CHAPTER 6.3 - PRECIPITATION Flashcards
1
Q
•combination of (?) plays an important role in the laboratory in diagnosing many different diseases.
A
antigen with specific antibody
2
Q
have been developed to detect either antigen or antibody, and they vary from easily
A
Immunoassays
3
Q
Immunoassays are based on the principles of
A
precipitation or agglutination
4
Q
Initial force of attraction that exist between singe Fab site (paratope) and a single epitope on the corresponding antigen
A
Affinity
5
Q
1 fab + 1 epitope
(Ab) (Ag)
A
Affinity
6
Q
Sum of all attractive forces between an Ag and Ab
A
Avidity
7
Q
Dictates the overall stability of the Ag-Ab complex
A
Avidity
8
Q
Multivalent Ab=Multivalent Ag
A
Avidity
9
Q
Types of Affinity
A
10
Q
electrovalent bond (ionic charge in a chemical soln)
A
Ionic bonds
11
Q
Hydrogen bridging
A
Hydrogen bonds
12
Q
major bond demonstrated by an Ag and Ab
A
Hydrophobic bonds
13
Q
combination between an ionic and a hydrophobic bond
A
Van der Waals forces
14
Q
Antibody
A
Precipitin
15
Q
Soluble antigens
A
Precipitinogen
16
Q
Insoluble complexes formed by the union of the two aforementioned
A
Precipitate
17
Q
Natural clumping
A
Flocculation
18
Q
Major Immunoglobulins Involved
A
19
Q
Much better precipitating Ab than IgM
A
IgG
20
Q
Much better agglutinating Ab than IgG
A
IgM
21
Q
Precipitation:
A
IgG>IgM>IgA
22
Q
Nonprecipitating
A
IgE
23
Q
FACTORS AFFECTING PRECIPITATION
A
24
Q
The pH of the medium used for testing should be near physiologic conditions, or an optimum pH of
A
6.5 to 7.5
25
Ideal: Temperature and Length of Incubation
Body temperature (37C/98.6F)
26
37C; warm reacting
IgG
27
40-45C; cold reacting
40-45C
28
Incubation time range from
15-60 minutes
29
involves combining soluble antigen with soluble antibody to produce insoluble complexes that are visible
30
first noted in 1897 by Kraus
31
: All antigen-antibody binding is reversible and free reactants are in equilibrium with bound reactants
Law of Mass Action
32
So that maximum precipitation occurs, Ag and Ab concentration must have an
optimum ratio
33
Ag and Ab are equal, therefore (?) occur
max precipitation
34
More Ab (patient) excess than Ag
prozone
35
remedy: Serum dilution
prozone
36
Ag excess, less Ab (Patient) may lead to false negative
postzone
37
Remedy: Repeat the test after a week to give time for antibody production
postzone
38
PRECIPITATION REACTIONS
PRECIPITATION IN A FLUID MEDIUM
PRECIPITATION BY A PASSIVE IMMUNODIFFUSION
PRECIPITATION BY ELECTROPHOTERIC TECHNIQUES
39
Precipitation in a Fluid Medium
40
measure of the turbidity or cloudiness of a solution
Turbidimetry
41
measures the reduction in light intensity due to reflection, absorption, or scatter
Turbidimetry
42
measures the light that is scattered at a particular angle from the incident beam
Nephelometry
43
amount of light scattered is an index of the solution’s concentration
Nephelometry
44
Ag-Ab complex may be observed in a support media (Agarose gel)
Precipitation by a Passive Immunodiffusion
45
Passive: No electric current is used to speed up reaction of the Ag and Ab combination, but through DIFFUSION
Precipitation by a Passive Immunodiffusion
46
Factors affecting rate of diffusion
o Size of the particles
o Temperature
o Gel viscosity
o Amount of Hydration
47
Precipitation in Gel Medium
48
• Only one reactant is moving
Single Diffusion
49
• Either Ag or Ab is moving
Single Diffusion
50
• Both Ag and Ab are moving through the medium
Double Diffusion
51
• Reaction in tubes- Ag or Ab migrate up and down
Single Dimension
52
• Petri dish – Ag or Ab diffuse radially
Double Dimension
53
• Ab is uniformly distribute in a support gel and Ag is applied to a well cut into gel.
