WO2023216517A1 - 计算样品的IgA免疫活动性指数的方法及装置 - Google Patents

计算样品的IgA免疫活动性指数的方法及装置 Download PDF

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WO2023216517A1
WO2023216517A1 PCT/CN2022/127454 CN2022127454W WO2023216517A1 WO 2023216517 A1 WO2023216517 A1 WO 2023216517A1 CN 2022127454 W CN2022127454 W CN 2022127454W WO 2023216517 A1 WO2023216517 A1 WO 2023216517A1
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plate
iga
activity index
immune activity
detection amount
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PCT/CN2022/127454
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French (fr)
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饶皑炳
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深圳市陆为生物技术有限公司
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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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
    • G01N21/3151—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths using two sources of radiation of different wavelengths
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • 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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • the present application relates to the field of immune detection technology, and in particular to methods and devices for calculating the IgA immune activity index of a sample.
  • IgA human immunoglobulin A
  • IgA complexes The downstream antibodies IgG and IgM, C3 complement and other molecules of the complement pathway participate to form IgA-IgG, IgA- IgM, IgA-C3, IgA-IgG-IgM, etc. are collectively referred to as IgA complexes.
  • IgA complexes During a normal immune response, these pathogen-bound complexes are generally cleared by the immune system, such as phagocytosis by macrophages.
  • IgA immune response when the IgA immune response is abnormal, the IgA complex cannot be effectively cleared by the immune system, but remains in the blood circulation system and urinary system for a long time, leading to two diseases. Specifically: the deposition and retention of IgA complex in the kidney will Causes IgA Nehphropathy (IgAN); the deposition of IgA complexes in blood vessels will lead to purpura, often called Henoch-Schonlein purpura (HSP) or IgA vasculitis (IgAV).
  • HSP Henoch-Schonlein purpura
  • IgAV IgA vasculitis
  • IgA detection is clinically routine immune three items (IgA, IgM, IgG) or immune five items (immunity three items + complement C3, C4 ) project.
  • IgA nephropathy and purpura the factors that increase total IgA may also be due to other diseases such as multiple myeloma, rheumatoid arthritis, thrombocytopenia, and infectious diseases. Therefore, total IgA plays an important role in IgA nephropathy and purpura. Specificity is low.
  • the applicant tried to proceed from the principle that since circulating IgA complexes in serum are the main factors inducing pathogenesis, the detection amount of IgA complexes is directly detected as an index of IgA immune activity to replace the three immune or immune Five tests.
  • this method has better sensitivity and specificity, it still inevitably brings some problems when faced with batch diagnosis of large sample volumes due to problems such as the operator's operation and the inter-plate variability of the enzyme plate itself. Therefore, it is necessary to provide a calculation method for IgA immune activity index that can further improve the accuracy of large-scale detection.
  • This application aims to solve at least one of the technical problems existing in the prior art. To this end, this application proposes a calculation method for IgA immune activity index that can further improve the accuracy of large-scale detection.
  • a first aspect of this application provides a method for calculating the IgA immune activity index of a sample, which method includes the following steps:
  • n ⁇ N are all positive integers.
  • the sample includes a test substance and a positive control substance
  • the intra-plate anchor value ⁇ n is the median detection amount of the sample in the n-th enzyme-titer plate or the n-th enzyme-titer plate. The detection amount of the positive control substance.
  • the inter-plate anchoring value ⁇ N is the median of the intra-plate anchoring values ⁇ n of N enzyme-titer plates.
  • the regression model is a linear regression model.
  • the absorbance value OD is converted into the detected amount ⁇ of the IgA complex according to the following formula:
  • each sample is provided with several replicate wells, and the deviation coefficient CV of the detection amount ⁇ of the several replicate wells of each sample is calculated.
  • the deviation coefficient CV ⁇ 20% the sample is determined to be Invalid sample.
  • the sample includes different amounts of standards and negative controls.
  • the absorbance value OD of each well is re-obtained:
  • the detection amount of negative control substance is > ⁇ 0 .
  • T 0 is 0.6
  • R 0 is 0.9
  • P 0 is 30, and ⁇ 0 is 0.1.
  • the second aspect of this application provides a method for judging IgA immune activity index, which method includes the following steps:
  • the subject's IgA immune activity index is judged to be within the normal basic range
  • the subject When m+2 ⁇ F ⁇ m+3 ⁇ , the subject’s IgA immune activity index is judged to be high within the basic range.
  • the subject When ⁇ F ⁇ m+3 ⁇ , the subject’s IgA immune activity index is judged to be high within the abnormal range;
  • m is the average value of the IgA immune activity index of normal people
  • ⁇ is the standard deviation of the IgA immune activity index of normal people.
  • the method further includes generating a physical examination report based on the results of judging the IgA immune activity index.
  • the physical examination report includes the IgA immune activity index and the interval standard of the index and the subject's corresponding IgA immune activity index data.
  • the physical examination report also includes the validity of the quality control results during the detection process.
  • the physical examination report also includes general information about the subject.
  • the physical examination report also includes general results of clinical experiments on IgA immune activity index for reference by doctors and patients.
  • the physical examination report also includes statistical characteristics, standard curves, etc. of healthy people for reference.
  • a third aspect of the present application provides a computer-readable storage medium, which stores computer-executable instructions.
  • the computer-executable instructions are used to cause the computer to execute the aforementioned method of calculating IgA immune activity index or to determine IgA immunity. Steps in the Activity Index Method.
  • a fourth aspect of the present application provides a device.
  • the device includes a processor and a memory.
  • the memory stores a computer program that can be run on the processor.
  • the processor runs the computer program, it implements the aforementioned method for calculating the IgA immune activity index. Or a method for judging IgA immune activity index.
  • the fifth aspect of this application provides a system, which includes:
  • the acquisition module is used to obtain the absorbance value OD of each well in the enzyme plate
  • the detection amount calculation module the detection analysis module is used to convert the absorbance value OD into the detection amount ⁇ of the IgA complex according to the regression model;
  • the system further includes a well quality control module, which is used to calculate the deviation coefficient CV of the detection amount ⁇ of several replicate wells of each sample.
  • a well quality control module which is used to calculate the deviation coefficient CV of the detection amount ⁇ of several replicate wells of each sample.
  • the system also includes a plate quality control module.
  • the plate quality control module is used to determine the following indicators in the enzyme plate. When any of the following conditions is met, the enzyme plate is determined to be invalid.
  • ELISA plate
  • the detection amount of negative standard substance is > ⁇ 0 ;
  • T 0 is 0.6
  • R 0 is 0.9
  • P 0 is 30, and ⁇ 0 is 0.1.
  • the system also includes an IgA immune activity index analysis module.
  • the IgA immune activity index analysis module is used to analyze the average value m of the IgA immune activity index ⁇ F and the normal human IgA immune activity index. Relationship,
  • the subject's IgA immune activity index is judged to be within the normal basic range
  • the subject's IgA immune activity index is judged to be high within the abnormal range
  • ⁇ is the standard deviation of the IgA immune activity index of normal people.
  • the system also includes a physical examination report generation module, which is used to determine the subject's IgA based on the relationship between the IgA immune activity index and the average value m of the normal human IgA immune activity index. Immune activity index.
  • the physical examination report includes the IgA immune activity index and the interval standard of the index and the subject's corresponding IgA immune activity index data.
  • the physical examination report also includes the validity of the quality control results during the detection process.
  • the physical examination report also includes general information about the subject.
  • the physical examination report also includes general results of clinical experiments on IgA immune activity index for reference by doctors and patients.
