Immunoassay Techniques

Version: 20210908
CONTENTS

01
PART ONE Basic Knowledge of Immunization

02
PART TWO
Brief Introduction of Immunoassay

03
PART THREE
Snibe Immunoassay Technique
01
PART ONE
Basic Knowledge of Immunization

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01
PART ONE
Basic Knowledge of Immunization
Common Phenomena Related to Immunity

Vaccination against SARS-COV-2 → Planned immunization

Suffered from shock after penicillin injection → Allergic reaction
Hemolytic reaction after blood transfusion → Allergic reaction
Organ or tissue transplantation → Rejection reaction

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01
PART ONE
Basic Knowledge of Immunization
What is Antigen?
In immunology, an antigen is a molecule capable of inducing an immune
response on the part of the host organism, though sometimes antigens
can be part of the host itself. In other words, an antigen is any substance
that causes the immune system to produce antibodies against it.

Examples:

Bacteria Virus Pollen Serum

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01
PART ONE
Basic Knowledge of Immunization
Characteristics of Antigen

1 2 3
• Foreignness • Macromolecular • Specificity

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01
PART ONE
Basic Knowledge of Immunization
What is Antibody?
An antibody (Ab), also known as an immunoglobulin (Ig),is a large, Y-shaped protein produced
mainly by plasma cells that is used by the immune system to neutralize pathogens such
as bacteria and viruses.

Antibody Structure Functional Classification

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01
PART ONE
Basic Knowledge of Immunization
How does the Immune Reaction Work?

In vivo

In vitro

Reaction Process

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01
PART ONE
Basic Knowledge of Immunization
Characteristics of Immune Reaction

• Specificity
1

• Reversibility
2

• Proportionality
3

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01
PART ONE
Basic Knowledge of Immunization
External Influence Factors of Immune Reaction

Temperature
• In a certain temperature range, the temperature can
accelerate the reaction process, normally is 37- 45℃

pH
• Change the physical and chemical properties of
antigen or antibody, affect the reaction results, pH 6-8

Electrolyte
• There must be a suitable electrolyte environment in the
reaction, otherwise there will be no visible reaction

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02
PART TWO
Brief Introduction of Immunoassay

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02
PART TWO
Brief Introduction of Immunoassay
History Overview

Chemiluminescence (CLIA, cLEIA, ECLI) 1990s

Enzyme-linked immunosorbent assay
(ELISA) 1970s

Immune colloidal gold technique 1970s

Radio immunoassay (RIA) 1960s

Fluorescent antibody technique 1940s

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02
PART TWO
Brief Introduction of Immunoassay
Fluorescent Antibody Technique
Use Activate Ag-Ab Observe
Bind antigen fluorescent complex to fluorescent
on the slide to label generate staining by
antibody fluorescence microscope

Fluorescent-labeled
antibody Advantage:
Rapid(several minutes). Low
Antigen cost and convenient.

Disadvantage:
poor specificity and sensitivity.
Qualitative or semi quantitative.
Testing Principle Fluorescent Microscope

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02
PART TWO
Brief Introduction of Immunoassay
Radio Immunoassay (RIA)
Radioactive
Bind specific Detect Calculate the
(125I, 3H)
antibody intensity of content of
labeled
competitively radioactitity antigen
antigen

Advantage:
Accurate,quantitative,complete parameters.

Disadvantage:
Radioactive pollution，
short valid period of reagent(one month)，
tedious operation process (2 hours).

1
Testing Principle
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02
PART TWO
Brief Introduction of Immunoassay
Immune Colloidal Gold Technique
Observe the
Bind specific
coloration
colloidal gold Bind antibody antigen on the
result on the
labeled antigen in the sample Nitrocellulose
test line and
membrane
control line

Sample flow direction

Advantage:
Rapid (several minutes)
No equipment required
Longer valid period of reagent (over 1 year).

Disadvantage:
Generally for qualitative detection, quantitative
detection sensitivity is not enough.

1
Testing principle

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02
PART TWO
Brief Introduction of Immunoassay
Enzyme-linked Immunosorbent Assay (ELISA)
Catalytic
Enzyme- Bind antigen coloration by Detect signal
labeled adsorbed on adding the by spectro-
antibody solid surface substrate of photometer
enzyme

Advantage:
No radioactive pollution. Longer valid

E
period of reagent (over 6 months).

