CN111458522A - Detection reagent and kit for detecting natural antibody of plasma interleukin6 and application of detection reagent and kit - Google Patents

Detection reagent and kit for detecting natural antibody of plasma interleukin6 and application of detection reagent and kit Download PDF

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CN111458522A
CN111458522A CN202010312663.2A CN202010312663A CN111458522A CN 111458522 A CN111458522 A CN 111458522A CN 202010312663 A CN202010312663 A CN 202010312663A CN 111458522 A CN111458522 A CN 111458522A
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CN111458522B (en
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徐宏海
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Zhongshan Jiuxing Biotechnology Co ltd
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Abstract

The invention relates to a detection reagent and a kit for detecting Natural antibodies (Natural antibodies for interleukin-6, NAI L6) of plasma interleukin6 and application thereof, and realizes qualitative and relative quantitative detection of NAI L6 in plasma by utilizing linear antigen polypeptide highly complementary to target NAI L6, and the detection reagent and the kit can be used for treating and researching inflammation-related diseases such as Novel Coronavir pnuemonia, type II diabetes, cardiovascular and cerebrovascular infarction and the like infected by Coronavirus.

Description

Detection reagent and kit for detecting natural antibody of plasma interleukin6 and application of detection reagent and kit
Technical Field
The invention belongs to the field of clinical application of immunological technology, and relates to a detection reagent and a kit for detecting Natural antibody for Interleukin 6(I L6) of plasma Interleukin6 (Natural antibody for Interleukin6, NAI L6) and an application method thereof.
Background
Inflammation is a defensive immune response generated in human and animals, and the inflammatory response may be accompanied by tissue and cell damage in the body to cause various common diseases.
The existence of 'inflammatory cytokine storm' which is mainly characterized by the rise of I L6 in patients with obvious pulmonary lobe lesions in the novel coronavirus pneumonia in 2019 can lead to the rapid increase of the disease condition of the patients and even death of the patients, meanwhile, the influence of the inflammatory cytokine is hidden in the conditions of diabetes, atherosclerosis, obesity, tumors, rheumatoid arthritis and the like, and the research of the causal kingdom is not made into consideration, the research on more inflammatory cytokines mainly comprises interleukin 1(interleukin 1 α and β, I L1- α 0 and I L1- β), interleukin 6(interleukin 6, I L6), interleukin 8(interleukin 8, I L8) and tumor necrosis factor (tumor necrosis factor α - α), the research of the causal kingdom that the inflammatory cytokine is almost involved in all inflammatory reaction processes and related to the occurrence and development of various diseases is acknowledged that the increase of the inflammatory cytokine is related to the development of the patients with the development of various diseases, and the research on the development of the health factors is partly reported that the increase of the inflammatory cytokine is related to the development of the patients with the diseases and the development of the diseases.
In the clinical application of anti-inflammatory cytokine technology, the new approach to immunotherapy of coronavirus pneumonia (trial seventh edition) is considered to treat severe patients with elevated levels of I L6 detected in the laboratory, and in the prior art, it is also common to treat rheumatoid arthritis with monoclonal antibody drugs, such as Adalimumab (Adalimumab or Humira) and Infliximab (Infliximab or Remicade), interleukin-1 β monoclonal antibody-canamab (Canakinumab or Ilaris) and interleukin-6 receptor monoclonal antibody-tollizumab (actemmra or Tocilizumab), but generally have large toxic and side effects and are prone to generate anti-antibodies with drug resistance, and are not suitable for long-term continuous use.
At present, no stable, accurate, cheap and easy-to-use I L6 detection means exists clinically, which leads to that a novel coronavirus pneumonia diagnosis and treatment scheme points to a 'laboratory detection of a heavy patient with an increased I L6 level', and is actually uncertain.
Disclosure of Invention
The present inventors have found that Interleukin 6(Interleukin 6, I L) Natural antibody (Natural antibody for Interleukin6, NAI L6) is present in human plasma, and the Natural antibody may be an important factor for controlling physiological levels of inflammatory cytokine I L in vivo, and detection of NAI L level in plasma may predict the onset risk of inflammation-related diseases. there is no stable, accurate, inexpensive and readily available means for detecting NAI L in clinical practice the present invention can accurately determine NAI L level by using a relatively quantitative means for detecting the rising or falling levels of Interleukin 6(Interleukin 6, I L) Natural IgG antibody (Natural antibody for Interleukin6, NAI 5636) because the biological half-life of IgG antibody in plasma is 3-5 weeks, and the NAI L level is stable during the corresponding detection period (e.g. 2-3 weeks), thus the condition of NAI L can be specifically defined as NAI L.
