IMMUNOASSAY TECHNIQUES

Jill Yeakel

History

1940s Originally designed for use in the medical

community for diagnostic tests

1950s RIA developed by Rosalyn Yalow and

Solomon Berson

Colorimetric measurements of enzymes and metabolites

Awarded Nobel Prize 1977 for ability to detect blood

glucose levels in diabetic patients

1960s Radio-isotopes replaced with enzymes

Safer, faster, higher specificity, longer shelf-life

Forensic Analysis

Screening Tests

Presumptive tests that allow the scientist to eliminate

groups of components within a sample
Used

to exclude drugs prior to confirmation

Quick, easy, sensitive

Confirmation Tests

Analyses that identify specific compounds within a

sample

Immunoassays

Laboratory technique using the binding between an

antigen and its homologous antibody to identify
and quantify the specific antigen or antibody in a
sample

Antibodies immunoglobulins capable of binding to a

variety of natural and synthetic antigens

Antigens immunogens are proteins or substances

coupled to a carrier that elicit antibody formation when
introduced into a host

Antibodies

Immunoglobulins

Y-shaped gamma globulin

proteins composed of:
4

polypeptides

2 Heavy chains
2 Light chains

Paratope variable region

where antigen binds
110-130

amino acids
Determines specificity

Antibody Regions

Constant Region

Determines the mechanism used to destroy antigen

IgM, IgG, IgA, IgD, IgE

Variable Region

Hypervariable (HV)
Complementarity

determining regions, directly contact portion of

antigens surface
HV1, HV2, HV3

Framework (FR)
4

regions that have stable amino acid sequences and separate

HV regions
-sheet structure serves as scaffold to hold HV regions in position

Antigens

Hapten

<2000 Daltons
Small molecular weight compound that elicits immune
response ONLY when attached to large carrier
Necessary to trick animals immune system into reacting by
binding the small molecule with a large antigenic molecule

Epitope

Place on antigen where it is recognized by antibody

Binds with antibody with induced fit, highly specific
interaction

Two Types of Antibodies

Polyclonal

Different types of antibodies are present in the

antisera
Each has a different affinity for the antigen
Stronger Response

Monoclonal

Antibody produced from a hybridoma

All antibodies have identical binding properties
More Specific

Polyclonal Antibody
Production
1.

2.

3.

4.

Inject compounds of
interest (antigen) into
animal
Animal system reacts to
form antibodies to antigen
Antibodies are in animals
blood
Scientists collect animal
blood (serum)

Y Y
Y

Y
Y
Y

Y
Y
Y

Polyclonal Antibodies

Antibodies are derived from different B cell lines

Produces heterogeneous mixture of antibodies that
recognize different epitopes

Have variety of binding affinities and specificities

Generally have broader based cross-reactivity

Easiest, cheapest, and quickest to produce

Monoclonal Antibody
Production
Spleen of Mouse

Monoclonal Antibodies

Sensitized B lymphocytes are fused with immortal

myeloma cells to form hybrid cells (hybridomas)
Hybridomas are capable of secreting antibodies and
reproducing in culture
Highly specific antibodies are produced

Binding Definitions

Affinity the strength of binding between an

antibody and its antigenic determinant
Avidity the strength of binding between antiserum
or an antibody mixture, and an antigen

Antiserum serum with antibodies specific to antigen

Immunoassay Uses

Primary screen of biological samples for drugs

Forensic Urine Drug Testing

Human Performance Testing
Postmortem Investigation

Used for high throughput analyses

Uses less volume of sample

Immunoassay Choice

Choice of immunoassay for laboratories depends on:

Assay sensitivity
Assay specificity
Format and ease of use
Specimen volumes
Analytical equipment available
Cost

Types of Immunoassays

ELISA Enzyme Linked ImmunoSorbent Assay

RIA Radio-ImmunoAssay
EMIT Enzyme Multiplied Immunoassay Technique
FRET Fluorescent Resonance Energy Transfer
FPIA Fluorescent Polarized ImmunoAssay
KIMS Kinetic Interaction of Microparticles in Solution
CEDIA Cloned Enzyme Donor ImmunoAssay
Lateral Flow Immunoassays

