ELISA: Basics & Technical Tips

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ELISA
Basics and Technical Tips

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ELISA: Basics & Technical Tips

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CONTENTS
3

Definition

4–8

ELISA Types

9

ELISA Factors

10

ELISA Results

11

FAQs

12

Troubleshooting

13

Contact Us

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ELISA: Basics & Technical Tips

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DEFINITIONELISA: Enzyme-Linked Immuno-Sorbent Assay–– First described in 1971 by Engvall and Perlmann.
–– Characteristic feature of an ELISA: Antigen is directly coatedto 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.

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ELISA: Basics & Technical Tips

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ELISA TYPESThe 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 theantigen, forming a "sandwich." The complex is then recognized
by a secondary labelled antibody.

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ELISA: Basics & Technical Tips

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ELISA TYPES
Direct ELISA–– The antigen is immobilized on the ELISA plate.
–– The primary antibody carries the label.
–– Remaining surface blocked.
–– Detection: Dependent on label.

Side view ofan ELISA Well.

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ELISA: Basics & Technical Tips

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ELISA TYPES
Indirect ELISA–– The antigen is immobilized on the ELISA plate.
–– The secondary antibody carries the label.
–– Remaining surface blocked.
–– Detection: Dependent on label.

Side view ofan ELISA Well.

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ELISA: Basics & Technical Tips

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ELISA TYPES
Sandwich ELISA–– 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 ofan ELISA Well

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ELISA: Basics & Technical Tips

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ELISA TYPES
Advantages& Disadvantages

Direct ELISAIndirect ELISA

Sandwich ELISA

Costs+/---

Cross-reactivity------

Availability+++

Adsorption effectsof Antigen--+

Signal amplification-++

Assay time+--www.ptglab.com

ELISA: Basics & Technical Tips

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ELISA FACTORS
Factors That
Influence an ELISAELISA Factor

Factor Characteristics

PlateMaterial, 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.

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ELISA: Basics & Technical Tips

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ELISA RESULTS–– The standards are specified on the x-axis.
–– The reading of each standard is specified onthe y-axis.
–– This standard curve is used to determine theunknown concentration.
–– The results should be in the range given by
the kit manufacturer.

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Residue Number

ELISA: Basics & Technical Tips

11FAQsWhat Is The
Purpose Of
Different Controls?
Positive Controls

Negative Controls

Zero Blank& Chromogen Blank

Chromogen Blank
Useful When

• Standard.

• Zero standard.

• Sample positive 		control (some 		companies provide).

• Chromogen blank.

• OD>0.25 (ELISA 		dependent) for 		zero blank means
high background.

• Background causedby reservoir.

• Comparing zero blankand chromogen blankhelps to find thelikely cause.

• Background causedby metal.
• Background causedby chromogen.

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ELISA: Basics & Technical Tips

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TROUBLESHOOTING
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.
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ELISA: Basics & Technical Tips

13CONTACT US
Proteintech GroupUS Head Officeproteintech@ptglab.com

Proteintech Europe
United Kingdom

europe@ptglab.com

Proteintech
China Officeservice@ptglab.com

SupportAvailable 24 hours via Live Chat and 9–5(CDT) via phone.
Please visit us at www.ptglab.com for
more information about our antibodies
and technical tips.

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