CN108427003B - Polypeptide sequence, kit and method for detecting natural antibody of anti-interleukin 2receptor subunit A - Google Patents
Polypeptide sequence, kit and method for detecting natural antibody of anti-interleukin 2receptor subunit A Download PDFInfo
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- 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
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Abstract
The invention relates to an antigen polypeptide for detecting an Anti-interleukin-2receptor subunit A (AIL2RA) natural antibody, a kit containing the antigen polypeptide, a method for detecting the concentration of an AIL2RA natural antibody in human plasma by using the antigen polypeptide or the kit, and application of the antigen polypeptide or the kit in detecting the human plasma AIL2RA natural antibody. The invention utilizes 3 linear antigen polypeptides which can be completely complementary with target AIL2RA natural antibodies to respectively realize qualitative and quantitative detection of AIL2RA natural antibodies in plasma, and can be used for distinguishing plasma (positive) rich in AIL2RA natural antibodies from plasma (negative) free of AIL2RA natural antibodies.
Description
Technical Field
The invention belongs to the technical field of immunology, and relates to 3 polypeptide antigens with amino acid sequences, which can be applied to preparation of an enzyme-linked immunosorbent assay (ELISA) antibody detection kit for detecting the concentration of natural antibodies against interleukin 2receptor subunit A (Anti-interleukin-2receptor subunit alpha, AIL2RA) in plasma.
Background
Malignant tumors are one of the most major killers of health in humans. Based on the continuous existence of environment pollution conditions such as dust, haze and the like, the incidence rate of main cancers of Chinese people is on the trend of rising year by year. According to statistics, the average annual incidence rate of the cancer in China currently reaches more than 3/1000. Early tumor treatment methods, such as surgical resection or radiotherapy, have been developed and are mature, and can significantly prolong the survival time of patients. However, how to predict the risk of tumor onset and diagnose the tumor at an early stage is an important way for effectively preventing and treating the tumor.
Previous studies have shown that high concentrations of autoantibodies against tumor associated antigens can be present in the blood of patients 3-5 years before malignant tumor volumes have progressed to the point where they can be detected using modern imaging techniques. Therefore, the detection of the autoantibody to the tumor-associated antigen in blood has important values in predicting the risk of tumor onset and early diagnosis of tumor. In foreign countries, diagnostic kits for early diagnosis of lung cancer and breast cancer are commercially available. For example, Early CDT, introduced by Oncommune Inc., of nottingham, UKTMThe Lung diagnostic kit has been used in North America and Europe for the early clinical diagnosis of Lung cancer for nearly 8 years. However, the currently reported antibody detection methods have relatively low sensitivity, poor specificity and false negative rate as high as more than 60%. The main reason is that the positive detection rate of each tumor-associated antigen autoantibody in the blood of a patient is about 10% on average, and even if a plurality of antigens are mixed, the positive detection rate is difficult to break through 50%.
In recent years, natural antibody research reports show that more than 50% of antibodies in human blood belong to natural antibodies, are mainly produced by B1 lymphocytes, and do not need specific antigen stimulation. Natural antibodies are involved in various physiological regulation and immune function stabilization of the body, and serve as a bridge between innate and specific immune systems. It is noted that some natural antibodies have immune surveillance function, and eliminate malignant cells formed in vivo at any time to maintain homeostasis. Therefore, natural antibodies that maintain a certain level may play a role in preventing cancer. It is speculated that the risk of developing tumors in natural antibody deficient or negative patients may be significantly higher than in normal populations. Numerous studies have demonstrated that Regulatory T cells (Treg cells) are closely associated with tumor immune escape mechanisms. Treg cell proportion is increased in peripheral blood lymphocytes and tumor infiltrating lymphocytes of patients with lung cancer, breast cancer, ovarian cancer and melanoma. Treg cells by direct contact with the cells or by release of cytokinesInhibitory Effect of CD4+And CD8+T cell activation and proliferation play a role. For example, Treg cells suppress tumor-specific antigen activated CD4+The effector cells secrete IL-2 and can limit the release of CD8+ T cytotoxic particles in the tumor microenvironment, allowing tumor cells to escape the killing of the immune system.
