WO2022001090A1 - 一种判断均相时间分辨荧光免疫分析钩状效应的方法 - Google Patents

一种判断均相时间分辨荧光免疫分析钩状效应的方法 Download PDF

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WO2022001090A1
WO2022001090A1 PCT/CN2021/073019 CN2021073019W WO2022001090A1 WO 2022001090 A1 WO2022001090 A1 WO 2022001090A1 CN 2021073019 W CN2021073019 W CN 2021073019W WO 2022001090 A1 WO2022001090 A1 WO 2022001090A1
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signal
value
concentration
hook effect
sample
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刘涛
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中国科学院苏州生物医学工程技术研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

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  • 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.
  • 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.
  • FRET Fluorescence resonance energy transfer
  • TRF Time Resolved Fluorescence
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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).
  • the excitation wavelength adopts the central wavelength of 320 nm
  • the detection wavelength adopts the central wavelengths of 620 nm and 665 nm
  • the ratio of the fluorescence intensity of 665 nm and 620 nm is used as the absolute value of the signal intensity
  • 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.
  • 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.
  • the signal from a single measurement or the average value of the signals from multiple measurements can be used.
  • the rate a single rate value or the average value of the rate values from multiple measurements can be used.
  • the sample does not have hook effect.
  • 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.
  • 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).
  • 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
  • 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.
  • the concentration value of the unknown sample can be calculated.
  • two concentration values can be calculated.
  • the concentration value of the unknown sample can be calculated.
  • two concentration values can be calculated.
  • 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.
  • the quantitative range of the final reading is 1-1000 ng/mL, those outside this range need to be diluted and determined.
  • 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. .
  • 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.
  • 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.
  • 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.
  • the concentration value of the unknown sample can be calculated.
  • two concentration values can be calculated.
  • 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.
  • the quantitative range of the final reading is 1-1000 ng/mL, those outside this range need to be diluted and determined.
  • 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. .
  • 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.

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Abstract

本申请涉及一种判断均相时间分辨荧光免疫分析钩状效应的方法，步骤是：（1）测定荧光供体和受体在同一激发波长下、两个检测波长处的荧光强度，然后以两个波长的荧光强度比值作为信号值；（2）通过测量反应起始阶段至少两个时刻的信号值，通过线性拟合、四参数拟合，分别建立信号强度的绝对值、信号变化的速率值与样品浓度的标准曲线；（3）检测未知样本的信号值，通过标准曲线，根据未知样本的荧光强度，获得未知样本的浓度，从而避免hook效应。利用信号强度绝对值和信号变化速率值与背景信号的比值，计算相对信号强度和信号变化速率相对值，在不同仪器上实现一致的结果。可以在既定时间内完成更多样品的浓度判读，从而使***具有更高检测通量。

