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IS FINALS AND REMOVALS > SEROLOGICAL TESTS > Flashcards

SEROLOGICAL TESTS Flashcards

1
Q

how to preserve serum

A

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

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2
Q

how to inactivate serum

A

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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3
Q

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

A

sensitivity

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4
Q

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

A

Specificity

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5
Q

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

A

Affinity

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6
Q

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

A

Avidity

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7
Q

IMMUNOLOGIC REACTIONS

A
  1. PRIMARY AG-AB REACTION
  2. SECONDARY AG-AB REACTION
  3. TERTIARY AG-AB REACTION
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8
Q

RESULTS OF IMMUNOLOGIC REACTIONS

A

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

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9
Q

EXAMPLE OF IMMMUNOLOGIC REACTIONS

A

. PRIMARY AG-AB REACTION - MOST SENSITIVE
–No visible reaction, first union of Ag-Ab
–Farr, Equilibrium, Dialysis, ELISA, RIA , IFA

  1. SECONDARY AG-AB REACTION - LESS
    SENSITIVE
    –Visible reaction
    –Precipitation, Agglutination, Neutralization, complement fixation
  2. TERTIARY AG-AB REACTION
    –In vivo Ag-Ab reaction
    –Phagocytosis, Opsonization, chemotaxis
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10
Q

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

A

AGGLUTINATION

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11
Q

Particulate Ag:

A

Bacteria, RBC, WBC, Latex
particle

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12
Q

Agglutinins

A

Antibodies that agglutinate
antigen

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13
Q

 – Associated antigen

A

Agglutinogen

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14
Q

STAGES OF AGGLUTINATION

A
  1. SENSITIZATION
  2. ELUTION
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15
Q

Physical attachment of antibody molecules to
antigen on erythrocyte membrane

A

SENSITIZATION
- 1ST PHASE OF AGLGUTINATION
- REVERSIBLE

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16
Q

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

A

ELUTION

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17
Q

FACTORS AFFECTING AGGLUTINATION

A
  1. PARTICLE CHARGE
    2.. ELECTROLYTE CONCENTRATION AND VISCOSITY
  2. ANTIGEN-TO-ANTIBODY RATIO
  3. ANTIGEN DETERMINANT
  4. PHYSICAL CONDITON
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18
Q

ANTIGEN ANTIBODY RATIO TYPES

A
  • ZONE OF EQUIVALENCE- no. of multivalent sites of antigen and
    antibodies are equal
  • PROZONE- excess antibody
  • POST ZONE- excess antigen
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19
Q

PHYSICAL CONDITION

A
  • pH – 6.5-7.5
  • Temperature- cold reacting, warm reacting
  • Time- 15 -60 minutes depending on the antibody
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20
Q

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

A

LATTICE FORMATION

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21
Q

Forces involved in Antigen-Antibody
Binding:

(LATTICE FORMATION)

A
  1. Electrostatic Forces (Ionic Bonds)
  2. Van der Waals Forces (London Dispersion
    Forces)
  3. Hydrogen Binding
  4. Hydrophobic Binding
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22
Q

TYPES OF AGGLUTINATION

A
  1. DIRECT
  2. INDIRECT
  3. AGGLUTINATION INHIBITION
  4. COAGLUTTINATION
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23
Q

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

A

DIRECT

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24
Q

Agglutination of particulate Ag (latex, charcoal, bentonite)

A

INDIRECT

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25
Q

– Blocking principle
– Positive result: No agglutination

A

AGGLUTINATION INHIBITION

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26
Q

Carrier: BacteriuM

A

COAGGLUTINATION

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27
Q

TYPES OF HEMAGLUTTINATION

A

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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28
Q

PRECIPITATION

A

 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

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29
Q

TYPES OF PRECIPITATION

A
  1. Gel precipitation
     Immunodiffusion (ID)
    * RID (Single ID)
     Fahey
     Mancini
    * Ouchterlony (Double)
     Electroimmunodiffusion (EID)
  2. Liquid precipitation
     Lancefield
     Neufield quellung
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30
Q

SINGLE IMMUNODIFFUSION
TECHNIQUE

A

– 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

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31
Q

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

A

DOUBLE IMMUNODIFFUSION

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

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32
Q

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

A

RADIAL IMMUNODIFFUSION
(RID)