Interleukin 2receptor subunit A (IL 2RA, Interleukin-2receptor subunit alpha), also known as CD25, is a major surface marker for Treg cells. The Treg cells are thymus-derived CD4+T cells, whereas CD25 is an important marker for Treg cell activation. CD25, a type I transmembrane protein, also plays a key role in a variety of T cell and B cell activation. CD25, CD122(IL2RB) and CD132(IL2RG) together constitute a high affinity IL 2receptor, and their soluble receptor (sIL 2R) is associated with various malignant tumorigenesis and has become a biological marker of the tumorigenesis and progression. Domestic and foreign studies show that the Treg cells play an important role in maintaining tumor immune tolerance by inhibiting the generation of activated T cells to limit immune response. Animal experiments prove that the anti-CD 25 monoclonal antibody can inhibit the activity of Treg cells in a tumor-bearing mouse body and improve the anti-tumor immune function of the mouse. In vitro experiments prove that the activity of cytotoxic cells (including CTL, LAK/NK cells and the like) can be enhanced by removing Treg cells in peripheral blood. Recent studies have shown that patients with brain gliomas develop CD4 as the disease progresses+/FOXP3+Positive Treg cells exhibited a time-dependent increase with high expression of CD 25. The application of the monoclonal antibody for resisting CD25 can obviously inhibit CD4+/FoxP3+Proliferation of positive Treg cells.
Disclosure of Invention
The long-term research of the inventor finds that various tumor-associated antibodies, such as natural anti-interleukin 2receptor A (AIL2RA) antibody associated with lung cancer, are naturally formed in healthy human bodies. Therefore, the main object of the present invention is to provide a group of linear antigen polypeptides that can be used for detecting the concentration of the natural antibody of AIL2RA in human plasma respectively, and a kit prepared therefrom, and a method for detecting the natural antibody of AIL2RA in human plasma by using the antigen polypeptides or the kit, and applications thereof. The antigenic polypeptide is derived from human interleukin 2receptor A protein. Interleukin 2receptor A is Interleukin 2receptor subunit A (IL 2RA), also known as CD25, the sequence of human Interleukin 2receptor subunit A is shown in Table 1. As shown in Table 1, the antigenic polypeptide is derived from the amino acid sequence of the italic region in the sequence structure of human IL2RA protein. These epitopes are capable of specifically binding to the natural antibody of AIL2RA in human plasma.
TABLE 1 human interleukin 2receptor subunit A protein sequences
The terms "natural antibody against interleukin 2receptor A" or "natural antibody of AIL2 RA" as defined herein are used interchangeably and refer to an antibody or mixture of antibodies naturally present in a healthy human that may be involved in the development and progression of a variety of malignancies, including lung cancer. The detection of the natural antibody of AIL2RA has the value of prompting the risk of tumor onset, and is favorable for further developing the technical means for early diagnosis and tumor treatment. In a particular embodiment of the invention, the AIL2RA natural antibody is a natural antibody or a mixture of natural antibodies that recognize any one or more of the epitope sequences of SEQ ID NOS: 1-3.
The inventor analyzes the epitope of Human leukocyte antigen II (HLA-II) on the IL2RA protein sequence by using an immune informatics method and an epitope mapping technology, screens an amino acid sequence with high affinity, and further designs HLA-II restricted epitopes and linear antigen polypeptides which can be recognized by most Human antigen presenting cells.
It is well recognized that binding of antigen to antibody occurs primarily between antigenic determinants and the antibody binding site. Therefore, the closer the two are to complete complementarity in spatial structure and configuration, the more stable the binding of antigen-antibody, the stronger the specificity, and the higher the binding efficiency, and therefore, the target antibody (antibody in the sample to be detected) and its binding site structure are the prerequisite factors, and the antigenic determinant can represent the binding state and affinity property of the whole protein antigen and antibody.
According to the biological characteristics of the IL2RA protein, the invention carries out immune informatics prediction and simulation aiming at a plurality of epitopes, and designs 3 linear antigen polypeptides which are completely complementary with a target antibody in human plasma in space structure and configuration respectively through analyzing various parameters related to antigenicity, and the amino acid sequences of the linear antigen polypeptides are shown in Table 2.