E
Enzyme-labeled colored substrate
Antigen antibody
Solid-phase Ab Disadvantage:
E Repeatability is not good, more
interference factors. The greatest
impact is temperature and time.
Testing Principle Tedious operation process (2 hours)

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02
PART TWO
Brief Introduction of Immunoassay
Chemiluminescence
Catalyzer

Luminous
CLIA Excited signal
state measuring
instrument
Oxidant Photon

Ground
state

Basic Principle

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02
PART TWO
Brief Introduction of Immunoassay
Chemiluminescence
Chemiluminescent
Chemiluminescence Electrochemiluminescence
enzymeimmunoassay
(CLIA) (ECLI)
(cLEIA)
Snibe / Abbott / Beckman/
Representive company Roche
Siemens(Bayer) Siemens(DPC)
Marker ABEI / acridiniumester ALP / HRP Ru(byp)2+3

substrate NaOH / H2O2 AMPPD TPA

Nano magenatic Nano magenatic Nano magenatic
Separation technology
microbeads microbeads microbeads
Light signal Flash Glow Flash

Reagent cost Low High High

Influence factor Few Mutiple, lack of stability Few, biotin interference

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02
PART TWO
Brief Introduction of Immunoassay
Chemiluminescence

Application • Applicable to a variety of clinical assay

scope tests quantitatively or qualitatively

• High sensitivity, wide linear dynamic range,

simple and fast analysis method, good
Advantage stability, good safety and security, longer
reagent valid period (over 1 year)

Shortcoming • Varies according to different Luminescent

substances and methodology

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02
PART TWO
Brief Introduction of Immunoassay
Comparison of Immunoassay Techniques
Fluorescent antibody technique
Radio immunoassay (RIA)
Immune colloidal gold technique
Enzyme-linked immunosorbent assay
Chemiluminescence
Advantage
Rapid(several minutes), low cost and
convenient
Accurate,quantitative,complete
parameters
Rapid (several minutes), No equipment
required, Longer valid period of reagent
(over 1 year)
No radioactive pollution，Longer valid
period of reagent (over 6 months).
High sensitivity, wide linear dynamic
range, simple and fast analysis method,
good stability, good safety and security,
longer reagent valid period (over 1 year)
Disadvantage
Poor specificity and sensitivity,
qualitative or semi quantitative
Radioactive pollution, short valid
period of reagent(one month), tedious
operation process(two hours)
Generally for qualitative detection,
quantitative detection sensitivity is not
enough
Repeatability is not good, more
interference factors, the greatest
impact is temperature and time,
tedious operation process(two hours)
Varies according to different
Luminescent substances and
methodology
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03
PART THREE
Snibe Immunoassay Technique

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03
PART THREE
Snibe Immunoassay Technique

Snibe
Immunoassay
Technique

Key Reagent Test Test Calibration

Technique Component Methodology Procedure Principle

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03
PART THREE
Key Technique

• Labeling Technique-ABEI

• Separation Technique-Microbeads

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 03
PART THREE
Key Technique

Immune Separation Labeling Immuno-

technique technique technique assay

Antigen Antibody Magenatic Microbeads ABEI Maglumi X8

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03
PART THREE
Key Technique
Chemiluminescence Principle of ABEI

NaOH O2

N2 hv
ABEI double anion excited intermediate the ground state

 ABEI reacts with hydroxide to form a double anion.

Advantage:
 It can be oxidized by oxygen from the decomposition of Enhance Sensitivity and reaction efficiency
hydrogen peroxide, the product is an organic peroxide. Good stability
Easier to label the antibody
 The peroxide is very unstable and immediately breaks Shorten the reaction time
down nitrogen to form the excited intermediate.

 In the transition from the excited state to the ground

state, the energy released is in the form of photons with
wavelengths in the blue part of visible light.
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03
PART THREE
Key Kechnique
Characteristic of ABEI

delay 2.55s delay 0.1s

ABEI Shorten the reaction time

NaOH H2O2
(Starter 1) (Starter 2)

Enhance Sensitivity and reaction efficiency

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 03
PART THREE
Key Technique
Separation Technique: Microbeads
Advantage:
 Enlarging the reaction area of antigen and
Abundant surface antibodies
active groups  Shorten the reaction time and reduce the
sample volume
 Better and faster capture of the antigens and
Fe3O4 nanoparticle antibodies to enhance the sensitivity
Organic materialoror
Organic material
inorganic
inorganic material
material

Characteristic of the Microbeads:

superparamagnetism

With magnetic field Without magnetic field

Ferromagnetism Paramagnetism

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03
PART THREE
Reagent Component
Reagent Component