  • the physical examination report also includes statistical characteristics, standard curves, etc. of healthy people for reference.
  • This method obtains the original OD value of each well on the enzyme plate from, for example, an enzyme-linked immunoreaction, first performs quality control at the well level, and obtains a calculation formula for converting OD into sample quantification through standard curve fitting, and calculates the IgA complex in the well. The detection amount of the substance is then carried out at the plate level for quality control, the data at the enzyme plate level is normalized, and the inter-plate differences of the enzyme plate are removed. Finally, the calculation is repeated for the sample to determine the final quantitative result of each sample. In this way, the accuracy of large-volume detection can be further improved.
  • Figure 1 is a flow chart for calculating the IgA immune activity index provided by an embodiment of the present application.
  • Figure 2 is another flow chart for calculating the IgA immune activity index provided by an embodiment of the present application.
  • Figure 3 is the result of 1 using different methods to construct a linear regression model in the embodiment of the present application.
  • a is to directly construct a linear regression model using the OD value and the detection amount
  • b is to construct a linear regression model by taking the logarithm of the OD value and the detection amount. regression model.
  • Figure 4 is the distribution of the IgA immune activity index of 33 healthy people in Example 2 of the present application.
  • a is its probability density distribution.
  • the horizontal axis is the value of the IgA immune activity index, and the vertical axis is the number of people.
  • Figure 5 is a schematic diagram of a physical examination report provided in Embodiment 3 of the present application.
  • the method includes the following steps:
  • the enzyme-labeled plate refers to the enzyme-linked immunosorbent (ELISA) experiment used for IgA complexes.
  • the enzyme-labeled plate is equipped with several wells, such as 6-well, 12-well, 24-well, 48-well, 96-well, etc. Kong et al.
  • the IgA complex is immobilized by binding to the probe in the well, and then combined with an enzyme-labeled secondary antibody for color development.
  • the probe of the IgA complex is the Fc receptor protein of IgA, such as FCAR/CD89 protein, which can specifically bind to the IgA complex.
  • FCAR/CD89 protein which can specifically bind to the IgA complex.
  • it is usually necessary to set up at least one of standards, controls, etc.
  • Standard refers to a sample containing a known detectable amount (calibrated content) of IgA complex, which can be used to provide a gradient of known detected amounts, or to obtain different gradients of known detected amounts through dilution. The relationship with the corresponding absorbance value OD was used to obtain the standard curve.
  • the control substance generally includes at least one of a negative control substance and a positive control substance.
  • the negative control substance does not contain IgA complexes or its content is lower than the detection limit.
  • the positive control substance contains IgA complexes and has been eliminated through various methods. Interfering substances are used to evaluate whether the test results are valid, as well as their stability and comparability. Both are quality control materials.
  • the absorbance value OD of each well refers to the reaction result of the enzyme-labeled secondary antibody that binds to the IgA complex fixed in the well and catalyzes the colored product formed by the enzyme reaction substrate. It can be understood that during the detection process, usually each sample (including standard substance, test substance, control substance, etc.) will be repeated several times (for example, two to three).
  • the regression model refers to a quantitative model of the statistical relationship between the absorbance value OD and the detected amount of IgA complex ⁇ .
  • the value of the correlation coefficient R 2 of the second model is closer to 1, so the goodness of fit is better. Therefore, according to the second model we get Among them, the specific values of ⁇ and ⁇ can be obtained based on the calibration amount of the standard and the corresponding absorbance value.
  • the detected amount ⁇ of IgA complex in the remaining wells can be calculated using the absorbance values OD of the remaining wells.
  • the detected amount and the maximum amount below may refer to mass, concentration or other measurements.
  • the detection amount of IgA complexes in different wells in the same plate is normalized to the difference from the in-plate anchoring value ⁇ n , It is convenient to align different microplates to the same level in S140.
  • the intra-plate anchor value ⁇ n can be chosen to have a relatively stable value between different plates.
  • the detection amount of the positive control substance is used as the intra-plate anchor value ⁇ n , or it is used as the in-plate anchor value ⁇ n .
  • the intermediate value of the detection amount of each test product within the effective gradient is used as the anchor value ⁇ n within the plate.
  • the inter-plate anchoring value ⁇ N can preferably be the median of the intra-plate anchoring values ⁇ n corresponding to each of the N enzyme-titer plates. For example, for ⁇ 1 , ⁇ 2 & ⁇ k in total k The median of the intra-plate anchoring values is taken as the inter-plate anchoring value ⁇ N . It can be understood that ⁇ N can also adopt other values consistent with this concept as the inter-plate anchoring value ⁇ N .
  • the median value of the test products between different plates will vary with the amount of the test products in the plate.
  • the number of samples increases and gradually stabilizes.
  • the positive control substance also has a relatively stable IgA complex content. Therefore, through the selection of the intra-plate anchoring value ⁇ n and the inter-plate anchoring value ⁇ N , the Stable anchor points align different microplates to the same level, normalize data, and eliminate differences between plates. That is, first the detection amount of the enzyme plate is normalized to a level close to 0, and then in order to get closer to the actual detection amount, the anchor point normalized to close to 0 is raised to the actual level.
  • quality control is carried out from two aspects, including quality control at the well level within the microplate and quality control at the microplate level.
  • the IgA complex of each sample is calculated.
  • the threshold values of the deviation coefficient can be 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, Values vary from 10%, 8%, 5%, 3%, etc.
  • the samples for quality control at the well level may include standards and positive controls.
  • the detection quantity of the product to be tested can also be quality controlled in this way.
  • Quality control at the enzyme plate level can be carried out in the following ways, including the following determinations: a) The absorbance value of the maximum detection amount standard ⁇ T 0 ; b) The correlation coefficient of the regression model ⁇ R 0 ; c) The detection of the standard The output deviates from the calibrated amount >P 0 %; d) The detection amount of the negative control substance is > ⁇ 0 .
  • T 0 is 0.6
  • R 0 is 0.9
  • P 0 is 30, and ⁇ 0 is 0.1ng.
  • the embodiments of the present application also provide a method for judging IgA immune activity index, which method includes the following steps:
  • the subject's IgA immune activity index is judged to be within the normal basic range
  • the subject When m+2 ⁇ F ⁇ m+3 ⁇ , the subject’s IgA immune activity index is judged to be high within the basic range.
  • the subject When ⁇ F ⁇ m+3 ⁇ , the subject’s IgA immune activity index is judged to be high within the abnormal range;
  • m is the average value of the IgA immune activity index of normal people
  • ⁇ is the standard deviation of the IgA immune activity index of normal people.
  • the range of the IgA immune activity index of the subject can be estimated by comparing it with the value of a normal person. Further, based on the range of the IgA immune activity index directly or in combination with other indicators, it can be understood whether the subject suffers from IgA nephropathy or purpura.
  • m and ⁇ can be obtained by detecting a certain number of normal people. The number of normal people can be more than 20, 30, 50, 100, or 200 people.
  • the normal population specifically refers to people who have been determined by various standard tests to not have IgA nephropathy, purpura, or other diseases that may affect the detection of this indicator. , or healthy people.
  • the normal population should preferably include people of all ages from children to the elderly, and it is best for men and women to account for half each or close to 1:1.
  • m and ⁇ can be obtained by any method known in the art based on the IgA immune activity index of each individual in the normal population, for example, by direct mathematical calculation, or by fitting a normal distribution.
  • the standards of 2 ⁇ and 3 ⁇ are not unique and can be appropriately adjusted according to the actual situation.
  • the physical examination report can also contain relevant results of other test items, which will not be discussed here. Repeat.
