Immuno Part 2 Lec Flashcards
Immunoassays have been developed to detect either antigen or
antibody, and they vary from easily performed manual tests to
highly complex automated assays.
Precipitation Reaction
involves combining soluble antigen with soluble antibody to
produce insoluble complexes that are visible.
Precipitation
is the process by which particulate antigens aggregate to form
larger complexes when a specific antibody is present.
Agglutination
is the initial force of attraction that exists between a
single Fab site on an antibody molecule and a single epitope or
determinant site on the corresponding antigen.
Affinity
School-based laboratory manual test
Precipitation reaction
Most efficent immmunoglobulin in this type of reaction because of its small molecules which is much efficient in Precipitation reaction
IgG
indicator for agglutination reaction
Rbc
most efficient; antibody involved in large agglutination reaction. Has 10 valence
IgM
Large complex of agglutination
Clumping
Antibodies are capable of reacting with antigens that are structurally similar to the original antigen that induced antibody production.
Cross-reactivity
It is inline to affinity
Cross-reactivity
initial force of attraction of antigen and antibody is called
Cross-reactivity
represents the sum of all the attractive forces between an antigen and an antibody.
Avidity
involves the strength with which a multivalent antibody binds a multivalent antigen, and it is a measure of the overall stability of an antigen–antibody complex
Avidity
is essential to detecting the presence of an unknown, whether it is antigen or antibody
Avidity
A high avidity can actually compensate for a ______ (low or high) affinity.
Low
stabilization ng antigen-antibody complex binding
Avidity
antigen-antibody binding
Affinity
Precipitin curve, we are dealing with the
Zone of equivalence
Zone of equivalence where it is inlined with the
Prozone and postzone
the optimum precipitant curve in which the number of the multivalent sites of the antigen and antibody are approximately equal
Zone of equivalence
phenomenon wherein we are dealing with antibody excess.
Prozone
The antigen combines with only 1 or 2 antibody molecules and no cross linkages are formed
Prozone
antigen excess. No lattice network formed.
Postzone
Every available antibody site is bound with single antigen and no crosslinks are formed
Postzon
Measurement of precipitation by
light scattering
Determination of Precipitation via
Passive immunodiffusion techniques
Immuno electrophoretic techniques
Light scattering test involves
Turbidimetry
Nephelometry
Passive immunodiffusion techniques test involve
Radial immunodiffusion
Ouchterlony double diffusion/immunodiffusion
Immunoelectrophoretic technique test involved
Rocket immunoelectrophoresis
Immunoelectrophoresis
Immunofixation electrophoresis
Countercurrent immunoelectrophoresis
is a measure of the turbidity or cloudiness of a solution.
Turbidimetry
A detection device is placed in direct line with the incident light.
Collecting light after it has passed through the solution.
Turbidimetry
turbidimetry has the intensity of light transmitted thru the medium, so that the unscattered light is measured at _______ degrees angle from the incident light beam
180 degrees
the intensity of scattered light is measured. It is usually not necessarily at right angles to the incident light beam
Nephelometry
It can detect antigen or antibody but we usually use antibody as reagent and the patient sample to detect the unknown antigen
Nephelometry
Nephelometers measure light scatter at angles ranging from ______degrees to about ______degrees.
10 degrees to 90 degrees
This would be the reaction allowed to run essentially to completion
which means that large particles tend to fall out of solution and decreases the amount of the scatter
End point nephelometry
provides accurate and precise quantitation of serum proteins, and due to automation, the cost per test is typically lower than other methods.
nephelometry
Nephelometry Can also be used as a quantitation of immunoglobulins such as _____, ___, and _____, ______ even the Kappa and Lambda light chain and it is exclusively done by nephelometry.
IgG, IgA, and IgM, IgE
Order of nephelometry device
light source→ monochromator → cuvette → lens → detector
The precipitation of antigen–antibody complexes can also be determined in a support medium such as a ______
Gel
Passive immunodiffusion techniques A high-molecular-weight complex polysaccharide derived from __________, and ___________, a___________, are used for this purpose.
seaweed, agarose and purified agar
The precipitation of antigen–antibody complexes can also be determined in a support medium such as a gel.
Passive immunodiffusion technique
The rate of diffusion is affected by the size of the particles, the temperature, the gel viscosity, and the amount of hydration.