Radial Immunodiffusions
54
Types of Radial Immunodiffusions
55
Oudin
Single DiffusionSingle Dimension
56
Macini, Fahey, and MacKelvey
Single DiffusionDouble Dimension
57
Single DiffusionSingle Dimension (Oudin)
Procedure:
1. Ab mixed in (?)
2. Antigen dilution is overlaid ([?]must always be greater to achieve zone of equivalence)
3. (?) diffuses through the gel, containing immobilized Ab forming insoluble Ag-Ab Complexes.
4. At equivalence concentration, the Ag stops moving and a (?) is formed.
agarose
Ag
Mobile Ag
stabilized band
58
Single DiffusionDouble Dimension (Macini, Fahey, and MacKelvey)
Procedure:
1. Ab is mixed with liquid agar and poured into the (?)
2. Circular wells cut in (?)
3. Ag is loaded into the (?)
4. (?) expands from the well as Ag diffuses toward its equilibrium concentration
5. (?) is measured.
petri dish
gel
wells
Ring precipitate
Diameter of the disc
59
Single DiffusionDouble Dimension (Macini, Fahey, and MacKelvey) Types:
A. Mancini/Endpoint Method
B. Fahey and McKelvey/Kinetic method
60
Diameter= Ag Concentration
Mancini/Endpoint Method
61
Diameter= Logarithm Ag Concentration
Fahey and McKelvey/Kinetic method
62
Both Ag and Ab diffuse independently through a semisolid medium in 2 dimension
Ouchterlony Double Diffusion
63
Ouchterlony Double Diffusion
Procedure
1. Pattern of well in cut in an (?)
2. (?) are loaded
3. Incubated until lines are (?)
agarose gel in petri dish
Reactants
precipitated
64
Possible Patterns in Ouchterlony Double Diffusion:
A. Serological Identity: Identical Ag
B. Non-Indentity: Ag are serologically distinct
C. Partial Identity: Ag are not identical but do possess common determinants.
65
: Identical Ag
A. Serological Identity
66
: Ag are serologically distinct
B. Non-Indentity
67
: Ag are not identical but do possess common determinants.
C. Partial Identity
68
Important in lab tests
Specificity
69
technique in which molecules with a net charge are separated when an electric field applied
Electrophoresis
70
Negative charged particles (anions) migrate to the
ANODE (Postive (+) Pole)
71
Positive charged particles (cations) migrate to the
CATHODE (Negative (-) pole)
72
FACTORS THAT INFLUENCE RATE OF PROTEIN MIGRATION
73
size, harder to migrate
↑
74
Solubility of the protein
AMOUNT OF SOLVATION
75
viscous, harder to migrate
↑
76
PH OF BUFFER
>8
77
• Room temperature
TEMPERATURE
78
• Protein will denature once it is exposed to high temperature
TEMPERATURE
79
flow of ions goes toward the cathode and can impede movement of proteins toward the anode
ENDOOSMOSIS
80
DIFFERENT TESTS FOR ELECTROPHORESIS
81
Single reactant moving in one dimension
1. ROCKET IMMUNOELECTROPHORESIS
82
Laurell Technique (1960)
1. ROCKET IMMUNOELECTROPHORESIS
83
Radial immunodiffusion (RID) + electrophoresis
1. ROCKET IMMUNOELECTROPHORESIS
84
End result: precipitin line that is conical in shape, resembling a rocket
1. ROCKET IMMUNOELECTROPHORESIS
85
The height of the rocket, measured from the well to the apex, is directly in proportion to the amount of antigen in the sample.
1. ROCKET IMMUNOELECTROPHORESIS
86
This technique has been used to quantitate immunoglobulins, using a buffer of pH 8.6
1. ROCKET IMMUNOELECTROPHORESIS
87
1. ROCKET IMMUNOELECTROPHORESIS
Procedure:
1. Antigen is pushed through antibody containing gel under influence of an applied (?)
2. When they are equivalence, precipitation will occur forming a (?)
electric field (EC)
cone/ rocket shape band
88
Reactant: Ag (mobile; sample) o Cut thru the gel to make wells where the Ags will be placed o Apply EC, allowing migration of Ag to the anode
Reagent: Ab (immobile; incorporated in the gel)
1. ROCKET IMMUNOELECTROPHORESIS
89
Single reactant moving in 2 dimensions
2. CROSSED IMMUNOELECTROPHORESIS
90
Either Ag or Ab will move (radially)
2. CROSSED IMMUNOELECTROPHORESIS
91
Ressler’s method
2. CROSSED IMMUNOELECTROPHORESIS
92
Either Ag or Ab will move (up or down)
1. ROCKET IMMUNOELECTROPHORESIS
93
2. CROSSED IMMUNOELECTROPHORESIS
Procedure:
