Immunoassay

and its validation

verification
process
Dr. Ali
Mirjalili
Pishtaz Teb
Diagnostics
Razi Vaccine
& Serum
Research
Ins.

1. History
2. Introduction
3. Principle
4. Classification
5. Application
6. Designing Steps
7. Quality control
8. Instrument
9. Training video

Contents
History of immunoassay
40 years ago Radioimmunoassay
developed for human Insulin
immunoassays have been developed with
emphasis on fast and sensitive detection
technologies and automated systems
History


40 years ago Radioimmunoassay
developed for human Insulin
1971 Engvall and Perlmann,
VanWeeman and Schuurs
introducing ELISA
In the 1972, the development of
EMIT Enzyme Multiplied
Immunoassay (Homogenous
Immunoassay)

Introduction
Laboratory Diagnosis based on:
Basic method (Culture,
Microscopy, etc)
Ag, Ab Detection
methods (Immunoassay)
Nucleic Acid methods
(Amplification methods)
Principle
(1) The specific antigen used to capture
target antibody;
(2) Specific Antibody, and the detector or
secondary antibody used for indirect
detection of antibody;
(3) the detection method
Definitions
Antibodies (also known as
immunoglobulins abbreviated Ig) are
gamma globulin proteins that are found in
blood and are used by the immune system
to identify and neutralize foreign objects,
such as bacteria and viruses.

Definitions- cont
Antigens
A substance that when introduced into the
body
stimulates the production of an antibody

Immunoassay
A laboratory technique that makes use of the
binding between an antigen and its
homologous antibody in order to identify
and quantify the specific antigen or
antibody in a sample

Definitions- cont
Analyte
The sample being analyzed and in
immunoasssays the analyte is either
Antibody or Antigen

Antibody Production

Specific antibodies are produced by
injecting an antigen into a mammal, such
as a mouse, rat or rabbit for small
quantities of antibody, or goat, sheep, or
horse for large quantities of antibody.
Blood isolated from these animals contains
polyclonal antibodiesmultiple antibodies
that bind to the same antigenin the
serum, which can now be called antiserum.



Properties of the antibody-antigen
bond
Non-covalent
Reversible
Intermolecular forces
Coulombic interactions (hydrogen bonds)
Hydrophobic interactions
van der Waals (London) forces
Classification of immunochemical
detection methods
Precipitation
Immunodiffusion
Immunoelectrophoresis
Agglutination
Latex
Hemagglutination
Light scattering
Nephelometry
Turbidimetry
Particle
methods
Colorimetric
Radiometric
Chemiluminescent
Fluorescent
Label
methods
Precipitation of antibody/antigen
complexes
Detection of the
antibody/antigen
complex depends on
precipitation
No label is involved
Many precipitation
methods are
qualitative, but
there are
quantitative
applications, too
Factors affecting solubility
Size Charge
Temperat
ure
Solvent
ionic
strength
Zone of equivalence
The precipitin reaction
P
r
e
c
i
p
i
t
a
t
e

Antibody/Antigen
etc.
Single radial immunodiffusion
Ag
Single radial immunodiffusion
] [Ag r
r
Electroimmunodiffusion
+
-
Immunoelectrophoresis
Electrophoresis provides separation
Immunoprecipitation provides
detection
Combines serum
protein
electrophoresis with
immunometric
detection
Immunoelectrophoresis
Immunofixation electrophoresis
Two related
applications:
Immunoelectrophoresis
Specimen
-human serum
+
-
Immunoelectrophoresis
P
C P C P C

+
-
Immunofixation electrophoresis
SPE IgG IgA IgM
Agglutination
Agglutinins
Antibodies that
produce such
reactions
Involves two-step
process:
Sensitization or initial
binding
Lattice formation or
formation of large
aggregates


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Agglutination
Types of particles that
participate in such reactions:
Erythrocytes
Bacterial cells
Inert carriers such as latex
particles
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Agglutination tests
Antibodies can agglutinate multivalent
particulate antigens, such as Red Blood Cells
(RBCs) or bacteria
Some viruses also have the ability to
agglutinate with RBCs.
This behavior is called agglutination.
Serological tests based on agglutination are
usually more sensitive than those based on
precipitation
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Examples
Slide Agglutination Test
Plate Agglutination Test
Tube Agglutination Test
Passive Agglutination Test
Microscopic Agglutination Test
Haemagglutination test (HAT)
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Steps in Agglutination
Primary phenomenon
(SENSITIZATION)
First reaction involving Ag-Ab combination
Single antigenic determinant on the surface
particle
1) Initial reaction: rapid and reversible
2) Cross link formation visible aggregates
(stabilization)