The invention designs and synthesizes 2 linear antigen polypeptides highly complementary with target NAI L by utilizing advanced sequence analysis means and epitope mapping technology, each polypeptide contains more than 10 high-specificity overlapped epitopes, and on the basis, an epitope-specific enzyme-linked immunosorbent assay (E L ISA) method is established to realize qualitative and relative quantitative detection of NAI L6 in plasma, and further lay an experimental foundation for researching the low-toxic-side effect adjuvant therapy of Novel coronavirus infection pneumonia (Novel Coronavir pneumoconia), low-toxic-side effect long-term accurate therapy of II-type diabetes, cardiovascular and human vascular infarction and other inflammation-related diseases.
In the present invention, "natural antibody" is used interchangeably with "autoantibody" and refers to a mixture of a plurality of antibodies (monoclonal and/or polyclonal antibodies) that are naturally present in the body and that recognize an epitope of a substance of interest (e.g., a protein of interest, such as an inflammatory cytokine), "interleukin 6 natural antibody" or "NAI L" as defined herein is a mixture of a plurality of antibodies (monoclonal and/or polyclonal antibodies) that are naturally present in the human body and that recognize an epitope of interleukin6, e.g., may be a natural IgG antibody that recognizes an epitope of interleukin 6. in some embodiments of the invention, the "interleukin 6 natural antibody" or "NAI L" refers to a mixture of antibodies (monoclonal and/or polyclonal antibodies) that recognize one or two polypeptide sequences selected from SEQ ID NO:1 and SEQ ID NO: 2. plasma enriched with NAI 3506 may have a high risk of treating or preventing an I L-related disease (i.e. a disease characterized by an increase in I L), or may be detected at a high relative low level of plasma levels of NAI 466 negative as a risk of entering plasma related NAI 736, or a high plasma counter-related disease in humans.
It is well recognized that binding of an antigen to an antibody occurs primarily between an antigenic determinant (i.e., an epitope) and the binding site of the antibody. 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 to be detected) and its binding site structure are the prerequisite factors, and the antigenic determinant affects the binding state and affinity characteristics of the whole protein antigen and antibody.
The invention relates to a method for carrying out immune informatics prediction and simulation on a plurality of antigen epitopes of a protein according to the biological characteristics of I L protein, and designs two linear antigen epitope polypeptides which are completely complementary with a target antibody in space structure and configuration through analyzing various parameters related to antigenicity, wherein the amino acid sequences of the linear antigen epitope polypeptides are shown in Table 1.
TABLE 1 detection of two linear epitope polypeptide sequences of NAI L6 in human plasma
Figure BDA0002458438620000041
Full-length amino acid sequence (212 amino acids) of I L6 molecule and I L6 a linear epitope polypeptide region
Figure BDA0002458438620000042
Full-length amino acid sequence (212 amino acids) of I L6 molecule and I L6 b Linear antigen polypeptide region
Figure BDA0002458438620000043
The antigen polypeptide is synthesized by a solid phase chemical method, and can be used for preparing an E L ISA antibody detection kit respectively or by mixing, and the concentration of NAI L6 in human plasma is detected according to a set flow specification, the detection reagent comprising one or two antigen polypeptides can be prepared into a simple and easy-to-use convenient kit in practical application, is packaged in a vacuum sealing mode by non-metal materials such as glass, medical plastics and the like, and can be stored for more than 6 months under the environment of 4 ℃ and 4 ℃.
Therefore, according to one aspect of the present invention, there is provided a detection reagent for detecting human plasma NAI L6, comprising at least one or two antigenic polypeptides selected from the group consisting of:
H-Y L QNRFESSEEQARAVQMSTKV L ICH-OH (SEQ ID NO:1), and
H-DLTKLQAQNQWLQDMTTHLILRSFKC-OH(SEQ ID NO:2)。
in some embodiments, the detection reagent consists of one or two antigenic polypeptides selected from any of the two antigenic polypeptides.