Labeled Antigens

Must be immunologically similar to the compound of

interest
Must lend themselves to sensitive detection
Usually prepared by attaching a radioactive
compound, fluorescent compound, or enzyme to the
compound of interest

Methodological Principles

Competitive

Based on competition between the analyte in the

sample and a labeled analyte for a limited amount of
antibody
Measurement of signal infers target compound

Noncompetitive

Sample is allowed to interact with the excess of

antibody
Measurement of signal is used to determine the amount
of analyte present in the sample

Methodological Principles

Heterogeneous

Requires separation of antigen bound to antibody from

the unbound antigen after the reaction occurs
Separation may occur by precipitation, liquid-phase, or
solid-phase adsorption

Homogeneous

Signal from the label is modulated by binding reaction,

thus allowing binding to be monitored without a
separation step

Heterogeneous Assays
ELISA
RIA

ELISA Procedure
1.
2.
3.
4.
5.
6.
7.
8.
9.

Add sample to antibody bound to plate

Add enzyme labeled drug
Incubate
Decant
Wash
Add Color Reagent
Incubate
Add Stop Solution
Analyze

ELISA
H

NH2

H +

NH2

Tetramethylbenzidine

HRP

OH- + OH-

NH + 2H2O

NH

Blue Color Complex

ELISA
Antibody
Drug

H+

Enzyme Bound Drug

H+

H+
H+
H+

H + HRP

OH- + OH-

2H2O
H2SO4

Step 4: Add acid stop (sulfuric acid)

2H+ + SO4-

ELISA
Drugs Present

Enzyme washed
away, nothing to
oxidize substrate

No color

Enzyme free to
oxidize substrate
Drugs
NOT Present

Color

MORE Drug = LESS Color

ELISA

Strengths:

Oriented antibody
Optical enzyme conjugates
Small sample size
Long shelf-life
Non-isotopic

Weaknesses:

Cost per sample is high

Heterogeneous
Common false positive (Sodium azide)

RIA Procedure
1.

2.
3.
4.
5.

6.

Mix known quantity of radio-labeled

antigen/drug with known amount of antibody
Incubate
Add sample containing drug/antigen
Incubate
Separate bound and unbound radioactive
antigens
Measure radioactivity of unbound antigens

RIA
Drugs Present

Radioactive antigen
in Solution
Radioactivity in
Solution

No radioactive
antigen in solution
Drugs
NOT Present

No Radioactivity in
Solution

MORE Drug = MORE Radioactivity

RIA

Strengths:

Sensitive
Resists matrix effect

Weaknesses:

Radioactive waste
Expensive
Short shelf-life
Heterogeneous
False positives from adulterants

Homogeneous Assays
EMIT
FRET
FPIA
KIMS
CEDIA
Lateral Flow

EMIT Procedure
1.
2.
3.
4.
5.
6.
7.

Mix sample containing drug with antibody

Incubate
Add fixed quantity of enzyme bound drug
Incubate
Add substrate and cofactor
Incubate
Measure absorbance 15-45 seconds after
substrate addition

EMIT

EMIT
Enzyme free to
oxidize substrate
Drugs Present

Color

Enzyme CANNOT
oxidize substrate
Drugs
NOT Present

No Color

MORE Drug = MORE Color

EMIT

Strengths:

Homogeneous
Long shelf-life
Assays are well established
More specialty assays are available

Weaknesses:

Many interferences
False negatives are common
Tolmetin

metabolites
Aspirin metabolites

FRET Procedure
1.

2.
3.
4.
5.

