SEROLOGICAL TESTS Flashcards
how to preserve serum
PHYSICL = ref for 72 hours at 4-6C
CHEM = add 0.001 mg methiolate powder per ml of serum or 5% phenol or tricresol at 0.1ml/ml of serum
how to inactivate serum
Physical- heat serum at 56C for 30 mins
or heat at 60-62C for 3-4 mins
Chemical- Add choline chloride
–Frequency of positive result obtained in the testing of a
population who are truly positive
sensitivity
–Proportion of negative test results obtained on the population
who lack the antibody being detected
Specificity
–Reaction between a single antigenic determinant (epitope)
and single antibody
Affinity
–Strength of binding between an Antigen with many
determinants and multivalent Antibod
Avidity
IMMUNOLOGIC REACTIONS
- PRIMARY AG-AB REACTION
- SECONDARY AG-AB REACTION
- TERTIARY AG-AB REACTION
RESULTS OF IMMUNOLOGIC REACTIONS
1.. PRIMARY AG-AB REACTION - MOST SENSITIVE
–No visible reaction, first union of Ag-Ab
2. SECONDARY AG-AB REACTION - LESS
SENSITIVE
–Visible reaction
3. TERTIARY AG-AB REACTION
–In vivo Ag-Ab reaction
EXAMPLE OF IMMMUNOLOGIC REACTIONS
. 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, chemotaxis
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
Particulate Ag:
Bacteria, RBC, WBC, Latex
particle
Agglutinins
Antibodies that agglutinate
antigen
– Associated antigen
Agglutinogen
STAGES OF AGGLUTINATION
- SENSITIZATION
- ELUTION
Physical attachment of antibody molecules to
antigen on erythrocyte membrane
SENSITIZATION
- 1ST PHASE OF AGLGUTINATION
- REVERSIBLE
Subsequent release of antibody into
surrounding medium by manipulation of the
physical condition to break antigen-antibody
complex
ELUTION
FACTORS AFFECTING AGGLUTINATION
- PARTICLE CHARGE
2.. ELECTROLYTE CONCENTRATION AND VISCOSITY - ANTIGEN-TO-ANTIBODY RATIO
- ANTIGEN DETERMINANT
- PHYSICAL CONDITON
ANTIGEN ANTIBODY RATIO TYPES
- ZONE OF EQUIVALENCE- no. of multivalent sites of antigen and
antibodies are equal - PROZONE- excess antibody
- POST ZONE- excess antigen
PHYSICAL CONDITION
- pH – 6.5-7.5
- Temperature- cold reacting, warm reacting
- Time- 15 -60 minutes depending on the antibody
Establishment of cross-links between
sensitized particles and antibodies ~
agglutination
LATTICE FORMATION
Forces involved in Antigen-Antibody
Binding:
(LATTICE FORMATION)
- Electrostatic Forces (Ionic Bonds)
- Van der Waals Forces (London Dispersion
Forces) - Hydrogen Binding
- Hydrophobic Binding
TYPES OF AGGLUTINATION
- DIRECT
- INDIRECT
- AGGLUTINATION INHIBITION
- COAGLUTTINATION
Agglutination of natural Ag (ABO, Rh, Cold agglutination)
DIRECT
Agglutination of particulate Ag (latex, charcoal, bentonite)
INDIRECT
– Blocking principle
– Positive result: No agglutination
AGGLUTINATION INHIBITION
Carrier: BacteriuM
COAGGLUTINATION
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
PRECIPITATION
Aggregation of soluble test antigens
Interaction of Ag with Ab in correct proportion
Layering of antigen in solution over a small volume of
antisera
Mechanism : sensitization and Lattice formation
TYPES OF PRECIPITATION
- Gel precipitation
Immunodiffusion (ID)
* RID (Single ID)
Fahey
Mancini
* Ouchterlony (Double)
Electroimmunodiffusion (EID) - Liquid precipitation
Lancefield
Neufield quellung
SINGLE IMMUNODIFFUSION
TECHNIQUE
– 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
TECHNIQUE
– Both reactants (Ag and Ab) diffuse within a gel
– Both reagents are added after gel has set
DOUBLE IMMUNODIFFUSION
– Both reactants (Ag and Ab) diffuse within a gel
– Both reagents are added after gel has set
A simple , specific method for identification and quantification of a number
of proteins found in serum and other body fluids
RADIAL IMMUNODIFFUSION
(RID)
PRINCIPLE:
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
SINGLE IMMUNODIFFUSION MEDTHOD AND RESULT
METHOD:
* Antibody in gel
* Antigen in well
RESULT : PRECIPITIN RING
INTERPRETATION : DIAMETER OF THE RING IS PROPORTIONAL TO
THE CONCENTRATION
QUANTITATIVE : IG LEVELS, SERUM CHONS, COMPLEMENT
COMPONENTS
METHODS OF RID
- FAHEY (KINETIC DIFFUSION)
* Kinetic approach in which the ring diameter is
read at a specified time 18 hours to 24 hours - MANCINI (ENDPOINT DIFFUSION)
* Ring diameter is read after diffusion is
completed, usually after 48 hours of incubation
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
PUCHTERLONY METHOD ND RESULT
RESULTS:
–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 determinant
Immunodiffusion reaction in a support medium with the
use of electric current to enhance mobility of reactants
