NEWS CENTER

Enzyme-linked immunosorbent assay (ELISA): A technology in which a known antigen or antibody is coated on the surface of a solid phase carrier, so that enzyme-labeled(mainly HRP) antigen and antibody reactions are carried out on the solid phase surface. This technology can be used to detect macromolecular antigens and specific antibodies, and has the advantages of fast, sensitive, simple and easy to standardize the carrier.

ELISA Principle diagram

 

Timely, efficient, and economical detection and quantification of antigens or antibodies present in samples is a crucial step in many situations in the life sciences field.

ELISA has proven to be an invaluable research and diagnostic tool for detecting antibodies or antigens in biological samples by the coated antigens or antibodies on the surface of solid phase carriers, enabling enzyme-labeled(mainly HRP) antigens and antibodies to react on the solid phase surfaces. Traditional ELISA avoids the use of isotopes and eliminates the hidden danger of safety, but the external conditions have a great influence on the color change of the solution and the linear range of the effective OD value is too low, that the sensitivity and dynamic range of ELISA detection are greatly limited by the defects of light absorption technology.

 

High sensitivity fluorescence——Fluorescent age

In 1982, Halonen et al. published an article entitled “Time-resolved fluoroimmunoassay: a new test for rubella antibodies”, marking the official step of immunoassay into the era of fluorescence, which is also the prototype of DELFIA's technology.

 

In short, the two differences between DELFIA and traditional ELISA experiments are:

 

1. The enzyme HRP on the antibody was replaced by lanthanide chelate (Eu, Sm, Tb, Dy).

 

2. The detection method is fluorescence.

 

DELFIA technology ----"Highly sensitive fluorescent ELISA kit". In short, the enzyme HRP used in traditional ELISA for detecting antibodies is replaced with lanthanide chelate (Eu, Sm, Tb, Dy), as shown below:

DELFIA (Dissociation-enhanced Lanthanide Fluorescence Immunoassay) technology: using the lanthanide chelates (Eu, Sm, Tb, Dy) labeled reagents, by washing to remove unbound reagents, combined with time-resolved fluorescence (TRF) detection to detect a labeled specific purpose compound or biomolecular in the system. The fluorescence lifetime of lanthanides can reach microseconds or even milliseconds, and the combination of time-resolved detection can significantly reduce the background interference of spontaneous fluorescence, and it has a wide wavelength difference between excitation light and emission light (Strokes' shift), which can greatly improve the detection signal-to-noise ratio and sensitivity.

 

Attenuation curve of ordinary fluorescence excitation:

Detection time window:

Attenuation curve of lanthanide chelates after excitation:

Ultra-long decay time of lanthanides:

 

DELFIA Technology application 1: Detection and quantification of biomarkers in various complex samples

 

Example 1: Comparing the traditional ELISA and DELFIA techniques by detecting the TNFA in mouse

 

 

Comparison of sample compatibility and sensitivity of DELFIA technology is as follows

 

Comparison of sensitivity between DELFIA and traditional ELISA technology:

 

Comparison of operation process between DELFIA and traditional ELISA technology:

 

TRF technology using the characteristics of the lanthanide chelates emitted by the fluorescence decay time is very long, the sensitivity is high, the dynamic range is wide. Several features of TRF technology:

1. Wide Stroke's shift of lanthanides;

2. Attenuation curve of lanthanide chelates after excitation;

3. Attenuation curve of ordinary fluorescence excitation;

4. Ultra-long decay time of lanthanides;

5. Large Strokes shift, reduce the interference of excitation light and emission light, reduce self-quenching, improve sensitivity;

6. The emitted light lasts for a long time, up to tens of thousands of times that of traditional fluorescein, so that the detection can be delayed and the background fluorescence can be deducted;

7. High sensitivity, because the background fluorescence is deducted, so can get much higher sensitivity than conventional fluorescence;

8. Multiple labeling, the emission wavelengths of europium, terbium, dysprosium and samarium in lanthanides are significantly different, and the maximum can be quadrupled labeling. Related technologies derived from the principle of time-resolved fluorescence, including DELFIA, LANCE, and TruPoint.

 

Applications: cAMP（cyclic adenosine 3’, 5’–monophosphate）detection

HTRF cAMP HiRange kit can quantify cAMP in cell samples. cAMP is a key second messenger in G protein-coupled receptor (GPCR) signaling. After the ligand binds to the GPCR, conformational changes occur that activate the receptor, which in turn activates the G protein. Further signal transduction depends on the type of G protein activated. The activation of Gs protein leads to the up-regulation of cAMP by adenylate cyclase. Free cAMP produced by cells competes with D2-labeled cAMP to bind anti-Camp cavitation compounds, so an increase in cellular cAMP leads to a decrease in FRET, which can be detected by a decrease in fluorescence emitted at 665 nm (figure below).

Conclusion:

DELFIA time-resolved fluorescence (TRF) analysis for mouse TNFA was developed and used as a model to evaluate the compatibility of DELFIA technology with various biological sample types. Linearity and standard recovery tests are used to determine the appropriate diluent for standard curve and overall sample compatibility. DELFIA analysis is compatible with complex sample matrices, including cell lysates, cell supernatants, urine, tissue, serum, plasma, and whole blood. Analytical performance is also measured by evaluating the sensitivity, dynamic range, and signal-to-noise ratio of each type of sample. DELFIA has proven to be a highly sensitive assay with a wide dynamic range and excellent background signals across a wide range of sample types (Reference: DELFIA Immunoassay Development Guide, PerkinElmer, 2019).