  • the physical examination report can provide the IgA immune activity index, the standards for each interval corresponding to the index, and the classification corresponding to the interval within which the subject's index falls, which belongs to the normal basic range. Or the basic range is too high, or it's abnormally high, etc. It is understandable that the physical examination report also contains the results of the quality control during the testing process, and the effectiveness of the quality control is given to prove that the test is effective. Of course, it also includes general information about the subject, such as name, etc. In addition, the general results of clinical experiments on IgA immune activity index, or the statistical characteristics and standard curves of normal populations can also be provided in the physical examination report for reference.
  • Embodiments of the present application also provide a computer-readable storage medium.
  • the computer-readable storage medium stores computer-executable instructions.
  • the computer-executable instructions are used to cause the computer to execute the aforementioned method of calculating IgA immune activity index or to determine IgA immunity. Steps in the Activity Index Method.
  • An embodiment of the present application also provides a device.
  • the device includes a processor and a memory.
  • the memory stores a computer program that can be run on the processor.
  • the processor runs the computer program, it implements the aforementioned method for calculating the IgA immune activity index. Or a method for judging IgA immune activity index.
  • the memory as a non-transitory computer-readable storage medium, can be used to store non-transitory software programs and non-transitory computer executable programs, such as the process of determining the IgA immune activity index described in the embodiments of this application.
  • the processor realizes the judgment of the subject's IgA immune activity index by running non-transient software programs and instructions stored in the memory.
  • the memory may include a program storage area and a data storage area, wherein the program storage area may store an operating system and an application program required for at least one function; the storage data area may store the computer program for executing the above.
  • the memory may include high-speed random access memory and may also include non-transitory memory, such as at least one disk storage device, flash memory device, or other non-transitory solid-state storage device.
  • the memory may optionally include memory located remotely relative to the processor, and these remote memories may be connected to the processor through a network.
  • Examples of the above-mentioned networks include but are not limited to the Internet, intranets, local area networks, mobile communication networks and combinations thereof.
  • the non-transitory software programs and instructions required to implement the above determination are stored in the memory, and when executed by one or more processors, the above determination is performed.
  • the embodiment of the present application also provides a system, which includes:
  • the acquisition module is used to obtain the absorbance value OD of each well in the enzyme plate
  • the detection amount calculation module the detection analysis module is used to convert the absorbance value OD into the detection amount ⁇ of the IgA complex according to the regression model;
  • Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, Digital Versatile Disk (DVD) or other optical disk storage, magnetic cassettes, tapes, disk storage or other magnetic storage devices, or may Any other medium used to store the desired information and that can be accessed by a computer.
  • communication media typically embodies computer readable instructions, data structures, program modules or other data in a modulated data signal such as a carrier wave or other transport mechanism, and may include any information delivery media.
  • the kit includes molecular probe freeze-dried powder, coating buffer, enzyme-labeled plate, standard substance, enzyme-labeled secondary antibody, chromogenic solution, stop solution, washing solution, Diluent and blocking solution.
  • the molecular probe lyophilized powder is recombinant FCAR protein lyophilized powder
  • the enzyme plate is a 96-well plate
  • the standard is commercial human serum IgA containing IgA complex
  • the negative control substance is human serum without IgA complex
  • the positive control substance is human serum containing IgA complex, and 25ng of commercial human serum IgA is selected;
  • the enzyme-labeled secondary antibody is horseradish peroxidase (HRP)-labeled mouse anti-human IgA;
  • the chromogenic liquid includes chromogenic liquid A—3,3’,5,5’-tetramethylbenzidine (TMB) and chromogenic liquid B—hydrogen peroxide solution;
  • the stop solution is 10% sulfuric acid
  • the washing solution is 0.15M phosphate buffer containing 0.05% Tween-20;
  • the diluent is washing solution + 1 ⁇ BSA;
  • the blocking solution is washing solution + 5% BSA.
  • the first wavelength is 450nm and the second wavelength is 630nm.
  • Tables 1 and 2 respectively show the sample mapping matrix of the microplate and the part of the OD value matrix obtained based on the sample mapping matrix of the microplate and the detection results.
  • STD1 to 6 represent diluted standards with different detection volume gradients
  • nCtrl is the negative control substance
  • B000001 to 5 is the sample to be detected
  • F at the end indicates whether the plasma sample is frozen (F: Frozen , are frozen samples), used to compare the differences between samples under different storage conditions; Q2, Q4, Q7, and Q8 are healthy human control samples.
  • Q2, Q4, Q7, and Q8 are healthy human control samples.
  • the deviation coefficients of the standard, control and test products are all less than 20%, which meet the quality control requirements at the well level and are all valid.
  • the other wells of the microplate and the other 17 microplates all met the quality control requirements at the well level of the microplate.
  • the absorbance value of the standard STD1 with the maximum detection amount is greater than 0.6, and the coefficient of the regression model is greater than 0.9.
  • the detection amount of the standard substance deviates from its calibrated amount by no more than 30% of the calibrated amount.
  • the detection amount of the negative control substance Less than 0.1, therefore, it meets the quality control requirements at the enzyme plate level.
  • the intra-plate anchoring values of these enzyme-titer plates obtained by the same method were 4.93, 5.28, 3, 3.34, 5.07, 5.11, 4.66, 6.21, 4.23, 4.94, 5.07, 5.14, 5.23, 5.3, 3.85, 4.85, 4.97, 4.74. Therefore, the inter-plate anchoring value ⁇ N of the enzyme plate is the middle value of the 18 intra-plate anchoring values, which is 4.96.
  • the results from Q2 to Q4 are the IgA immune activity index of the corresponding subject.
  • the interval of the IgA immune activity index detection value ⁇ is defined as follows:
  • the basic range is higher m+2 ⁇ m+3 ⁇ , that is, 15.18 ⁇ 18.27;
  • the IgA immune activity index of 33 normal people conforms to a normal distribution.
  • the characteristics of the normal distribution are that 97.72% of people satisfy ⁇ m+2 ⁇ , and 99.87% of people satisfy ⁇ m+2 ⁇ .
  • This embodiment provides a system, which includes:
  • the acquisition module is used to obtain the absorbance value OD of each well in the enzyme plate
  • the detection amount calculation module the detection analysis module is used to convert the absorbance value OD into the detection amount ⁇ of the IgA complex according to the regression model;
  • the physical examination report generation module is used to provide the subject's IgA immune activity index, the standards for each interval corresponding to the index, and the classification corresponding to the interval within which the subject's index falls.
  • the physical examination report contains three parts.
  • the patient's IgA immune activity index value was 26.661 (the solid circle in the figure), which is greater than 18.27, so it is "abnormally high.”
  • the values of the IgA immune activity index of other different sample types are also listed as background reference (dashed circle in the figure), and are given different color marks according to the risk classification of the index: red represents abnormally high, and brown represents "basic range deviation”. High", green represents "normal basic range”. And marked the two thresholds for dividing risk groups, 15.18 and 18.27.
  • the second part presents the clinical evidence and references related to the IgA immune activity index so far.
  • the third part gives the standard curve shown in Figure 2 and the fitting results of the healthy human normal distribution shown in Figure 3.

