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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 antigenspecific, attributing to the high sensitivity and
specificity of the IMMUNOASSAY.
Process Creation
Defi n ition of assay form at & ch aracteristics
Reag en t Defi n ition an d Develop m ent
– en zym e targ et, sub strate, lab elled
reag en ts d epend ing on assay d esign
Process Development
Determ in ation of p aram eters (LoD, LoQ)
Reag en t p erform ance
Workin g ran g e & sen sitivity
Lin earity of en zym e activity
Rep rod ucib ility Assessmen t
ProcessValidation
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.
Rob ustn ess verifi cation & system suitab ility
(scale -up , autom ation , d ay-to-d ay)
Precision sp ecificity
In ter an d in tra-assay rep rod ucib ility
Valid ation rep ort
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.
Process Documentation
Prep aration an d im p lem en tation of
SOPs an d p rotocols
FIGURE 1: IMMUNOASSAY DEVELOPMENT PHASES
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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.
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 molecule-conjugated 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
Signal Detection
andQuantification
Substrate
A
ColourDevelopment
Wavelength(λ)Ecitation(Amax)
Flourescence(Emax)
B
Substrate
C
Chemiluminescence
FIGURE 2: IMMUNOASSAYS REQUIRE THE ADSORPTION OF AN ANTIGEN ONTO A SOLID SUPPORT (MICROTITRE PLATE WELL), A HIGHLYSPECIFIC PRIMARY ANTIBODY RAISED AGAINST THE ANTIGEN AND A LABELLED SECONDARY ANTIBODY. ENZYME-LABELLED SECONDARY
ANTIBODIES ARE USED IN THE EIA (A) AND CLIA (C) WHICH UPON ADDITION OF THE ASSAY-SPECIFIC 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
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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).
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 enzymeconjugated 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.
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Immobilised
Antigen
Labelled
SecondaryAntibody
PrimaryAntibody
Wash
SubstrateAddition
Signal DetectionandQuantification
Wash
FIGURE 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.
Additionof Antigen
Labelled
SecondaryAntibody
SubstrateAddition
SignalDetectionandQuantification
Signal Strength
Immobilised
CapturingAntibody
Wash
Wash
AnalyteConcentration
Signal strengthisdirectlyproportional
totheconcentrationof 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
antigen-conjugate is added to individual wells – competing for available capturing antibody
binding sites.
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
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.
Additionof AntigenandAntigen-conjugatedEnzymecomplex
BindingcompetitionbetweentheAntigenandAntigenconjugate
Signal DetectionandQuantification
Signal Strength
Immobilised
CapturingAntibody
AnalyteConcentration
Signal strengthisinverselyproportional
totheconcentrationof theAnalyte
Wash
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.
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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.
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