TABLE 2 detection of the Linear antigen polypeptide sequence of the natural antibody of AIL2RA in human plasma
The 3 antigen polypeptides are synthesized by a solid phase chemical method and used for preparing an ELISA antibody detection kit, and the concentration of the natural antibody of AIL2RA in the plasma of a healthy individual is detected according to a set process specification. The antigen polypeptide can be prepared into a simple and easy-to-use convenient kit in practical application, is packaged in a vacuum sealing way by non-metallic materials such as glass, medical plastic and the like, and can be stored for more than 6 months at the temperature of 4 ℃. Briefly, any one, any two or three of the 3 antigenic polypeptide species were coated on a Maleimide (Maleimide) activated 96-well microtiter plate. Drying in 40-45 deg.C (40-45 deg.C) oven, vacuum sealing and packaging with nonmetal packaging material, and making into kit. Preferably each antigenic polypeptide is a preparation with a purity > 90%.
Thus, according to one aspect of the present invention, there is provided a detection reagent for detecting a native antibody to AIL2RA, comprising any one, any two or all three antigenic polypeptides selected from the group consisting of the following antigenic polypeptides:
H-RGPAESVCKMTHGKTRWTQPQLICTGEMEH-OH(SEQ ID NO:1)；
H-TGEMETSQFPGEEKPQASPEGRPESETSCH-OH (SEQ ID NO: 2); and
H-TSCLVTTTDFQIQTEMAATMETSIFTTEYQH-OH(SEQ ID NO:3)。
in some embodiments, the detection reagent consists of any one, any two, or all three of the three antigenic polypeptides.
In some embodiments, the 3 antigenic polypeptides are highly pure preparations, preferably chemically synthesized with a purity > 90%.
In some embodiments, when the detection reagent comprises two or three antigenic polypeptides, the two or three antigenic polypeptides are used separately.
In some embodiments, when the detection reagent comprises two or three antigenic polypeptides, the two or three antigenic polypeptides are used in admixture. When used in combination, the two or three antigenic polypeptides may be combined in any weight ratio. In a preferred embodiment, the two or three antigenic polypeptides are mixed in equal weight proportions.
In some embodiments, when the detection reagent comprises three antigenic polypeptides, one antigenic polypeptide of the three antigenic polypeptides is used alone and the other two antigenic polypeptides are used in admixture. The antigenic polypeptide used alone may be any of the three antigenic polypeptides. In some embodiments, the antigenic polypeptides used in combination may be combined in any weight ratio. In a preferred embodiment, the antigenic polypeptides used in admixture are mixed in equal weight proportions.
Thus, in some embodiments, when the detection reagent comprises two or three antigenic polypeptides, the two or three antigenic polypeptides are each present alone.
In other embodiments, when the detection reagent comprises two or three antigenic polypeptides, the two or three antigenic polypeptides are mixed together. In other embodiments, when the detection reagent comprises three antigenic polypeptides, one antigenic polypeptide of the three antigenic polypeptides is present alone and the other two antigenic polypeptides are mixed together. In some embodiments, the antigenic polypeptides mixed together may be mixed in any weight ratio. In a preferred embodiment, the antigenic polypeptides mixed together are mixed in equal weight proportions.
According to another aspect of the present invention, there is provided a kit comprising the above-mentioned detection reagent.
In some embodiments, the kit includes a microplate, the detection reagents being coated within the wells of the microplate.
In some embodiments, in the kit, the microplate coated with the detection reagent is dried and then vacuum-sealed with a non-metallic medical packaging material. In some embodiments, the microplate is a Maleimide (Maleimide) activated 96-well microplate.
In other preferred embodiments, the non-metallic medical packaging material is glass or medical plastic.
In other embodiments, the kit further comprises a positive control and/or a negative control. In some embodiments, the positive control and/or the negative control are coated on a microtiter plate.
In some embodiments, the three antigenic polypeptides are arranged as shown in figure 1 on a 96 well microplate.
According to another aspect of the present invention, there is provided a method for detecting the native antibody to AIL2RA in a sample using the above-described detection reagent or the above-described kit, preferably the method is an in vitro, non-diagnostic detection technique.