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Abstract

提供一种计算IgA免疫活动性指数的方法及装置。方法包括以下步骤：步骤1：获取吸光值（S110）；步骤2：根据回归模型将吸光值转换为IgA复合物的检出量(S120)；步骤3：设定酶标板上检出量的板内锚定值，计算得到IgA复合物的板内归一化检出量(S130)；步骤4：设定若干酶标板之间的检出量的板间锚定值，计算板间归一化检出量(S140)。获取酶标板上各个孔的原始OD值，先进行孔层面的质量控制，拟合计算出孔中IgA复合物的检出量，再进行板层面的质量控制，将酶标板层面的数据归一化，去除酶标板的板间差，能够更进一步提高大批量检测准确性。

Description

计算样品的IgA免疫活动性指数的方法及装置 技术领域
本申请涉及免疫检测技术领域，尤其是涉及计算样品的IgA免疫活动性指数的方法及装置。
背景技术
在人体的自身免疫反应过程中，人体免疫球蛋白A(IgA)参与时，IgA与病原体结合启动免疫反应，下游抗体IgG与IgM、补体通路的C3补体等分子的参与形成IgA-IgG、IgA-IgM、IgA-C3、IgA-IgG-IgM等，统称IgA复合物。在正常免疫反应过程中，这些结合了病原体的复合物一般被免疫系统清除，比如被巨噬细胞吞噬等。但当IgA免疫反应异常时，IgA复合物无法被免疫系统有效清除，而是在血液循环系统及泌尿系统长期滞留，并导致两种疾病，具体而言：IgA复合物在肾脏的沉积与滞留将导致IgA肾病(IgA Nehphropathy，IgAN)；IgA复合物在血管的沉积将导致紫癜，通常称为过敏性紫癜(Henoch-Schonlein purpura，HSP)或者IgA血管炎(IgAV)。
现有方法中通常通过对血清学总IgA的免疫检测来进行诊断，总IgA检测为临床上为常规的免疫三项(IgA、IgM、IgG)或免疫五项(免疫三项+补体C3、C4)项目中的任一种。然而，总IgA升高的因素除了IgA肾病与紫癜之外，还可能是因为多发性骨髓瘤、类风湿性关节炎、血小板减少、感染性疾病等其它疾病，因此总IgA对IgA肾病与紫癜的特异性偏低。为此，申请人尝试从其原理出发，由于血清中循环IgA复合物是主要的诱导发病因素，因此直接检测IgA复合物的检出量作为IgA免疫活动性的指数来代替对免疫三项或免疫五项的检测。然而，这种方式虽然具有更好的灵敏度和特异性，但由于实验员的操作以及酶标板本身的板间差异性等问题，在面临大样本量的批量诊断时仍然不可避免地带来了一些问题，因此，有必要提供一种能够更进一步提高大批量检测准确性的IgA免疫活动性指数的计算方法。
发明内容
本申请旨在至少解决现有技术中存在的技术问题之一。为此，本申请提出一种能够更进一步提高大批量检测准确性的IgA免疫活动性指数的计算方法。
本申请的第一方面，提供一种计算样品的IgA免疫活动性指数的方法，该方法包括以下步骤：
获取第n个酶标板上各个孔的吸光值OD；
根据回归模型将吸光值OD转换为IgA复合物的检出量μ；
设定第n个酶标板上检出量的板内锚定值ω n，根据以下公式计算得到IgA复合物的板内归一化检出量μ n＝μ-ω n；
设定第1到N个酶标板的检出量的板间锚定值ω N，根据以下公式计算得到第n个酶标板上IgA复合物的板间归一化检出量μ F＝μ n+ω N，记为IgA免疫活动性指数；
其中，n≤N且均为正整数。
在本申请的一些实施方式中，样品包含待测品和阳性对照品，板内锚定值ω n为第n个酶标板中样品的检出量的中位数或第n个酶标板中阳性对照品的检出量。
在本申请的一些实施方式中，板间锚定值ω N为N个酶标板的板内锚定值ω n的中位数。
在本申请的一些实施方式中，回归模型为线性回归模型。
在本申请的一些实施方式中，根据以下公式将吸光值OD转换为IgA复合物的检出量μ：
Figure PCTCN2022127454-appb-000001
在本申请的一些实施方式中，每个样品设有若干重复孔，计算每个样品的若干重复孔的检出量μ的偏离系数CV，当偏离系数CV≥20％时，所述样品判定为无效样品。
在本申请的一些实施方式中，样品包括不同量的标准品、阴性对照品，酶标板满足以下至少一种条件下，重新获取各个孔的吸光值OD：
a)最大量标准品的吸光值<T 0；
b)回归模型的相关系数<R 0；
c)标准品的检出量偏离标定量>P 0％；
d)阴性对照品的检出量>Δ 0。
在本申请的一些实施方式中，T 0为0.6，R 0为0.9，P 0为30，Δ 0为0.1。
本申请的第二方面，提供一种判断IgA免疫活动性指数的方法，该方法包括以下步骤：
根据前述的方法计算受试者的IgA免疫活动性指数μ F；
根据受试者的IgA免疫活动性指数μ F执行以下判断：
当μ F<m+2σ，判断受试者的IgA免疫活动性指数为正常基础范围，
当m+2σ≤μ F<m+3σ，判断受试者的IgA免疫活动性指数为基础范围内偏高，
当μ F≥m+3σ时，判断受试者的IgA免疫活动性指数为异常范围内偏高；
其中，m为正常人IgA免疫活动性指数的平均值，σ为正常人IgA免疫活动性指数的标准差。
在本申请的一些实施方式中，该方法还包括根据判断IgA免疫活动性指数的结果生成体检报告。
在本申请的一些实施方式中，体检报告包括IgA免疫活动性指数及指数的区间标准和受试者对应的IgA免疫活动性指数数据。
在本申请的一些实施方式中，体检报告还包括检测过程中质控结果的有效性。
在本申请的一些实施方式中，体检报告还包括受试者的一般信息。
在本申请的一些实施方式中，体检报告还包括IgA免疫活动性指数的临床实验的一般性结果，供医生与病人参考。
在本申请的一些实施方式中，体检报告还包括健康人群的统计特性、标准曲线等，以供参考。
本申请的第三方面，提供计算机可读存储介质，该计算机可读存储介质存储有计算机可执行指令，计算机可执行指令用于使计算机执行前述的计算IgA免疫活动性指数的方法或判断IgA免疫活动性指数的方法中的步骤。
本申请的第四方面，提供设备，该设备包括处理器和存储器，存储器上存储有可在处理器上运行的计算机程序，处理器在运行计算机程序时实现前述的计算IgA免疫活动性指数的 方法或判断IgA免疫活动性指数的方法。
本申请的第五方面，提供系统，该系统包括：
采集模块，采集模块用于获取酶标板中各个孔的吸光值OD；
检出量计算模块，检测分析模块用于根据回归模型将吸光值OD转换为IgA复合物的检出量μ；
板内归一化检出量计算模块，板内归一化检出量计算模块用于根据以下公式计算得到IgA复合物的板内归一化检出量μ n＝μ-ω n，其中，ω n为板内锚定值；
板间归一化检出量计算模块，板间归一化检出量计算模块用于根据以下公式计算得到IgA复合物的板间归一化检出量μ F＝μ n+ω N，其中，ω N为板间锚定值。
在本申请的一些实施方式中，该系统还包括孔质控模块，孔质控模块用于计算每个样品的若干重复孔的检出量μ的偏离系数CV。当偏离系数CV≥20％时，该样品被判定为无效样品。
在本申请的一些实施方式中，该系统还包括板质控模块，板质控模块用于判断酶标板中以下的指标，当满足以下任一种条件时，该酶标板被判定为无效酶标板：
a)最大检出量标准品的吸光值<T 0；
b)回归模型的相关系数<R 0；
c)阳性对照品的检出量与标定量的偏离系数>P 0％；
d)阴性标准品的检出量>Δ 0；
在本申请的一些实施方式中，T 0为0.6，R 0为0.9，P 0为30，Δ 0为0.1。
