–Phagocytosis, Opsonization, chemotaxi

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Process whereby specific antigens aggregate
to form larger visible clumps when the
corresponding specific antibody is present in
the serum. Clumping and sedimentation of
Particulate Ag/Ab complexes

agglutination

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Agglutinins vs
Agglutinogen

 Agglutinins- Antibodies that agglutinate
antigen
 Agglutinogen – Associated antige

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stages of agglutination

sensitization
elution

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– First phase of agglutination
– Physical attachment of antibody molecules to antigen on erythrocyte membrane
– Reversible

SENSITIZATION

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Subsequent release of antibody into
surrounding medium by manipulation of the
physical condition to break antigen-antibody
complex

ELUTION

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FACTORS AFFECTING HEMAGLUTTINATIOBN

PARTICLE CHARGE
* Zeta potential
ELECTROLYTE CONCENTRATION AND VISCOSITY
ANTIGEN-TO-ANTIBODY RATIO
* ZONE OF EQUIVALENCE- no. of multivalent sites of antigen and
antibodies are equal
* PROZONE- excess antibody
* POST ZONE- excess antigen
ANTIGENIC DETERMINANTS
PHYSICAL CONDITION
* pH – 6.5-7.5
* Temperature- cold reacting, warm reacting
* Time- 15 -60 minutes depending on the antibod

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Establishment of cross-links between
sensitized particles and antibodies ~
agglutination

LATTICE FORMATION

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Forces involved in Antigen-Antibody
Binding:
1. Electrostatic Forces (Ionic Bonds)
2. Van der Waals Forces (London Dispersion
Forces)
3. Hydrogen Binding
4. Hydrophobic Binding

Electrostatic Forces (Ionic Bonds)
Van der Waals Forces (London Dispersion
Forces)
Hydrogen Binding
Hydrophobic Binding

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TYPES OF AGGLUTINATION

DIRECT
INDIRECT
b) Passive
AGGLUTINATION INHIBITION
COAGGLUTINATION
HEMAGGLUTINATION

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– Agglutination of natural Ag (ABO, Rh, Cold agglutination)

DIRECT AGGLUTINATION

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–Agglutination of particulate Ag (latex, charcoal, bentonite)

INDIRECT AGGLUTINATION

a) Reverse Passive – for Ag detection ; Uses antibody
coated particles (CRP latex test)

b) Passive – for Ab detection ; Uses antigen coated
particles (ASO latex test)

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CARRIWER OF COAGLUTINATION

Carrier: Bacterium

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BLOCKING PRINCIPLE

AGGLUTINATION INHIBITION

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TYPES OF HEMAGLUTTINATION

a) Direct – natural Ag

b) Indirect - Needs AHG to effect hemagglutination
(Coomb’s)

c) Passive Hemagglutination – uses passive Ag ; (+)
Hemagglutination:carpet / Mat cells)

d) Hemagglutination inhibition- Blocking ; (+) No
hemagglutination : Button cell

e) Virus Hemagglutination – influenza, mumps

f) Virus hemagglutination inhibition – influenza, mumps, german measles

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 Aggregation of soluble test antigens
 Interaction of Ag with Ab in correct proportion
 Layering of antigen in solution over a small volume of
antiserA

PRECIPITATION

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MECHANISM OF PRECIPITATIOB

 Mechanism : sensitization and Lattice formation

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TYPES OF PRECIPITATION

Gel precipitation
 Immunodiffusion (ID)
* RID (Single ID)
 Fahey
 Mancini
* Ouchterlony (Double)
 Electroimmunodiffusion (EID)
Liquid precipitation
 Lancefield
 Neufield quellung

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– One reactant (Ag or Ab) remains fixed in gel – Other reactant is allowed to moved – Interaction with the reagent that is immobilized – Reagents become fixed in the gel if they are added to the gel medium while it is in liquid form

SINGLE IMMUNODIFFUSION TECHNIQUE

– Both reactants (Ag and Ab) diffuse within a gel – Both reagents are added after gel has set

DOUBLE IMMUNODIFFUSION TECHNIQUE

A simple , specific method for identification and quantification of a number of proteins found in serum and other body fluids

RADIAL IMMUNODIFFUSION (RID)

Internal reactants like specific antibodies added to buffered agarose medium, and serum containing standard volume of CHON or Ag is placed in well, centered in agarose

RADIAL IMMUNODIFFUSION (RID)

RID MEHOD

1. FAHEY (KINETIC DIFFUSION) * Kinetic approach in which the ring diameter is read at a specified time 18 hours to 24 hours 2. MANCINI (ENDPOINT DIFFUSION) * Ring diameter is read after diffusion is completed, usually after 48 hours of incubation

RID RESULT : INTERPRETATION : QUANTITATIVE :

RESULT : PRECIPITIN RING INTERPRETATION : DIAMETER OF THE RING IS PROPORTIONAL TO THE CONCENTRATION QUANTITATIVE : IG LEVELS, SERUM CHONS, COMPLEMENT COMPONENTS

First method used in establishing relationship of HBsAg to Hepa B. Both reactants (Ag and Ab) diffuse within a gel. Thus, both reagents are added after the gel has set

OUCHTERLONY

Ag/ab complexes form and precipitate when the 2 reactants meet at the equivalence zone

OUCHTERLONY

Use to determine the relationship between Ag and Ab Both Ag and Ab diffuse

OUCHTERLONY

OUCHTERLONY RESULT

–Identity : SINGLE SMOOTH ARC/ CURVE LINE Ab is precipitating identical Ag specificities –Non-identity : CROSS- No common antigen determinants –Partial identity : SPUR LINE- Antigens are Not identical but Common Antigen determinan