In some embodiments, the method comprises detecting the concentration of the native antibody of AIL2RA in the test sample by an antigen-antibody binding reaction using the detection reagent described above.
In a preferred embodiment, the "detection of the concentration of the natural antibody of AIL2RA in the sample to be tested by antigen-antibody binding reaction" is carried out by an enzyme-linked immunosorbent assay (ELISA) method.
In a more preferred embodiment, the enzyme-linked immunosorbent assay is a sandwich ELISA.
In some embodiments, the method is performed by: (1) performing a step-by-step loading analysis using a microplate coated with the detection reagent and detecting an Optical Density (OD) value in each well; (2) antibody levels were expressed as Positive Sample Ratios (PSR).
In some embodiments, the microplate in the above step is a Maleimide (Maleimide) -activated 96-well microplate. In some embodiments, the step of detecting the optical density value in each well is performed using a microplate reader. .
In a more preferred embodiment, the step (1) comprises setting a sample to be tested in duplicate wells, setting 2 negative control wells and 2 positive control wells at the same time, diluting plasma with an analysis solution, diluting horseradish peroxidase-labeled goat anti-human IgG antibody, washing the plate, adding 100. mu.l of a mixture of 3,3',5,5' -Tetramethylbenzidine (TMB) and peroxidase to each well, keeping the plate away from light at room temperature for 20-30 minutes, and adding 50. mu.l of a 10% sulfuric acid solution (12% H) to each well2SO4) Then, the Optical Density (OD) value was measured with a microplate reader, the detection wavelength was 450nm, and the reference wavelength was 630 nm.
In a more preferred embodiment, the sample is human plasma, more preferably single individual plasma.
In a more preferred embodiment, the individual's plasma is plasma from a single healthy individual.
In a more preferred embodiment, the specific operation steps of the step (1) are as follows:
1. before the operation, the antigen polypeptide used was dissolved in 67% acetic acid to give a 5 mg/ml (mg/ml) stock solution, which was then stored in a refrigerator at-20 ℃ (within ± 2 ℃).
2. When the operation is started, the antigen polypeptide is diluted into 10-50 micrograms/milliliter (mu g/ml) of working solution by using a coating solution, wherein the coating solution is 0.1M phosphate buffer solution containing 0.15M sodium chloride and 10mM EDTA, and the pH value is 7.0-7.4.
3. A96-well microplate (Thermo Scientific, USA) activated by Maleimide (Maleimide) is coated with a working solution, and after overnight incubation at 4 ℃, the plate is washed 3 times with a washing solution, wherein the washing solution is 0.1M phosphate buffer solution containing 0.15M sodium chloride and 0.1% TWEEN-20, and the pH value is 7.0-7.4.
4. Then the sample is loaded and analyzed step by step according to the following steps:
a) the plasma samples to be tested were prepared in duplicate wells, 2 negative control wells (NC, reference is a negative control solution without AIL2RA natural antibody, such as bovine serum albumin (Sigma-Aldrich Co., Ltd.), which can reflect the experimental index of the antigen polypeptide used in the AIL2RA natural antibody negative reaction system) and 2 positive control wells (PC, reference is a mixture of anti-human IL2RA antibodies, which can reflect the experimental index of the antigen polypeptide used in the AIL2RA natural antibody positive reaction system).
b) Diluting a blood plasma sample to be detected by 1:200 by using an analysis solution, wherein the analysis solution is the same as an antigen coating solution, namely 0.1M phosphate buffer solution containing 0.15M sodium chloride and 10mM EDTA, the pH is 7.0-7.4, 100 mu l of the analysis solution is added into each hole, incubation is carried out for 1-2 hours at 25 ℃, and then the plate is washed for 3 times.
c) And (3) diluting the horseradish peroxidase-labeled goat anti-human IgG antibody (used for verifying whether a substance to be detected in the plasma is a specific antibody) by using an analysis solution (namely 0.1M phosphate buffer solution containing 0.15M sodium chloride and 10mM EDTA and having the pH value of 7.0-7.4), wherein the dilution ratio of the antibody is 1: 10000-1: 50000, 100 mu l of the antibody is added into each hole, and incubating for 1-2 hours at 25 ℃.
d) After washing the plate 3 times with a washing solution (i.e., 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, pH 7.0-7.4), 100. mu.l of a mixture of 3,3',5,5' -Tetramethylbenzidine (TMB) and peroxidase was added to each well, and the plate was protected from light at room temperature for 20-30 minutes.
e) Add 50. mu.l of stop solution 10% sulfuric acid solution (10% H) to each well2SO4) Then, the Optical Density (OD) value was measured with a microplate reader, the detection wavelength was 450nm, and the reference wavelength was 630 nm. The detection process was completed within 10 minutes after the addition of the stop solution, thereby quantitatively analyzing the level of the natural antibody of AIL2RA in the plasma of the subject.