Mix sample containing drug with rhodamine

labeled antibody
Incubate
Add known quantity of fluorescein-labeled drug
Incubate
Measure fluorescence of solution

FRET Principle

Depends on the amount of spectral overlap

between donor and acceptor

FRET is related to the distance separating the

donor-acceptor pair
Donor fluorescein labeled antigen
Acceptor rhodamine labeled antibody

FRET
Acceptor and donor
DO NOT interact
Drugs Present

Acceptor quenches
fluorescence
Drugs
NOT Present

MORE Drug = MORE fluorescence

Fluorescence

Less Fluorescence

FRET

Strengths:

Homogeneous
Sensitive
Fast
Robust
Accurate

Weaknesses:

Poor stability of rhodamine labeled antibodies

Interference of scattered light may limit sensitivity
Limited intensity of rhodamine emission

FPIA Procedure
1.
2.
3.
4.
5.

Mix sample containing drug with antibody

Incubate
Add known quantity of fluorescein-labeled drug
Incubate
Measure fluorescence of solution

FPIA
Unbound
Drugs Present
Depolarized
Emission

Bound
Polarized
Light

Polarized
Emission

Drugs
NOT Present
Step 3: Measure fluorescence of solution

MORE Drug = LESS Emission/Polarization

FPIA

Strengths:

Fluorescent tag stability

Low limits of detection
Long shelf-life

Weaknesses:

Expensive
Not easily adapted to automation
False positives:
Ibuprofen
Bile

salts

KIMS Procedure
1.

2.
3.
4.
5.

Mix sample containing drug with antibody (bound

with microparticle)
Incubate
Add fixed quantity of drug polymer conjugate
Incubate
Measure absorbance change over time

KIMS
Drugs Present
No Aggregation

No change in
absorbance

Aggregation
Change in
absorbance

Drugs
NOT Present

Step 3: Measure absorbance change over time

Aggregates formed will scatter light

MORE Drug = LESS Absorbance Change

KIMS

Strengths:

Inexpensive
Homogeneous
Long shelf-life
Microparticle drug conjugate stability

Weaknesses:

Special system maintenance

Small linear range

CEDIA Procedure
1.
2.
3.
4.

5.
6.
7.
8.

Prepare enzyme donor and enzyme acceptor

Mix sample containing drug with antibody
Incubate
Add fixed quantity of enzyme donor ligand conjugate
labeled drug
Incubate
Add substrate and enzyme acceptor
Incubate
Measure absorbance three times over 30 to 60
seconds

CEDIA

Split the enzyme -galactosidase from E. coli

bacteria

Enzyme acceptor 95% of polypeptide sequence

Enzyme donor remaining 5% of polypeptide
sequence

EA and ED combine, then aggregate to form

tetramers which expresses enzymatic activity

Step 1: Prepare enzyme donor and enzyme acceptor

CEDIA

-galactosidase
Chlorophenol red--galactopyranoside

Chlorophenol red
+
-galactopyranoside
Step 4: Observe/measure color

CEDIA
Enzyme donor and
acceptor complement
Drugs Present
RED Color
Cleave substrate

Drugs
NOT Present

Enzyme donor and

acceptor CANNOT
complement

CANNOT cleave substrate

MORE Drug = MORE Color

NO Color

CEDIA

Strengths:

Homogeneous
Long shelf-life
Specialty assays

Weaknesses:

False-negatives common from urine adulterants

Lateral Flow Immunoassay

One-step, competitive membrane-based

immunochromatographic assay
Detector reagent (Ab coated with colloidal gold or
other label) dried onto sample pad interacts with
added sample as it wicks through pads or membranes
of test strip
At a capture line the detector reagent interacts with
the capture reagent that has been immobilized on the
membrane (Ag/drug-derivative) to form a line

One-Step Procedure
Patient Info
COCAINE

OPIATES

THC

METH

AMP
C

Test Strip Interactions

NEGATIVE

C
YYYYYYY

T
YYYYYYY

Test Strip Interactions

POSITIVE

YYYYYYY
C
YYYYYYY

YYYYYYY

Lateral Flow

Strengths:

Fast
Simple
Can be performed at point of collection
Only non-negatives are forwarded to lab for testing
Multiple drugs tested at once

Weaknesses:

