#26 immunochemistry

8/9/2019 #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

#26 immunochemistry

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