In some embodiments, either or both of the two antigenic polypeptides are high purity preparations, preferably chemically synthesized with a purity > 95%.
In some embodiments, the two antigenic polypeptides may be used in admixture, and when used in admixture, the two antigenic polypeptides may be mixed in equal mass proportions. Thus, in some embodiments, the detection reagent is a mixture of the two antigenic polypeptides, e.g., can be a mixed solution. In some embodiments, the ratio of the two antigenic polypeptides in the detection reagent is 1:1 (ratio of mass to volume concentrations).
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 some preferred embodiments, the non-metallic medical packaging material is glass or medical plastic.
In some embodiments, the positive control may also be referred to as a positive standard, such as human anti-I L6 antibody, and the negative control may be any reagent that does not contain the I L6 antibody, such as an albumin solution.
In some embodiments, the method comprises detecting the level of NAI L in the test sample by an antigen-antibody binding reaction using the above-described detection reagent.
In some embodiments, the method comprises allowing an antigen-antibody binding reaction between the detection reagent and the NAI L in the test sample, and determining the level of NAI L in the test sample the techniques for detecting or determining the level of antibodies in the test sample by antigen-antibody binding reaction using antigenic polypeptides are well known in the art, such as the enzyme-linked immunosorbent assay (E L ISA) method.
In a more preferred embodiment, the enzyme-linked immunosorbent assay is the sandwich method E L ISA.
In some embodiments, the method is performed by (1) incubating the detection reagent with the test sample, a negative control, and a positive control, respectively, under conditions suitable for an antigen-antibody binding reaction between the detection reagent and the NAI L6 in the test sample, followed by addition of an enzyme-labeled secondary antibody and incubation, followed by addition of a chromogenic agent, and then terminating the reaction after development and detecting the optical density value (OD), and (2) determining the relative level of NAI L6, which is the relative level of NAI L6 in the test sample relative to the positive control.
In some embodiments, the relative level of NAI L6 is determined using the Specific Binding Ratio (SBR) in some embodiments, the SBR calculation is:
SBR (sample to be tested OD value-NC OD value ]/[ positive standard OD value-NC OD value ]
Where NC is a negative control. The antibody concentration in a sample (e.g. plasma) is expressed as the SBR mean and standard deviation.
In some embodiments, a NAI L positive sample threshold may be set, a NAI 736 positive sample threshold is used, and a NAI L positive sample threshold is determined by a percentile method, a sample detection result below the threshold is negative indicating that the NAI L content in the sample is low, a sample detection result above the threshold is positive indicating that the NAI L content in the sample is high, in some embodiments, the normal population may be a normal adult, the normal population may also be referred to as a healthy population, which is a population determined to have no diabetes and other serious inflammation-related diseases in clinical examination, the percentile may be, for example, the 95 th percentile method, the 90 th percentile method, etc. the determination of the NAI 8656 positive sample threshold is to screen out a sample having a relatively high NAI 5836 content, which is not limited to a specific normal population when the threshold is determined, and is not limited to a specific percentile, which is not limited to a specific percentile, when the NAI 6336 content in the sample threshold is determined, for example, when the NAI 636 positive sample threshold is determined to be not lower, the sample of the normal population, which may be a higher NAI 636 content, when the sample is not lower than the percentile, which may be a higher than the number of the sample, which may be a patient having a higher risk of a higher than the number of a patient, which may be determined, which may be a patient having a higher number of NAI 636, which may be a higher number of a patient, which may be a patient having a higher number of a patient, such as a patient having a higher number of a patient, such as a patient having a higher number of a patient, such as a patient having a higher number of a patient, such as a patient having a higher number of a patient having a.
The conditions suitable for the antigen-antibody binding reaction between the detection reagent and the NAI L6 in the sample to be tested are readily known to those skilled in the art, and may be known, for example, from conventional antigen-antibody reaction conditions and/or routine experimentation.
In some embodiments, the mixing incubation in step (1) above comprises coating the detection reagent above on a Maleimide (Maleimide) activated microplate and adding the sample to be tested to the wells of the microplate.