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Secondary phenomenon:
LATTICE FORMATION
Ab + multivalent Ag stable network (visible
reaction)
conc. of Ag and Ab
Governed by physiochemical factors:
Ionic strength of milieu
pH
temperature

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Secondary Phenomenon
Lattice Formation
The Fab portion of the Ig molecule attaches to antigens on
2 adjacent cells-visible results in agglutination
If both antigen and antibody are SOLUBLE reaction will
become visible over time, ie, precipitation
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DIRECT AGGLUTINATION
- Test patient serum against large, cellular
antigens to screen for the presence of
antibodies.
Antigen is naturally present on the
surface of the cells.
In this case, the Ag-Ab reaction forms an
agglutination, which is directly visible.
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DIRECT AGGLUTINATION
The particle antigen may be a bacterium.
e.g.: Serotyping of E. coli, Salmonella using a
specific antiserum
The particle antigen may be a parasite.
e.g.: Serodiagnosis of Toxoplasmosis
The particle antigen may be a red blood cell.
e.g.: Determination of blood groups
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DIRECT AGGLUTINATION
These reactions
can be performed
on slides (rapid
tests) or on
microliter plates
or tubes for
Antibody titration
if required.

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Positive Negative
Ag-Ab complex
Direct agglutination
Principle
combination of an insoluble
particulate antigen with its soluble
antibody
forms antigen-antibody
complex
particles clump/agglutinate
used for antigen detection
Examples
bacterial agglutination tests
for sero-typing and sero-
grouping e.g., Vibrio cholerae,
Salmonella spp

Slide Agglutination Test
Used for serotyping (e.g. Salmonella)
Antigen: isolated Salmonella in suspension
Antibody: specific antisera against Salmonella
Place test Salmonella in a drop of saline on a
slide
Add a drop of antiserum, mix and rock slide
for approx. 1 minute
Examine for agglutination
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Slide Agglutination Test
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Tube Agglutination Test
Also known as the standard agglutination test or
serum agglutination test (SAT)
Test serum is diluted in a series of tubes (doubling
dilutions)
Constant defined amount of antigen is then added to
each tube and tubes incubated for ~20h @37C
Particular antigen clumps at the bottom of the test
tube
Test is read at 50% agglutination
Quantitative
Confirmatory test for ELISA reactors
Example: Brucellosis screening , Widal Testing
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Tube Agglutination Test
No agglutination Agglutination
1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl
In this case, the titre is 1/40
Tube Agglutination Test
Passive Agglutination
An agglutination reaction that employs
particles that are coated with antigens not
normally found in the cell surfaces
Particle carriers include:
Red blood cells
Polystyrene latex
Bentonite
charcoal

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Passive Agglutination
Passive agglutination has been used
in the detection of :
Rheumatoid factor
Antinuclear antibody in LE
Ab to group A streptococcus antigens
Ab to Trichinella spiralis

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Passive Agglutination Test
Converting a precipitating test to an
agglutinating test
Chemically link soluble antigen to inert
particles such as LATEX or RBC
Addition of specific antibody will cause the
particles to agglutinate
Reverse PAT: antibody linked to LATEX
e.g. Lancefield grouping in Streptococci.

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REVERSE PASSIVE
Agglutination Tests

Antibody rather than antigen is attached to a carrier particle
For the detection of microbial antigens such as:
Group A and B streptococcus
Staphylococcus aureus
Neisseria meningitides
Haemophilus influenza
Rotavirus
Cryptococcus neoformans
Mycoplasma pneumoniae
Candida albicans
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Quantitative Micro
Hemagglutination Test (HA)
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Haemagglutination Test (HA)
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Haemagglutination
RBC
Viral Haemagglutination
Some viruses and microbes contain proteins which
bind to erythrocytes (red blood cells) causing them
to clump together