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33
Q

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

A

RADIAL IMMUNODIFFUSION
(RID

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34
Q

SINGLE IMMUNODIFFUSION MEDTHOD AND RESULT

A

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

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35
Q

METHODS OF RID

A
  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
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36
Q

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

A

OUCHTERLONY

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37
Q

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

A

OUCHTERLONY

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38
Q

PUCHTERLONY METHOD ND RESULT

A

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

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39
Q

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

A

GEL PRECIPITATION:ELECTROIMMUNODIFFUSION

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40
Q

PRINCIPLE OF GEL PRECIPITATION: ELECTROIMMUNODIFFUSION

A

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

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41
Q

METHODS OF EID

A

COUNTERCURRENT IMMUNOELETROPHORESIS
(CIE)

ROCKET ELECTROPHORESIS/ LAURELL
TECHNIQUE

42
Q
  • Antigen and antibody migrate toward each other by
    electrophoresis
  • Used only when Ag and Ab have opposite charges
A

COUNTERCURRENT IMMUNOELETROPHORESIS
(CIE)

43
Q

When RID is applied with current, it becomes

A

ROCKET ELECTROPHORESIS/ LAURELL
TECHNIQUE

44
Q

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

A

ROCKET ELECTROPHORESIS/ LAURELL
TECHNIQUE

45
Q

IMMUNOELECTROPHORESIS

Method, interpretation, adv and application

A

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

46
Q
  • 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
A

IMMUNOFIXATION OR RESSLER’S TECHNIQUE

47
Q

WESTERN BLOT

A

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

48
Q

PPRINCIPLE OF NEUTRALIZATION

A
  • Virus (Ag) + Serum (Ab) -> Injected (test animal) -> Observed for
    reaction
  • (+) Neutralization: Animal does not die
  • (-) Neutralization: Animal died
49
Q

TOXIN NEUTRALIZATION

A
  • ASO titration test (In vitro)
  • Animal protection test (In Vivo)
  • Schick and Dick test (In Vivo)
50
Q

VIRUS NEUTRALIZATION

A
  • Pock and Plaque reduction
  • Tissue culture technique
  • Metabolic inhibition test
  • In vivo and In ovo test
51
Q

PRINCIPLE, METHOD AND RESULT OD COMPLEMENT FIXATION

A

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

52
Q

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

A

FLOCCULATION

53
Q

TYPES OF FLOCCULATION

A

VDRL
RPR
TRUST
USR

54
Q
  • any substance that will complex
    to another substance; the substance to be
    measured
A

LIGAND

55
Q
  • one reactant is labeled
    so that the amount of binding can be
    measured
A

LIGAND ASSAY

56
Q

TYPE OF IMMUNOASSAY

A

HETEROGENOUD AND HOMOGENOUES

57
Q
  • Involve a solid phase (microwell, beads)
  • Require washing step to remove unbound
    antigens or antibodies
  • Competitive or Non competitive
A
  1. HETEROGENOUS
58
Q
  • Liquid phase only
  • Do not require washing
  • Faster and easier to automate
A
  1. HOMOGENOUS
59
Q

CONSTITUENTS OF LABELED IMMUNOASSAY

A
  1. LABELED ANALYTES
  2. ANTIBODIES
  3. STANDARDS OR CALIBRATORS
  4. SEPARATION METHODS
  5. LABEL DETECTION
60
Q

LABELED ANALYTES

A
  • 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

61
Q

is a very specific antibody derived from a
single antibody – producing cell that has been cloned or
duplicated

A

Monoclonal Antibody

62
Q

If ______is used, it has to be purified to cut down on
cross-reactivity with other substance in the patient specimen

A

polyspecific serum

63
Q

Unlabeled analytes with known concentrations of the substance to
be measured

A

STANDARDS OR CALIBRATOR

64
Q

 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

A

SEPARATION MNETHODS

65
Q

LABEL DETECTION

A

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

66
Q

COMPETITIVE RIA

A

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.

67
Q

NON-COMPETITIVE RIA

A

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

68
Q

2 METHODS OF RADIOIMMNIASSAY

A

LIQUID ANS OLID PHASE RIA

69
Q
  • Determination of Radioactivity of analytes
A

LIQUID PHASE RIA

70
Q
  • Hormones like insulin, GH, ACTH, T3, T4 and
    estrogen
  • Serum proteins like CEA, anti-DNA
A

SOLID PHASE RIA

71
Q

APPLICATION OF RIA

A
  1. RADIOALLERGO SORBENT TEST
    (RAST)
    2.RADIOIMMUNOSORBENT TEST (RIST)
72
Q

 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

A

RADIOALLERGO SORBENT TEST
(RAST)

73
Q

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

A

RADIOIMMUNOSORBENT TEST (RIST)

74
Q

ADVANTAGE AND DISADVANTAGE OF RIA

A

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

75
Q

It can be used to assay both antigens and
antibodies.

