Assay Development
FOR 40 YEARS, ROCKLAND IMMUNOCHEMICALS INC. HAS SUPPORTED THE LIFE SCIENCE
INDUSTRY WITH A FULL RANGE OF THE HIGHEST QUALITY PRIMARY AND SECONDARY
ANTIBODIES, FUSION PROTEINS, SUBSTRATES, STANDARDS AND CONTROLS FOR BASIC
RESEARCH, ASSAY DEVELOPMENT AND PRE-CLINICAL STUDIES.
TO FURTHER ENABLE INNOVATIVE BIOMARKER DEVELOPMENT FOR DRUG DISCOVERY AND
DIAGNOSTIC APPLICATION, ROCKLAND OFFERS HIGHLY CUSTOMISED SOLUTIONS TO MEET
YOUR BASIC, APPLIED AND CLINICAL RESEARCH DEMANDS. WHETHER IT IS THE GENERATION
OF HIGHLY SPECIFIC ANTIBODIES OR THE DEVELOPMENT OF UNIQUE IN VITRO ASSAY SYSTEMS,
ROCKLAND PROVIDES THE SCIENTIFIC EXPERTISE AND CGMP COMPLIANT MANUFACTURING
FACILITIES NECESSARY TO SUPPORT YOUR GLOBAL BIOMARKER EVALUATION AND
DEVELOPMENT NEEDS.
IMMUNOASSAY DEVELOPMENT AT ROCKLAND INC.
Modern laboratory techniques in molecular biology allow for the
testing and/or measurement of different biochemical substances in
an organism and/or biological sample. The substance hereon designated “THE ANALYTE” might be present at very low concentrations
not detectable by other tests.
IMMUNOASSAYS are specialized assays regularly employed in biological, virological, cellular secretion (Elispot) and/or drug studies and
make use of ANTIBODIES that have been produced by immune cells
in response to continued stimulation by an antigen. These antibodies are highly antigen-specific, attributing to the high sensitivity and
specificity of the IMMUNOASSAY.
The development of a customised Immunoassay is initiated by the
user-defined specifications pertaining to their overall research-related
design goals and is a process characteristically divided into 4 phases,
as illustrated in Figure 1.
ROCKLAND’s experienced staff with over 20 years experience in the
development and validation of multiple assay formats has shown
a long line of success in delivering customer solutions and have the
expertise in optimizing every aspect of an immunoassay.
Most IMMUNOASSAYS typically display the same strategy in which
generated antigen–specific antibodies are used to capture the antigen
present in a biological sample or vice versa where the antigen functions to detect circulating antigen-specific antibodies. The addition
of a measurable label that directly or indirectly indicates the presence of the analyte is thereafter included in the assay. Dependent on
the client’s requirements, the assay format and characteristics are
defined during the Process Creation Phase shown in Figure 1.
P ro c ess Cre a tio n
D e fi n ition of assay form at & c h arac te ristic s
Re ag e n t D e fi n ition an d D e velop m ent
– e n zym e targ e t, sub strate , lab e lle d
re ag e n ts d e pe nd ing on assay d esign
P ro c ess D ev elo pm en t
D e te rm in ation of p aram e te rs (LoD, LoQ )
Re ag e n t p e r form ance
W orkin g ran g e & se n sitivity
Lin e arity of e n zym e ac tivity
Re p rod uc ib ility A sse ssmen t
P ro c ess Va lida tio n
Rob ustn e ss ve rifi c ation & system suitab ility
(sc ale -up , autom ation , d ay-to-d ay)
Pre c ision sp e c ific ity
In te r an d in tra-assay re p rod ucib ility
Valid ation re p ort
P ro c ess D o c u m en ta tio n
Pre p aration an d im p le m e n tation of
SO Ps an d p rotoc ols
FIGURE 1:
IMMUNOASSAY DEVELOPMENT PHASES
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•
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The measurable label to be used in the immunoassay will define the Assay’s characteristics. Enzymes
conjugated to either antibodies or antigens are commonly found to be the measurable label in the
ENZYME IMMUNOASSAY (EIA). Enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) function to chemically modify compounds known as THE SUBSTRATE, resulting in the
development of a colourimetric signal (Figure 2A). Similarly, fluorescent or chemiluminescent moleculeconjugated measuring labels resulting in fluorescence or chemical light emission in the FLUORESCENT
IMMUNOASSAY (FIA, Figure 2B) and CHEMILUMINESCENT IMMUNOASSAY (CLIA or ChLIA, Figure
2C) respectively, display a greater analytical sensitivity than the EIA.