NDV
Adenovirus III
AIV
IBV
Mycoplasma



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Viral Hemagglutination
the attachment of viral particles by their receptor sites to
more than 1 cell.
As more and more cells become attached in this manner
agglutination becomes visible
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Titer = 32 HA units/ml
Hemagglutination test: method
1:8
1:2 1:2 1:2 1:2 1:2
8 16 32 64 128 256
virus
serial dilution
mix with red
blood cells
side view
top view
One HA unit :minimum amount of virus that causes
complete agglutination of RBCs
In the absence of anti-virus
antibodies
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Erythrocytes
Virus
Virus agglutination of
erythrocytes
In the presence of anti-virus
antibodies
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Erythrocytes
Virus
Anti-virus
antibodies
Viruses unable to bind to
the erythrocytes
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What is Antibody Titer
Is the lowest
concentration
of antibodies
against a
particular
antigen.
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Figure 18.6
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Readings
The end point is the well with the lowest
concentration of the serum where a clear button is seen.
2 4 8 16 32 64 128 256 512 1024 2048 4096



The antibody titer in this row will be 512 (2
9
).
(the lowest concentration of Abs which inhibit HA caused by the virus )

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Coombs Test an Agglutination Test
The Coombs test is
actually two
separate tests: the
"direct" and
"indirect" Coombs
tests. Both aim to
identify autoimmune
haemolysis of red
blood cells
(erythrocytes).

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Coombs (Antiglobulin)Tests
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Incomplete Ab
Direct Coombs Test
Detects antibodies on erythrocytes
+


Patients RBCs Coombs Reagent
(Antiglobulin)
Coombs Test
Direct ant globulin test (also called the Coombs
test,

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Coombs (Antiglobulin)Tests
Indirect Coombs Test
Detects anti-erythrocyte antibodies in serum
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Patients
Serum
Target
RBCs
+


Step 1
+

Coombs Reagent
(Ant globulin)
Step 2
Application of Coombs (Antiglobulin)Tests
Applications
Detection of
anti-Rh Ab
Autoimmune
hemolytic
anemia

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Agglutination Inhibition
Based on the competition between
particulate and soluble antigens for
limited antibody combining site
Lack of agglutination is indicator of a
positive reaction
Usually involves haptens complexed with
proteins
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Agglutination Inhibition
Tests

Pregnancy Testing
-classic example of
agglutination inhibition
Human chorionic
gonadotropin
(hCG)
Appears in serum
and urine early in
pregnancy

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Agglutination Inhibition
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Urine Antiserum
No hCG in urine:
Anti-hCG free
hCG in urine:
Anti-hCG neutralized
Carriers coated with hCG added
Carriers coated with hCG
added
AGGLUTINATION of carriers:
Negative test for hCG
NOT PREGNANT
NO AGGLUTINATION of carriers:
Positive test for hCG
PREGNANT
Co-agglutination
Co agglutination is similar to the latex
agglutination technique for detecting antigen
(described above). Protein A, a uniformly
distributed cell wall component of
Staphylococcus aureus, is able to bind to the
Fc region of most IgG isotype antibodies
leaving the Fab region free to interact with
antigens present in the applied specimens.
The visible agglutination of the S. Aureus
particles indicates the antigen-antibody
reactions
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Coagglutination
Name given to systems
using inert bacteria as
the inert particles to
which the antibody is
attached
S.aureus: most
frequently used
because it has protein A
in its outer surface that
naturally adsorbs the Fc
portion of the antibody


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Highly specific but not very sensitive in detecting
small quantities of antigen


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Co agglutination Test
Agglutination test in
which inert particles
(latex beads or heat-
killed S aureus Cowan
1 strain with protein
A) are coated with
antibody to any of a
variety of antigens
and then used to
detect the antigen in
specimens or in
isolated bacteria.

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Complement fixation Test
The complement fixation test (CFT) was extensively
used in syphilis serology after being introduced by
Wasserman in 1909.
Complement is a protein (globulin) present in normal
serum.
Whole complement system is made up of nine
components: C1 to C9
Complement proteins are heat labile and are
destroyed by heating at 56C for 20 30 minutes.
Complement binds to Ag-Ab complex
When the Ag is an RBC it causes lysis of RBCs.
Principle
Complement takes part in many of the immunological reactions.
It gets absorbed during the combination of antigens and
antibody.

This property of antigenantibody complex to fix the
complement is used in complement fixation test for the
identification of specific antibodies.

The hemolytic system containing sheep erythrocytes (RBC) and
its corresponding antibody (Amboceptor) is used as an indicator
which shows the utilization or availability of the complement.