Description

一种判断均相时间分辨荧光免疫分析钩状效应的方法
相关申请的援引
本申请要求在2020年6月29日提交中国专利局、申请号为202010603405.X、发明名称为《一种判断均相时间分辨荧光免疫分析钩状效应的方法》的中国专利申请的优先权，记载于上述申请中的全部内容通过引用结合在本申请中。
技术领域
本申请涉及一种判断均相时间分辨荧光免疫分析钩状效应的方法，属于荧光免疫检测技术领域。
背景技术
均相时间分辨荧光免疫分析技术结合了荧光共振能量转移(FRET，FluorescenceResonance Energy Transfer)和时间分辨荧光(TRF，Time Resolved Fluorescence)两种技术。该技术是利用了具有穴状结构的稀土元素的螯合标记物作为荧光供体，以及与荧光供体具有良好光谱重叠的短寿命荧光分子作为荧光受体，在稀土元素穴状化合物的供体与受体(第二荧光标记物)之间发生荧光共振能量转移(FRET)。在荧光共振能量转移中，受体发射荧光的寿命等同于供体的发射荧光的寿命。因为供体荧光衰减周期较长，所以供体会诱导受体长时间地发射荧光，受体激发后产生的荧光便能持续较长时间，这样通过时间分辨就可以区分那些短寿命的自身散射的荧光，这样从短寿命荧光背景中就很容易区分出FRET信号。
钩状效应(hook effect)是类同于沉淀反应中抗原过剩的后滞现象，当标本中待测抗原浓度相当高时，过量抗原分别和固相抗体及酶标抗体结合，而不再形成夹心复合物，所得结果将低于实际含量。钩状效应严重时甚至可出现假阴性结果。正常浓度下信号随浓度升高，产生钩状效应时，信号不再随浓 度增加。
均相时间分辨荧光免疫分析采用一步法测试，受到钩状效应影响尤为明显，如图1所示，该图是反应终点时信号与浓度的关系，显然高浓度会造成测量结果偏低，为避免这种结果，需要判断钩状效应的存在。应用均相时间分辨荧光免疫分析技术用于临床诊断，由于临床标本浓度变化动态范围大，因此必须要开发一种识别高浓度标本即hook效应的技术，才能满足临床使用要求。
(Elimination of"hook-effect"in two-site immunoradiometric assays bykinetic rate analysis，K L Hoffman et.al.)《Clin.Chem.》30/9，1499-1501(1984)公开了利用两个时间点的定标曲线浓度差异判断放射免疫法钩状效应的方法，该方法采用2min和10min两个时刻定量曲线，判断钩状效应，利用了放免的特点在第一次读数时不清洗，第二次终读数时清洗。缺点是需在两个固定时刻测量，且只适合放免。公开号为CN105339794A的中国专利申请涉及用于检测光度测定法的前带效应的方法，该方法适用于光度法(免疫浊度法)，在反应初始阶段测量四次信号，通过一定算法进行判读。缺点是适用于浊度法，四次测量次数较多，占用检测***。公开号为CN102944672A的中国专利申请涉及采用光激发化学发光免疫分析对血清中的待测靶物质进行定性与定量检测的方法，该方法通过在反应终点读数后，加入一定靶物质再继续反应后二次读数，通过两次读数差异判断。缺点：加入靶物质带来额外试剂消耗，增加成本；在反应结束时才能判断。以上技术适用于特定的免疫学方法，并不适合于均相时间分辨荧光免疫分析。目前用于临床的国外产品仅有赛默飞世尔的Kryptor系列产品，尚未检索到其判断hook效应的专利，从公开资料看，其采用检测免疫反应初始阶段60s内反应动力学曲线进而判断样品是否浓度过高需要稀释，需要在9个时间点进行读数，这会造成检测***长时间被占用，影响检测通量。
发明内容
为了克服现有技术的不足，本申请的目的是提供一种判断均相时间分辨荧光免疫分析钩状效应的方法，较少使用检测***，可以在既定时间内完成更多样品的浓度判读，从而使***具有更高检测通量。
为实现上述申请目的，本申请采用以下技术方案：
一种判断均相时间分辨荧光免疫分析钩状效应的方法，同时测定荧光供体和受体在两个检测波长的荧光强度，然后以两个波长的荧光强度比值作为本申请信号强度的绝对值。
进一步，可以利用信号强度绝对值和信号变化速率值与背景信号的比值，计算相对信号强度和信号变化速率相对值，可以在不同仪器上实现一致的结果(图2)。
具体而言，激发波长采用中心波长320nm，检测波长采用中心波长620nm和665nm，以665nm和620nm的荧光强度比值，作为信号强度的绝对值；
通过测量反应起始阶段至少两个时刻的信号值，通过线性拟合、四参数拟合，分别建立信号强度的绝对值和信号变化的速率值与浓度的标准曲线；检测未知样本的信号强度的绝对值，通过标准曲线，根据未知样本的荧光强度，获得未知样本的浓度，从而避免hook效应。
测量时间是在反应的起始阶段，在至少两个时刻，可以是20s到10分钟，优选的是20s到2分钟。
两个时刻的差值是计算反应动力学速率的时间差，时间差取值范围从2秒到10分钟，优选6-60秒。
通过比较两条曲线得到的待测物质的浓度差异，可以判断样本的浓度水平。
为使拟合曲线在不同仪器上适用，将测量信号除以阴性标本的背景信号，可以获得相对信号，相对信号只与反应进行的程度有关。
计算测量信号的绝对值与浓度的关系时，可采用单次测量的信号，或者 多次测量的信号平均值，测量速率时可采用单个速率值或多次测量的速率值的平均值。
当未知样品的信号值和速率值均低于预设标准时，判断为浓度处于正常范围。
当未知样品按照两条测量曲线进行计算时，如果得到的浓度值差异，小于预设值则样品不存在钩状效应。
当两条曲线计算得到的浓度存在显著差异时(超出预设值)，则样品中存在钩状效应。
存在钩状效应时，利用钩状效应时，信号与浓度的关系，可计算浓度水平。