在本申请的一些实施方式中，该系统还包括IgA免疫活动性指数分析模块，IgA免疫活动性指数分析模块用于分析IgA免疫活动性指数μ F与正常人IgA免疫活动性指数的平均值m的关系，
当μ F<m+2σ，判断所述受试者的IgA免疫活动性指数为正常基础范围，
当m+2σ≤μ F<m+3σ，判断所述受试者的IgA免疫活动性指数为基础范围内偏高，
当μ F≥m+3σ时，判断所述受试者的IgA免疫活动性指数为异常范围内偏高；
其中，σ为正常人IgA免疫活动性指数的标准差。
在本申请的一些实施方式中，该系统还包括体检报告生成模块，体检报告生成模块用于根据IgA免疫活动性指数与正常人IgA免疫活动性指数的平均值m的关系判断受试者的IgA免疫活动性指数。
在本申请的一些实施方式中，体检报告包括IgA免疫活动性指数及指数的区间标准和受试者对应的IgA免疫活动性指数数据。
在本申请的一些实施方式中，体检报告还包括检测过程中质控结果的有效性。
在本申请的一些实施方式中，体检报告还包括受试者的一般信息。
在本申请的一些实施方式中，体检报告还包括IgA免疫活动性指数的临床实验的一般性结果，供医生与病人参考。
在本申请的一些实施方式中，体检报告还包括健康人群的统计特性、标准曲线等，以供参考。
根据本申请实施例的计算IgA免疫活动性指数的方法，至少具有如下有益效果：
该方法从例如酶联免疫反应得到酶标板上各个孔的原始OD值，先进行孔层面的质量控制，通过标准曲线拟合得到从OD转换成样本定量的计算公式，计算出孔中IgA复合物的检出量，再进行板层面的质量控制，将酶标板层面的数据归一化，去除酶标板的板间差，最后针对样本重复进行计算以确定每个样本的最终定量结果。通过这种方式能够更进一步提高大批量检测准确性。
本申请的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本申请的实践了解到。
附图说明
图1是本申请的实施例所提供的一种计算IgA免疫活动性指数的流程图。
图2是本申请的实施例所提供的一种计算IgA免疫活动性指数的另一流程图。
图3是本申请的实施例中1采用不同的方式构建线性回归模型的结果，a为直接以OD值和检出量构建线性回归模型，b为取OD值和检出量的对数构建线性回归模型。
图4是本申请的实施例中2中33例健康人群的IgA免疫活动性指数的分布情况，a为其概率密度分布，横轴为IgA免疫活动性指数的值，纵轴为所占人数的比例；b为其Q-Q图；c为其累积分布图；d为其P-P图。
图5是本申请的实施例3所提供的体检报告的示意图。
具体实施方式
以下将结合实施例对本申请的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本申请的目的、特征和效果。显然，所描述的实施例只是本申请的一部分实施例，而不是全部实施例，基于本申请的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本申请保护的范围。
下面详细描述本申请的实施例，描述的实施例是示例性的，仅用于解释本申请，而不能理解为对本申请的限制。
在本申请的描述中，若干的含义是一个以上，多个的含义是两个以上，大于、小于、超过等理解为不包括本数，以上、以下、以内等理解为包括本数。如果有描述到第一、第二只是用于区分技术特征为目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量或者隐含指明所指示的技术特征的先后关系。如果在流程图中描述到了逻辑顺序，但是在某些情况下，可以以不同于流程图中的顺序执行所描述或示出的步骤。
本申请的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本申请的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
参考图1，示出了本申请的实施例中计算IgA免疫活动性指数的方法，该方法包括以下步骤：
S110、获取第n个酶标板上各个孔的吸光值OD。
其中，酶标板是指用于进行IgA复合物的酶联免疫吸附(ELISA)实验的酶标板，酶标板上设有若干孔，例如6孔、12孔、24孔、48孔、96孔等。在本申请实施例中，IgA复合 物通过结合孔内的探针而固定，随后结合酶标二抗进行显色。具体的，IgA复合物的探针为IgA的Fc受体蛋白，例如FCAR/CD89蛋白，其能够特异性结合IgA复合物。在ELISA过程中，通常需要设置标准品、对照品等其中至少一种。标准品是指含有已知检出量(标定的含量)的IgA复合物的样品，可以通过其给出的已知检出量的梯度，或自行通过稀释得到不同的已知检出量的梯度和对应吸光值OD的关系得到标准曲线。对照品一般包括阴性对照品和阳性对照品中的至少一种，阴性对照品中不含IgA复合物或其含量低于检出限，阳性对照品含有IgA复合物并且通过多种方法剔除了其中的干扰性物质，用于评价检测结果是否有效以及其稳定性和可比性，两者均为质控品。各个孔的吸光值OD是指与固定到孔内的IgA复合物结合的酶标二抗，催化酶反应底物所形成的有色产物的反应结果。可以理解的是，在检测过程中，通常每一个样品(包括标准品、待测品、对照品等)都会通过设置若干个重复(例如两到三个)。
S120、根据回归模型将吸光值OD转换为IgA复合物的检出量μ。
该步骤中，回归模型是指对吸光值OD和IgA复合物检出量μ之间统计关系的定量模型。具体的，回归模型通常采用线性回归模型对标准曲线进行拟合，例如直接以两者的值进行拟合：OD＝κ×μ+δ；或者将两者取对数后进行拟合：log 2(OD)＝κ×log 2(μ)+δ。在其中一些具体的实施方式中，两个线性回归模型比较后，第二个模型的相关系数R 2的值更接近于1，因而拟合优度更好。因而，根据第二个模型得到
Figure PCTCN2022127454-appb-000002
其中，δ和κ的具体取值根据标准品的标定量和对应的吸光值可以得到。根据这一模型，可以利用其余孔的吸光值OD计算得到其余孔中IgA复合物的检出量μ。其中，检出量以及下文中的最大量，可以是指质量，也可以是指浓度或者其它量度。
S130、设定第n个酶标板上检出量的板内锚定值ω n，根据下述公式计算得到IgA复合物的板内归一化检出量μ n＝μ-ω n。
在该步骤中，通过板内锚定值ω n的使用，将同一个板内不同孔中的IgA复合物的检出量归一化后变化为与板内锚定值ω n的差值，方便随后S140中将不同酶标板对齐到同一水平。在这之中，板内锚定值ω n可选任意的在不同板间具有相对稳定的值，例如，以阳性对照品的检出量作为板内锚定值ω n，或者以在标准曲线的有效梯度内的各个待测品检出量的中间值作为板内锚定值ω n。
S140、设定第1到N个酶标板的检出量的板间锚定值ω N，根据以下公式计算得到第n个酶标板上IgA复合物的板间归一化检出量μ F＝μ n+ω N，记为IgA免疫活动性指数。
在这一步骤，通过第1到N个酶标板的板间锚定值ω N的运用将归一化后变为差值的检出量重新上调至实际水平(n≤N且均为正整数)。为了实现这一目的，板间锚定值ω N优选可以采用N个酶标板各自对应的板内锚定值ω n的中位数，例如，对于ω 1、ω 2……ω k共k个板内锚定值取其中位数作为板间锚定值ω N。可以理解的是，ω N也可以采用符合这一构思的其它值作为板间锚定值ω N使用。虽然不同酶标板内所测的待测品的实际检出量存在一定差别，导致在归一化处理上存在一定困难，但不同板间待测品的中位数会随着板内待测品的数量增多而逐渐稳定下来，同样的，阳性对照品也具有相对稳定的IgA复合物的含量，因此，通过板内锚定值ω n和板间锚定值ω N的选择，设定出稳定的锚定点，把不同酶标板对齐到同一个水平，进行数据归一化，消除板间差。即，首先将酶标板的检出量归一化为接近0的水 平，然后为了接近实际的检出量，再把归一化为接近0的锚定点上调至实际水平。