Passive immunodiffusion techniques
involves the stabilization and visualization
Agar or agarose gel
A modification of the single-diffusion technique was the
Radial immunodiffusion
who was the first to use gels for precipitation reactions, and he pioneered the technique known as single diffusion.
James Oudin
Radial immunodiffusion has
positive result would be the formation of the
precipative bonds
Radial Immunodiffusion is also called as
RID
Antibody is uniformly distributed in the support gel, and antigen is applied to a well cut into the gel.
RID
The area of the ring obtained is a measure of antigen concentration, and this can be compared to a standard curve obtained by using antigens of known concentration.
RID
Two techniques for measurement of RID
Mancini method
Fahey and Mckelvey method
DIAMETER OF THE RING = antigen concentration
Mancini method
Mancini method occurs between _____ and ______ hours
24 and 72 hours
Mancini method determines
IgM and IgG
Determination of IgM:
completed at 50-72hrs
Mancini method forms:
Ring formation
Determination of IgG:
Complete at 24 hours
Mancini method involve in method of
End point method
the diameter is proportional to the log of the concentration
Fahey and mckelvey method
Fahey and mckelvey method involves method We are using measurement taken before the point of equivalence is reached
Kinetic method
Fahey and mckelvey methods Readings is taken for about
18 hours
Sources of error include in radial immunodiffusion
● overfilling or under filling the wells,
● nicking the side
● spilling sample
● improper incubation time and temperature,
● incorrect measurement.
Radial immunodiffusion has been used to measure
IgG, IgM, IgA, and complement components.
In this technique, both antigen and antibody diffuse independently through a semisolid medium in two dimensions, horizontally and vertically.
Ouchterlony double diffusion
has a smooth curve which means it have the same antigen that is present
Category A
demonstrates a cross line pattern which shows two separate reactions and indicates that the compared antigens share no common epitotes
Category B
there’s a fusion of 2 lines with a spur indicating partial identity.
Category C
Also called as ‘Laurel Technique” because it was developed by Laurel in early 1960s
Rocket immunoelectrophoresis
Rocket immunoelectrophoresis Also called as ‘_____________” because it was developed by Laurel in early 1960s
Laurel Technique
The end result of rocket immunoelectrophoresis
Conical line
Double-diffusion technique that incorporates electrophoresis current to enhance results.
Immunoelectrophoresis
Immunoelectrophoresis Introduced by __________ and __________ in 1953, this is performed as a two-major step process and can be used for semi quantitation of a wide range of antigens.
Grabar and Williams
this is performed as a two-major step process and can be used for semi quantitation of a wide range of antigens.
Immunoelectrophoresis
2 Step process of immunoelectrophoresis
Electrophoresis
Diffusion of antigen and antibodies
Immunoelectrophoresis Reaction takes place within_________
18-24hrs.
procedure has been used as screening tool for the differentiation of many serum proteins including the major classes of immunoglobulins.
Immunoelectrophoresis
Immunoelectrophoresis used in the laboraratory for the determination of
Myelomas
Immunoelectrophoresis can detect
waldenstroms
macroglobulinemia
malignant lymphomas
other lymphoproliferative disorders
causes increased bacterial infection
IgG deficiency
Either increase value of antigen or deficient
B lymphocyte deficiency
as first described by Alper and Johnson.
Immunofixation electrophoresis
Similar to immunoelectrophoresis
Immunofixation electrophoresis
Immunofixation electrophoresis meduim can be use
Agarose
Cellulose acetate
Immunofixation electrophoresis Reaction Time:
Less than 1 hour
Immunodiffusion takes place in a shorter time and results in a higher resolution than when antibody diffuses from a trough
Immunofixation electrophoresis
Modification of immunoelectrophoresis.
Countercurrent immunoelectrophoresis
Based on the movement of antigen towards the
anode and of the antibody towards the cathode during the passage of the electric current through agar.
Countercurrent immunoelectrophoresis
Countercurrent immunoelectrophoresis reaction to take place
30-60 mins
are designed for antigens and antibodies that may be small in size or present in very low concentrations.