本申请有以下积极的效果：通过测量反应起始阶段至少两个时刻的信号值，分别建立信号强度的绝对值和信号变化的速率值与浓度的关系，计算样本浓度，从而判断hook效应。进一步可以利用信号强度绝对值和信号变化速率值与背景信号的比值，计算相对信号强度和信号变化速率相对值，可以在不同仪器上实现一致的结果，使***具有更高检测通量。
附图说明
图1是钩状效应检测结果示意图(当浓度小于1000ng/mL时，信号与浓度呈正比；当浓度大于1000ng/mL时，信号与浓度呈反比)。
图2是信号绝对值和反应速率与浓度的关系图。
图3是实施例1的信号绝对值与浓度关系(浓度达到80000ng/mL时产生钩状效应)。
图4是实施例1的速率值与浓度的关系(浓度达到16000ng/mL时产生钩状效应)。
图5是实施例2不同***(仪器)间信号绝对值与浓度关系(浓度达到80000ng/mL时产生钩状效应)。
图6是信号绝对值与速率值与背景值比值与浓度的关系(■为RS，●为 RRR)。
具体实施方式
下面结合具体实施方式对本申请做进一步的详细说明。
以下实施例所需检测条件：
1、仪器：
具有多波长时间分辨荧光，检测功能的仪器，进行测定，例如帝肯的多功能酶标仪F200，也可采用具备全自动加样功能的测量仪器
2、测量程序：
检测体系包括待测样本、反应试剂r1和反应试剂r2组成，两种试剂分别标记荧光供体和荧光受体，其中，荧光供体可发出长寿命的620纳米荧光，荧光受体本身只发出短寿命荧光，但是当接受620纳米荧光时也可发出长寿命的荧光，只有当形成免疫复合物时这一荧光共振能量转移过程才能发生，按照均相时间分辨荧光的标准测量方法可以进行测量。
2.1移液方案
将2.5uL样品和9uL测定试剂r1和9uL测定试剂r2到反应孔中通过吹打混合，形成反应混合物。
2.2信号测量
测量开始时刻(时刻一)的620纳米和665纳米的时间分辨荧光，然后间隔12秒钟重复测量，计算665纳米和620纳米荧光强度的比值作为信号的绝对强度值，计算绝对强度的平均值和变化值，由于间隔时间是固定的，因此变化值可以作为相对反应速率值。
2.3生成校准曲线
分别利用测量信号绝对值和速率值与浓度的关系，可以建立两条校准曲线，在浓度与信号正相关的范围内，采用四参数法进行拟合，生成校准曲线。
2.4利用校准曲线计算未知样品浓度
利用拟合的四参数曲线方程，可以计算出未知样品的浓度值，采用两条 曲线，可以计算出两个浓度值。
实施例1：用于AFP测定的钩状效应检测
2.3生成校准曲线
检测一系列已知浓度样品的信号绝对值，利用测量信号绝对值和速率值与浓度的关系，可以建立两条校准曲线，在浓度与信号正相关的范围内(见图2)，采用四参数进行拟合，生成校准曲线，信号绝对值与浓度关系拟合曲线见图3，速率值与浓度关系拟合曲线见图4。
2.4利用校准曲线计算未知样品浓度
利用拟合的四参数曲线方程，可以计算出未知样品的浓度值，采用两条曲线，可以计算出两个浓度值。
浓度ng/mL 按信号绝对值曲线计算 按速率值曲线计算 比值
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利用浓度值判断钩状效应
当浓度测量值低于267ng/mL时，不存在钩状效应。
当浓度在267-16000ng/mL范围时，差异小于2倍，两者一致，计算浓度为正常值，不存在钩状效应。但由于最终读数时定量范围为1-1000ng/mL，因此超出这一范围的需要稀释后测定。
当浓度在16000-80000ng/mL范围时，比值显著大于2，速率法曲线出现钩状效应，但信号绝对值与浓度不存在钩状效应，因此可根据信号绝对值曲 线计算浓度，判断合理稀释倍数。
当浓度超过80000ng/mL时，比值大于2，两条曲线均存在钩状效应，计算浓度显著偏低，可直接稀释5000倍后重新测量，也可以根据速率法曲线中速率值与浓度反相关关系计算。
实施例2：在不同***间，用于AFP测定法的钩状效应检测
2.3生成校准曲线
在不同***(仪器)之间测量得到的信号值和速率值是不同的，图5展示了信号值在两个仪器间的***偏差。与实施例1相比，实施例2引入空白样本的665/620nm测定比值作为背景值，校准信号在不同***(仪器)之间的测量***偏差，计算方法如下：将信号绝对值和速率值除以背景值，分别得到相对信号绝对值(RS，relative signal)和相对速率值(RRR,relative reaction rate)。在同样的温育条件下，RS和RRR曲线只与免疫反应程度有关，与测量***偏差无关，分别利用测量信号的相对值(RS)和速率相对值(RRR)与浓度的关系，可以建立两条校准曲线，在浓度与信号正相关的范围内，采用四参数进行拟合，生成校准曲线(图6)，适用于同样温育条件的不同***(仪器)。
2.4利用校准曲线计算未知样品浓度
利用拟合的四参数曲线方程，可以计算出未知样品的浓度值，采用两条曲线，可以计算出两个浓度值。
浓度ng/mL 按信号绝对值曲线计算 按速率值曲线计算 比值
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利用浓度值判断钩状效应
当浓度测量值低于267ng/mL时，不存在钩状效应。
当浓度在267-16000ng/mL范围时，差异小于2倍，两者一致，计算浓度为正常值，不存在钩状效应。但由于最终读数时定量范围为1-1000ng/mL，因此超出这一范围的需要稀释后测定。
当浓度在16000-80000ng/mL范围时，比值显著大于2，速率法曲线出现钩状效应，但信号绝对值与浓度不存在钩状效应，因此可根据信号绝对值曲线计算浓度，判断合理稀释倍数。
当浓度超过80000ng/mL时，比值大于2，两条曲线均存在钩状效应，计算浓度显著偏低，可直接稀释5000倍后重新测量，也可以根据速率法曲线中速率值与浓度反相关关系计算。
2.6一条标准曲线适用于多个***，不需要在每个***上单独建立标准曲线。上述具体实施方式不以任何形式限制本申请的技术方案，凡是采用等同替换或等效变换的方式所获得的技术方案均落在本申请的保护范围。