In the case of random population sampling, the data obtained from each individual assay can be analyzed to determine the relative levels of the native antibodies to AIL2RA in plasma using the Positive Sample Ratio (PSR).
In some embodiments, the Positive Sample Ratio (PSR) in step (2) above is calculated as follows:
PSR ═ OD value-OD of sample to be measuredNCValue of]/[ Positive standard OD value-ODNCValue of]NC is a negative control for each sample.
In another aspect of the present invention, there is provided the use of the above-mentioned detection reagent, the above-mentioned kit or the above-mentioned method for detecting the native antibody of AIL2RA in a sample. In some embodiments, the use is for in vitro, non-diagnostic purposes.
In another aspect of the invention, the application of the detection reagent in preparing a reagent for detecting the AIL2RA natural antibody in a sample is also provided.
In a preferred embodiment, the sample is human plasma, more preferably single individual plasma.
In a more preferred embodiment, the individual's plasma is plasma from a single healthy individual.
Based on the scheme, the invention provides the AIL2RA natural antibody detection technology with high precision, simple operation and moderate cost, and further provides a semi-quantitative (or relative quantitative) analysis and application scheme for the AIL2RA natural antibody on the basis, thereby laying an important foundation for developing brand-new early diagnosis tumor and early implementation treatment strategies based on the healthy human plasma AIL2RA natural antibody.
The invention provides a simple and convenient human plasma AIL2RA natural antibody detection means, which can be used for auxiliary qualitative and quantitative detection of the level of AIL2RA natural antibodies and auxiliary quantitative determination of the level of plasma AIL2RA natural antibodies of different individuals. The level of the natural antibody of the AIL2RA is reduced or negative individuals are likely to have higher tumor onset risk, and the method has important values of predicting the tumor onset risk and diagnosing the tumor at an early stage. The plasma AIL2RA natural antibody negative individual detected by the product of the invention has high risk of tumor onset, can be clinically followed up and traced, and is suitable for further developing technical means for early tumor discovery and treatment implementation.
The AIL2RA natural antibody method can be used for tumor biology research, and discussing tumor occurrence mechanism, tumor immunity escape mechanism and immunity monitoring mechanism.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The primary objects and other advantages of the invention may be realized and attained by the structure particularly pointed out in the written description and claims hereof.
Drawings
FIG. 1 shows the arrangement of three IL2RA antigen polypeptides on a 96-well plate. Wherein NC represents a negative control well; PG represents a positive sample well; sn is a sample to be detected. The 3 polypeptide antigen sequences of the coating are SEQ ID NO. 1(Ag1), SEQ ID NO. 2(Ag2) and SEQ ID NO. 3(Ag 3).
FIG. 2 is a ROC plot of the IgG levels of the natural antibody of AIL2RA in plasma from lung cancer patients (SEQ ID NO:2 antigen polypeptide sequence).
Examples
1. Sample collection
23 plasma samples of early stage non-small cell lung cancer (squamous lung carcinoma of stage I and glandular epithelial lung carcinoma) patients before surgical treatment and 199 healthy human plasma samples were collected for detection of plasma AIL2RA natural antibody. All plasma samples were stored at minus 80 degrees (-80 ℃) prior to manipulation, and verified to have a storage time of no more than two years, and no repeated freeze-thawing (no more than 3 times).
2. Sample detection
Plasma samples were thawed at 4 ℃ and 3 polypeptide antigens (see table 2) used in this experiment were synthesized by chemical solid phase synthesis with a purity of 95%, and specifically the following steps were performed:
(1) prior to the procedure, each antigen was dissolved in 67% acetic acid to a 5.7mg/ml stock solution, then mixed in equal volumes and stored in a-20 ℃ freezer.
