Immunology test 2
Direct coomb's test
(in vivo) attachment of antibody or complement to an individual's RED blood cells A positive Coomb's test (DAT) can indicate autoimmune HEMOLYTIC anemia, HDN, sensitization of RBCs caused by certain DRUGS or a TRANSFUSION reaction Called direct because red cells are used as they come from the body
Southern blot
ANALYSIS of DNA fragments using PROBES. DNA fragments are separated out by gel electrophoresis Pieces are denatured using ALKALI Transferred to a nitrocellulose or nylon membrane HYBRIDIZATION reaction takes place Once the DNA is on the membrane a labeled probe is added (looking for complementary single strands)
Bases of DNA
AT (2 hydrogen bonds) and GC (3 hydrogen bonds
Agglutination Quality control
Agglutination is a fairly SIMPLE reaction FALSE negatives and false positive Techniques must be STANDARDIZED CROSS REACTIVITY may occur (HETEROPHILE antibodies and RF are notorious) STORAGE and outdates Specificity and SENSITIVITY COST
Amplification caution
All amplification techniques are subject to FALSE POSITIVE reactions (contamination or background reactivity) Molecular methods in general are beginning to be used routinely in the CLINICAL laboratory Strict quality control must be adhered to for each test
Antiglobulin medicated agglutination
Also known as the Coomb's test Detects NON-agglutinating antibody by means of coupling with ANOTHER antibody blood banking Key component is antibody to HUMAN globulin (made in animals) antibody REACTS with the Fc portion of the immunoglobulin
Reverse passive agglutination **
Antibody RATHER than antigen is ATTACHED to carrier particle Active sites facing OUTWARD Principle: Latex particles coated with antibody are allowed to react with the patient sample containing the suspected antigen.
Capture assays **
Antibody is BOUND to the solid phase Called sandwich immunoassays or capture assays Antigens captured must have MULTIPLE epitopes excess antibody attached to solid phase is combined with TEST SAMPLE Enzyme labeled antibody is ADDED SECOND antibody recognizes same or different epitope. Enzymatic activity directly PROPORTIONAL to the amount of ANTIGEN in the test sample This assay is best suited to antigens that have multiple determinants
Single diffusion
Antibody is distributed in the support gel and antigen is applied to a cut into the gel. As the antigen diffuses out from well, antigen/antibody combinations occur until zone of equivalence is reached
sensitization
Antigen antibody combination through a SINGLE epitope on the particle SURFACE Rapid and reversible
Non competitive process
Antigen or antibody is BOUND to solid phase When antigen is bound, then PATIENT antibody is ADDED and given time to react Used for measuring the IMMUNITY to infectious agents and autoantibody testing Viral infections are easily diagnosed using this method More sensitive than fluorescent methods
Homogenous enzyme assay **
Any antigen-antibody system where there is NO separation step Usually LESS sensitive rapid and simple to perform NO washing steps Based on the principle of change in enzyme activity as a specific antigen-antibody reaction occurs Reagent antigen labeled with an enzyme TAG
coagglutination
BACTERIA are used as particles to which the antibody is attached STAPH AUREUS is the most frequently used (Protein A) Active site faces OUTWARD
Agglutination inhibition **
Based on COMPETITION between particle and soluble antigen for limited antibody BINDING SITES Looking for HAPTENS complexed to proteins agglutination is a NEGATIVE result. Latex particles would NOT be able to form the lattice work
Hybridization
Binding of TWO complementary strands, takes place in either a solid support MEDIUM or in a solution
End point method
CONCENTRATION is in proportion to diameter SQUARED
RIA Advantages/Disadvantages
Can detect hCG, FSH, gastrin, insulin, CEA, thyroxine, TSH, estrogens, androgens, and IgE DISADVANTAGES: HEALTH hazards in working with RADIOACTIVE substances GOVERNMENT regulation DISPOSAL problems
Choosing enzymes as labels
Certain number of substrate molecules converted per molecule of enzyme Purity Sensitivity Ease and speed of detection Stability Absence of interfering factors
Chemiluminescent assays **
Chemiluminescence - emission of light caused by chemical reaction, producing an EXCITED molecule that DECAYS back to its original ground state. Can be heterogeneous or homogeneous EXCELLENT sensitivity Reagents STABLE and non toxic iINEXPENSIVE, good turn around time False results may occur- QC must be performed every time test is run commonly used in the clinical IMMUNOLOGY laboratory
Precipitation
Combination of soluble antigen with soluble antibody Produces insoluble complexes can be visualized. Not Agglutination.