参考图2，为了保证对样品的检测准确性，在进行S130和S140的归一化处理之前，还可以增加判别的步骤，由于酶标板上的部分孔或部分酶标板存在质量问题或检测过程中步骤失误等问题，使其检测出的结果无法使用而认定为无效进行排除。需要在后续重新进行测试。
为此，从两个方面进行质量控制，包括酶标板内在孔层面的质量控制和酶标板层面的质量控制。参考图2，酶标板内在孔层面的质量控制通过步骤S121实现，由于每个样品都会有若干个孔进行重复，因此，针对同一样品的不同重复孔的检出量进行分析，计算其偏离系数CV，当偏离系数高于20％时，这一样品的不同重复孔之间的误差过大，认定其为无效样本，在剔除所有的无效样本后进行下一步骤。具体的，偏离系数CV的计算公式CV＝标准差/平均数×100％，因此，根据这一公式和每个样品的所有重复孔的检出量μ的结果，计算每个样品的IgA复合物检出量的偏离系数，当偏离系数高于20％时，认定为无效样本。可以理解的是，为了追求更好的实验准确性，偏离系数的阈值可以为20％、19％、18％、17％、16％、15％、14％、13％、12％、11％、10％、8％、5％、3％等数值不等。其中，进行孔层面质量控制的样品可以是包括标准品和阳性对照品。当然，可以理解的是，待测品的检出量也可以通过这种方式进行质控。
在完成酶标板内的孔层面质量控制后，进一步需要在酶标板层面进行质量控制。酶标板层面的质量控制可以通过以下方式进行，包括进行以下判定：a)最大检出量标准品的吸光值<T 0；b)回归模型的相关系数<R 0；c)标准品的检出量偏离标定量>P 0％；d)阴性对照品的检出量>Δ 0。当满足以上任一种条件时，该酶标板将被认定为无效酶标板。具体的，例如T 0为0.6，R 0为0.9，P 0为30，Δ 0为0.1ng。
本申请实施例还提供一种判断IgA免疫活动性指数的方法，该方法包括以下步骤：
根据前述的计算IgA免疫活动性指数的方法计算受试者的IgA免疫活动性指数μ F；
根据受试者的IgA免疫活动性指数μ F执行以下判断：
当μ F<m+2σ，判断受试者的IgA免疫活动性指数为正常基础范围，
当m+2σ≤μ F<m+3σ，判断受试者的IgA免疫活动性指数为基础范围内偏高，
当μ F≥m+3σ时，判断受试者的IgA免疫活动性指数为异常范围内偏高；
其中，m为正常人IgA免疫活动性指数的平均值，σ为正常人IgA免疫活动性指数的标准差。
在得到每个具体受试者样品的IgA免疫活动性指数μ F后，可以通过与正常人的数值进行比较，从而预估该受试者的IgA免疫活动性指数所处的范围。进一步根据其IgA免疫活动性指数所处的范围直接或者与其它指标相结合，可以了解受试者是否患有IgA肾病或紫癜。上述判断过程中，m和σ可以由满足一定数量的正常人群检测后得到。其中正常人群的数量可以是20、30、50、100、200人以上，正常人群具体指的是通过各项标准检测确定没有患有IgA肾病与紫癜、或者其它可能影响该指标检测的疾病的人，或者是健康人群。可以理解的是，为了增强m和σ的代表性，正常人群最好包含从儿童到老年等各个年龄段的人，最好男性和女性各占一半或接近于1：1。m和σ的得出可以根据正常人群每个个体的IgA免疫活动性指数通过任何本领域所知晓的方法得到，例如通过直接的数学计算得到，或者通过 正态分布进行拟合而得到。当然，上述划分区间中，2σ和3σ的标准并非是唯一，可以根据实际情况进行适当的调整。
在得出判断的结果后，可以针对每个受试者生成对应的体检报告，该体检报告上除了IgA免疫活动性指数的相关信息外，还可以含有其它检测项目的相关结果，在此不再赘述。对于IgA免疫活动性指数的部分，体检报告中可以提供IgA免疫活动性指数、指数所对应的各个区间的标准，以及受试者的指数落入其中的区间所对应的分类，属于正常基础范围，或基础范围偏高，或异常偏高等。可以理解的是，在体检报告中还含有对检测过程中质控的结果，给出质控的有效性结果以证明此次检测有效。当然也包括受试者的一般信息，如姓名等。此外，还可以在体检报告中提供IgA免疫活动性指数的临床实验的一般性结果，或者正常人群的统计特性、标准曲线等以供参考。
本申请实施例还提供一种计算机可读存储介质，该计算机可读存储介质存储有计算机可执行指令，计算机可执行指令用于使计算机执行前述的计算IgA免疫活动性指数的方法或判断IgA免疫活动性指数的方法中的步骤。
本申请实施例还提供一种设备，该设备包括处理器和存储器，存储器上存储有可在处理器上运行的计算机程序，处理器在运行计算机程序时实现前述的计算IgA免疫活动性指数的方法或判断IgA免疫活动性指数的方法。
其中，存储器作为一种非暂态计算机可读存储介质，可用于存储非暂态软件程序以及非暂态性计算机可执行程序，如本申请实施方式中描述的判断IgA免疫活动性指数的过程。处理器通过运行存储在存储器中的非暂态软件程序以及指令，从而实现对受试者IgA免疫活动性指数的判断。
存储器可以包括存储程序区和存储数据区，其中，存储程序区可存储操作系统、至少一个功能所需要的应用程序；存储数据区可存储执行上述计算机程序。此外，存储器可以包括高速随机存取存储器，还可以包括非暂态存储器，比如至少一个磁盘存储器件、闪存器件、或其他非暂态固态存储器件。
在本申请的一些实施方式中，存储器可选包括相对于处理器远程设置的存储器，这些远程存储器可以通过网络连接至该处理器。上述网络的实例包括但不限于互联网、企业内部网、局域网、移动通信网及其组合。
实现上述判断所需的非暂态软件程序以及指令存储在存储器中，当被一个或者多个处理器执行时，执行上述上述判断。
本申请实施例还提供一种系统，该系统包括：
采集模块，采集模块用于获取酶标板中各个孔的吸光值OD；
检出量计算模块，检测分析模块用于根据回归模型将吸光值OD转换为IgA复合物的检出量μ；
板内归一化检出量计算模块，板内归一化检出量计算模块用于根据以下公式计算得到IgA复合物的板内归一化检出量μ n＝μ-ω n，其中，ω n为板内锚定值；
板间归一化检出量计算模块，板间归一化检出量计算模块用于根据以下公式计算得到IgA复合物的板间归一化检出量μ F＝μ n+ω N，其中，ω N为板间锚定值。
以上所描述的系统实施仅仅是示意性的，其中作为分离部件说明的单元可以是或者也可 以不是物理上分开的，即可以位于一个地方，或者也可以分布到多个网络单元上。可以根据实际的需要选择其中的部分或者全部模块来实现本实施例方案的目的。
可以理解的是，上文中所公开的全部或某些步骤、系统可以被实施为软件、固件、硬件及其适当的组合。某些物理组件或所有物理组件可以被实施为由处理器，如中央处理器、数字信号处理器或微处理器执行的软件，或者被实施为硬件，或者被实施为集成电路，如专用集成电路。这样的软件可以分布在计算机可读介质上，计算机可读介质可以包括计算机存储介质(或非暂时性介质)和通信介质(或暂时性介质)。可以理解的是，计算机存储介质包括在用于存储信息(诸如计算机可读指令、数据结构、程序模块或其他数据)的任何方法或技术中实施的易失性和非易失性、可移除和不可移除介质。计算机存储介质包括但不限于RAM、ROM、EEPROM、闪存或其他存储器技术、CD-ROM、数字多功能盘(DVD)或其他光盘存储、磁盒、磁带、磁盘存储或其他磁存储装置、或者可以用于存储期望的信息并且可以被计算机访问的任何其他的介质。