If the complement is fixed then there will be no lysis of sheep
erythrocytes, thus denoting a positive test.

If the complement is available then there will be hemolysis
which is a property of complement, denoting a negative test.
Components of CFT
Test System
Antigen: It may be soluble or particulate.

Antibody: Human serum (May or may not contain Antibody
towards specific Antigen)

Complement: It is pooled serum obtained from 4 to 5 guinea
pigs. It should be fresh or specially preserved as the complement
activity is heat labile (stored at -30 C in small fractions). The
complement activity should be initially standardized before using
in the test.

Indicator System (Hemolytic system)
Erythrocytes: Sheep RBC

Amboceptor (Hemolysins): Rabbit antibody to sheep red cells
prepared by inoculating sheep erythrocytes into rabbit under
standard immunization protocol.

Positive Test
Step 1:
At 37C
Antigen + Antibody + Complement Complement gets fixed
(from serum) 1 Hour



Step 2:
At 37C
Fixed Complement complex + Hemolytic system No Hemolysis
1 Hour (Test Positive)
Negative Test

Step 1:
At 37C
Antigen + Antibody absent + Complement Complement not fixed
1 Hour



Step 2:
At 37C
Free Complement + Hemolytic system Hemolysis
1 Hour (Test Negative)
Results and Interpretations:
No hemolysis is considered as a positive test.
hemolysis of erythrocytes indicative of a negative test.
1 2 3 4

A


B


Microtiter plate showing Hemolysis (Well A3, A3 and B4) and
No Hemolysis (Well
Light reflection
Distribution of scattered radiation
Nephelometry vs. Turbidimetry
0-90
Terms
Transmittance (T) - The ratio of the radiant flux
transmitted by the test substance to that of the
incident radiant flux. Terms formerly used include
transmittancy and transmission.
Turbidance (S) - A measure of the light-scattering effect
of suspended particles.
Turbidity () - In light-scattering measurements, the
turbidity is the measure of the decrease in incident
beam intensity per unit length of a given suspension.
Analytical methods using labeled antigens/antibodies
What are desirable properties of labels?
Easily attached to antigen/antibody
Easily measured, with high S/N
Does not interfere with antibody/antigen reaction
Inexpensive/economical/non-toxic
Principle

Colorimetric, Radiometric,
Chemiluminescent, Fluorescent
Immobilized antibody methods
Coated tube
Coated bead
Solid phase antibody methods
Coated tube methods
Specimen Labeled antigen
Wash
Coated bead methods
Microparticle enzyme immunoassay
(MEIA)
Labeled antibody
E
E E
S P
Glass fiber matrix
Magnetic separation methods
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Magnetic separation methods
Fe Fe Fe Fe Fe
Aspirate/Wash

Colorimetric
ELISA
Immunoblotting
Lateral Flow Diffusion
(handheld assay)


Contact
Zone
Detection
Zone
Internal
Control
Zone
Fluorescent
Latex
Ab-coated
Immobilized Ab
Sample
Pad
Wicking Pad
Ag
Ag
Ag
Ag
Ag
Ag
Radioisotope labels
Advantages
Flexibility
Sensitivity
Size
Disadvantages
Toxicity
Shelf life
Disposal costs
Radiometric
RIAs use 125 I, 14 C,
or 3 H labeled Ag/Ab
as so- called tracer.
Enzyme labels
Advantages
Diversity
Amplification
Versatility
Disadvantages
Lability
Size
Heterogeneity
93
Enzyme Immunoassay (EIA)
Typical enzyme labels include alkaline
phosphatase, horseradish peroxidase and b-
galatosidase.
Substrate: TMB: 3,3', 5,5'-tetramethyl benzidine
OPD: o-phenylene diamine
ABTS: 2, 2'-azino-bis(3-ethylbenzthiazoline-6-
sulfonic acid)
94
Fluorescent labels
Advantages
Size
Specificity
Sensitivity
Disadvantages
Hardware
Limited selection
Background
Fluorescent Immunoassays
(1) direct Flu. assay (DFA),
(2) indirect immunouorescence (IFA),
(3) Flow cytometry (FC)
(4) multianalyte profile (xMAP) technology /multiplexed
particle-based fow cytometric assays technology,
Two Approaches
Planar microarrays
Encoded micoparticle assay (Suspension)
xMAP technology by Luminex, the CBA
technology by Becton Dickinson BioSciences,
and the VeraCode technology by Illumina, as