A

ELISA

76
Q

Either antigen or antibody can be linked to an enzyme :

A

 Horse Radish Peroxidase – Horse Radish
 Alkaline Phosphatase – E.coli
 G6PD
 Glucose Oxidase – Aspergillus niger
 Urease
 Beta-d-galactosidase – E.coli

77
Q

Cross linker to join amino groups

A

Glutaraldehyde

78
Q

Types of Enzyme Immunoassay

A
  1. HETEROGENOUS ENZYME
    IMMUNOASSAY
    2 TYPES: competitive elisa and nonceompetitive elisa
  2. HOMOGENOUS ENZYME
    IMMUNOASSAY
79
Q
  • 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.
A

Competitive ELISA

  • The enzyme activity is inversely proportional to
    the concentration of the test substance
80
Q
  • 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
A

B. Non-Competitive ELISA

81
Q
  • 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
A

HOMOGENOUS ENZYME
IMMUNOASSAY

82
Q

double antibody sandwich elisa

A

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

83
Q

elisa advantage and disadvantage

A

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

84
Q

 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

A

flourescence immunoassay

85
Q

FLUORESCENT DYE:

A

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

86
Q

Emission of light caused by a chemical reaction
producing an excited molecule that decays back to
its original ground state measured using a
luminometer

A

chemoliluminescence immunoassay

87
Q

CHEMILUMINESCENT SUBSTANCE

A

 Luminal
 Acridium esters
 Peroxyoxalates
 Rutherium derivatives
 Dioxetanes.

88
Q
  1. NITROBLUE TETRAZOLIUM
A
  • Intracellular killing activity of neutrophil
  • Positive in CGD
  • Positive: YELLOW
89
Q
  1. NEPHELOMETRY
A
  • Scattered Light
  • Protein (sample) + antisera = insoluble complex = scattered light (measured in
    photodiode)
  • For CRP, Ig, Complement
90
Q
  1. FLOW CELL CYTOMETRY
A
  • 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
91
Q

MICROLYMPHOCYTOTOXICITY TEST/ COMPLEMENT DEPENDENT CYTOLYSIS

A
  • Detects HLA-A,B,C and E antigens
  • Lymphocyte + Anti- HLA-BB Typing Sera + complement
    (rabbit serum) + stain with trypan blue or Eosin Y.
92
Q

5.MIXED LYMPHOCYTE CULTURE

A
  • Responder Cell (Recipient lymphocyte)
  • Irradiate Stimulator (donor) lymphocyte – mitomycin
    treated
  • 3H Thymidine (label)
  • (+) Uptake of radioactive – dead cell
  • (-) No uptake of dye = intact cells
93
Q

TUBERCULIN TEST

A
  • 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
94
Q

VOLLMER’S PATCH TEST

A
  • 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
95
Q

FREI TEST

A
  • For Lymphogranuloma venerum (LGV)
  • Chlamydial Ag – ID
  • (+) 7mm diameter papule (read after 48 hours)
96
Q

SCHICK TEST

A
  • Diphteria anti toxin
  • Diphteria toxin ID
  • (+) Redness (24-36 hours)
  • (+) indicates lack of immunity to diphteria
97
Q

DICKS TEST

A
  • Erythrogenic toxin→ID
  • (+) Redness (24-36 hours)
98
Q

BRUCELLERGIN test

A
  • Brucella Ag →ID
  • (+) Edematous plaque (1-6mm)
  • (+) Brucellosis
99
Q

 Histoplasmin , Coccidiodin

A
  • Ag → ID
  • (+) Wheal (48 hours)
100
Q

Toxoplasmin

A
  • Ag →ID →(+) Induration (48 hours)
101
Q

 Trichinella skin test

A
  • Allergen →ID →wheal (20mins)
  • Delayed HPS skin test
  • Measure T cell function
  • Most simple procedure to evaluate T cell function
102
Q
A

IS FINALS AND REMOVALS (5 decks)

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