(2) At the beginning of the procedure, 3 antigens were first diluted to 30. mu.g/ml with a coating solution of 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, respectively, and the pH (pH) was found to be 7.2.
(3) The 3 antigenic polypeptides were then coated onto a Maleimide (Maleimide) activated 96-well assay plate (Thermo Scientific, USA), one antigenic polypeptide per well, in the arrangement shown in FIG. 1, and after incubation overnight for 15 hours at 4 ℃ the plate was washed 3 times with a wash solution of 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, and the pH was measured to be 7.2.
(4) The fractional loading analysis was then as follows:
a) for each antigen test, duplicate wells were set for each plasma sample, and 2 negative control wells (NC, bovine serum albumin as a reference, supplied by Sigma-Aldrich) and 2 positive control wells (PC, a mixture of anti-human AIL2RA as a reference, supplied by Sigma-Aldrich) were set.
b) Plasma was diluted 1:200 with the same assay as the antigen coating, 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, and the pH was 7.2, 100. mu.l was added per well, and incubated at 25 ℃ for 1.5 hours.
c) After washing the plate 3 times with the aforementioned wash solution (i.e., 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, pH 7.2 measured), horseradish peroxidase-labeled goat anti-human IgG (supplied by Sigma-Aldrich) was diluted with the assay solution at an antibody working concentration of 1:30000, 100. mu.l per well and incubated at 25 ℃ for 1.5 hours.
d) After washing the plate 3 times with the aforementioned wash solution (i.e., 0.1M phosphate buffer containing 0.15M NaCl and 0.1% TWEEN-20, pH 7.2), 100. mu.l of a mixture of 3,3',5,5' -Tetramethylbenzidine (TMB) and peroxidase (supplied by Life Technologies) was added to each well, and incubated at room temperature in the dark for 25 minutes.
e) Add 50. mu.l stop solution 12% sulfuric acid solution (12% H) per well2SO4) And then detecting the Optical Density (OD) value by using an enzyme-labeling instrument, wherein the detection wavelength is 450nm, the reference wavelength is 630nm, the detection is finished within 10 minutes after the stop solution is added, and the subsequent steps are to perform quantitative comparative analysis on the AIL2RA natural IgG antibody for each individual according to the result.
3. Results of the experiment
When analyzing the data obtained by the above detection, Positive Sample Ratio (PSR) is used to determine the level of AIL2RA natural IgG in plasma, and the PSR calculation formula is: PSR ═ OD value-OD of sample to be measuredNCValue of]/[ Positive standard OD value-ODNCValue of]NC is a negative control for each sample. The PSR mean and standard deviation are used to represent the antibody concentration in plasma. In contrast to previous studies of the same type, the threshold for determining positive samples for the level of native IgG in plasma AIL2RA was defined as the mean value of PSR minus two standard deviations for the control group, and IgG antibody levels below this threshold were defined as positive samples. In other words, plasma with negative native levels of AIL2RA IgG was defined as a positive sample.
The level of native IgG of AIL2RA in the plasma of lung cancer patients was significantly lower than that of the control group, in which anti-SEQ ID NO:1 and: SEQ ID NO: the changes in IgG levels of the 2 two polypeptide antigens were statistically significant (see table 3).
TABLE 3 analysis of the levels of AIL2RA native IgG in the plasma of lung cancer patients and controls
And (3) analyzing and comparing whether the lung cancer patient has significant difference with the control group in the plasma natural IgG level by using a T test method. The data was further analyzed using Receiver Operating Characteristics (ROC) graphs. The ROC curve is a curve which is drawn by taking the true positive rate (sensitivity) of a lung cancer patient plasma sample as an ordinate and the false positive rate (1-specificity) of a healthy human plasma sample as an abscissa, and determining a cut-off value or a decision threshold according to a series of different two classification modes. The area under the ROC curve (AUC) value should be between 1.0 and 0.5. In the case of AUC >0.5, the closer the AUC is to 1, indicating better diagnostic accuracy. The ROC curve graphically combines sensitivity and specificity to accurately reflect the relationship between specificity and sensitivity of an assay.