Chemiluminescent labels
Advantages
Size
Sensitivity
S/N
Disadvantages
Hardware
?
Chemiluminescent labels
+ 2H
2
O
2
+ OH
-
COO
-
COO
-
O
-
O
-
+ h (
max
= 4 3 0 nm)
+ N
2
+ 3H
2
O
NH
2
L umi n o l
Pe r o x i d a s e
O
O
N
N
H
NH
2
H
O
O
*
NH
2
Chemiluminescent labels
CH
3
N
+
CO
2
H
O O
B r
-
Ac r i d i n i um e s t e r
O
-
CO
2
H
+ H
2
O
2
+ OH
-
+
+ CO
2
+ h
O
CH
3
N
Electrochemiluminescence (ECL)
similar to ELISA except that the secondary
antibody is labeled with a chemiluminescent
label ruthenium (Ru)
Electron transfer between the Ru atom and
the substrate tripropylamine (TPA) results in
photon production
Introduction to Heterogeneous versus
Homogenous Immunoassay
They require separation of
bound and free ligands
What is the
distinguishing feature of
heterogeneous
immunoassays?
Yes
Do heterogeneous
methods have any
advantage(s) over
homogeneous methods?
Sensitivity
Specificity
What are they?
Introduction to Homogeneous
Immunoassay
What is the distinguishing feature of
homogeneous immunoassays?
They do not require separation of bound and free ligands
Do homogeneous methods have any
advantage(s) over heterogeneous methods?
Yes
What are they?
Speed
Adaptability
Homogeneous immunoassays
Virtually all homogeneous immunoassays are one-
site
Virtually all homogeneous immunoassays are
competitive
Virtually all homogeneous immunoassays are
designed for small antigens
Therapeutic/abused drugs
Steroid/peptide hormones
Typical design of a homogeneous
immunoassay
No signal
Signal
Enzyme-multiplied immunoassay
technique (EMIT)
Developed by Syva Corporation (Palo Alto, CA) in 1970s--now owned by
Behring Diagnostics
Offered an alternative to RIA or HPLC for measuring therapeutic drugs
Sparked the widespread use of TDM
Adaptable to virtually any chemistry analyzer
Has both quantitative (TDM) and qualitative (DAU) applications; forensic
drug testing is the most common use of the EMIT methods
EMIT method
Enzyme
S
S P
No signal
Signal
Enzyme
S
Immunoassay Different Format
Immunoassay
Indirect Ag Sandwich Ab Competition
Antibody Detection
Antigen Detection
Ab Sandwich Ag Competition
Application

ELISA Manufacturing
1. Development
2. Optimization
3. Validation
Basic Steps for Developing and
Running an Immunoassay
1 Establish assay critical
success factors.
2 Ensure appropriate
antibody and antigen
reagents are available.
3 Adsorb antigen or primary
antibody to a solid surface.
4 Block nonspecific binding
sites to reduce background.
5 Incubate the primary
antibody with the sample.
6 Wash off unbound
reagents.
7 Incubate secondary
antibody-conjugate with
sample.
8 Wash off unbound
reagents.
9 Incubate substrate to
generate signal.
10 Calibration curve fitting,
data analysis and
quantitation by non linear
regression
IMMUNOASSAY PARAMETERS

1. Analyte (hapten or antigen) to be measured.
2 .Sample matrices in which measurements will be
made (serum, plasma, cell lysates, culture media etc.)
3 .Source of antibody, analyte standards and detection
reagents (labeled antibody, enzyme substrates etc).
Availability of these reagents is a critical requirement.
4 . Detection mode (colorimetric, fluorescence or
chemiluminescence) and appropriate plate readers.
5 .Type of immunoassay to develop (sandwich,
competitive).
6 .Expected analyte concentration ranges to be
measured (pg/ml, ng/ml or g/ml) in the sample matrix
of choice.
IMMUNOASSAY PARAMETERS

7. Data analysis models and format for reporting
results.
8. Validation and optimization criteria using
statistical experimental design tools.
9. Recovery, accuracy and precision expected at the
limits of quantification and the measurable range.
10. Sample throughput, frequency of use,
automation and the number of laboratories that
would run the assay.
11 Control samples that would be used for
optimization, validation and quality control runs.
Design
Optimization
ELISA Kit
Results
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OIE principles and Methods of
Diagnostic Test Validation

