PRECI RESET Flashcards
1
Q
Scientist who discovered precipitation reaction
A
Kraus (1897)
2
Q
Key observation in precipitation discovery
A
Precipitate formed when enteric bacteria culture mixed with specific antibody
3
Q
Reactants needed for precipitation
A
Soluble antigen+Soluble antibody
4
Q
Product of soluble antigen and antibody interaction
A
Insoluble Ag-Ab complex (precipitate)
5
Q
Optimal temperature for precipitation
A
40°C-45°C
6
Q
Requirement for visible precipitation
A
Equal concentration of antigen and antibody (zone of equivalence)
7
Q
Phenomenon describing optimal antigen-antibody ratio for precipitation
A
Zoning phenomenon+Zone of equivalence
8
Q
Effect of antigen or antibody excess on precipitation
A
Reduces lattice formation and precipitation
9
Q
Factors affecting precipitation
A
Affinity+Avidity of binding sites
10
Q
Definition of affinity
A
Initial force of attraction between single Fab site of antibody and single epitope of antigen
11
Q
Definition of avidity
A
Sum total of all attractive forces between antigen and antibody+Determines stability of Ag-Ab complex
12
Q
Possibility associated with low affinity
A
Cross-reactivity with structurally similar antigens
13
Q
Law describing equilibrium in precipitation
A
Law of Mass Action
14
Q
Equation for equilibrium constant in precipitation
A
K = K1/K2 = (Ag-Ab complex)/(Ab)(Ag)
15
Q
What K1 represents in precipitation
A
Rate constant for forward reaction (complex formation)
16
Q
What K2 represents in precipitation
A
Rate constant for reverse reaction (complex dissociation)
17
Q
Significance of high equilibrium constant in precipitation
A
Greater stability and visibility of Ag-Ab complexes
18
Q
Effect of increased K1 on precipitation
A
More Ag-Ab complex formed+More visible precipitate
19
Q
Effect of increased binding strength on dissociation
A
Decreased tendency for Ag-Ab complex to dissociate
20
Q
Process leading to visible precipitate in precipitation
A
Lattice formation between Ag and Ab+Loss of solubility+Formation of insoluble complexes
21
Q
Structural requirement for antibody in precipitation
A
Bivalent antibody (must have at least two binding sites)
22
Q
Structural requirement for antigen in precipitation
A
Bivalent or polyvalent antigen (must have at least two epitopes)
23
Q
Difference between precipitation and agglutination
A
Precipitation involves soluble antigens+Agglutination involves insoluble antigens or particles[6]
24
Q
Antibodies that bind soluble antigens to form insoluble complexes
A
Precipitins
25
Requirement for precipitation to occur
Antigen and antibody must be multivalent or at least bivalent
26
Immunoglobulin classes involved in precipitation
IgG+IgM+IgA
27
Most efficient immunoglobulin in precipitation
IgG
28
Immunoglobulin more efficient in agglutination than precipitation
IgM
29
Immunoglobulin that can participate in precipitation but less prominent
IgA
30
Immunoglobulins not involved in precipitation
IgE+IgD
31
Reason IgD is not involved in precipitation
Low serum concentration+Short half-life+Early class switching
32
Main function of IgE
Allergic reactions+Parasitic infections
33
Effect of increased temperature on precipitation
Improves initiation of precipitate formation (40–45°C)
34
Effect of cold temperature on precipitation
Enhances lattice formation+Allows visible precipitate to settle (0–4°C)
35
Disadvantage of precipitation curve formation
Prozone and postzone effects can cause false negatives
36
Zone of equivalence in precipitation
Optimal antigen-to-antibody ratio for maximal lattice formation and visible precipitate
37
Prozone effect cause
Antibody excess prevents proper lattice formation
38
Correction for prozone effect
Serial dilution of patient serum
39
Postzone effect cause
Antigen excess prevents lattice formation
40
Correction for postzone effect
Repeat sample after one week+Cell washing to reduce free antigen
41
Patient samples used in serologic precipitation testing
Serum+Plasma
42
Types of reagents used in serologic testing
Lab-prepared or commercially available standardized reagents
43
Source of antiserum for serologic reactions
Animals immunized with specific antigens
44
Key components of serologic reactions
Patient serum/plasma+Known reagents+Animal-derived antisera
45
Antisera produced by rabbits with stable precipitation and no change with excess antibody
R-type antisera
46
Effect of adding excess antibody to R-type antisera
No change in precipitate because antigen sites are already saturated
47
Effect of adding excess antigen to R-type antisera
Partial dissolution of precipitate due to formation of soluble antigen-antibody complexes (prozone effect)
48
Antisera produced by horses with unstable precipitation that dissolves with excess antibody or antigen
H-type antisera
49
Effect of adding excess antibody to H-type antisera
Complete dissolution of precipitate as all antigen binding sites become saturated
50
Effect of adding excess antigen to H-type antisera