In some embodiments, the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-human IgG antibody, and in some embodiments, the chromogenic agent is 3,3',5,5' -Tetramethylbenzidine (TMB). In some embodiments, the reaction is terminated using a sulfuric acid solution, preferably having a concentration of 10-12% (v/v). In some embodiments, the Optical Density (OD) value is detected at a wavelength of 450nm and the reference wavelength is 630 nm.
In some embodiments, the sample is human plasma. In some embodiments, the sample is a healthy or normal human plasma. In some embodiments, the sample is more preferably a single individual plasma. The healthy or normal person may be a person determined by a clinical physical examination to have not had a diabetic patient and other serious inflammation-related diseases. In some embodiments, the subject's plasma is plasma from a single healthy subject or a single normal subject.
In a more preferred embodiment, the above step (1) comprises:
dissolving two antigen polypeptides listed in Table 1 with 67% acetic acid to obtain 5 mg/ml stock solutions, storing in-20 deg.C refrigerator, and standing alone or mixing the two stock solutions in equal volume;
diluting the single storage solution or the mixed solution into 10-50 micrograms/ml working solution by using 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;
coating a 96-hole micro-detection plate activated by Maleimide (Maleimide) with a working solution, incubating overnight at 4 ℃, and washing the plate for 3 times by using 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;
the plasma sample to be tested is provided with double wells, and 2 Negative Control (NC) wells (which are negative control liquid without anti-I L6 antibody, such as bovine serum albumin, so that the experimental index value of the two polypeptide antigens in the NAI L6 negative reaction system shown in the table 1 can be reflected) and 2 Positive Control (PC) wells (which are a mixture of human anti-I L6 (SEQ NO:1) antibody and human anti-I L6 (SEQ NO:2) antibody, so that the experimental index value of the two polypeptide antigens in the NAI L6 positive reaction system shown in the table 1 can be reflected) are additionally arranged;
diluting a to-be-detected plasma sample by 1:100-200 with 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 value is 7.0-7.4, and 100 mu l of the analysis solution is added into each hole; incubating for 1-2 hours at 20-25 ℃, and then washing the plate for 3 times;
diluting a horseradish peroxidase-labeled goat anti-human IgG antibody (used for verifying whether a substance to be detected in 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 the pH value of the buffer solution is between 7.0 and 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 20-25 ℃;
washing the plate for 3 times by using washing liquor (namely 0.1M phosphate buffer solution containing 0.15M sodium chloride and 0.1 percent TWEEN-20 and having the pH value of 7.0-7.4), adding 100 mu l of 3,3',5,5' -Tetramethylbenzidine (TMB) and peroxidase mixed liquor into each hole, and keeping the plate away from light at room temperature for 20-30 minutes;
add 50. mu.l stop solution 12% sulfuric acid solution (12% H) per well2SO4) Then, the Optical Density (OD) value was measured with a microplate reader at a detection wavelength of 450nm and a reference wavelength of 630nm, and the detection was completed within 10 minutes after the addition of the stop solution, thereby relatively quantitatively analyzing the NAI L6 levels in the plasma of different individuals.
In some embodiments, plasma NAI L6 levels can be measured in vitro for type II diabetic patients and the data obtained from each individual measurement can be analyzed when performing a population random sampling analysis.
In another aspect of the invention there is provided the use of a detection reagent or kit as described above in the detection of Natural antibody for Interleukin 6(Interleukin 6, I L) Natural antibody (NAI L) in a sample.
In another aspect of the present invention, there is also provided a use of the above detection reagent for preparing a reagent for detecting a Natural antibody for Interleukin 6(Interleukin 6, I L6) Natural antibody (NAI L6) in a sample.
In a preferred embodiment, the sample is human plasma, more preferably single individual plasma.
In a more preferred embodiment, the subject's plasma is plasma from a healthy subject.
In another aspect of the invention, the application of the detection reagent or the kit in screening human plasma rich in Natural antibody for interleukin6 (NAI L6) of I L6 is also provided.
Based on the scheme, the invention provides the NAI L6 detection technology with high precision, simple operation and moderate cost, and further provides a relatively quantitative analysis and application scheme for the NAI L6 on the basis, thereby laying an important foundation for predicting the disease risk of inflammation-related diseases and developing a brand-new immunotherapy strategy with low side effect.
The detection system is an accurate relative quantification method, can detect the NAI L6 in the plasma at reasonable cost, and plays a key role in the application of the plasma rich in NAI L6 and the gamma globulin rich in NAI L6 in the field of low toxic and side effects and inflammation resistance.