Immunodiffusion reaction in a support medium with the use of electric current to enhance mobility of reactants and to increase movement toward one another

ELECTROIMMUNIDIFFUSION

MEHODS OF EID

COUNTERCURRENT IMMUNOELETROPHORESIS (CIE) ROCKET ELECTROPHORESIS/ LAURELL TECHNIQUE

PRINCIPLE: Antibody : Positively charged : Migrates toward the cathode (-) Antigen : negatively charged : Migrates toward the anode (+)

ELECTROIMMUNIDIFFUSION

* Antigen and antibody migrate toward each other by electrophoresis * Used only when Ag and Ab have opposite charges

COUNTERCURRENT IMMUNOELETROPHORESIS

* Ag and Ab different charges at selected pH * Band coverage finally meet at the middle, when all antigen is precipitated , the precipitin patter resembles that of a shooting rocket

ROCKET ELECTROPHORESIS/ LAURELL TECHNIQUE

IMMUNOELECTROPHORESIS RESULT

METHOD * Ag is separated by electrophoresis * Ab is placed in trough cut in the agar INTERPRETATION * Precipitin represent individual antigen ADVANTAGE * Reliable and accurate method fro detecting structural abnormalities and concentration changes in proteins * Useful in screening circulating immune complexes/ Myeloma proteins APPLICATIONS * Serum- detection of monoclonal gammopathy * Urine- detection of Bence Jones Protein

A cellulose acetate strip impregnated with antiserum is placed over the separate proteins after serum , urine or CSF are electrophoresed * Diffusion of antiserum into the gel occurs rapidly, resulting in precipitation * The cellulose acetate strip are then removed and the precipitin bands are stained

IMMUNOFIXATION/RESSLER TECHNIQUE

Antigens are separated by Poly Acrylomide Gel Electrophoresis (PAGE) and trans-blotted onto nitrocellulose/nylon membranes

WESTERN BLOT

* Antibodies in serum react with specific antigens * Signals are detected according to the principles of test systems * Antibodies against microbes with numerous cross-reacting antibodies identified more specifically

WESTERN BLOT

NEUTRALIZATION RESULT

* Virus (Ag) + Serum (Ab) -> Injected (test animal) -> Observed for reaction * (+) Neutralization: Animal does not die * (-) Neutralization: Animal died

TOXIN NEUTRALIZATION

* ASO titration test (In vitro) * Animal protection test (In Vivo) * Schick and Dick test (In Vivo)

VIRUS NEUTRALIZATION

* Pock and Plaque reduction * Tissue culture technique * Metabolic inhibition test * In vivo and In ovo test

COMPLEMENT FIXATION PRINCIPLE METHOD AND RESULT

PRINCIPLE :  Ag + Ab = Ag-Ab complex + COMPLEMENT = (FIXED) unable to react to other cells METHOD : * Rgt (Ag) + Unknown (Ab) + Guinea Pig serum + Sensitized sheep RBC + Rabbit anti sera * Guinea pig serum - best source of complement * Rabbit anti-sera- best source of hemolysin/ amboceptor (use to sensitized the indicator cell) * Sensitized sheep’s RBC – indicator cells RESULT : (+)- No Hemolysis (-) – Hemolysi

A specific type of precipitation that occurs over a narrow range of Ag concentration Aggregation of colloidal particles (clumping) in a serological reaction

FLOCCULATION

FLOCCULATION TESTS

VDRL RPR TRUST USR

any substance that will complex to another substance; the substance to be measured

LIGAND

one reactant is labeled so that the amount of binding can be measured

LIGAND ASSAY

2 TYPES OF MMUNOASSAY

1. HETEROGENOUS 2. HOMOGENOUS

* Involve a solid phase (microwell, beads) * Require washing step to remove unbound antigens or antibodies * Competitive or Non competitive

HETEROGENOUS

* Liquid phase only * Do not require washing * Faster and easier to automate

HOMOGENOUS

CONSTITUENT OF LBELED IMMUNOASSAY

1. LABLED ANALYTES 2. ANTIBODIES 3. STANDARD OR CALIBRATION 4.SEPARATION METHODS 5.LABEL DETECTION

A. This method uses LABELED ANTIBODY that is present in excess. B. The amount of radioactivity is directly proportional to the concentration of antige

NON-COMPETITIVE IMMUNORADIOMETRIC ASSAYS (IRMA)

2 METHOD OF RADIOMMMUNOASSAY

LIQUID PHASE RIA * Determination of Radioactivity of analytes SOLID PHASE RIA * Hormones like insulin, GH, ACTH, T3, T4 and estrogen * Serum proteins like CEA, anti-DNA

APPLICATION FO RIA

1. RADIOALLERGO SORBENT TEST (RAST) 2.RADIOIMMUNOSORBENT TEST (RIST)

 Used to detect allergenic antibodies (allergic)  Employs a paper disc + Patient serum → Washed with saline → Radioactively labeled anti serum specific for human IgE is applied to the disc → Measure the IgE

RADIOALLERGO SORBENT TEST (RAST)

 Measures the total IgE concentration in serum (allergic and parasitism)  Uses a paper + Patient serum → IgE in the serum reacts specifically with disc → Disc is washed → Addition of radiolabeled antibody (sheep or rabbit) to human IgE → Measure the total Ige

RADIOIMMUNOSORBENT TEST (RIST)

ADV AND DIS OF RIA

ADVANTAGE: 1. Sensitive and Precise technique for determining trace amounts of analytes that are small in size DISADVANTAGE: 1. Health hazard is involved in working radioactive substance 2. Disposal of radioactive wastes in an environmental problem 3. RIA require expensive equipment

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