WO2022001090A1 - Hook effect determination method for homogeneous time-resolved fluorescence immunoassay - Google Patents

Abstract

The present application relates to a hook effect determination method for a homogeneous time-resolved fluorescence immunoassay. The method comprises the steps of: (1) measuring, under the same excitation wavelength, fluorescence intensities of a fluorescence donor and acceptor at two detection wavelengths, and using a fluorescence intensity ratio between the two wavelengths as a signal value; (2) respectively establishing, by means of measuring signal values of at least two time instances in a reaction initiation phase and performing linear fitting and four-parameter fitting, reference curves of an absolute signal intensity value, a signal change rate value, and a sample concentration; and (3) detecting a signal value of an unknown sample, and obtaining, by means of the reference curves, a concentration of the unknown sample according to a fluorescence intensity of the unknown sample, so as to avoid the hook effect. The invention enables calculation of a relative signal intensity and a relative signal change rate value by means of ratios of the absolute signal intensity value and signal change rate value to background signals, thereby achieving consistency of results across different instruments. The invention increases the number of sample concentration readings which can be completed within a given period of time, thereby improving the detection throughput of systems.

Description

A method for judging the hook effect of homogeneous time-resolved fluorescence immunoassay

Citation of related applications

This application claims the priority of the Chinese patent application filed on June 29, 2020 with the Chinese Patent Office, the application number is 202010603405.X, and the invention name is "A Method for Determining Homogeneous Time-Resolved Fluorescence Immunoassay Hook Effect", The entire contents described in the above application are incorporated herein by reference.

technical field

The present application relates to a method for judging the hook effect of homogeneous time-resolved fluorescence immunoassay, and belongs to the technical field of fluorescence immunoassay.

Background technique

Homogeneous time-resolved fluorescence immunoassay technology combines fluorescence resonance energy transfer (FRET, Fluorescence Resonance Energy Transfer) and time-resolved fluorescence (TRF, Time Resolved Fluorescence) two technologies. This technology utilizes the chelate label of rare earth elements with a cryptic structure as a fluorescent donor, and a short-lived fluorescent molecule with good spectral overlap with the fluorescent donor as a fluorescent acceptor. Fluorescence resonance energy transfer (FRET) occurs with the acceptor (second fluorescent label). In fluorescence resonance energy transfer, the lifetime of the fluorescence emitted by the acceptor is equal to the lifetime of the fluorescence emitted by the donor. Because the donor fluorescence decay period is long, the donor will induce the acceptor to emit fluorescence for a long time, and the fluorescence generated after the acceptor is excited can last for a long time, so that those short-lived self-scattered fluorescence can be distinguished by time resolution. , so that the FRET signal can be easily distinguished from the short-lived fluorescent background.

The hook effect is a hysteresis phenomenon similar to the excess antigen in the precipitation reaction. When the concentration of the antigen to be tested in the sample is quite high, the excess antigen binds to the solid-phase antibody and the enzyme-labeled antibody, respectively, instead of forming a sandwich. compound, the result will be lower than the actual content. False-negative results can even occur when the hook effect is severe. Under normal concentration, the signal increases with the concentration, and when the hook effect occurs, the signal no longer increases with the concentration.

Homogeneous time-resolved fluorescence immunoassay is tested by one-step method, which is particularly affected by the hook effect. As shown in Figure 1, the graph is the relationship between the signal and the concentration at the end of the reaction. Obviously, high concentrations will cause low measurement results. To avoid For this result, it is necessary to judge the existence of the hook effect. Homogeneous time-resolved fluorescence immunoassay technology is used for clinical diagnosis. Due to the large dynamic range of changes in the concentration of clinical samples, it is necessary to develop a technology to identify high-concentration samples, namely the hook effect, in order to meet the requirements of clinical use.

(Elimination of "hook-effect" in two-site immunoradiometric assays bykinetic rate analysis, K L Hoffman et.al.) "Clin. Chem." 30/9, 1499-1501 (1984) discloses the use of two time points The method of judging the hook effect of the radioimmunoassay by the concentration difference of the calibration curve, this method adopts the quantitative curve of 2min and 10min to judge the hook effect, and uses the characteristics of radioimmunoassay to not wash in the first reading, and the second time ends. Wash when reading. The disadvantage is that it needs to be measured at two fixed times, and it is only suitable for radiography. The Chinese patent application with publication number CN105339794A relates to a method for detecting the prozone effect of photometry, the method is suitable for photometry (immunoturbidimetry), and the signal is measured four times in the initial stage of the reaction, and is interpreted by a certain algorithm. The disadvantage is that it is suitable for turbidity method, the number of four measurements is more, and the detection system is occupied. The Chinese patent application with publication number CN102944672A relates to a method for qualitative and quantitative detection of a target substance to be tested in serum by using light-excited chemiluminescence immunoassay. The number of readings is judged by the difference between the two readings. Disadvantages: The addition of the target substance brings additional reagent consumption and increases the cost; it can only be judged at the end of the reaction. The above techniques are suitable for specific immunological methods and are not suitable for homogeneous time-resolved fluorescent immunoassays. At present, the only foreign products used in clinical practice are Thermo Fisher's Kryptor series products, and its patent for judging the hook effect has not yet been retrieved. From the public information, it uses the reaction kinetic curve within 60s of the initial stage of the detection of the immune response to determine the sample. If the concentration is too high, it needs to be diluted and read at 9 time points, which will cause the detection system to be occupied for a long time and affect the detection throughput.

