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Transcript of #26 Immunochemistry
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Immunochemistry 101Immunochemistry 101
Chris White, PhD
Scientific Affairs Manager
Beckman Coulter, Inc.
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The Science of Clinical ChemistryThe Science of Clinical Chemistry
The science involving chemical analysis ofbody tissues/fluids to diagnose disease.
McGraw-Hill Dictionary of Scientific & Technical Terms, 6E, Copyright 2003 by The McGraw-Hill Companies, Inc.
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Measurements and Methods
Blood- most frequently analyzed specimen.
Whole Blood Blood Alcohol Serum BMP, CMP
Urine Creatinine
Spinal Fluid Protein and Glucose
Other fluids/specimens Synovial fluid
Wound abscesses
Fecal samples
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WhatWe Measure
Proteins Enzymes- biological catalyst substance that can cause a
change in the rate of a chemical reaction without itselfbeing consumed in the reaction. AST, ALT, ALP Liver
Troponin Heart
Hormones- secretory substance carried from one gland or
organ of the body via the bloodstream to more or lessspecific tissues, where it exerts some influence upon themetabolism of the target tissue. HCG Pregnancy
TSH Thyroid
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WhatWe Measure
Elements
Sodium Potassium
Calcium
Tumor Markers
PSA
OV Monitor
Vitamins
Vitamin D
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How We Measure
Most Common
Spectrophotometry Clinical Chemistry Instruments
Immunoassay Chemiluminescence
Other Detection Systems
Electrochemical Detection ECD- HPLC Chromatography- Toxicology
Electrophoresis- Proteins
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Absor
bance of N
ADH
How reactions work
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Absorbance in a Reaction
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Immunochemistry - 101Chris White, PhD.
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What are Antibodies?
Antibodies are glycoproteins called immunoglobulins
Five Classes IgG, IgM, IgA, IgD and IgE Y-shape
Two heavy chains
Two light chains
Fab Antibody Binding Sites
Fc- complement binding site
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What are Antibodies?
Key reagent in all immunoassays
Physically binds to an antigen
Strength of binding determines sensitivity
Specificity allows detection in complex matrix
(minimal sample preparation)
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What is an antibody?Any of a large variety of proteins normally present in thebody or
produced in response to an antigen which it neutralizes, thus
producing an immune response.
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Key Terms
An antigen is any molecule that is able to elicit
specific immune response. An antibody- protein produced by the immune
system in response to an invading foreign
substance.
An epitope part of the antigen which binds toantibody (usually 5 10 amino acids). Aparatope Hyper variable part of the antibody
that binds with the antigen.
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What is an Immunoassay?
An analytical method that uses antigen-antibody
interactions to quantitate very low concentrationof analytes.
Yalow & Berson - Awarded Nobel Prize in 1977
-Uses
detection, quantitation,monitoring, diagnosis
-Analyte (Targets)
Proteins, Hormones , Peptides, Carbohydrates, Vitamins,Pharmaceutical agents, Infectious agents, Lipids
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Standardization
Antibody selection
Sample Interferences
Factors AffectingImmunoassay Performance
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Standards / Calibration
Source of pure or purified analyte is essential for
calibration and antibody production For small analytes, pure materials are available
steroids, drugs, peptides etc
Larger proteins are more heterogeneous
International standards are desirable and are availablefor few analytes only to calibrate new methods (NIH,
WHO, NIBSC)
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Standardization
In clinical chemistry, well established
methods have been chosen as definitive orreference method, against which a new
methods are calibrated (CLSI)
For protein immunoassay standardization, no
reference methods exist
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Calibration material:
Protein standards are heterogeneous and differ from sample
analyte
International reference standards desirable
Biological samples often contain proforms, splice variants,
fragments and aggregates
Glycoprotein analytes - heterogeneity due to variable glycosylation,
sialylation, polymerization, aggregation and decomposition
Standardization Issues
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Reasons forMethod Bias
Key Reasons:
Different companies use different antibodies that recognize
different glycoforms to some (or significant) different degree
The different companies use different calibration material
which contains the different glycoforms in different amounts
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Key Property - Specificity
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Antibody Specificity
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Homogeneous andHeterogeneous Assays
Homogeneous
Do not require separation of immune complexes
Detection occurs directly in the reaction vessel on your
chemistry system.
Examples: EMIT, Turbidometric
Use Simple assay protocol - easy to automate
Short incubation times, limited sensitivity and measuringrange
Highly suitable for small molecular weight compounds: Drugs,Steroids, Thyroid hormones, Peptides
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Heterogeneous Assays
Heterogeneous
Requires physical separation of immune complexes prior todetection
Examples: ELISA, FIA, LIA, ECLIA, ECIA, DELFIA, EIA
Use
The concentration of immune complexes is too small for
direct detection A solid phase separation system is needed
A sensitive molecular label is needed
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Immune Complex Separation Methods
Coated magnetic beads
Latex beads Coated tubes, micro-wells, capillary Precipitation (PEG, Double antibody) Gel filtration, electrophoresis, liquid phase separation Adsorption - Dextran coated charcoal Membranes
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Key PointsHeterogeneous Immunoassay
Generally more complex to automate
Higher sensitivity, wider dynamic measuring range
Allows us to measure extremely small amounts of
antigen in blood (10-9 - 10-15 mol/L)
Suitable for large variety of compounds:
Proteins, virus, bacterial antigens, antibodies,drugs, steroids
Variety of assay formats possible
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Heterogeneous Competitive Format
Competition between labeled and unlabeledantigen for a limited number of binding sites
(antibody).