Matrix limitations
Subjective
Not automated

IMMUNOASSAY
PERFORMANCE AND
TROUBLESHOOTING

Performance Characteristics

Discrimination Point
Thresholds
Precision and Accuracy
Dynamic Range
Specificity and Cross-Reactivity
Correlation
Interferences

Discrimination Point

Point at which dose response curve is most linear

and discrimination between concentrations is highest
Used to determine the volume of sample to add
Generally in picogram range
Too much volume = decreased discrimination
Too little volume = decreased sensitivity

Setting Thresholds

Concentration of drug below which all specimens

are considered to be negative

Generally above limit of detection

Based on technology and clinical relevance

To minimize issues that have an impact on interpretation

of results
WUDT

Opiates and THC levels

Cross-reactivity of more than 1 target compound

(amp/meth) may increase cutoff

Precision and Accuracy

Measured in terms of a cutoff calibrator

Whether results are correct relative to the cutoff (+/- 25%)

Repeated analysis for samples of known [ ] , both above and
below cutoff [ ]

Precision and Accuracy

Precision is measured both within a plate and between

plates
Precision is affected by:

Reagents, analyzer, temperature and other factors that can

influence the repeatability of the results

Accuracy is affected by:

Components of test specimens that may interfere

Dynamic Range

Linearity and sensitivity are measures of the

proportionality of the analytical response to changes in
analyte [ ]
Curve shape will vary depending on type of
immunoassay used
Ability of test to distinguish (-) and + around cutoff [ ]
can be inferred from slope of the line at that point

Specificity

Specificity

The degree to which an assay correctly identifies only

the compound of interest

Cross Reactivity

Describes the degree of assay response to compounds

other than the one the assay was designed to detect

Correlation

Vendor package inserts provide some data regarding

comparison studies

Data is generally derived from relatively small n

Comparison between initial screening test and

confirmatory test
Evaluated by:

Are specimens identified as +/- confirmed by GC/MS

Can [ ] be predicted by initial test result relative to cutoff

Interferences

Any factor causing bias in an assay result other than the

presence of a target compound
Types of Assay Interferences:

Photometric
Enzyme Activity
Antigen-Antibody Binding behavior
Additional Sources of Interference

Photometric Interferences

Most non-radioisotopic assays rely on photometric

measurement to produce results
Wavelength is specific to assay design

Salicylate metabolites, metronidazole and mefenamic acid

produce initial excessive absorbance at 340nm, interfering
with EMIT assays

Compounds that fluoresce can produce interference in

FPIA, FRET assays

Radiologic imaging dyes

Enzyme Interferences

Physiochemical parameters that affect enzyme activity

Most enzymes have a pH range for optimal activity,

variations may have an impact on performance

Presence of compounds that degrade the enzyme

and/or the presence or absence of specific enzyme
cofactors or inhibitors

Ab-Ag Binding Behavior

Antibody-antigen binding behaviors are governed by

basic chemical principles

Van der waals, etc.

Binding activity can be disrupted by significant changes

in pH, ionic strength, specimen viscosity and other factors
affecting the affinity or conformation of the binding site

Addition of high concentrations of NaCl, sodium bicarbonate,

sodium hypochlorite and H2O2 to urine specimens affect
reagent performance

Sources of Interference

Endogenous compounds
Interference from structurally similar compounds
Cross-reactivity from structurally unrelated compounds
Adulterants
High dose hook effect

Endogenous Compounds

Greater concern when plasma and serum are matrices

Affected by physical or disease state of individual

Proteinuria, nitrites, ketones, blood, bacteria, fungi are

potential sources of nonspecific cross-reactivity

Structurally Similar
Compounds

The activity of sympathomimetic amine compounds in

amphetamine class assays is most commonly observed
Antibodies used in individual assay systems exhibit wide
range of cross-reactivities

Ephedrine, phenylpropanolamine
Significant improved specificity of amphetamine
immunoassays by introducing monoclonal antibodies
High

concentration may still give positive result

Structurally Unrelated
Compounds

False Positive Examples:

Venlafaxine and o-desmethylvenlafaxine with PCP

immunoassays
Rifampin and quinolones with opiate immunoassays
Verapamil and phenothiazines with methadone immunoassays
Oxaprozin with benzodiazepines immunoassays
Sustiva (reverse transcriptase inhibitor) with cannabinoids
immunoassays

False Negative Examples:

Aspirin metabolites decrease signal in enzyme immunoassays

Adulterants

Interference with enzyme activity:

Interference with binding:

Table salt
Bleach
Visine - For EMIT, THCC partitions between the aqueous
solvent and hydrophobic interior of benzalkonium chloride
micelles, reducing availability of THCC to bind to antibody

Interference by breaking down analyte:

Pyridium chlorochromate UrineLuck THC metabolites

Potassium nitrite Klear THC metabolites
Peroxidase, Hydrogen Peroxide Stealth THC Metabolites

High-Dose Hook Effect

More likely to occur in non-competitive assay designs

Lead to decreased signal in the presence of very high
analyte [ ]

When high [ ] of analyte simultaneously saturate both

capture and detection Antibodies, preventing formation of
detectable capture/detecting Antibody complexes

Hook effect refers to the shape of

the dose-response curve when
signal is plotted against [ ]

Applications

In WUDT >90% specimens are negative and can be

reported from initial immunoassay result

Sample Types Urine, but can use oral fluid, hair and sweat
Specimen Volumes Small volume required
Format Qualitative and semiquantitative
Federally regulated labs MUST do initial screen using
immunoassay

In Postmortem

Sample Types Urine, but can use blood, stomach contents,

vitreous, bile, etc.

Instrument Selection

Important considerations:

Lab environment, available space and labor,

compatibility with existing systems (LIMS)
Current and anticipated sample volumes
Analyzer speed (# reportable tests per hour, sample
throughput)
Costs
Availability of other lab references to consult
+/- of using a single or multiple vendors

Quality Control

Record lot # for reagents, calibrators, daily

calibrations
Test QC specimens above and below cutoff
May be isolated or developed as bias over time

Random usually from deterioration or insufficient

volume of QC material in sample vessel and are
resolved by replacing QC material
Shift or ongoing bias systematic issue improper
reagent preparation, storage or shipping conditions

Reagent Considerations

Development of monoclonal antibodies has resulted

in enhanced lot-to-lot consistency in reagents

Shifts in antibody affinity and reagent formulation can

impact performance

Monitoring of calibration, reaction curves, and QC

results provides insight into reagent shifts

Proficiency Testing
Performance

External PTs provide lab with independent

assessment of assay performance and comparison to
other users and reagent systems
Errors or significant bias in results may indicate
problems not identified by routine monitoring systems
Screening errors may indicate a calibration bias or
error, or may also identify reagent integrity or crossreactivity issues
PT sample matrix or analyte composition may also
contribute to performance problems

Troubleshooting

Need to fully understand each assay individually

When a problem is realized, look at the whole
process and watch

Determine if there is really a problem or if

expectations are inaccurate

Review normal performance and compare to

pinpoint issue

Troubleshooting in ELISA

Most critical step is washing

If washer isnt working properly may cause one cell to

perform incorrectly or entire strip
Check by running a blank plate and watching what
happens during the delivery and aspiration steps

To get lowest variance on each plate, make sure no

liquid is left in plate or very little amount that is
consistent throughout the plate

Troubleshooting in EMIT

Major issue is in contaminating Reagent B with

Reagent A

Reagent A: Glucose-6-phosphate dehydrogenase

Reagent B: Glucose-6-phosphate and NAD
Substrate becomes depleted

Large concentrations of drugs depletes NAD

Identified because first absorbance is high and last

absorbance is high (difference between the two is low)

Plate Drift

Process issue, not a plate issue

If absorbance changes across the plate, could cause
false +/

Usually due to washing time with a strip washer

To prevent or check, calibrate at beginning and end of
plate