and to increase movement toward one another
GEL PRECIPITATION:ELECTROIMMUNODIFFUSION
PRINCIPLE OF GEL PRECIPITATION: ELECTROIMMUNODIFFUSION
PRINCIPLE:
Antibody : Positively charged : Migrates toward the
cathode (-)
Antigen : negatively charged : Migrates toward the anode
(+)
METHODS OF EID
COUNTERCURRENT IMMUNOELETROPHORESIS
(CIE)
ROCKET ELECTROPHORESIS/ LAURELL
TECHNIQUE
- Antigen and antibody migrate toward each other by
electrophoresis - Used only when Ag and Ab have opposite charges
COUNTERCURRENT IMMUNOELETROPHORESIS
(CIE)
When RID is applied with current, it becomes
ROCKET ELECTROPHORESIS/ LAURELL
TECHNIQUE
PRINCIPLE:
* 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
Method, interpretation, adv and application
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 OR RESSLER’S TECHNIQUE
WESTERN BLOT
RINCIPLE
* Antigens are separated by Poly Acrylomide Gel
Electrophoresis (PAGE) and trans-blotted onto
nitrocellulose/nylon membranes
* 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
PPRINCIPLE OF NEUTRALIZATION
- 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
PRINCIPLE, METHOD AND RESULT OD COMPLEMENT FIXATION
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
(-) – Hemolysis
A specific type of precipitation that occurs
over a narrow range of Ag concentration
Aggregation of colloidal particles
(clumping) in a serological reaction
FLOCCULATION
TYPES OF FLOCCULATION
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
TYPE OF IMMUNOASSAY
HETEROGENOUD AND HOMOGENOUES
- 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
CONSTITUENTS OF LABELED IMMUNOASSAY
- LABELED ANALYTES
- ANTIBODIES
- STANDARDS OR CALIBRATORS
- SEPARATION METHODS
- LABEL DETECTION
LABELED ANALYTES
- RADIOACTIVE ELEMENTS
– 125I, 131I, 3H –RIA - ENZYMES – Horse radish peroxidase and alkaline phosphatase– EIA
- FLUORESCENT DYES – FITC / TRITC – FIA
*CHEMILUMINESCENT TAGS – Luminal,
Acridium esters, peroxyoxalates, ruthenium, derivates and dioxetanes - CIA
is a very specific antibody derived from a
single antibody – producing cell that has been cloned or
duplicated
Monoclonal Antibody
If ______is used, it has to be purified to cut down on
cross-reactivity with other substance in the patient specimen
polyspecific serum
Unlabeled analytes with known concentrations of the substance to
be measured
STANDARDS OR CALIBRATOR
Adsorption on Particles such as dextran-coated charcoal, tarc, silica or
cellulose
Precipitation of antigen-antibody complex
Solid phase vehicle – Polystyrene test tubes, microtiter plates and
glass
SEPARATION MNETHODS
LABEL DETECTION
RIA – Label Detection is done by a counting syste
called scintillation counters
EIA – Label Detection is done by a Spectrophotometer
FIA and CIA – Label detection is done by
spectrofluorometer, fluorometer, flow cytometer or
fluorescence microscope
COMPETITIVE RIA
A. Analyte being detected competes
with a radio labeled analyte for a
limited number of binding sites
B. The concentration of the
radioactive analyte is in excess, so
that all binding sites on antibody
will be occupied
C. If the patient is present, some of
the binding site will be filled with
unlabelled analyte, thus
decreasing the amount of bound
radioactive label
D. Amount of label in the bound
phase is indirectly proportional to
the amount of patient antigen
present.
NON-COMPETITIVE RIA
A. This method uses LABELED ANTIBODY that is
present in excess.
B. The amount of radioactivity is directly
proportional to the concentration of antigen
2 METHODS OF RADIOIMMNIASSAY
LIQUID ANS OLID PHASE RIA
- Determination of Radioactivity of analytes
LIQUID PHASE RIA
- Hormones like insulin, GH, ACTH, T3, T4 and
estrogen - Serum proteins like CEA, anti-DNA
SOLID PHASE RIA
APPLICATION OF RIA
- 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)
ADVANTAGE AND DISADVANTAGE 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
It can be used to assay both antigens and
antibodies.
ELISA
Either antigen or antibody can be linked to an enzyme :
Horse Radish Peroxidase – Horse Radish
Alkaline Phosphatase – E.coli
G6PD
Glucose Oxidase – Aspergillus niger
Urease
Beta-d-galactosidase – E.coli
Cross linker to join amino groups
Glutaraldehyde
Types of Enzyme Immunoassay
- HETEROGENOUS ENZYME
IMMUNOASSAY
2 TYPES: competitive elisa and nonceompetitive elisa - HOMOGENOUS ENZYME
IMMUNOASSAY
- Enzyme-labeled antigen competes with
unlabeled patient antigen for a limited number
of binding sites on antibody molecules that are
attached to a solid phase.