All reactants are mixed simultaneously or
sequentially. The amount of antigen in the test sample is
inversely proportional to the signal measured.
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Competition Assay Small Targets
EE
Light
1
2 E +
+
Substrate
Target
ALP- hapten
Conjugate
Magneticbead withantibody
Hapten-antibodyComplex
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Skit!
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Comments - Competitive Assays
The limiting reagent is the antibody
If the molecule is too small to bind two antibodies, aCOMPETITIVE assay design is used
Molecular musical chairs
Works well for small molecules, drugs and hormones
Homogeneous and heterogeneous assay formats
Less sensitive than non-competitive
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H
eterogeneous Non-competitive Format(Immunometric, Sandwich)
An excess amount of antibody captures the analyte
from the sample. A labeled second antibody binds to the first antigen-
antibody complex to form a sandwich. This complex
is measured.
Can be one or two step methods.
Provide the highest level of assay sensitivity and
specificity.
The amount of antigen in the test sample is directly
proportional to the signal measured.
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1 Step Assay
Two antibodies are
incubated with thespecimen with no washsteps between additions.
One antibody labelled with
an nzyme or conjugate.
One antibody bound tomagnetic particles.
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Immunometric (sandwich) Assays
Generally uses two antibodies
One antibody attached to solid phase
(paramagnetic particles)
Another antibody conjugated to labeled marker
e.g. enzyme ALP
One-step and two step assay format possible
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2 Step Assay
Sandwiches are formed with
the desired Target as filling.
Additional wash steps are
done to remove unwanted
specimen components.
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AMPPD(Substrate)
AlkalinePhosphatase
(Enzyme)
Alkaline Buffer
+ + Light
530 nm
Immunoassay SystemChemiluminescence Detection
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Immunoassay Labels & Detection Limits*
Label Type Detection Limit (M)
Fluorescence
ChemiluminescenceFluorescence Polarization
Radioactive
Time Resolved Fluorescence
lectrochemiluminescence
nzymeb
ased:Photometric
Fluorescence
Coupled nzyme Reaction
Chemiluminescence
*ClinicalDiagnostic Technology,AACC Press 2005
1 x 10 10
1 x 10 13
1 x 10 14
1 x 10 15
1 x 10 17
2 x 1020
1 x 10 16
1 x 10 19
1 x 10 20
1 x 10 21
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HAMA (Human Anti-Mouse Antibody)
Heterophile Antibody
Hook ffect
Sample Interferences
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Presence of HAMA in human sample
Produces falsely elevated (false positive) or suppressed (false
negative) results in immunoassays
Sandwich assays are usually the most susceptible to HAMA
interference
Manufacturers utilize various techniques to minimize the impact
from HAMA including:
True two step design to wash away interference
Blockers to reduce if not eliminate interference by occupying
the binding sites on HAMAs
Human Anti-Mouse Antibody HAMAInterference
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HAMA Interference Sandwich
Assay
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Immunoassay Data Reduction
Dose response curves are NOT linear
Complex mathematical manipulation of datais required (software)
Data reduction varies with assay
The high dose hook effect is very dangerous
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Hook ffect
High Dose Hook ffect: Antigen xcess (Prozone ffect)
Very high concentrations of antigen in the patient sample bind to allavailable sites saturating them - on both the antibody-solid phase and
the antibody-labeled conjugate and thereby prevent the sandwich
formation
Under these conditions, the measured level of analyte may be
significantly lower than the actual level present in the sample
Phenomenon inherent with one-step sandwich assay designs
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High Dose Hook
Very dangerous normal result for a veryabnormal result!! Because the dose response
curve is altered
Can eliminate with pre-dilution is the onlypreventative measure.
Not caused by Interfering substances
Tumor markers can be present in very highlevels
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Suspect Interference?
Delta checks: new results compared to
previous results. Results do not match the condition.
Lack of proper relationship, e.g. high T4 and
high TSH.
Serial dilution does not result in a linearresponse.
Different methods give different results.
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Interference
ffect of Interferences:
False Negatives:
False sense of security
Delayed therapy or intervention
False Positives:
Unnecessary therapy or clinical intervention
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Monoclonal
Lot-to-lot consistency
Indefinite supply
Highly specific
Longer lead time
Higher initial costs
Polyclonal
Lot-to-lot variability
Limited supply
More broadly reactive
Shorter lead times
Lower initial costs
Often more sensitive
Selection is based on application, time and money
Monoclonal vs. Polyclonal