HTRF® ratio and data reduction

Ratiometric data reduction: a straightforward way to eliminate compound interference

HTRF technology uses either Eu3+ or Tb3+cryptate as the donor fluorophore, and either XL665 or d2 as the acceptor. For these fluorophores, we recommend measuring the fluorescence emission at 620 nm for the donor and at 665 nm for the acceptor. When a green acceptor is used in combination with Lumi4®-Tb cryptate, e.g. in some Tag-lite® assays, the acceptor emission must be measured at 520 nm.

The measurement of HTRF emissions at two different wavelengths (620 nm and 665 nm) allows the ratiometric reduction of data. This feature of HTRF is extremely advantageous, particularly for reducing well-to-well variations that may arise in homogeneous assay formats where a separation or wash step is not performed. Compounds and/or media additives left in the plate may change the photophysical properties in a given sample, and the degree to which this occurs can vary from sample to sample. By using the ratio of the donor and acceptor emission signals, it is possible to eliminate compounds that are simply interfering with detection.

Cisbio Bioassays has developed a ratiometric measurement that uses both the emission wavelength of the donor and of the acceptor to correct for well-to-well variability and signal quenching from assay components and media. Emissions at 620 nm (donor) are used as an internal reference, while emissions at 665 nm (acceptor) are used as an indicator of the biological reaction being assessed. Measurement of the total (positive control) and background (negative control) signal was carried out as well as the signal generated in the presence of a colored compound possessing quenching capabilities. As expected, the positive and negative controls show a clear difference in the absolute signal intensity at 665 nm and not at 620nm (see diagrams on the right).

This corresponds to an appropriate change in the HTRF ratio for the two sample types. However, in the sample containing the colored compound, a similar decrease in both 620 nm and 665 nm signals is observed. Since the degree of sample quenching in both emissions is similar, the HTRF ratio remains relatively unchanged. This shows that the compound is nonspecifically interfering with the total emission in the sample and demonstrates the benefit of using the ratiometric data reduction to eliminate interfering compounds in an assay.

If the ratio had not been calculated, the sample would be falsely identified as an inhibitor of the biological reaction being tested in the assay. This clearly demonstrates the usefulness of calculating the acceptor/donor ratio for each well.

Channel A
665 nm
(acceptor)
Channel B
620 nm
(donor)
Ratio Mean
ratio
Fig
1459
1392
35178
35547
415
392
403 (1)
69584
70245
34331
34612
20269
20295
20282 (2)
29949
28490
14952
14213
20030
20045
20038 (3)

Case study: standard curve in a competitive assay

Ratiometric data reflects the "raw" results

The ratio must be calculated for each well individually. The mean and standard deviation can then be worked out from replicates. The 104 multiplying factor is introduced for easier data processing:

  Channel A
665 nm
Channel B
620 nm
Ratio
(A/B) x 104
Background 2,040 40,765 500
Std 0 45,999 41,442 11,100
Std 1 40,615 41,000 9,906
Std 2 29,212 41,732 7,000
Std 3 15,249 40,124 3,800
Std 4 6,258 39,124 1,600

Delta ratio reflects the "specific signal"

The delta ratio is obtained by subtracting the background from the signal of each positive point.

  Channel A
665 nm
Channel B
620 nm
Ratio
(A/B) x 104
ΔR
Background 2,040 40,765 500  
Std 0 45,999 41,442 11,100 10,599
Std 1 40,615 41,000 9,906 9,406
Std 2 29,212 41,732 7,000 6,499
Std 3 15,249 40,124 3,800 3,300
Std 4 6,258 39,124 1,600 1,099

Assay window (Signal max/Signal min)

The window is obtained by dividing the maximum signal ratio value by the minimum signal ratio value.

  Channel A
665 nm
Channel B
620 nm
Ratio
(A/B) x 104
Assay Window
Std 0 45,999 41,442 11,100  
Std 1 40,615 41,000 9,906  
Std 2 29,212 41,732 7,000  
Std 3 15,249 40,124 3,800  
Std 4 6,258 39,124 1,600 7

 

In-depth use of the normalized signal

Delta F for inter-assay comparisons

Delta F is used for the comparison of day-to-day runs of the same assay. It reflects the signal to background of the assay. The negative control plays the role of an internal assay control.

Assay
day 1
Channel A
665 nm
Channel B
620 nm
Ratio
(A/B) x 104
ΔF
Background 2,228 48,765 457  
Module 1 8,294 45,442 1,825 299%
Module 2 14,999 46,000 3,261 614%
Assay
day 2
Channel A
665 nm
Channel B
620 nm
Ratio
(A/B) x 104
ΔF
Background 8,021 49,875 1,608  
Module 1 31,315 48,997 6,391 297%
Module 2 57,466 50,001 11,493 615%

Delta F / Delta F max enables the comparison of two experiments

This calculation is used for normalizing the signal in competitive assays.

Assay 1 Channel A
665 nm
Channel B
620 nm
Ratio
(A/B) x 104
ΔF ΔF/Δ Fmax
Background 2,040 40,765 500    
std 0 45,999 41,442 11,100 2,118% 100%
Std 1 40,615 41,000 9,906 1,880% 89%
Std 2 29,212 41,732 7,000 1,299% 61%
Std 3 15,249 40,124 3,800 659% 31%
Std 4 6,258 39,124 1,600 220% 10%
Assay 1 Channel A
665 nm
Channel B
620 nm
Ratio
(A/B) x 104
ΔF ΔF/Δ Fmax
Background 2,140 42,765 500    
std 0 75,241 43,242 17,400 3,377% 100%
Std 1 69,319 45,100 15,370 2,971% 88%
Std 2 49,115 44,732 10,980 2,094% 62%
Std 3 25,098 43,124 5,820 1,063% 31%
Std 4 9,991 43,924 2,275 355% 10%

Determination of the negative control

Depending on the assay type, the negative ratio may be either the negative control of the assay for sandwich formats or the cryptate blank for direct binding partner assays (e.g. immunocompetitive assays).