Competitive ELISA
- The enzyme activity is inversely proportional to
the concentration of the test substance
- They are referred to as indirect ELISA tests
because the enzyme-labelled reagent does not
participate in the initial antigen-antibody
binding reaction - Either antigen or antibody may be bound to
solid phase - The amount of enzyme label is directly
proportional to the amount of the test substance
B. Non-Competitive ELISA
- These are antibody-antibody system in which no
separation step is necessary - Based on principle of change in enzyme activity
as specific antigen-antibody combination occurs - Rapid, simple to perform and adapt easily to
automation but generally less sensitive than
heterogenous assay
HOMOGENOUS ENZYME
IMMUNOASSAY
double antibody sandwich elisa
This is used for the assay of HBV antigens.
PRINCIPLE :
HB Antigen on plastic surface (polystyrene complex) +
Serum containing antibodies → Washed → Addition of
enzyme-labeled specific antibody → Washed →
Addition of enzyme substrate is added → Degradation
of substrate by enzyme = intensity of colored product
→ Measured by spectrophotometer
elisa advantage and disadvantage
ADVANTAGE:
1. Sensitivity of EIA is similar to RIA without
creating health hazard or causing disposal
problem
2. No need for expensive instrumentation
because most assays can be ready by
spectrophotometer or by simply noting the
presence or absence of color
Disadvantages
1. Some specimens contain inhibitors for enzyme
2. The enzymes are sensitive to temperature
Assays that uses fluorescent compounds known as fluorophores or
fluorochromes as labels or conjugates of known antibody molecules
to allow visualization of ag-ab reactions via ultraviolet light and
fluorescence microscope
Unknown antigens can be detected either in fixed tissue sections or
live cells suspensions with sensitivity and specificity
flourescence immunoassay
FLUORESCENT DYE:
FLUORESCEIN ISOTHIOCYANATE – Absorbs maximally
at 490 to 495nm and emits a green color at 517nm
TETRA METHYL RHODAMINE ISOTHIOCYANATE –
Absorbs at 550nm and emits bright red light at 580-585nm
PHYCOBILIPROTEINS – Derived from algae, porphyrins
and chlorophylls all of which exhibit red fluorescence at
over 600nm
Emission of light caused by a chemical reaction
producing an excited molecule that decays back to
its original ground state measured using a
luminometer
chemoliluminescence immunoassay
CHEMILUMINESCENT SUBSTANCE
Luminal
Acridium esters
Peroxyoxalates
Rutherium derivatives
Dioxetanes.
- NITROBLUE TETRAZOLIUM
- Intracellular killing activity of neutrophil
- Positive in CGD
- Positive: YELLOW
- NEPHELOMETRY
- Scattered Light
- Protein (sample) + antisera = insoluble complex = scattered light (measured in
photodiode) - For CRP, Ig, Complement
- FLOW CELL CYTOMETRY
- Fluorescent immunoassay
- FITC – dye conjugated to specific antibody on cell
- Cell + fluorescent Ab = cell fluoresce
- Scattered light
- T and B cell, NK cell , granulocyte, RBC count
MICROLYMPHOCYTOTOXICITY TEST/ COMPLEMENT DEPENDENT CYTOLYSIS
- Detects HLA-A,B,C and E antigens
- Lymphocyte + Anti- HLA-BB Typing Sera + complement
(rabbit serum) + stain with trypan blue or Eosin Y.
5.MIXED LYMPHOCYTE CULTURE
- Responder Cell (Recipient lymphocyte)
- Irradiate Stimulator (donor) lymphocyte – mitomycin
treated - 3H Thymidine (label)
- (+) Uptake of radioactive – dead cell
- (-) No uptake of dye = intact cells
TUBERCULIN TEST
- PPD (purified protein derivative) ID Area of definite
palpable induration of edema after 48 hours - (+) More than or equal 10mm ~ TB but not definite
- (-) Less than 1-mm ~ rule out TB
VOLLMER’S PATCH TEST
- Painless skin test for tuberculosis
- PPD tape patch on arm
- (+) Red area with tiny vesicle (48 hours)
- (+) TB infection but not definite
- (-) Cannot rule out TB
- For children
FREI TEST
- For Lymphogranuloma venerum (LGV)
- Chlamydial Ag – ID
- (+) 7mm diameter papule (read after 48 hours)
SCHICK TEST
- Diphteria anti toxin
- Diphteria toxin ID
- (+) Redness (24-36 hours)
- (+) indicates lack of immunity to diphteria
DICKS TEST
- Erythrogenic toxin→ID
- (+) Redness (24-36 hours)
BRUCELLERGIN test
- Brucella Ag →ID
- (+) Edematous plaque (1-6mm)
- (+) Brucellosis
Histoplasmin , Coccidiodin
- Ag → ID
- (+) Wheal (48 hours)
Toxoplasmin
- Ag →ID →(+) Induration (48 hours)
Trichinella skin test
- Allergen →ID →wheal (20mins)
- Delayed HPS skin test
- Measure T cell function
- Most simple procedure to evaluate T cell function