SUMMARY OF THE INVENTION

In order to overcome the deficiencies of the prior art, the purpose of this application is to provide a method for judging the hook effect of a homogeneous time-resolved fluorescence immunoassay, which uses less detection system and can complete the concentration interpretation of more samples within a given time, thereby Make the system have higher detection throughput.

In order to realize the above-mentioned application purpose, the application adopts the following technical solutions:

A method for judging the hook effect of homogeneous time-resolved fluorescence immunoassay, simultaneously measuring the fluorescence intensities of the fluorescence donor and the acceptor at two detection wavelengths, and then taking the ratio of the fluorescence intensities of the two wavelengths as the absolute value of the signal intensity of the present application .

Further, the absolute value of the signal intensity and the ratio of the signal change rate value to the background signal can be used to calculate the relative signal intensity and the relative signal change rate value, which can achieve consistent results on different instruments (Figure 2).

Specifically, the excitation wavelength adopts the central wavelength of 320 nm, the detection wavelength adopts the central wavelengths of 620 nm and 665 nm, and the ratio of the fluorescence intensity of 665 nm and 620 nm is used as the absolute value of the signal intensity;

By measuring the signal values at at least two moments in the initial stage of the reaction, through linear fitting and four-parameter fitting, the absolute value of the signal intensity and the standard curve of the signal change rate value and concentration were established respectively; The absolute value, through the standard curve, obtains the concentration of the unknown sample according to the fluorescence intensity of the unknown sample, so as to avoid the hook effect.

The measurement time is at the initial stage of the reaction, at least two times, and can be 20s to 10 minutes, preferably 20s to 2 minutes.

The difference between the two moments is the time difference for calculating the kinetic rate of the reaction, and the time difference ranges from 2 seconds to 10 minutes, preferably 6-60 seconds.

The concentration level of the sample can be determined by comparing the concentration difference of the substance to be tested obtained from the two curves.

In order to make the fitted curve applicable on different instruments, the relative signal can be obtained by dividing the measured signal by the background signal of the negative sample, which is only related to the degree of reaction progress.

When calculating the relationship between the absolute value of the measured signal and the concentration, the signal from a single measurement or the average value of the signals from multiple measurements can be used. When measuring the rate, a single rate value or the average value of the rate values from multiple measurements can be used.

When both the signal value and the rate value of the unknown sample are lower than the preset standard, it is determined that the concentration is in the normal range.

When the unknown sample is calculated according to the two measurement curves, if the difference of the obtained concentration value is less than the preset value, the sample does not have hook effect.

When there is a significant difference between the calculated concentrations of the two curves (beyond the preset value), then there is a hook effect in the sample.

When the hook effect is present, the concentration level can be calculated using the relationship between the signal and the concentration when the hook effect is present.

The present application has the following positive effects: by measuring the signal values at at least two moments in the initial stage of the reaction, the relationship between the absolute value of the signal intensity and the rate value of the signal change and the concentration is established respectively, and the sample concentration is calculated, thereby judging the hook effect. Further, the absolute value of the signal intensity and the ratio of the signal change rate value to the background signal can be used to calculate the relative signal intensity and the relative value of the signal change rate, which can achieve consistent results on different instruments and make the system have a higher detection throughput.

Description of drawings

Figure 1 is a schematic diagram of the detection results of the hook effect (when the concentration is less than 1000ng/mL, the signal is proportional to the concentration; when the concentration is greater than 1000ng/mL, the signal is inversely proportional to the concentration).

Figure 2 is a graph of absolute value of signal and reaction rate versus concentration.

Figure 3 shows the relationship between the absolute value of the signal and the concentration in Example 1 (a hook effect occurs when the concentration reaches 80,000 ng/mL).

Figure 4 is the relationship between the rate value and the concentration of Example 1 (a hook effect occurs when the concentration reaches 16000 ng/mL).

Figure 5 is the relationship between the absolute value of the signal and the concentration between different systems (instruments) in Example 2 (the hook effect occurs when the concentration reaches 80,000 ng/mL).

Figure 6 is the relationship between the absolute value of the signal and the ratio of the rate value to the background value and the concentration (■ is RS, ● is RRR).

detailed description

The present application will be further described in detail below in conjunction with specific embodiments.