Competitive ELISA Enzyme linked immunosorbent assays **
Competive assay based on RIA principles Enzyme linked antigen COMPETES with unlabeled patient antigen for a limited number of BINDING sites on the antibody molecule that are attached to SOLID phase. WASH to remove any nonspecifically bound antigen Enzyme activity is DETERMINED Enzyme activity INVERSELY proportional to the CONCENTRATION of test substance
Western Blot **
Confirmatory test to detect antibodies to HIV-1 Mixture of HIV antigens placed on a gel and electrophoresed transferred by "blotting" Patient serum is applied to nitrocellulose gel and allowed to react Strip is washed and stained to detect precipitin
source of error in electrophoresis
Current in the WRONG direction incorrect pH of the BUFFER incorrect TIME CONCENTRATIONS of antigen and antibody Amount of CURRENT WELLS properly filled
RFLP
DIFFERENCES in restriction patterns are known as restriction fragment length polymorphisms (RFLP) caused by VARIATIONS in the nucleotides within the genes that CHANGE where the restriction enzymes cleave the DNA
Immunoelectrophoresis
DOUBLE DIFFUSION technique source of the antigen is in the SERUM which is electrophoresed to separate out main protein fractions Next, A TROUGH is cut in the gel PARALLEL to the line of separation and ANTISERUM is placed in the trough Gel incubated 18-24 hours Used for detection of MYELOMAS
Indirect coomb's test
Determines the PRESENCE of a particular antibody in a patient or can look for a specific antigen on the patient's RBCs 2 step process RBCs and the antibody COMBINE at 37C Cells are washed to remove unbound antibody Anti-human globulin is added antibody is present = agglutination
Categories of agglutination **
Direct Passive Reverse passive Agglutination inhibition coagglutination
PCR process **
Each cycle of PCR consists of 3 steps High HEAT (95C) DENATURE DNA Cooled to anneal DNA strands (primers and pieces go together) 55C once the primers have attached, 72C allows the four nucleotide bases in solution to pair in a very specific manner and completes the strand BETWEEN the primers. DNA polymerase used ADDS nucleotides to the 31 end of each primer
Classification of enzymes
Either heterogeneous or homogeneous. Heterogeneous does REQUIRE a separation step Homogeneous: NO SEPARATION STEP
Quality control for assays
Extremely critical when testing for such SMALL quantities Running a BLANK will help rule out random error UNACCEPTABLE results will indicate a background result that is too HIGH Negative control as well as high and low controls should be run EACH time the test is performed
Quantitative fluorescent immunoassays **
FIAs some heterogeneous requiring a separation step, others are homogeneous Heterogeneous based on the SAME principles as the enzyme immunoassays label is a FLUOROCHROME rather than an enzyme
Advantages and disadvantages of fluorescent immunoassays **
Fairly simple methodology Main problem comes in SEPARATING the signal from the background NONSPECIFIC binding can occur EXPENSIVE
Homogenous fluorescent immunoassay **
Fluorescence polarization immunoassay (FPIA) Based on the CHANGE in polarization of fluorescent light emitted from labeled molecule when it is BOUND to antibody This technique is limited to SMALL molecules Used to determine certain DRUG levels and hormone levels (THYROID) More patient antigen, polarization DECREASED
Fluorescent immunoassay **
Fluorophores or fluorochromes can convert the energy absorbed from a LIGHT into a LONGER wavelength Fluorescent probes- fluorescein, rhodamine presence of specific antigen determined by the appearance of color in a BLACK background Direct (antibody CONJUGATED with fluorescent tag) vs. indirect (fluorescent tag added AFTER antigen and antibody reaction has taken place)
Direct agglutination
Found when antigens are found NATURALLY on a particle Example: bacterial typing looking for different antigens
Separation
Fractions are usually separated by PHYSICAL means centrifugation, filtration, decanting followed by a WASH step to remove unbound analyte (critical step)
Antibodies
HIGH affinity for the antigen in question Sensitivity determined largely by the magnitude of AFFINITY very SPECIFIC for antigen Monoclonal antibodies (IMPROVED technique) DECREASED the need for any pretreatment of the antibody
parts of labeled assay **
Labeled and unlabeled ANALYTES Specific ANTIBODY STANDARDS/ CALIBRATORS separation of the bound from the free components Means of detecting the LABEL
Sandwich hybridization