In conclusion, the invention discovers a group of brand-new antigen polypeptides and further designs the antigen polypeptides into a practical kit, thereby providing an effective and convenient human plasma NAI L detection means which can be used for assisting in qualitatively and relatively quantitatively detecting autoantibodies of inflammatory cytokines such as NAI L and quantitatively determining NAI L levels of different individual plasmas, and distinguishing plasma rich in NAI L (strong positive) from plasma free of NAI L (negative).
Detailed description of the preferred embodiments
In specific embodiments, the following are 1 of the two antigenic polypeptides listed in table 1:1 the technical scheme of the invention is concretely illustrated by taking the mixed solution as a detection reagent. If the detection reagent containing only one antigen polypeptide is used, the mixed solution of the two antigen polypeptides is changed to the storage solution containing only one antigen polypeptide, and other implementation operation steps are the same.
The specific operation steps of the detection are as follows:
1. prior to the procedure, each antigen was dissolved in 67% acetic acid as a 5 mg/ml stock solution, then mixed in equal volumes and stored in a-20 deg.C (error within + -2 deg.C) refrigerator.
2. At the beginning of the operation, the mixed solution of 2 antigen polypeptides listed in table 1 is diluted to 10-50 micrograms/ml of working solution by coating solution, 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 (Corning, USA) activated by Maleimide (Maleimide) is coated with a working solution, and after overnight incubation at 4 ℃, the plate is washed for 3 times by 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:
(1) the plasma samples to be tested were prepared in duplicate wells, 2 Negative Control (NC) wells (the reference is a negative control solution without anti-I L6 antibody, such as bovine serum albumin (Sigma-Aldrich), which can reflect the experimental index of 2 polypeptide antigens in NAI L6 negative reaction system as shown in Table 1) and 2 Positive Control (PC) wells (the reference is a polyclonal antibody mixture of human anti-I L6 full-length protein, which can reflect the experimental index of 2 polypeptide antigens in NAI L6 positive reaction system as shown in Table 1).
(2) Diluting a blood plasma sample to be detected by 1:100-200 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 value is 7.0-7.4, 100 mu l of the buffer solution is added into each hole, incubation is carried out for 1-2 hours at the temperature of 20-25 ℃, and then the plate is washed for 3 times.
(3) 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 is carried out for 1-2 hours at the temperature of 20-25 ℃.
(4) 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 3,3',5,5' -Tetramethylbenzidine (TMB) was added to each well, and the plate was protected from light at room temperature for 20-30 minutes.
(5) Add 50. mu.l stop solution 12% sulfuric acid solution (12% H) per well2SO4) And then detecting the Optical Density (OD) value by using a microplate reader, wherein the detection wavelength is 450nm, and the reference wavelength is 630nm, and the detection process is completed within 10 minutes after the stop solution is added, so that the NAI L6 levels in the blood plasma of different individuals are relatively quantitatively analyzed.
(6) Definition of Positive threshold for anti-I L6-enriched Natural antibody plasma IgG level
(7) When analyzing the data obtained by the detection, the Specific Binding Ratio (SBR) is adopted to judge the level of IgG of the natural antibody rich in I L6 in the plasma, and the calculation formula of the SBR is as follows:
SBR (sample to be tested OD value-NC OD value ]/[ positive control OD value-NC OD value ]
The SBR threshold (cut-off) of a sample positive for the I L6 natural antibody IgG level in healthy human plasma was determined using the percentile method, samples with plasma IgG levels above this threshold were defined as positive samples enriched with anti-I L6 natural antibody plasma, samples with plasma IgG levels below this threshold were defined as negative samples of anti-I L6 natural antibody plasma, anti-I L6 natural antibody-enriched plasma was packaged in standard storage bags (150 and 200 ml plasma per bag) and stored in a freezer at minus 80 degrees (-80 ℃) for no more than 6 months.
(8) In some embodiments, the relative level of NAI L in the plasma is determined using Specific Binding Ratio (SBR). the SBR calculation is as follows:
SBR ═ value of sample to be tested-NC OD value/[ positive control OD value-NC OD value ].