Immobilised
Antigen
PrimaryAntibody
Labelled
SecondaryAntibody
SignalDetection
andQuantification
Substrate
A
ColourDevelopment
Wavelength (λ) Ecitation(Amax)
B
Flourescence (Emax)
Substrate
C
Chemiluminescence
FIGURE 2:
IMMUNOASSAYS REQUIRE THE ADSORPTION OF AN ANTIGEN ONTO A SOLID SUPPORT (MICROTITRE PLATE WELL), A
HIGHLY-SPECIFIC PRIMARY ANTIBODY RAISED AGAINST THE ANTIGEN AND A LABELLED SECONDARY ANTIBODY. ENZYMELABELLED SECONDARY ANTIBODIES ARE USED IN THE EIA (A) AND CLIA (C) WHICH UPON ADDITION OF THE ASSAYSPECIFIC SUBSTRATE, RESULT IN MEASURABLE COLOUR DEVELOPMENT OR CHEMICAL LIGHT EMISSION RESPECTIVELY.
FLUOROPHORE-LABELLED SECONDARY ANTIBODIES ARE USED IN FLUORESCENT IMMUNOASSAYS OR FIAS (B) AND EMIT
MEASURABLE LIGHT UPON FLUOROPHORE EXCITATION AT A PARTICULAR WAVELENGTH. COLOUR OR EMITTED LIGHT
INTENSITY DIRECTLY CORRELATES TO THE CONCENTRATION OF THE PRIMARY ANTIBODY AND THE RESPECTIVE ANTIGEN.
ROCKLAND INC. extensive range of high-quality primary, including custom-generated primary antibodies and secondary antibodies offers a variety of alternatives in the developmental process of an immunoassay. An HRP-conjugated secondary antibody is generally used for the development of colourimetric
immunoassays (EIA), in which the enzyme converts the added TMB (3,3´,5,5´-tetramethylbenzidine)
substrate yielding a blue colour when oxidized with hydrogen peroxide (catalyzed by the HRP) displaying major absorbencies at 370nm and 652nm. Upon addition of sulfuric or phosphoric acid, the colour
changes to yellow with a maximum absorbance at 450nm (Figure 2A).
2
For the development of fluorescent immunoassays, ROCKLAND INC., most commonly utilizes a Oregon
Green 488 fluorophore-conjugated secondary antibody emitting measurable light at 520nm upon excitation of the fluorophore at 493nm. The HRP enzyme-conjugated secondary antibody (described above)
displays a dual role: This antibody conjugate is also applied in the developmental process of the chemiluminescent assay. Following the addition of the substrate Luminol, the enzyme catalyses the formation
of an excited intermediate product, which upon decay emits chemically produced light or chemiluminescence. The colour of the emitted light is dependent on the utilized dye.
IMMUNOMETRIC ASSAYS: ANTIGEN OR ANTIBODY
Different Immunoassay methodologies are applied to detect and quantify either the antigen or antibody
concentration in a biological sample. The conventional IMMUNOMETRIC ASSAY approach for the measurement of an antibody analyte is shown in Figure 3.
Individual steps in this Immunometric Assay are:
•The adsorption of the capture antigen to the inner well surface of a 96-well microtitre
plate followed by a blocking step of the remaining uncoated surface area.