Company Logo
Manufacturer Standards
Harmonized Standard
Directive 98/79/EC
Laboratory Standards
ISO 15189, ISO guide 25
ISO 13485
Quantitative QC - Module 7 120
The Quality Management System
Organization Personnel Equipment
Purchasing
&
Inventory

Process
Control
Information
Management
Documents
&
Records
Occurrence
Management

Assessment
Process
Improvement
Customer
Service
Facilities
&
Safety
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Five M of Quality
Man
Machine
FACILITY (SIZE, CONSTRUCTION, LOCATION)
Qualifications,
Organization, Job
description,
Training, etc.
Qualification, Calibration
Manual
Methodology
Material/
Sample
Storage
Label
Motivation
SOP, Mfr
Bruchure
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METHOD VALIDATION
Specificity
Mfr claimed
Performance
Characteristics
(Quantitative, Qualitative)
Reference
Interval
Stability
Known
Panels
Quantitative QC - Module 7 123
Accuracy and Precision
Accurate = Precise but not Biased
Accurate
and Precise

Precise
but Biased
Imprecise
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Measurement of Precision
1 2 3 4
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Validation methods 126
Accuracy
Closeness of determined value to the true value.
Represent Systemic Error. or Bias (X-)

The acceptance criteria is mean value 15%
deviation from true value.

At LOQ, 20% deviation is acceptable.

Accuracy (%) = 100 x
Found value - Theoretical value
Theoretical value
Poor Precision
Good Accuracy
Good Precision
Poor Accuracy
Poor Precision
Poor Accuracy
Gold
Standard
Silver
Standard
Off-Base
Model
Hit or
Miss Model
Good Precision
Good Accuracy
Graph
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1. Recovery test: Adding a known amount
of analyte to a base and measuring the
concentration
2. Specificity (cross-reactivity)
3. Interferences
4. Parallelism (Linearity)
Correlation
Comparing the
results to a
reference values
obtained from a
definitive method,
How Accuracy Determined
Direct
Indirect
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Correlation
3. Parameters like m,
Y intercept, r, Bias, etc
2. Samples: At least 40 samples
(~200-300 serum samples)
1. Reference method
Regression Statistics Review:
Correlation Coefficient (r) - characterizes the
dispersion of results around the line of best fit.
Slope - The lean of the line of best fit
(proportional bias)
Y-Intercept - the point at which the line of best fit
intersects the Y axis. (constant bias)
Acceptability Criteria:
Correlation Coefficient (r) - the closer to 1.0 the
better
Slope - The closer to 1.0 the better
Y-Intercept - the closer to zero the better
m b Sy Bias SDd t r
0.92 -0.01 0.65 -0.32 0.67 0.00 0.98
Random Constant Proportional Proportional Error(Calibration)
m No No Yes 8.24%
b No Yes No Random Error (Precision)
Sy Yes No No 0.65 0.67
Bias No Yes Yes Constant Error (Specificity)
SDd Yes No Yes -0.01 -0.32
r Yes No No
Indirect Accuracy Detection
Recovery (C-B/A)x100
Matrix Effect
Precision
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Linearity Measurement
1
2 3
Requires a
minimum of
4-5 concent.
levels

0, 25%, 50%,
75%, 100%
solution
3 replicates
of each
solution
tested
Linearity Graph
0
10
20
30
40
50
60
70
80
90
0% 25% 50% 75% 100%
Di l ut i on %
C
o
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c
.
SAMPLES Mean OD Mean Con. Mean Exp.Con. RECOVERY
0% 0.011 0.007 0.01
0.009
0
0.0
100%
25% 0.362 0.35 0.304
0.339
3
3.3
92%
50% 0.736 0.752 0.757
0.748
6.7
6.6
102%
75% 1.066 1.08 1.052
1.066
10.2
9.8
104%
100% 1.322 1.379 1.303
1.335
13.1
13.1
100%
OD
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Sensitivity
Definition: Smallest amount of analyte that can
be detected under the conditions of the assay
1. Lower limit of detection, ie., The least or minimum detection dose (LDD)
2. Minimum distinguishable difference in concentration, Resolution (MDDC)