The 95 th percentile method was used to determine the SBR threshold (cut-off) for NAI L6 positive plasma in normal adult plasma samples, and samples below this SBR threshold were NAI L6 free (negative) plasma.
Application example one screening of NAI L6-enriched plasma
1. 200 healthy human plasma samples were provided from a central blood station in a city, all plasma samples were stored at minus 80 degrees (-80 ℃) and thawed in a 4 degree (4 ℃) freezer 12 hours prior to use.
(1) Sample detection: the 2 polypeptide antigens listed in table 1 used in this experiment were synthesized by the british SEVERN BIOTECH co ltd, with a purity of 95%, and the specific operating procedures were as follows:
(2) before the operation, each antigen was dissolved in 67% acetic acid to give a stock solution of 5.0mg/ml, and the two antigens were mixed in equal volumes and stored in a refrigerator at-20 ℃ (error within. + -. 2 ℃).
(3) At the beginning of the operation, the mixture of 2 antigens was first diluted to 20. mu.g/ml with a coating solution of 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, and the pH was measured to be 7.3.
(4) After incubation with a 96-well Maleimide (Maleimide) activated microtiter plate (Thermo Scientific, USA) at 4 ℃ overnight (18 hours) with the coating solution containing the 2 antigen mixtures obtained in step (2) above, 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 7.3.
(5) The post fractional sample application analysis was as follows:
a) the test plasma samples were prepared in duplicate wells, 2 Negative Control (NC) wells (bovine serum albumin, supplied by Sigma-Aldrich) and 2 Positive Control (PC) wells (human anti-I L6 whole molecule antibody cocktail, supplied by Sigma-Aldrich).
b) The plasma was diluted 1:150 with the same assay as the antigen coating solution, 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, pH 7.3, 100. mu.l per well and incubated at 25 ℃ for 1.5 hours.
c) After washing the plate 3 times with a wash solution (i.e., 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, pH 7.3 for pH), horseradish peroxidase-labeled goat anti-human IgG antibody (supplied by Sigma-Aldrich) was diluted with an assay solution (0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, pH 7.3 for pH) at a dilution ratio of 1:25000, 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 sodium chloride and 0.1% TWEEN-20, pH 7.3 for pH), 100. mu.l of 3,3',5,5' -Tetramethylbenzidine (TMB) was added to each well and protected from light at room temperature for 25 minutes.
e) Adding 50 μ l of 12% sulfuric acid solution (12% H2SO 4) of stop solution into each well, detecting Optical Density (OD) value with an enzyme-linked immunosorbent assay, wherein the detection wavelength is 450nm, the reference wavelength is 630nm, the detection is completed within 10 minutes after the stop solution is added, and performing relative quantitative comparative analysis of NAI L6 for each individual according to the result in the subsequent steps.
(6) Analyzing data obtained by the detection, and judging the level of IgG containing I L6 natural antibodies in plasma by using a Specific Binding Ratio (SBR), wherein the SBR is calculated by the formula of SBR (SBR- [ sample to be detected OD value-NC OD value ]/[ positive control standard product OD value-NC OD value ], NC is negative control of each sample, the SBR average value and standard difference are used for representing antibody concentration in plasma, and a percentile method is applied to the detection results of the antibody concentration of 200 healthy human plasma samples to determine plasma NAI L6 positive and strong positive threshold values, wherein the plasma IgG antibody level lower than the positive threshold value is defined as a negative sample, and the plasma IgG antibody level higher than the strong positive threshold value is defined as a strong positive sample.
2. Results of the experiment
In 200 randomly drawn normal adult plasma samples in the example, the mean value of NAI L6 concentration is SBR (0.78 +/-0.42), the coefficient of variation is 53.8%, the 5 th percentile method is used for determining the negative threshold value of plasma NAI L6 to be 0.25, and the 95 th percentile method is used for determining the strong positive threshold value of plasma NAI L6 to be 1.55 (shown in Table 1).
TABLE 1 results of the concentration test of anti-I L6 antibody in 200 normal adult plasma samples from a blood station
Figure BDA0002458438620000141
As can be seen from Table 1, the number of patients who were strongly positive for NAI L6 was 12 (6%) among 200 normal adult plasma samples examined.