•A washing step precedes the addition of the antigen-specific detection antibody to the
well.
•Plates are washed before a second species-specific antibody linked to an enzyme (the
conjugate) is added and incubated.
•Plates are washed to remove all unbound components before the substrate is added.
Bound enzyme conjugated antibody reacts and chemically modifies the substrate resulting in colour development.
•The enzymatic reaction is stopped by addition of stop buffer to ensure a consistent
reaction time period for all wells before the colour intensity is measured.
•The amount of coloured product is measured spectrophotometrically and this value
directly relates to the quantity of bound antibody.
Immobilised
Antigen
PrimaryAntibody
Wash
Labelled
SecondaryAntibody
SubstrateAddition
Signal Detection andQuantification
Wash
FIGURE 3:
3
IMMUNOMETRIC COLOURIMETRIC ASSAY TO DETERMINE THE ANTIBODY CONCENTRATION.
Immunometric Assays that are routinely used for the detection and quantitation of different ANTIGEN ANALYTES
display a different strategy (Figure 4).
In this assay, designed to measure the antigen concentration, the individual wells are coated with an antigen-specific
antibody before addition of the biological sample. Analyte present in the sample is bound by the capturing antibody
before removal of all unbound components in a washing step. Addition of a non-competing enzyme-conjugated antibody binds to a different location/epitope on the target antigen before plates are washed and the substrate is added.
The bound conjugated enzyme reacts with the substrate resulting in signal development.
Addition ofAntigen
Labelled
SecondaryAntibody
SubstrateAddition
SignalDetection andQuantification
SignalStrength
Immobilised
CapturingAntibody
Wash
Wash
AnalyteConcentration
Signal strength isdirectly proportional
totheconcentration of theAnalyte
FIGURE 4:
IMMUNOMETRIC CHEMILUMINESCENT ASSAY FOR THE QUANTITATION OF AN ANTIGEN ANALYTE IN A BIOLOGICAL SAMPLE.
The obtained signal strength in this Immunometric assay is directly proportional to the concentration of the analyte.
Binding of the capturing and detecting antibodies occurs at different locations on the surface of the antigen and
because the antigen is wedged in between two antibodies, this assay is commonly referred to as a Sandwich ELISA.
Immunometric assays are suitable for determining the concentrations of analytes that facilitate binding of multiple
antibodies on their surface; however, a different kind of assay has to be designed if the analyte is a small molecule
not permitting the binding of multiple antibodies. This immunoassay known as the COMPETITIVE ASSAY requires
the methodology shown in Figure 5.
•As in the Immunometric assay designed for the quantitation of an antigen; the wells of the competitive assay are coated with a capturing antibody.
•The analyte is conjugated to a reporting enzyme and a proportional mixture of antigen and antigenconjugate is added to individual wells – competing for available capturing antibody binding sites.
•A washing step to remove all unbound antigen and antigen-conjugate precedes the addition of
substrate.
•Bound enzyme-conjugated antigen reacts and chemically modifies the substrate resulting in colour
development before the reaction is stopped and the optical density of the colour development is
measured.
4
Addition ofAntigen andAntigen-conjugatedEnzymecomplex
Bindingcompetition between theAntigen andAntigenconjugate
SignalDetection andQuantification
SignalStrength
Immobilised
CapturingAntibody
Wash
AnalyteConcentration
Signal strength isinversely proportional
totheconcentration of theAnalyte
FIGURE 5:
COMPETITIVE FLUORESCENCE IMMUNOASSAY
Competitive immunoassays can be designed in two different ways. When the ratio of
ANALYTE > ANALYTE CONJUGATE, the resulting signal will be lower
ANALYTE CONJUGATE > ANALYTE, the resulting signal will be higher
therefore the signal in the competitive assay is inversely proportional to the antigen concentration in the sample
(Figure 5).