1. Proteins are separated by (?)
2. Proteins are subjected to a 2nd electrophoresis where they will move through a (?) until rocket is formed (AgAb reach equivalence)
electrophoresis
Ab-containing agarose
94
Reactant: Ag (immobile)
Reagent: Ab (mobile)
2. CROSSED IMMUNOELECTROPHORESIS
95
Anode: Ab – migrates toward the cathode
3. COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
96
Cathode: Ag – migrates toward the anode
3. COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
97
Zone of equivalence will form a precipitate
3. COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
98
SPECIFICITY is important
3. COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
99
Voltage Facilitated double immunodiffusions
3. COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
100
DOUBLE: Double reactants moving in one dimension
3. COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
101
Use: Identify bacterial, fungi or virus in fluids
3. COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
102
3. COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
Procedure:
1. Ag and Ab are added to separate parallel wells cut out in an (?)
2. When an electric field is applied, the Ag will migrate to the (?) and Ab to the (?)
3. Zone of equivalence will form a (?)
agar gel
Anode; cathode
precipitate
103
Cathode (+) to Anode (–)
Albumin (fastest, lightest) → a-1 globulin → a-2 globulin → b- globulin → y- globulin (immunoglobulin)
104
Grabar and Williams
4. CLASSIC IMMUNOELETROPHORESIS
105
Double reactants moving in 2 dimensions
4. CLASSIC IMMUNOELETROPHORESIS
106
Both Ag or Ab will move (radially)
4. CLASSIC IMMUNOELETROPHORESIS
107
Use: Differentiate the Ig Class, identify abnormal proteins, myeloma proteins (BJP), Monitor purity of pharmaceutical products
4. CLASSIC IMMUNOELETROPHORESIS
108
(+) Result: Any change in the shape of the arcs indicate abnormality
4. CLASSIC IMMUNOELETROPHORESIS
109
4. CLASSIC IMMUNOELETROPHORESIS
Procedure:
1. Ag is introduced in a well and an electric field is applied resulting in (?)
2. Ab is introduced in a (?) parallel to the separated protein
3. (?) form
separation of proteins
trough
Ag-Ab complex
110
Process by which particulate antigens (agglutinogen) such as cell aggregate to form larger complexes when a specific antibody (agglutinin) is present
111
published the first report about the ability of antibody to clump cells, based on observations of agglutination of bacterial cells by serum
1896: Gruber and Durham
112
This finding gave rise to the use of serology as a tool in the diagnosis of disease, and it also led to the discovery of the ABO blood groups
(Karl Landsteiner)
113
2 STAGES OF AGGLUTINATION
114
Antigen-Antibody reaction
SENSITIZATION
115
No agglutination yet
SENSITIZATION
116
Cross linking
LATTICE FORMATION
117
Visible agglutination
LATTICE FORMATION
118
Represents binding of Ag and Ab
SENSITIZATION
119
Stabilization of antigen–antibody complexes with the binding together of multiple antigenic determinants.
SENSITIZATION
120
Forms bridges between Ab and Ag
LATTICE FORMATION
121
FACTORS THAT AFFECT AGGLUTINATION
122
Buffer pH Routine:
pH 7 (close to physiological pH) 7.35 – 7.45
123
Affects the zoning phenomenon
Relative concentration of Ag and Ab
124
Abs will not detect determinants buried within the particle
Location and concentration of Antigenic determinants of the particle
125
More number of determinants, the higher the likelihood of cross bridging
Location and concentration of Antigenic determinants of the particle
126
Electrostatic interactions between particles
Non covalent interaction
127
in the buffer plays an important role in agglutination
Electrolyte concentration (ionic strength)
128
: charge bet RBC and the elctrolyte/ECF
Zeta potential
129
Zeta potential
in RBC
outside the body
↑
↓
130
reduce electrostatic charges that interfere with lattice formation
Electrolytes
131
Antibody isotope Best: (dealing w/ particulate Ag)
IgM
132
: Cold reacting (range 4-22oC)
IgM
133
: Warm reacting with optimum temperature at 37oC
IgG
134
Incubation times ranges from
15-60 minutes
135
No agglutinates
0
136
Dark, turbid, homogenous
0
137
Many tiny agglutinates, many free cells, may not be visible without microscope
W+
138
Dark, turbid
W+
139