Label immunoassay
The presence of such antigens or antibodies is determined indirectly by using a
Labeled reactant
The substance to be measured is known as the
Analyte
On this Labelled immunoassays here are some examples of analytes:
Bacterial antigen hormone
Drugs ( test kits)
Tumor markers
Specific immunoglobulins
is an antibody bonded to an analyte.
Labelled immunoassay
Labeled immunoassays have made possible rapid quantitative measurement of many important entities such as
Viral antigen
All the test kits that we encounter in immunology and serology are examples of Labelled immunoassays:
Mallaria test kits
Dengue NS1 for detecting recent dengue
infections
Dengue Duo for detecting IgM or IgG
Pregnancy test kits
Characteristic of labelled immunoassay
o Competitive Assay
o Non-Competitive Assay
Classification of labelled immunoassays
o Radioimmunoassay (RIA)
o Enzyme Immunoassay (EIA) o Fluorescent Immunoassay
All the reactants are mixed together simultaneously , and a labelled antigen competes with unlabeled patient antigen for a limited number of antibody-binding sites
Competitive immunoassay
Competitive immunoassay The amount of bound label is ______________ to the concentration of the patient antigen.
Inversely Proportional
Antibody, is first passively absorbed to a solid phase. (in solid phase we use wells)
Non-competitive immunoassay
Non-competitive immunoassay The amount of label measured is _________ to the amount of patient antigen
Directly proportional
Non-competitive immunoassay is often called as
Captured antibody
discovered by Georges Kohler and Cesar Milsten
Monoclonal antibodies
remains a constant source of highly specific antibody
Monoclonal antibodies
Antibodies in the Labelled immunoassays are characterized as
Monoclonal antibodies
Monoclonal antibodies aka
Calibrators
Are unlabeled analytes that are made up in known
concentrations of the substance to be measured
Calibrators
Most immunoassays used the change in absorbance measured by
Spectrophotometry
Quality Controls: should be run.
blank, negative control, and high and low positive control
Pioneered by Rosalyn Yalow and Solomon Berson.
Radioimmunoassay
Used radioactive substances as a label
Radio immunoassay
Most common Label for RIA is Iodine
125I (Iodine 125), 131I (Iodine 131), 3H (tritiated Hydrogen)
was originally based on the principle of competitive binding labelled antigen competes with unlabelled patient antigen for a limited number of antibody-binding sites.
Radioimmunoassay
Ria The amount of bound label is ______________ to the concentration of the labelled antigen.
Inversely Proportional
Advantages in using enzymes as label
cheap and readily available
Long shelf-life
easily adapted
Easy to dispose
little reagent is necessary
Common enzymes used in EIA:
Horseradish peroxidase (MOST COMMON)
ALP (MOST COMMON)
G-6-PD
Beta-D-galactosidase
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 Enzyme immuno assay
Similar to RIA
Enzyme immunoassay
Ezyme immunoassay aka
Direct elisa
referred to as indirect enzyme-linked immunosorbent assays (ELISA), because the enzyme-labeled reagent does not participate in
the initial antigen–antibody binding reaction.
Non-competitive enzyme immunoassay
Non-competitive EIA Enzymatic activity
Direct proportional
Membrane-based cassette assays are a relatively new type of enzyme immunoassay
Rapid immuno assay
The analyte is applied at one end of the strip and migrates toward the distal end, where there is an absorbent pad to maintain a constant capillary flow rate.
Immunochromatography
Cassette test 2 lines indicates
Positive results
1 line on control (C) means
Negative results
1 line on test (T) or no lines at all indicates
Invalid results
A form of immunoassay that uses label that fluoresce upon interaction with the desired analytes.
Fluorescent immunoassay
these fluorescent compounds are called
fluorophores or fluorochromes
fluorophores or fluorochromes compound
rhodamine, fluorescein, phycoerythrin
have a red-orange fluorescence
Phycoerythrin
Most commonly used fluorophores/fluorochromes:
Fluorescein
→ absorbs maximally at 490 to 495 nm and emits a “green color” at 517-520 nm
Fluorescein
absorbs at 550 nm and emits “ red colors” at 580 to 585 nm
Tetramethylrhodamine
there is a known antigen is fixed on the slide. This is for the antigen detection only.
Direct IFA
there is a known antigen fixed on the slide and it is used both for antigen and antibody detection. It involves a two step process.
Indirect IFA