Immunoassay Development at ROCKLAND INC. follows the stringent guidelines set forth by the National Accrediting
Agency for Clinical Laboratory Sciences (NAACLS). Throughout the development process of custom immunoassays
as indicated in the Development Process (Figure 1), the strengths and limitations of individually designed assay are
determined.
Given that dissimilar immunoassays perform differently in that the high selectivity and sensitivity which is attained for
some immunoassays is not reflected by others, experienced scientists at ROCKLAND INC. optimize all assay-specific
parameters (Figure 6) to guarantee optimal assay performance.
Among the many immunoassay parameters, the Limit of Detection or LoD for any analytical procedure is defined
by the point at which the analysis thereof is just feasible (Figure 6). “An Assay is simply not capable of accurately
measuring analyte concentrations down to zero. A sufficient analyte concentration must be present to produce an
analytical signal that can be reliably distinguished from the “analytical noise or Limit of Blank (LoB)” – the signal
produced in the absence of analyte”. The LoD of any Assay may be determined by either a statistical approach in
which negative (blank) samples are measured in replicate to determine the LoB – a reasonable starting point for
the estimation of the LoD or by the alternative empirical approach entailing the measurement of progressively more
dilute concentrations of the given analyte.
5
FIGURE 6:
CALIBRATION CURVE INDICATING THE LOD, THE LOQ, THE LIMIT OF LINEARITY (LOL), THE ASSAY SENSITIVITY AND THE DYNAMIC RANGE.
Furthermore, the Limit of Quantification or LoQ shown in Figure 6, is defined as the concentration of analyte at
which quantitative results can be reported with a high degree of confidence. Not only is it defined by the lowest analyte concentration that can be reliably detected, but it also fulfills predefined goals for bias and imprecision.
The Limit of Linearity or LoL of an immunoassay (Figure 6) is defined as the analyte concentration at which the
calibration curve departs from linearity by a specified amount. A deviation of approximately 5% is usually considered
the upper limit and is frequently observed at higher analyte concentrations. The range between the LoQ and LoL is
referred to as the Dynamic Range in which the sensitivity of the immunoassay is determined by the interplay between
analyte concentration and signal strength.
The Process Validation and Documentation (Figure 1) follow our meticulous Process Development and complete the
overall custom Immunoassay Development.
PUT ROCKLAND INC. EXTENSIVE EXPERIENCE AND SCIENTIFIC EXPERTISE IN IMMUNOASSAY DESIGN AND
DEVELOPMENT, VALIDATION AND PRODUCTION TO WORK FOR YOU.
OUR CGMP COMPLIANT MANUFACTURING FACILITIES ENSURE THE SYSTEMATIC DEVELOPMENT OF
YOUR IMMUNOASSAY, GUARANTEE THE METICULOUS PROCESS DOCUMENTATION AND ALLOW FOR THE
EFFICIENT TRANSFERAL TO LARGE-SCALE MANUFACTURING. OUR CUSTOM ASSAY DEVELOPMENT TEAM
WILL SCHEDULE REGULAR MEETINGS TO KEEP YOU ACTIVELY INVOLVED AND WILL PROVIDE REAL-TIME
UPDATES ON YOUR PROJECT(S) TO MEET YOUR REQUIREMENTS AND HELP YOU KEEP TRACK, THUS GIVING
YOU THE FREEDOM TO FOCUS ON YOUR SCIENTIFIC RESEARCH. CUSTOM IMMUNOASSAYS DEVELOPED AT
ROCKLAND INC. ARE AVAILABLE IN COLOURIMETRIC OR FLUORESCENT AND CHEMILUMINESCENT FORMAT
FOR ENHANCED SENSITIVITY AND DYNAMIC RANGE.
Rockland Immunochemicals Inc.
P.O. BOX 326 • Gilbertsville, PA 19525
www.rockland-inc.com • [email protected]
Phone: 610-369-1008
Toll Free (US and Canada):800-656-ROCK (7625)
Fax: 610-367-7825
Copyright© 2012 Rockland Immunochemicals Inc. OW9965 01/12