Claims (7)

  1. 一种判断均相时间分辨荧光免疫分析钩状效应的方法，其特征在于，
    测定荧光供体和受体在同一激发波长下、两个检测波长处的荧光强度，然后以两个波长的荧光强度比值作为信号值；
    通过测量反应起始阶段至少两个时刻的信号值，通过线性拟合、四参数拟合，分别建立信号强度的绝对值与样品浓度的标准曲线、信号变化的速率值与样品浓度的标准曲线；
    检测未知样本的信号值，通过标准曲线，根据未知样本的荧光强度，获得未知样本的浓度，从而避免hook效应。
  2. 根据权利要求1所述的判断均相时间分辨荧光免疫分析钩状效应的方法，其特征在于，利用信号强度绝对值和信号变化速率值与背景信号的比值，计算相对信号强度和信号变化速率相对值，在不同仪器上实现一致的结果。
  3. 根据权利要求1所述的判断均相时间分辨荧光免疫分析钩状效应的方法，其特征在于，所述的激发波长采用中心波长320nm，检测波长采用中心波长620nm和665nm，以665nm和620nm的荧光强度比值，作为信号值；
    测量时间是在反应的起始阶段，在至少两个时刻，可以是20s到10分钟；
    两个时刻的差值是计算反应动力学速率的时间差，时间差取值范围从2秒到10分钟。
  4. 根据权利要求1所述的判断均相时间分辨荧光免疫分析钩状效应的方法，其特征在于，测量时间是在反应的起始阶段，在至少两个时刻，可以是20s到2分钟，时间差取值范围从6-60秒。
  5. 根据权利要求1所述的判断均相时间分辨荧光免疫分析钩状效应的方法，其特征在于：
    (1)当未知样品的信号值和速率值均低于预设标准时，判断为浓度处于正 常范围；
    (2)当未知样品按照两条测量曲线进行计算时，如果得到的浓度值差异，小于预设值则样品不存在钩状效应；
    (3)当两条曲线计算得到的浓度存在显著差异，超出预设值时，则样品中存在钩状效应；
    (4)存在钩状效应时，利用钩状效应时信号与浓度的关系，计算浓度水平。
  6. 根据权利要求1所述的判断均相时间分辨荧光免疫分析钩状效应的方法，其特征在于，为使拟合曲线在不同仪器上适用，将测量信号除以阴性标本的背景信号，可以获得相对信号，相对信号只与反应进行的程度有关；
    通过比较两条曲线得到的待测物质的浓度差异，可以判断样本的浓度水平。
  7. 根据权利要求1所述的判断均相时间分辨荧光免疫分析钩状效应的方法，其特征在于，建立信号强度的绝对值与样品浓度的标准曲线时采用单次测量的信号，或者多次测量的信号平均值；建立信号变化的速率值与样品浓度的标准曲线时，可采用单个速率值或多次测量的速率值的平均值。
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