此外，可以理解的是，通信介质通常包含计算机可读指令、数据结构、程序模块或者诸如载波或其他传输机制之类的调制数据信号中的其他数据，并且可包括任何信息递送介质。
以下结合具体的实施例对本申请所提供的方法进行说明。
实施例1
以下计算和体检报告生成在开源开发环境RStudio Version 1.4.1717得以实现，其操作系统环境为macOS Big Sur Version 11.6.。
一、IgA复合物的检测
涉及一种检测IgA复合物的试剂盒，该试剂盒中包括分子探针冻干粉、包被缓冲液、酶标板、标准品、酶标二抗、显色液、终止液、洗涤液、稀释液和封闭液。
其中，分子探针冻干粉为重组FCAR蛋白冻干粉；
包被缓冲液为0.05M的碳酸盐缓冲液(pH＝9.6)；
酶标板为96孔板；
标准品为商品化的含IgA复合物的人血清IgA；
阴性对照品为无IgA复合物的人体血清；
阳性对照品为含IgA复合物的人体血清，选定25ng的商品化人血清IgA；
酶标二抗为辣根过氧化物酶(HRP)标记的小鼠抗人的IgA；
显色液包括显色液A—3,3’,5,5’-四甲基联苯胺(TMB)和显色液B—过氧化氢溶液；
终止液为10％的硫酸；
洗涤液为含0.05％吐温-20的0.15M的磷酸盐缓冲液；
稀释液为洗涤液+1‰BSA；
封闭液为洗涤液+5％BSA。
上述试剂盒的检测方法如下，具体流程参加图1：
(1)将分子探针冻干粉溶解于包被缓冲液中，得分子探针溶液；
(2)将分子探针溶液加入96孔酶标板的每个孔中，覆盖封口膜，置于4℃环境中过夜；甩去孔中的溶液，用洗涤液洗涤酶标板多次，向酶标板中加入封闭液，覆盖封口膜，置 于4℃环境中过夜，用洗涤液洗涤酶标板1次，干燥得包被有分子探针的酶标板；
(3)用稀释液稀释待测品得到待测品稀释液、标准品梯度稀释液(检出量依次为50、25、12.5、6.75、3.375、1.5625ng)，阳性对照品和阴性对照品不用稀释液稀释；然后将样本稀释液、标准品梯度稀释液、阴性对照液、阳性对照液分别加入包被有分子探针HQP001的酶标板中，覆盖封口膜进行孵育，孵育结束后用洗涤液洗酶标板3次；将HRP标记的二抗加入酶标板中孵育，孵育结束后洗涤酶标板3次；将显色液A和显色液B按1：1的比例混合加入酶标板中，室温避光显色，显色结束后加入终止液，轻轻震荡后将酶标板置于酶标仪中检测吸光值(OD值)，第一波长450nm，第二波长630nm。
表1和表2分别为酶标板的样本映射矩阵和根据酶标板的样本映射矩阵及检测结果得到OD值矩阵的局部。
表1.样本映射矩阵局部
STD1 STD1 B000001 B000001 B000001F B000001F
STD2 STD2 B000002 B000002 B000002F B000002F
STD3 STD3 B000003 B000003 B000003F B000003F
STD4 STD4 B000004 B000004 B000004F B000004F
STD5 STD5 B000005 B000005 B000005F B000005F
STD6 STD6 Q2 Q2 Q7 Q7
nCtrl nCtrl Q4 Q4 Q8 Q8
表2.OD值矩阵局部
1.863 2.123 0.626 0.709 0.691 0.601
1.44 1.767 1.05 1.081 1.015 0.849
0.989 0.946 1.272 1.319 1.229 1.132
0.563 0.61 0.988 1.035 1.006 0.992
0.331 0.346 0.991 0.954 1.009 0.861
0.173 0.16 0.84 0.891 0.499 0.521
0.020 0.011 0.448 0.531 0.626 0.653
如表1所示，其中STD1～6表示稀释后的不同检出量梯度的标准品，nCtrl为阴性对照品，B000001～5为待检测样本，尾端F表示该血浆样本是否冰冻(F：Frozen，为冰冻样本)，用于对比样本不同存储条件下的区别；Q2、Q4、Q7、Q8为健康人对照样本。根据上表，检测时，每个样品设置两个重复。
二、IgA免疫活动性指数的计算
根据按照试剂盒检测得到的含有标准品、对照品、待测品的OD值矩阵进行线性回归，以计算模型参数。分别使用原始的OD值及对数后的OD值分别进行线性回归建立模型，结 果如图3所示，其中，a为原始OD值的回归模型(OD＝0.0373×μ+0.331，rsq＝0.8908)，b为取对数后的OD值的回归模型(log 2(OD)＝0.7237×log 2(μ)-2.826，rsq＝0.9759)，该结果表明，b中的对数OD值模型更精确，因此，下面利用b中的对数OD值回归模型进行样本检出量的计算。对数线性模型中参数δ＝-2.826、κ＝0.7237，因此样本检出量计算公式为
Figure PCTCN2022127454-appb-000003
Figure PCTCN2022127454-appb-000004
根据此公式计算得到的样本的检出量见表三。
表3.检出量矩阵局部
35.39 42.39 7.84 9.31 8.99 7.41
24.79 32.9 16.02 16.68 15.29 11.95
14.75 13.87 20.89 21.96 19.92 17.78
6.77 7.57 14.73 15.71 15.1 14.81
3.25 3.46 14.79 14.04 15.17 12.18
1.33 1.19 11.77 12.77 5.73 6.08
0.07 0.03 4.94 6.25 7.84 8.31
对上述样本中标准品和待测品的各自重复进行统计，得到样本偏差，结果见表4。
表4.标准品的偏离系数
样品 平均值 标准差 偏离系数(％)
STD1 38.89 4.95 13
STD2 28.84 5.73 20
STD3 14.31 0.62 4
STD4 7.17 0.56 8
STD5 3.35 0.15 4
STD6 1.26 0.1 8
B000001 8.58 1.04 12
B000001F 8.2 1.11 14
B000002 16.35 0.46 3
B000002F 13.62 2.36 17
B000003 21.42 0.76 4
B000003F 18.85 1.51 8
B000004 15.22 0.69 5
B000004F 14.96 0.2 1
B000005 14.41 0.54 4
B000005F 13.67 2.11 15
Q2 12.27 0.71 6
Q4 5.59 0.92 17
Q7 5.91 0.25 4
Q8 8.08 0.33 4
从表4中的结果可以看出，标准品、对照品和待测品的偏离系数均小于20％，在孔层面上符合质控要求，均为有效。同理，对该酶标板的其它孔和其它17个酶标板检测均符合酶标板内孔层面的质控要求。另外，最大检出量的标准品STD1的吸光值大于0.6，回归模型的系数大于0.9，标准品的检出量偏离其标定量的值不超过标定量的30％，阴性对照品的检出量小于0.1，因此，符合酶标板层面的质量控制要求。
