ELISA Development Guide from Creative Diagnostics. Creative Diagnostics provides contract ELISA kit development services for the R&D and IVD community.

1. ELISA Development Guide
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2. Developing an ELISA
 ELISA plate-coating strategy
 Antigen resources and
Antibody pairs
 Conjugating/labeling strategy
 Enzyme and chromogen
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3. ELISA Coating Strategy
 When developing a new ELISA for a specific antigen, the
first step is to determine an immobilizing strategy and
optimize the plate-coating conditions for the antigen or
capture antibody.
 Although generally we use Polyvinyl/ Polystyrene 96/ 384-
well plate as the solid phase of ELISA, there are actually
variety of solid phase materials can be used.
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4. www.creative-diagnostics.com
Binding Capacity
Higher
High
Moderate
Low

5. ELISA Coating Strategy
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Immobilization can be defined as the attachment of molecules (antigen
and antibody) to a surface resulting in reduction or loss of mobility.
In some cases, immobilization may lead to partial or complete loss of
protein activity, due to random orientation and structural deformation.
Generally, the choice of a suitable immobilization strategy is
determined by the physicochemical and chemical properties of both
surface and protein.

6. ELISA Coating Strategy
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7. Antigen and Antibody
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 Antigen and antibody are two major factors determining the
sensitivity and specificity of an assay.
 The purity and stability of antigen are key parameters that affect the
performance of ELISA.
 The three dimensional configuration of the antigen-binding site found
in the Fab portion of the antibody controls the strength and specificity
of the interaction with antigen.

8. Antigen and Antibody
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 Either monoclonal or polyclonal antibodies can be used as the
capture and detection antibodies in sandwich ELISA systems.
 Monoclonal antibodies have an inherent mono-specificity toward a
single epitope that allows fine detection and quantification of small
differences in the antigen.
 A polyclonal is often used as the capture antibody to pull down as
much of the antigen as possible.

9. Antigen and Antibody
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10. Conjugate Strategy
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 Enzyme labeled antibody is the key of the ELISA signal output.
Conjugation of enzymes to antibodies involves the formation of a
stable, covalent linkage between an enzyme and an antigen-specific
monoclonal or polyclonal antibody in which neither the antigen-
combining site of the antibody nor the active site of the enzyme is
functionally altered.
 HRP and AP can be attached to antibodies and proteins through the
use of different coupling chemistries to ensure the maximum
retention of activity of both enzyme and protein.

11. Enzyme and Chromogen
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 The final stage of ELISA is dictation. The intensity of signal produced
when the substrate is added will be directly proportional to the
amount of antigen captured in the plate and bound by the detection
reagents.

12. Enzyme and Chromogen
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13. ELISA Kit Development
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 Creative Diagnostics provides contract ELISA development kit
services for the R&D and IVD community.
 We conduct ELISA kit development services to support regulatory
approval submission.
 Creative Diagnostics will carry out the approval proposal and deliver
the expected results and documents in a time and cost effective
manner.

14. Thanks
For more info about ELISA kit development
services or antigen/antibody products.
Please visit: www.creative-diagnostics.com
www.creative-diagnostics.com