The required detection conditions of the following examples:

1. Instrument:

Instruments with multi-wavelength time-resolved fluorescence and detection functions, such as Tecan's multi-function microplate reader F200, can also use measuring instruments with automatic sample loading function

2. Measurement procedure:

The detection system consists of the sample to be tested, the reaction reagent r1 and the reaction reagent r2. The two reagents label the fluorescent donor and the fluorescent acceptor respectively. The fluorescent donor can emit long-lived 620 nm fluorescence, and the fluorescent acceptor itself only emits short-term fluorescence. Long-lived fluorescence, but also long-lived fluorescence when subjected to 620 nm fluorescence, this fluorescence resonance energy transfer process can only occur when immune complexes are formed, and can be measured according to standard measurement methods for homogeneous time-resolved fluorescence.

2.1 Pipetting protocol

2.5uL of the sample, 9uL of the assay reagent r1 and 9uL of the assay reagent r2 were mixed into the reaction well by pipetting to form a reaction mixture.

2.2 Signal Measurement

Measure the time-resolved fluorescence at 620 nm and 665 nm at the start time (time 1), and then repeat the measurement at intervals of 12 seconds, calculate the ratio of the fluorescence intensity at 665 nm and 620 nm as the absolute intensity value of the signal, and calculate the average value of the absolute intensity and The change value, since the interval time is fixed, the change value can be used as the relative reaction rate value.

2.3 Generate calibration curve

Using the relationship between the absolute value of the measured signal and the rate value and the concentration, two calibration curves can be established. In the range where the concentration is positively correlated with the signal, the four-parameter method is used to fit and generate the calibration curve.

2.4 Calculate unknown sample concentration using calibration curve

Using the fitted four-parameter curve equation, the concentration value of the unknown sample can be calculated. Using two curves, two concentration values can be calculated.

Example 1: Hook effect detection for AFP assays

2.3 Generate calibration curve

Detect the absolute value of the signal of a series of samples of known concentration, and use the relationship between the absolute value of the measured signal and the rate value and the concentration to establish two calibration curves. Fitting is performed to generate a calibration curve. The fitting curve between the absolute value of the signal and the concentration is shown in Figure 3, and the fitting curve between the rate value and the concentration is shown in Figure 4.

2.4 Calculate unknown sample concentration using calibration curve

Using the fitted four-parameter curve equation, the concentration value of the unknown sample can be calculated. Using two curves, two concentration values can be calculated.

Concentrationng/mL Calculated according to the absolute value curve of the signal Calculated according to the rate value curve ratio 0 - -   3.3 - -   9.9 - -   29.6 - -   88.9 - -   267 185 187 0.99 533 901 624 1.44 1067 893 1028 0.87 3200 3144 3207 0.98 16000 16620 15992 1.04 80000 74008 4068 18.2 400000 2555 307 8.31

2.5 Using the concentration value to judge the hook effect

There was no hook effect when concentrations were measured below 267 ng/mL.

When the concentration is in the range of 267-16000ng/mL, the difference is less than 2 times, the two are consistent, the calculated concentration is a normal value, and there is no hook effect. However, since the quantitative range of the final reading is 1-1000 ng/mL, those outside this range need to be diluted and determined.

When the concentration is in the range of 16000-80000ng/mL, the ratio is significantly greater than 2, and the rate method curve has a hook effect, but there is no hook effect between the absolute value of the signal and the concentration, so the concentration can be calculated according to the absolute value of the signal. .

When the concentration exceeds 80000ng/mL, the ratio is greater than 2, and both curves have hook effect, and the calculated concentration is significantly lower. It can be directly diluted 5000 times and then measured again, or the inverse correlation between the rate value and the concentration in the rate method curve can be used. calculate.

Example 2: Hook effect detection for AFP assay across different systems

2.3 Generate calibration curve

The measured signal and rate values are different between different systems (instruments). Figure 5 shows the systematic deviation of the signal values between the two instruments. Compared with Example 1, Example 2 introduces the 665/620nm measurement ratio of the blank sample as the background value, the measurement system deviation of the calibration signal between different systems (instruments), the calculation method is as follows: divide the absolute value of the signal and the rate value. Using the background value, the absolute value of the relative signal (RS, relative signal) and the relative rate value (RRR, relative reaction rate) are obtained respectively. Under the same incubation conditions, the RS and RRR curves are only related to the degree of immune response and have nothing to do with the measurement system deviation. Using the relationship between the relative value (RS) of the measured signal and the relative value of the rate (RRR) and the concentration, two can be established. A calibration curve, in the range where the concentration is positively correlated with the signal, is fitted with four parameters to generate a calibration curve (Fig. 6), which is suitable for different systems (instruments) with the same incubation conditions.

