The document provides an overview of the enzyme-linked immuno-sorbent assay (ELISA), detailing its definition, types (direct, indirect, and sandwich), and the factors influencing its results. It includes troubleshooting tips and addresses common concerns regarding signal levels, control types, and interpretation of results. Additionally, the document contains contact information for technical support related to ELISA.

1. 1ELISA: Basics & Technical Tips
ELISA
Basics and Technical Tips
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2. 2ELISA: Basics & Technical Tips
CONTENTS
Definition
ELISA Types
ELISA Factors
ELISA Results
FAQs
Troubleshooting
Contact Us
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4–8
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10
11
12
13
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3. 3ELISA: Basics & Technical Tips
DEFINITION
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ELISA: Enzyme-Linked Immuno-Sorbent Assay
–– First described in 1971 by Engvall and Perlmann.
–– Characteristic feature of an ELISA: Antigen is directly coated
to a well.
–– Three main different types of ELISA are currently available.
Why is an ELISA a great research tool?
–– A biochemical test that detects and measures antibodies
in your blood and antibodies related to certain infectious
conditions. ELISA tests are mainly used in immunology.

4. 4ELISA: Basics & Technical Tips
ELISA TYPES
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The ELISA technique is divided into:
–– Direct ELISA:
The antigen is immobilized on the ELISA plate. The primary
antibody carries the label.
–– Indirect ELISA:
The antigen is immobilized on the ELISA plate. The secondary
antibody carries the label.
–– Sandwich ELISA:
Two primary antibodies (for capture and detection) embed the
antigen, forming a "sandwich." The complex is then recognized
by a secondary labelled antibody.

5. 5ELISA: Basics & Technical Tips
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Direct ELISA
ELISA TYPES
–– The antigen is immobilized on the ELISA plate.
–– The primary antibody carries the label.
–– Remaining surface blocked.
–– Detection: Dependent on label.
Side view of
an ELISA Well.

6. 6ELISA: Basics & Technical Tips
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Indirect ELISA
ELISA TYPES
–– The antigen is immobilized on the ELISA plate.
–– The secondary antibody carries the label.
–– Remaining surface blocked.
–– Detection: Dependent on label.
Side view of
an ELISA Well.

7. 7ELISA: Basics & Technical Tips
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Sandwich ELISA
ELISA TYPES
–– Two primary antibodies.
–– Capture antibody is bound to the well.
–– Remaining well surface blocked.
–– Antigen bound by capture antibody and detection antibody.
–– Detection: Dependent on label.
Side view of
an ELISA Well

8. 8ELISA: Basics & Technical Tips
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Advantages
& Disadvantages
ELISA TYPES
Direct ELISA Indirect ELISA Sandwich ELISA
Costs +/- - -
Cross-reactivity -- -- --
Availability + + +
Adsorption effects
of Antigen
- - +
Signal amplification - + +
Assay time + - -

9. 9ELISA: Basics & Technical Tips
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ELISA FACTORS
Factors That
Influence an ELISA
ELISA Factor Factor Characteristics
Plate Material, type.
Washing
Buffer type, frequency, time,
intensity, cross-reactions.
Target-of-interest
Conformation, epitope,
matrix effects.
Antibodies
Specificity, concentration,
cross-reactions.
Substrate Sensitivity, lot-to-lot variation.

10. 10ELISA: Basics & Technical Tips
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ELISA RESULTS
–– The standards are specified on the x-axis.
–– The reading of each standard is specified on
the y-axis.
–– This standard curve is used to determine the
unknown concentration.
–– The results should be in the range given by
the kit manufacturer.
Residue Number

11. 11ELISA: Basics & Technical Tips
FAQs
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Positive Controls Negative Controls Zero Blank
& Chromogen Blank
Chromogen Blank
Useful When
• Standard.
• Sample positive
control (some
companies provide).
• Zero standard.
• Chromogen blank.
• OD>0.25 (ELISA
dependent) for
zero blank means
high background.
• Comparing zero blank
and chromogen blank
helps to find the
likely cause.
• Background caused
by reservoir.
• Background caused
by metal.
• Background caused
by chromogen.
What Is The
Purpose Of
Different Controls?

12. 12ELISA: Basics & Technical Tips
TROUBLESHOOTING
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No/Low/High Signal
Potential Source Possible Solution
Too short/long incubation time. Incubate following manufacturer’s instruction.
Too much/not enough substrate. Follow manufacturer’s instruction.
Poor sample preparation. Ensure accurate sample type and preparation.
Too harsh handling of plates. Ensure gentle washing.
Antibody concentration. Titrate antibody concentration.
Target-of-interest not suitable for assay detection limit.
Double check detection limit of ELISA/
concentrate samples.
Good Standard But No Signal In Samples?
No fresh sample or sample storage incorrect.
Validated sample type?
Many cytokines in normal serum are difficult to detect.

13. 13ELISA: Basics & Technical Tips
CONTACT US
proteintech@ptglab.com
europe@ptglab.com
service@ptglab.com
Available 24 hours via Live Chat and 9–5
(CDT) via phone.
Proteintech Group
Proteintech Europe
Proteintech
Support
US Head Office
United Kingdom
China Office
Please visit us at www.ptglab.com for
more information about our antibodies
and technical tips.
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