Modification of dot blot TWO probes One probe is bound to MEMBRANE The other is annealed to a different site on the TARGET DNA sample nucleic acid is SANDWICHED between the capture on the membrane and signal generating probe
assay labels
Must NOT alter REACTIVITY of the molecule Should remain STABLE Radioactivity, enzymes, fluorescent and chemiluminescent tags have all been used
Ouchterlony (Double diffusion) **
Older method Both antigen and antibody diffuse INDEPENDENTLY through a semisolid medium in two dimensions (horizontal and vertical) PRECIPITIN line forms where the moving front of antigen meets that antibody The DENSITY indicates the amount of immune complex formed Used to identify fungal infections
Seperation methods
Once reaction of antigen and antibody has taken place there must be a "PARTITIONING step" SEPARATES reacted from unreacted analytes Several methods adsorption, precipitation, solid phase
Rocket Immunoelectrophoresis **
One dimension CONICAL shape resembling a rocket antigen being forced through the gel with ELECTRICAL current The HEIGHT of the rocket is proportional to the amount of ANTIGEN in sample Standards run and a standard curve constructed Advantages is the SPEED (results in a few hours) Extreme caution with NET CHARGES and pH because this determines the DIRECTION of migration in gel
Passive agglutination **
Particles coated with antigen NOT NORMALLY found on their surface Size of particle may vary from 7 microns to 0.05 microns May require chemical coupling in order to achieve attachment Can detect a variety of antigens
Target amplification **
Polymerase chain reaction (PCR) Capable of AMPLIFYING tiny quantities of nucleic acid PRIMERS (small segments of DNA) are added These complement segments of opposite strands that flank the target Only segments of the target DNA between the primers will be replicated
Antigen antibody binding
Primary union of binding sites on antibody with specific epitopes on an antigen. Dependent upon characteristics of the ANTIBODY.
Ligase chain reaction
Probe amplification DNA ligase enzyme Used to JOIN two pairs of oligonucleotide probes only after they have BOUND to the complementary target sequence
Northern blots
RNA is EXTRACTED and SEPERATED Used to determine the level of expression of a particular mRNA RNA does not have to be digested before electrophoresis, must be DENATURED to make sure it unfolds in to a linear piece
Membrane based cassette assay **
Rapid and EASY to perform ONE time use antigen or antibody can be COUPLED to the membrane Reaction is read by a COLOR reaction qualitative "waived" test
Restriction enzymes
Restriction endonucleases Enzymes that CLEAVE both DNA strands at specific RECOGNITION sites (4-6 bp) Fragments are SEPARATED out on the basis of size and charge (gel electrophoresis)
Electrophoresis **
SEPARATES molecules according to the differences in their electric charge Direct CURRENT is forced through the gel Causing antibody, antigen or both to migrate Distinct precipitin bands are formed (can be double or single)
Nucleic acid probe **
SHORT strand of known DNA or RNA that is used to IDENTIFY a complementary strand in a patient specimen
Dot blot
SIMPLEST clinical samples applied directly to membrane SURFACE Membrane heated to denature the DNA Labeled probes added Critical wash step removes unhybridized probe Presence of remaining probe detected by autoradiography or enzyme assays QUALITATIVE testing only
Lattice formation
Second step Sum of the interactions between ANTIBODY and MULTIPLE antigenic determinants on a particle "physiochemical" factors ENHANCED by DECREASING buffers IONIC strength (LISS), with enzyme treatment, increasing centrifugation and viscosity and agitation.
Reading ouchterlony test
Several patterns can occur: Fusion of lines at the junction = IDENTITY Crossed lines (no shared epitopes) = NON identity Fusion of two lines with a spur indicates PARTIAL identity (two antigens share a common epitope but some antibody molecules are not captured by antigen and continue to travel)
Immunofixation electrophoresis
Similar to immunoelectrophoresis, the antiserum is placed directly to the gels SURFACE rather than being sent through a trough CELLULOSE acetate of agarose works well Most often the antibody of a known concentration is used to determine if the patient has the antigen WESTERN BLOT is best known of these tests
Passive immunodiffusion
Support media is usually an agarose (preferred) gel, NO ELECTRICAL CURRENT is used Rate of diffusion is affected by size of particles, temperature, viscosity, amount of hydration, and interactions between the matrix and reactants.