Complete dissolution of precipitate indicating formation of soluble antigen-antibody complexes
51
Example of R-type antisera
7Sγ2-globulin from rabbits
52
Example of H-type antisera
7Sγ1 pseudo-globulin from horses
53
Principle measured by turbidimetry
Turbidity or cloudiness of solution due to suspended particles
54
What turbidimetry quantifies
Decrease in light intensity transmitted through a solution
55
Relationship between turbidity and antigen-antibody binding
Increased turbidity indicates more antigen-antibody lattice or precipitate formation
56
Principle measured by nephelometry
Light scattering by antigen-antibody complexes
57
Angle of scattered light measured in nephelometry
10° to 90° from incident beam
58
Relationship between scattered light and antigen-antibody complex concentration in nephelometry
Amount of scattered light is proportional to concentration of Ag-Ab complexes
59
Type of light scatter with particles much smaller than the wavelength of light
Rayleigh scattering
60
Direction of Rayleigh scattering
Forward and backward+wide range of angles
61
Main application of Rayleigh scattering
Light scatter testing for small molecules
62
Wavelength dependence of Rayleigh scattering
Greater scattered light intensity at shorter wavelengths
63
Type of light scatter for particles equal to or slightly smaller than wavelength
Rayleigh-Debye scattering
64
Direction of Rayleigh-Debye scattering
Mainly forward scattering+varied angles
65
Type of light scatter for particles larger than the wavelength of light
Mie scattering
66
Direction of Mie scattering
Strongly forward scatter
67
Types of light scatter relied upon in nephelometry
Rayleigh-Debye scattering+Mie scattering
68
Application of nephelometry
Quantification of immunoglobulins (IgG+IgA+IgM+IgE+kappa+lambda light chains)+serum proteins (complement+CRP+haptoglobin+ceruloplasmin)
69
Fixed-time nephelometry method
Measures light scatter at a specific time point after Ag-Ab mixing
70
Kinetic nephelometry method
Measures rate of change in light scattering over time
71
Flocculation test used to determine optimal antigen-antibody ratio
Dean and Webb procedure+Ramon procedure
72
Example of flocculation test for syphilis
VDRL (Venereal Disease Research Laboratory) test+RPR (Rapid Plasma Reagin) method
73
Qualitative precipitation test with ring formation at interface
Ring test
74
Positive result in ring test
Cloudy layer or distinct white band at reagent interface
75
Measurement of precipitation by gel medium involves
Movement of soluble antigen+antibody or both in a semisolid gel to form precipitate at the zone of equivalence
76
Most common gel medium for precipitation
Agarose (no charge+does not interfere with analytes)
77
Other gel media used for precipitation
Gelatin+Cellulose acetate+Polyacrylamide starch
78
General factors affecting diffusion rate in gel
Size and molecular weight of molecules
79
Effect of increased molecular size or weight on diffusion
Decreases diffusion rate
80
Reason IgG is more effective than IgM in gel precipitation
IgG is smaller and diffuses faster in gel
81
Type of diffusion where only one reactant moves
Single diffusion
82
Type of diffusion where both antigen and antibody move
Double diffusion
83
Diffusion direction in single dimension
Vertical (tube format)
84
Diffusion direction in double dimension
Vertical and horizontal (plate or petri dish)
85
Passive immunodiffusion technique
Precipitation in gel without electrical current
86
Single diffusion single dimension method
Oudin method (antibody in gel+tube+antigen layered on top+antigen diffuses down)
87
Result of antigen and antibody meeting at zone of equivalence in Oudin method
Precipitin band forms in gel
88
Single diffusion double dimension method
Radial immunodiffusion (Fahey and Mancini method)
89
Principle of radial immunodiffusion
Antibody in gel+antigen in well+antigen diffuses radially+precipitin ring forms
90
Relationship between precipitin ring diameter and antigen concentration
Diameter is proportional to antigen concentration
91
Kinetic/time method in radial immunodiffusion
Measures ring before zone of equivalence (18-19 hours)
92
End point method in radial immunodiffusion
Measures ring after zone of equivalence (24-72 hours)
93
Antibody incorporation in radial immunodiffusion
Antibody is mixed into gel medium
94
Antigen application in radial immunodiffusion
Antigen added to wells and diffuses outward
95
Measurement in radial immunodiffusion
Diameter of precipitin ring is measured and plotted against antigen concentration
96
Source of error: Overfilling or underfilling the well
Leads to inaccurate antigen or antibody concentration and affects precipitate formation
97
Source of error: Spilling sample outside the well
Reduces available reactant and causes weak or absent precipitin bands
98
Source of error: Nicking the well
Disrupts diffusion and leads to irregular or distorted precipitin rings
99
Source of error: Improper incubation time or temperature