Diluent(Assay Dependent)
Nano Magnetic
Microbeads
ABEI Label Antibody
Calibrator Low Buffer/FITC Label Antibody

Calibrator High

Displacing Reagent
03
PART THREE
Test Methodology

• Sandwich

• Competitive

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03
PART THREE
Test Methodology
Sandwich
1. Add TSH, ABEI, FITC, magnetic microbeads in 2. Incubate for 15 min and forming a “sandwich”
the cuvette. (Left pipetting position) immune complex.(Incubator)

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03
PART THREE
Test Methodology
Sandwich
3. Wash 3 times, and immune complex stay, other 4. Add Starter 1 and 2 in the cuvette, then ABEI emits
components are washed away.(Washer) light. PMT measures the signal.(Chamber)

H2O2

NaOH PMT

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03
PART THREE
Test Methodology
Sandwich

Starter 2 Starter 1

Sandwich Complex
ABEI

TSH

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03
PART THREE
Test Methodology
Sandwich
 Analyzer shows the results as RLU (Relative light unit), using a working
curve to calculate the concentration of the sample.

 In sandwich method, it is positive correlation between concentration and

RLU. Curve is monotone increasing.

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03
PART THREE
Test Methodology
Competitive
1. Add T4, ABEI, FITC, magnetic microbeads in 2. Incubate for 15 min, FITC label antigen and
the cuvette. (Left pipetting position) sample antigen competitively binds ABEI labeled
antibody.(Incubator)

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03
PART THREE
Test Methodology
Competitive
3. Wash 3 times, and immune complex stay, other 4. Add Starter 1 and 2 in the cuvette, then ABEI emits
components are washed away.(Washer) light. PMT measures the signal.(Chamber)

H2O2

NaOH
PMT

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03
PART THREE
Test Methodology
Competitive

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PART THREE
Test Methodology
Competitive

Stronger Light emission, lower concentration Less Light emission, higher concentration
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03
PART THREE
Test Methodology
Competitive
 Analyzer shows the results as RLU (Relative light unit), using a working
curve to calculate the concentration of the sample.

 In competitive method, it is negative correlation between concentration

and RLU. Curve is monotone decreasing.

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PART THREE
Test Procedure

• One-step Assay

• Two-step Assay

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PART THREE
Test Procedure
One-step Assay

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PART THREE
Test Procedure
One-step Assay

Sample ABEI FITC Magnetic beads Pipetting

Incubate

Wash

H2O2
Starter

NaOH Measure

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03
PART THREE
Test Procedure
Two-step Assay

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03
PART THREE
Test Procedure
Two-step Assay
First pipetting(sample,
FITC, microbeads)
Sample ABEI FITC Magnetic beads
Incubate

Wash

Second pipetting(ABEI)

Incubate
H2O2
Wash

Starter
NaOH
Measure

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03
PART THREE
Test Procedure
One-Step Assay for High Concentration Sample

One-Step Assay may occur

False-Negative result on high concentration

Sample
Sample

Sample
Sample Sample

Wash out
Sample
Sample

Sample
No light Emittion

N S
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03
PART THREE
Test Procedure
Two-Step Assay for High Concentration Sample
1. First Peppeting 3. Second Peppeting
2. Washing 4. Second Washing
5. Light Emission

AFP

ABEI
AFP AFP
Two-Step Assay was
applied to avoid the
AFP AFP False-Negative result
Starter 2
ABEI

AFP

ABEI
AFP

Starter 1
AFP

N S
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03
PART THREE
Calibration Principle
Calibration Curve
RLU
RLUMT

RLUwk

RLU W K - RLU MT LogC(C)

Dev  100 %
RLU MT
LogC(C)

Dev

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03
PART THREE
Calibration Principle
Calibration Principle

• The stored master curve is generally defined with 10 master curve base
points.

• Two calibrators with defined concentration values are measured. These

measured signals (RLU) are compared with the master curve signal of the
corresponding calibrator concentrations.

• The relative difference between the measured RLU and the master RLU of
the calibrators is calculated and a linear extrapolation is performed between
the recalculated RLU (Y-axis)and the logarithmic (Log) concentrations (X-axis).

• Based on appropriate compensation factors, a re-adjustment of the master

curve points is made in order to achieve, by a “cubic spline function”, the
working curve.

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As a professional IVD Manufacturer

Snibe has focused on immunoassay

for 26 years

As an excellent CLIA system

Maglumi
are distributed throughout the word

Welcome to join us
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 Thanks
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