2.4 Calculate unknown sample concentration using calibration curve

Using the fitted four-parameter curve equation, the concentration value of the unknown sample can be calculated. Using two curves, two concentration values can be calculated.

Concentrationng/mL Calculated according to the absolute value curve of the signal Calculated according to the rate value curve ratio 0 - -   3.3 - -   9.9 - -   29.6 - -   88.9 - -   267 185 187 0.99 533 901 624 1.44 1067 893 1028 0.87 3200 3144 3207 0.98 16000 16620 15992 1.04 80000 74008 4068 18.2 400000 2555 307 8.31

2.5 Using the concentration value to judge the hook effect

There was no hook effect when concentrations were measured below 267 ng/mL.

When the concentration is in the range of 267-16000ng/mL, the difference is less than 2 times, the two are consistent, the calculated concentration is a normal value, and there is no hook effect. However, since the quantitative range of the final reading is 1-1000 ng/mL, those outside this range need to be diluted and determined.

When the concentration is in the range of 16000-80000ng/mL, the ratio is significantly greater than 2, and the rate method curve has a hook effect, but there is no hook effect between the absolute value of the signal and the concentration, so the concentration can be calculated according to the absolute value of the signal. .

When the concentration exceeds 80000ng/mL, the ratio is greater than 2, both curves have hook effect, the calculated concentration is significantly lower, it can be directly diluted 5000 times and then re-measured, or the inverse correlation between the rate value and the concentration in the rate method curve can be used. calculate.

2.6 A standard curve is applicable to multiple systems, and there is no need to establish a separate standard curve on each system. The above specific embodiments do not limit the technical solutions of the present application in any form, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present application.

Claims (7)

A method for judging the hook effect of homogeneous time-resolved fluorescence immunoassay, characterized in that, Measure the fluorescence intensity of the fluorescence donor and the acceptor under the same excitation wavelength and at the two detection wavelengths, and then use the ratio of the fluorescence intensity of the two wavelengths as the signal value; By measuring the signal values at at least two moments in the initial stage of the reaction, through linear fitting and four-parameter fitting, a standard curve between the absolute value of the signal intensity and the sample concentration, and a standard curve between the rate value of the signal change and the sample concentration are established respectively; Detect the signal value of the unknown sample, and obtain the concentration of the unknown sample according to the fluorescence intensity of the unknown sample through the standard curve, thereby avoiding the hook effect. The method for judging the hook effect of homogeneous time-resolved fluorescence immunoassay according to claim 1, wherein the relative signal intensity and the relative signal change rate are calculated by using the absolute value of the signal intensity and the ratio of the signal change rate value to the background signal. value to achieve consistent results on different instruments. The method for judging homogeneous time-resolved fluorescence immunoassay hook effect according to claim 1, wherein the excitation wavelength adopts a central wavelength of 320 nm, the detection wavelength adopts central wavelengths of 620 nm and 665 nm, and the fluorescence of 665 nm and 620 nm is adopted. Intensity ratio, as signal value; The measurement time is in the initial stage of the reaction, at least two times, which can be 20s to 10 minutes; The difference between the two moments is the time difference for calculating the kinetic rate of the reaction, and the time difference ranges from 2 seconds to 10 minutes. The method for judging the hook effect of homogeneous time-resolved fluorescence immunoassay according to claim 1, characterized in that, the measurement time is in the initial stage of the reaction, at least two moments, can be 20s to 2 minutes, and the time difference is taken as Values range from 6-60 seconds. The method for judging homogeneous time-resolved fluorescence immunoassay hook effect according to claim 1, characterized in that: (1) when the signal value and the rate value of the unknown sample are both lower than the preset standard, it is judged that the concentration is in the normal range; (2) When the unknown sample is calculated according to the two measurement curves, if the obtained concentration value difference is less than the preset value, the sample does not have hook effect; (3) When there is a significant difference between the calculated concentrations of the two curves and exceeds the preset value, there is a hook effect in the sample; (4) When there is a hook effect, use the relationship between the signal and the concentration in the hook effect to calculate the concentration level. The method for judging the hook effect of a homogeneous time-resolved fluorescence immunoassay according to claim 1, wherein, in order to make the fitting curve applicable to different instruments, the measured signal is divided by the background signal of the negative sample, and the relative Signal, relative signal is only related to the degree of reaction; The concentration level of the sample can be determined by comparing the concentration difference of the substance to be tested obtained from the two curves. The method for judging the hook effect of homogeneous time-resolved fluorescence immunoassay according to claim 1, characterized in that, when establishing a standard curve between the absolute value of the signal intensity and the sample concentration, a single-measured signal, or a multiple-measured signal is used. Signal average value: When establishing a standard curve of the rate value of signal change versus sample concentration, a single rate value or the average value of the rate values of multiple measurements can be used.

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