HTRF® ratio and data reduction

Ratiometric data reduction: a straightforward way to eliminate compound interference

HTRF technology uses either Eu3+ or Tb2+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 and patented a ratiometric measurement that uses both the emission wavelength of the donor and of the acceptor (patent US 5,527,684 and foreign equivalents) 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
In-depth use of the normalized signal
Determination of the negative control

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	ΔR
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

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/td>	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/td>	2,094%	62%
Std 3	25,098	43,124	5,820	1,063%	31%
Std 4	9,991	43,924	2,275	355%	10%

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).