Solution hybridization
Target nucleic acid and the probe free to INTERACT in the mixture Sensitivity is higher than the solid Chemiluminescent label is common If the probe is ATTACHED to the target DNA, it is PROTECTED from hydrolysis Probes that remain bound to a specific target sequence give off light when exposed to a chemical trigger, can be measured
In situ hybridization **
Target nucleic acid is FOUND in intact cell Formalin fixed and paraffin embedded tissue sections Fluorescent labels (FISH) Enzyme labels used to detect malignancies linked to CHROMOSOMAL ABNORMALITIES Example: CML translocation of chromosome 9 to chromosome 22
Immunoassay necessity
The need for RAPID and SENSITIVE methods antigens and antibodies that are very SMALL or in very LOW concentrations Concentrations determined indirectly by labeled REACTANT Detects specific BINDING
Standards or calibrators
Unlabeled analytes made up of known concentrations of substance measured Usually determined by "line of best fit"
Precipitation and light scattering
When antigen and antibody solutions are mixed they are turbid The initial turbidity is followed by precipitation The precipitation can be measured by use of light scattering
Instrumentation
agglutination reactions Increases SENSITIVITY Many work using TURBIDIMETRY Nephelometry Particle counting immunoassay (PACIA)- Measures the number of NON agglutinating residual particles
competitive immunoassay
all reactants are MIXED together simultaneously and labeled antigen COMPETES with unlabeled patient antigen for a limited number of antibody BINDING sites
Radial immunodiffusion (RID) **
antibody is uniformly distributed in the support gel and antigen is applied to a WELL cut into the gel. As antigen DIFFUSES out antigen-antibody combination occurs in CHANGING proportions until zone of equivalence is reached. 2 techniques: end point and kinetic method Used to measure IgG, IgM, IgA and complement components
cross-reactivity
antigens capable of REACTING with antigens that are structurally SIMILAR to the original antigen that induced antibody production
Enzyme assays **
are naturally occurring molecules that CATALYZE biochemical REACTIONS produce BREAKDOWN products Breakdown products may be chromogenic, fluorogenic, or luminescent Cost efficient and readily available, long shelf life, easily adapted to automation
Hydrogen bonds
attraction between POLAR molecules
Ionic bonds
between OPPOSITELY charged particles
Radioimmunoassay ** (RIA)
counts radioactivity Analyte being tested COMPETES with a radiolabeled analyte for a limited number of BINDING sites on antibody. If antigen is PRESENT there will be a DECREASED number of radio labels, and the difference will be amount of antigen found in the patient serum.
kinetic method
diameter proportional to the LOG of the concentration
Noncompetitive ELISA
have a HIGHER SENSITIVITY Detect very low levels of antigen Also referred to as indirect ELISAs Enzyme labeled reagent does NOT participate in the initial antigen antibody binding reaction
Agglutination **
is the aggregation of particulate matter caused by a combination with SPECIFIC antibody to form larger complexes Reaction takes place on the SURFACE of the molecule 2 steps initial BINDING (sensitization) and aggregate (lattice) FORMATION
Nephelometry
measures the light from the incident beam as it passes through the suspension. Can be used to detect antigen or antibody
Precipitation curve
prozone (antibody excess), zone of equivalence(number of multivalent sites of antigen and antibody are equal), postzone (antigen excess)
Hemagglutination
reaction involves RED cells Used in blood banking and immunology (HBV, HCV, HIV I and II and infectious diseases)
Heterogenous enzyme assays **
sensitivity similar to RIA Test conditions carefully controlled NONSPECIFIC binding may occur may have to PRE ABSORB serum to remove IgM RHEUMATOID factor HOOK EFFECT may occur (too much analyte) and the reaction does not progress as expected serum may have to be DILUTED
Analyte
substance being MEASURED, bound by molecules that react SPECIFICALLY with them Typically this is an ANTIBODY One reactant (either antibody or antigen) is LABELED with a marker so the amount of binding can be measured
Avidity
sum of all the attractive forces between antigen and an antibody.
Affinity
the initial force of attraction that exists between Fab site on the antibody and epitope on the corresponding antigen.
Assay methodologies
• Direct immunofluorescence • Competitive ELISA • Noncompetitive ELISA • Indirect immunofluorescence • FPIA
Agglutination methodologies
• Passive • Direct • Hemagglutination • Agglutination inhibition • Particle counting immunoassay
Precipitation methodologies
• Radial immunodiffusion • Electrophoresis • Rocket immunoelectrophoresis • Immunofixation electrophoresis • Western Blot
Molecular methodologies
• Restriction endonuclease • Restriction length polymorphism • DNA chip • Ligase chain reaction (LGR) • Transcription mediated amplification