Many small agglutinates, many free cells (25% are agglutinated)
1+
140
Turbid
1+
141
Clear
2+
4+
2+
142
Many medium sized agglutinins, moderate number of free cells
2+
143
(50% are agglutinated)
2+
144
Several large agglutinates, few free cells
3+
145
(75% are agglutinated)
3+
146
One large solid agglutination, no free
4+
147
(100% are agglutinated)
4+
148
Newer tests performed in the serolab
149
categorized based on the labels being used
150
EIA -
RIA -
FIA -
- enzymes
- radio isotopes
- flourescence
151
Opposite charge attracts, negative charge repels
Ionic bonds
152
most of Ags are proteins (hydrophobic/waterfearing)
Hydrophobic bonds
153
Result: Precipitate (smaller complexes)
Precipitin
154
counterpart of precipitin
Precipitinogen
155
Precipitin + precipitinogen =
precipitates
156
Same w/ precipitation
Consistency: smoother
Result: Fleecy mass
Flocculation
157
Monomer: precipitates
IgG
158
Pentamer: agglutinates
IgM
159
Precipitation : best
IgG
160
lowest pH
physiologic conditions
optimum pH
6.5 to 7.5
161
Blood pH:(ideal)
7.35 – 7.45
162
IgG: warm-reacting
37oC or 98.6oF
163
IgM: cold-reacting
40-45oC
164
Much complicated procedure
PRECIPITATION
165
Reversible esp. in Ag-Ab binding
PRECIPITATION
166
Shows optimal reaction
Zone of equivalence
167
Ag and Ab are equal
Zone of equivalence
168
Leading to lattice formation (result: precipitates/agglutinates)
Zone of equivalence
169
may lead to false negative
Prozone phenomenon
Postzone phenomenon
170
Amount of light scattered =
Soln’s/analyte conc (Ag or Ab)
171
Same w/ agar used in culture media w/ the exception of additional nutrients or inhibitors to be selective
passive immunodiffusion
172
Ex. In chromatography – addition of soln to allow movement of reactant
DIFFUSION
173
slower
↑ Size of the particles - slower
↓ Temperature - slower
↑ Gel viscosity - slower
↓ Amount of Hydration - slower
174
Direction of movement
Single Dimension
175
Single diffusion: Ag (moves)
Mancini/Endpoint Method
176
Antigen is allowed to diffuse to completion
Mancini/Endpoint Method
177
↑ Ag diffusion = ↑ Ag conc
Mancini/Endpoint Method
178
occurs between 24 and 72 hours
Mancini/Endpoint Method
179
Measures diameter of ring before Ag diffuses
Fahey and McKelvey/Kinetic method
180
Antigen is not allowed to diffuse completely
Fahey and McKelvey/Kinetic method
181
Mnemonic:
“FAK ME”
FA→Kinetic Mancini→Endpoint
182
Ouchterlony Double Diffusion Types:
183
Single diffusion: Ag (moves)
Mancini/Endpoint Method
184
Antigen is allowed to diffuse to completion
Mancini/Endpoint Method
185
↑ Ag diffusion = ↑ Ag conc
Mancini/Endpoint Method
186
occurs between 24 and 72 hours
Mancini/Endpoint Method
187
Measures diameter of ring before Ag diffuses
Fahey and McKelvey/Kinetic method
188
Antigen is not allowed to diffuse completely
Fahey and McKelvey/Kinetic method
189
Performed using petri dish
Double Diffusion
190
Used to compare 2 Ags that are the same and capable of reacting w/ the
same Ab
Double Diffusion
191
Fusion of the lines at their junction to form a smooth arc represents serological identity or the presence of a common epitope
Serological Identity: Identical Ag
192
The arc indicates that the two antigens are identical.
Serological Identity: Identical Ag
193
Pattern of crossed lines demonstrates two separate reactions
Non-Indentity: Ag are serologically distinct
194
indicates that the compared antigens share no common epitopes
Non-Indentity: Ag are serologically distinct
195
Two crossed lines represent two different precipitation reactions
Non-Indentity: Ag are serologically distinct
196
The antigens share no identical determinants.
Non-Indentity: Ag are serologically distinct
197
“Spur formation”
Partial Identity: Ag are not identical but do possess common determinants.
198
Fusion of two lines with a spur indicates partial identity.
Partial Identity: Ag are not identical but do possess common determinants.
199
The two antigens share a common epitope
Partial Identity: Ag are not identical but do possess common determinants.
200
some antibody molecules are not captured by antigen and travel through the initial precipitin line to combine with additional epitopes found in the more complex antigen
Partial Identity: Ag are not identical but do possess common determinants.