该酶标板的板内锚定值取标准曲线检出值(去除高于20ng的前两个梯度)的中间值ω n＝(6.77+3.46)/2＝5.11。检测实验中共有18个酶标板，这些酶标板按照相同方法得到的板内锚定值分别为4.93、5.28、3、3.34、5.07、5.11、4.66、6.21、4.23、4.94、5.07、5.14、5.23、5.3、3.85、4.85、4.97、4.74，因此酶标板的板间锚定值ω N为18个板内锚定值的中间值，为4.96。根据ω n和ω N的结果和μ F＝μ-ω n+ω N计算得到各个样本最终的IgA免疫活动性指数，如表5所示。
表5.各个样本的IgA免疫活动性指数
样品 IgA免疫活动性指数
B000001 8.42
B000001F 8.04
B000002 16.19
B000002F 13.46
B000003 21.26
B000003F 18.69
B000004 15.06
B000004F 14.80
B000005 14.25
B000005F 13.51
Q2 12.11
Q4 5.43
Q7 5.75
Q8 7.92
Q2～Q4的结果即为对应的受试者的IgA免疫活动性指数。
实施例2：IgA免疫活动性指数风险区间阈值定义
参考实施例1，在其它的18个酶标板中的一个有20例健康儿童及13例健康成年人的待测样本，计算出这些健康人群各自的IgA免疫活动性指数后，再次计算这33个数值的平均值与标准差，平均值m＝9.0，标准差σ＝3.09。
因此，对IgA免疫活动性指数检出值μ的区间定义如下：
正常基础范围μ<m+2σ，即μ<15.18；
基础范围偏高m+2σ≤μ<m+3σ，即15.18≤μ<18.27；
异常偏高μ F≥m+3σ，即μ F≥18.27。
参考图4，33例正常人的IgA免疫活动性指数符合正态分布。正态分布的特征为97.72％的人满足μ<m+2σ，99.87％的人满足μ<m+2σ。
实施例3：IgA免疫活动性指数体检报告
本实施例提供一种系统，这一系统包括：
采集模块，采集模块用于获取酶标板中各个孔的吸光值OD；
检出量计算模块，检测分析模块用于根据回归模型将吸光值OD转换为IgA复合物的检出量μ；
板内归一化检出量计算模块，板内归一化检出量计算模块用于根据以下公式计算得到IgA复合物的板内归一化检出量μ n＝μ-ω n，其中，ω n为板内锚定值；
板间归一化检出量计算模块，板间归一化检出量计算模块用于根据以下公式计算得到IgA复合物的板间归一化检出量μ F＝μ n+ω N，其中，ω N为板间锚定值；
体检报告生成模块，体检报告生成模块用于提供受试者的IgA免疫活动性指数、指数所对应的各个区间的标准，以及受试者的指数落入其中的区间所对应的分类。
体检报告包含三个部分，第一部分参考图5，报告中病人检出IgA免疫活动性指数值为26.661(图中实心圆)，大于18.27，因此为“异常高”。同时，还列出了其它不同样本类型的IgA免疫活动性指数的值作为背景参考(图中虚圈)，并且按照指数的风险分类给予不同颜色标记：红色代表异常高，棕色代表“基础范围偏高”，绿色则代表“正常基础范围”。并且标记出划分风险人群的两个阈值，15.18与18.27。第二部分给出目前为止的IgA免疫活动性指数的相关临床证据及参考文献。第三部分给出图2所示的标准曲线以及图3中所示的健康人正态分布的拟合结果。
上面结合实施例对本申请作了详细说明，但是本申请不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本申请宗旨的前提下作出各种变化。此外，在不冲突的情况下，本申请的实施例及实施例中的特征可以相互组合。

Claims (10)

  1. 计算样品的IgA免疫活动性指数的方法，其特征在于，包括以下步骤：
    获取第n个酶标板上孔的吸光值OD；
    根据回归模型将所述吸光值OD转换为IgA复合物的检出量μ；
    设定第n个酶标板上检出量的板内锚定值ω n，根据以下公式计算得到IgA复合物的板内归一化检出量μ n＝μ-ω n；
    设定第1到N个酶标板的检出量的板间锚定值ω N，根据以下公式计算得到第n个酶标板上IgA复合物的板间归一化检出量μ F＝μ n+ω N，记为IgA免疫活动性指数；
    其中，n≤N且均为正整数。
  2. 根据权利要求1所述的方法，其特征在于，所述样品包含待测品和阳性对照品，所述板内锚定值ω n为第n个酶标板中待测品的检出量的中位数或第n个酶标板中阳性对照品的检出量。
  3. 根据权利要求2所述的方法，其特征在于，所述板间锚定值ω N为N个酶标板的板内锚定值ω n的中位数。
  4. 根据权利要求1所述的方法，其特征在于，回归模型为线性回归模型；
    优选地，根据以下公式将所述吸光值OD转换为IgA复合物的检出量μ：
    Figure PCTCN2022127454-appb-100001
    Figure PCTCN2022127454-appb-100002
  5. 根据权利要求1至4任一项所述的方法，其特征在于，每个样品设有若干重复孔，计算每个样品的若干重复孔的检出量μ的偏离系数CV，当偏离系数CV≥20％时，所述样品判定为无效样品。
  6. 根据权利要求1至4任一项所述的方法，其特征在于，所述样品包括不同量的标准品、阴性对照品，酶标板满足以下任一种条件时，重新获取各个孔的吸光值OD：
    a)最大量标准品的吸光值<T 0；
    b)回归模型的相关系数<R 0；
    c)标准品的检出量偏离标定量>P 0％；
    d)阴性对照品的检出量>Δ 0；
    优选地，T 0为0.6，R 0为0.9，P 0为30，Δ 0为0.1。
  7. 判断IgA免疫活动性指数的方法，其特征在于，包括以下步骤：
    根据权利要求1至6任一项所述的方法计算受试者的IgA免疫活动性指数μ F；
    根据受试者的IgA免疫活动性指数μ F执行以下判断：
    当μ F<m+2σ，判断所述受试者的IgA免疫活动性指数为正常基础范围，
    当m+2σ≤μ F<m+3σ，判断所述受试者的IgA免疫活动性指数为基础范围内偏高，
    当μ F≥m+3σ时，判断所述受试者的IgA免疫活动性指数为异常范围内偏高；
    其中，m为正常人IgA免疫活动性指数的平均值，σ为正常人IgA免疫活动性指数的标准差。
  8. 计算机可读存储介质，其特征在于，所述计算机可读存储介质存储有计算机可执行指令，所述计算机可执行指令用于使计算机执行权利要求1至7任一项所述的方法中的步骤。
  9. 设备，其特征在于，包括处理器和存储器，所述存储器上存储有可在所述处理器上运行的计算机程序，所述处理器在运行所述计算机程序时实现权利要求1至7任一项所述的方法。
  10. 系统，其特征在于，包括：
    采集模块，所述采集模块用于获取酶标板中各个孔的吸光值OD；
    检出量计算模块，所述检测分析模块用于根据回归模型将所述吸光值OD转换为IgA复合物的检出量μ；
    板内归一化检出量计算模块，所述板内归一化检出量计算模块用于根据以下公式计算得到IgA复合物的板内归一化检出量μ n＝μ-ω n，其中，ω n为板内锚定值；
    板间归一化检出量计算模块，所述板间归一化检出量计算模块用于根据以下公式计算得到IgA复合物的板间归一化检出量μ F＝μ n+ω N，其中，ω N为板间锚定值。
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