ROC curve analysis showed anti-SEQ ID NO:1 the polypeptide antigen natural IgG has an AUC value of 0.68 (95% confidence interval of 0.54-0.82), a sensitivity of 22.7%, an AUC value of the anti-SEQ ID NO:2 natural IgG in the plasma of the lung cancer patient of 0.70 (95% confidence interval of 0.57-0.82), a sensitivity of 27.7% (see FIG. 2), an AUC value of the anti-SEQ ID NO:3 natural IgG in the plasma of the lung cancer patient of 0.53 (95% confidence interval of 0.40-0.67), and a sensitivity of 4.5%. The specificity of detecting the natural IgG antibody levels of 3 plasma AIL2RA was 95.5% (see table 4).
TABLE 4 analysis of the plasma AIL2RA native IgG levels by the ROC curve method
Note: the antibody detection specificity was 95.5%.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that may be made by those skilled in the art within the technical scope of the present invention will be covered by the scope of the present invention.
Sequence listing
<110> Shanghai Ying far away Biotech Co., Ltd
CHANGCHUN HAILANSHEN BIOMEDICAL TECHNOLOGY Co.,Ltd.
<120> polypeptide sequence, kit and method for detecting natural antibody of anti-interleukin 2receptor subunit A
<160> 3
<170> SIPOSequenceListing 1.0
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<211> 30
<212> PRT
<213> Artificial Sequence
<400> 1
Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg
1 5 10 15
Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Glu Met Glu His
20 25 30
<210> 2
<211> 30
<212> PRT
<213> Artificial Sequence
<400> 2
Thr Gly Glu Met Glu Thr Ser Gln Phe Pro Gly Glu Glu Lys Pro Gln
1 5 10 15
Ala Ser Pro Glu Gly Arg Pro Glu Ser Glu Thr Ser Cys His
20 25 30
<210> 3
<211> 31
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<213> Artificial Sequence
<400> 3
Thr Ser Cys Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr Glu Met
1 5 10 15
Ala Ala Thr Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln His
20 25 30
Claims (10)
1. A detection reagent for detecting natural antibodies against interleukin 2receptor subunit A, comprising any one, any two or all three of the following three antigenic polypeptides:
H-RGPAESVCKMTHGKTRWTQPQLICTGEMEH-OH；
H-TGEMETSQFPGEEKPQ ASPEGRPESETSCH-OH; and
H-TSCLVTTTDF QIQTEMAATM ETSIFTTEYQH-OH。
2. the detection reagent according to claim 1, wherein when the detection reagent comprises two or three antigenic polypeptides, the two or three antigenic polypeptides are present separately.
3. The detection reagent of claim 1, wherein when the detection reagent comprises two or three antigenic polypeptides, the two or three antigenic polypeptides are mixed together.
4. The detection reagent according to claim 1, wherein when the detection reagent comprises three antigenic polypeptides, one antigenic polypeptide of the three antigenic polypeptides is present alone and the other two antigenic polypeptides are mixed together.
5. A kit comprising the detection reagent according to any one of claims 1 to 4.
6. The kit of claim 5, wherein said kit comprises a microplate, said detection reagents being coated within wells of the microplate.
7. An in vitro method for detecting natural antibodies against interleukin 2receptor subunit a for non-diagnostic purposes, comprising detecting natural antibodies against interleukin 2receptor subunit a in a sample using a detection reagent according to any one of claims 1 to 4 or a kit according to claim 5 or 6.
8. The method of claim 7, the step of performing comprising: (1) performing a step-wise loading analysis using a microplate coated with the detection reagent according to any one of claims 1-4; (2) optical density values (OD) in each well were measured and antibody levels were expressed as Positive Sample Ratios (PSR).
9. Use of a detection reagent according to any one of claims 1 to 4, or a kit according to claim 5 or 6, or a detection method according to claim 7 or 8 for the detection of natural antibodies against interleukin 2receptor subunit a for non-diagnostic purposes.
10. Use of a detection reagent according to any one of claims 1 to 4 in the preparation of a reagent for the detection of a natural antibody against interleukin 2receptor subunit a in a sample.
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| CN108427003A (en) | 2018-08-21 |
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