Prevents optimal Ag and Ab diffusion and precipitation (incubate at RT 20°C for 16-20 hours)
100
Principle of double diffusion-single dimension (Oakley & Fulthrope)
Both antigen and antibody diffuse toward each other in gel and form a precipitin band at the meeting point
101
Process in Oakley & Fulthrope method
Antibody in gel+plain agar above+antigen solution layered on top+Ag diffuses down+Ab diffuses up+precipitate forms in plain agar
102
Principle of double diffusion-double dimension (Ouchterlony)
Both antigen and antibody move in agar plate (vertical and horizontal)+precipitin lines form where they meet
103
Ouchterlony setup
Central well with antibody+outer wells with antigens+compare band positions for antigen identity
104
Ouchterlony result: Pattern of identity
Precipitin band forms a smooth arch indicating antigens are identical
105
Ouchterlony result: Pattern of partial identity
Bands fuse with spur formation indicating shared but non-identical epitopes
106
Ouchterlony result: Pattern of non-identity
Crossed bands indicate different antigens with no shared epitopes
107
What is the principle of electrophoretic technique in immunodiffusion
Combines immunodiffusion with electrical current to separate molecules by charge in an electric field
108
What is the main purpose of electrophoresis in immunoelectrophoresis
Separates antigen mixtures by their charge and size before immunodiffusion with antibody
109
What happens when direct current is applied to a gel matrix in immunoelectrophoresis
Antigen and antibody migrate and form precipitin bands
110
Who developed rocket immunoelectrophoresis
Laurell
111
What is measured in rocket immunoelectrophoresis
Serum proteins in agarose gel at pH 8.6
112
How are antibody and antigen arranged in rocket immunoelectrophoresis
Antibody is incorporated in the gel+antigen is placed in wells
113
What causes the "rocket" shape in rocket immunoelectrophoresis
Antigen migrates in electric field and forms conical precipitin line as it reacts with antibody
114
What does the height of the rocket indicate in rocket immunoelectrophoresis
It is proportional to the concentration of the antigen
115
What is Ressler's method in immunoelectrophoresis
Single reactant moves in two dimensions+first phase separates proteins by charge+second phase forms Ag-Ab reaction
116
What is countercurrent immunoelectrophoresis
Double diffusion with electrophoresis to speed up visible reaction+antigen moves to anode+antibody to cathode+precipitin line forms in the middle
117
What are common sources of error in electrophoretic techniques
Reversal of wells/current direction+improper pH+insufficient electrophoresis time+prozone or postzone+wells not parallel
118
What is immunoelectrophoresis
Two-step technique: first electrophoresis separates proteins by charge/size+then immunodiffusion with antibody forms precipitin arcs
119
What is the main application of immunoelectrophoresis
Identification and quantification of serum proteins and detection of abnormalities such as myeloma or lymphoproliferative disorders
120
What does the presence of elliptical precipitin arcs in immunoelectrophoresis indicate
Antigen-antibody interaction and identification of specific proteins
121
Two-step technique where proteins are first separated by electrophoresis and then specific antisera are applied directly to the gel surface to detect and identify immunoglobulins
Immunofixation electrophoresis
122
Method where patient serum is loaded into six lanes of the gel before electrophoresis
Immunofixation electrophoresis serum application
123
Process where five lanes are overlaid with specific antisera (anti-gamma, anti-alpha, anti-mu, anti-kappa, anti-lambda) and the sixth lane is overlaid with antisera to all serum proteins as a reference,
Post-electrophoresis step in immunofixation
124
Comparison of reactions in each of the five specific lanes to the reference lane to determine the presence and type of immunoglobulin
Immunofixation electrophoresis analysis
125
First described by Alper and Johnson
Immunofixation electrophoresis
126
Typical reaction time is less than one hour
Immunofixation electrophoresis
127
Primary use: detection and identification of monoclonal gammopathies such as multiple myeloma
Waldenstrom macroglobulinemia
128
Precipitin test to diagnose anthrax by detecting thermostable antigens from Bacillus anthracis using rabbit antiserum
Ascoli test
129
Test classifying streptococci into groups based on specific polysaccharide antigens in their cell walls resulting in visible precipitates with specific antisera
LANCEFIELD PRECIPITIN TEST
130
Test detecting antibodies by observing a precipitate ring at the interface of antigen and antibody solutions commonly used for pneumococcal pneumonia
CAPILLARY TUBE PRECIPITIN TEST
131
Protein detected in capillary tube test for pneumococcal pneumonia produced by the liver during acute inflammation
C polysaccharides of pneumococcus