The sensitivity of an analytical method is its
ability to give response to small changes in
the absolute amount of analyte present
1
2
3
High sensitivity
Concentration (X)
added quantity
Response (Y)
measured
quantity
Three analytical areas
1 2 3
Xb
not
detected
Area of
detection
Area of
quantification
or CV<20%
LOD LOQ
Indirect Accuracy Detection
Interferences
Matrix effect
Proteins,RF,Complement,
Heterophilic Ab, Mechanical ,
Nonspesific, Hook effect
:




Indirect Accuracy Detection
Specificity /Cross reactivity
Similarity of Biochemical Structures
Detection Methods
Qualitative Measurement
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Stability
Accelerated Stability
Full term stability
In use stability
Simulated stability
Transport stability


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Evaluation in Lab
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Lab Method Evaluation
1.Initial screening using available
information
a. Positive attributes of the product
1. Less cross-reactivity
2. Instrument features
3. Better service support
4. Cost
b. Gather information
1. Find out which methods are being used by your colleagues
2. External quality control schemes are a useful reservoir of
information
a. How many participants
b. Which methods are growing popularity
c. Estimates of precision, %CV, mean, outliers
d. Organizations Dept. of health and social security DHSS
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Method Evaluation
1.Initial screening using available
information (Continue)
c. Contact the manufacturers for
1. Brochures
2. Product information
Check whether accessory equipment or reagents are required
Check the labor requirements and turnaround times
Check environmental specifications, Laboratory temperature
and humidity

a. Basic test of product performance
b. Evaluate the quality of the instructions
c. How good the service is from the supplier


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Method Evaluation
Description (2 Kits) Replication
Non-specific binding (NSB) or no sample 2
Set of controls normally used by laboratory 4 x 2
Zero calibrator 10
Remaining Calibrators 5 X 2
Kit manufacturers controls 3 x 2
Patient samples 12 x 2
Sample dilutions (1/2, 1/5, 1/10) 3 x 2
Diluent 1 x 2
External QC scheme samples 5 X 2
Set of controls normally used by laboratory 5 x 2
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Method Evaluation
Analysis of results from initial kit
evaluation
The calibration curve should be fitted as
recommended by the manufacturer
Within assay precision
%CV for the controls, samples and calibrators
(Value not OD)
Between-assay differences and stored calibration
curve stability
% CV for control and sample
Compare the values generated by the stored
calibration curve with those derived from a manual
plot of all the calibrators

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Method Evaluation
Analysis of results from initial kit
evaluation (continue)
Drift
Plot the values of controls obtained at the
beginning, middle and end of the assay to detect
assay drift
Sensitivity
10 replicates of zero calibrator, Analytical
sensitivity is two SD above or below the zero
calibrator mean
Accuracy
Compare the results for the external QC scheme samples
with those obtained from other methods and all-laboratory
trimmed means
Compare the patient samples with current method

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Method Evaluation
Analysis of results from initial kit
evaluation (continue 2)
Dilution
Samples diluted by zero calibrator, Plot the dilution
curve, straight line
Other information
1. Check the appearance of the reagents,
2. Check the ease of using of packaging
3. Quality of the instructions
4. Estimate the total assay time
5. Telephone to customer service and ask
one/two questions to check the quality and the
speed of their responses


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Monitoring and maintenance of
Validation criteria; Daily QC program
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Analysis of Control Materials

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Levey-Jennings Chart
80
85
90
95
100
105
110
115
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
C
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V
a
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s

(
e
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g
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m
g
/
d
L
)
80
85
90
95
100
105
110
115
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
C
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V
a
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Time (e.g. day, date, run number)
Record Time on X-Axis and the Control Values on Y-Axis
80
85
90
95
100
105
110
115
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mean
+1SD
+2SD
+3SD
-1SD
-2SD
-3SD
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Example of QC Graph



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8x
12x
R4s

4 1s

3
1s
6x
9x
7
T

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Westgard Multi-Rule Procedure (examples for 1
3s
, 2
2s
,
R
4s
. 2
2s
, 4
1s
across two levels, and 4
1s
for one level )
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Westgard Multi-Rule Procedure
(examples for across two levels and for one level.)

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Errors Detected
Error Condition
1. No errors
1
2s

Random error
1
3s,
R
4s

Systematic error
2
2s
, 4
1s
, 8
x
, 10
x
, 12
x




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Thanks for your attention