Application example type II diabetes patients NAI L6 detection
1. Collecting samples: 420 plasma samples of II diabetics and normal adults were provided from a hospital, a blood station. The normal adult was identified as an individual who had not had diabetes and other serious inflammation-related diseases by clinical examination. All plasma samples were stored at minus 80 degrees (-80 ℃) and thawed in a 4 degree (4 ℃) freezer 12 hours prior to use.
2. Sample detection: the 2 polypeptide antigens listed in table 1 used in this experiment were synthesized by the british SEVERN BIOTECH co ltd, with a purity of 95%, and the specific operating procedures were as follows:
(1) prior to the procedure, each antigen was dissolved in 67% acetic acid to a stock solution of 5.0mg/ml, then mixed in equal volumes to form a coating solution and stored in a refrigerator at-20 ℃ (error within. + -. 2 ℃).
(2) At the beginning of the operation, the mixture of 2 antigens was first diluted to 20. mu.g/ml with a coating solution of 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, and the pH was measured to be 7.3.
(3) Then, a 96-well Maleimide (Maleimide) -activated microtiter plate (Thermo Scientific, USA) was coated with the coating solution containing the 2 antigen mixture obtained in the above step (2), and after overnight (14-18 hours) incubation 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 7.3.
(4) The fractional loading analysis was then as follows:
a. the test plasma samples were prepared in duplicate wells, 2 Negative Control (NC) wells (bovine serum albumin, supplied by Sigma-Aldrich) and 2 Positive Control (PC) wells (human anti-I L6 whole molecule IgG antibody cocktail, supplied by Sigma-Aldrich).
b. The plasma was diluted 1:150 with the same assay as the antigen coating solution, 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, pH 7.3, 100. mu.l per well and incubated at 25 ℃ for 1.5 hours.
c. After washing the plate 3 times with a wash solution (i.e., 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, pH 7.3 for pH), horseradish peroxidase-labeled goat anti-human IgG antibody (supplied by Sigma-Aldrich) was diluted with an assay solution (0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, pH 7.3 for pH) at a dilution ratio of 1:25000, 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 sodium chloride and 0.1% TWEEN-20, pH 7.3), 100. mu.l of 3,3',5,5' -Tetramethylbenzidine (TMB) (supplied by L area technologies) was added to each well and protected from light at room temperature for 25 minutes.
e. Add 50. mu.l stop solution 12% sulfuric acid solution (12% H) per well2SO4And 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 relative quantitative comparative analysis of NAI L6 is carried out on each individual according to the result in the subsequent steps.
(5) Analyzing the data obtained by the detection, and judging the NAI L6 level in the plasma by using Specific Binding Ratio (SBR), wherein the SBR is calculated by the formula of SBR ═ OD value-NC OD value of the sample to be detected ]/[ OD value-NC OD value of the positive control standard substance ]. the 95 th percentile method is used for determining the threshold value (cut-off) of the plasma without NAI L6 (negative).
3. The experimental results are as follows: this example220 randomly drawn plasma samples of normal adults had an NAI L6 concentration mean value of SBR of 1.07 +/-0.35, and the 5 th percentile method was used to determine plasma NAI L6 negative threshold of 0.25 and an artificial NAI L6 detection negative of SBR less than 0.25. As shown in Table 2, 20.5% of 200 diabetic patients tested had NAI L6 detection negative, and 4.5% of 220 normal persons had NAI L6 detection negative, and the Ka square (X square)2) The difference between the two groups is obvious (X) by inspection2＝25.0，P<0.001)。
TABLE 2 analysis and comparison of 420 type II diabetes mellitus with the NAI L6 negative detection rate of a normal adult human plasma sample
Figure BDA0002458438620000161
Represents Odds Ratio (OR)
4. The results evaluate that the 2 linear antigen polypeptides can be specifically combined with the NAI L6 in the blood plasma, namely, the amino acid sequences of the 2 linear antigen polypeptides are completely complementary with the NAI L6 molecules in the spatial structure and configuration.
The related embodiments and application examples are only for illustrating some specific embodiments of the technical solutions of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that may occur to those skilled in the art within the technical scope of the present invention disclosed herein should be covered within the scope of the present invention.
Sequence listing
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Claims (10)

1. The detection reagent for detecting the I L6 natural antibody is characterized by comprising any one or two antigen polypeptides selected from the following two antigen polypeptides:
H-Y L QNRFESSEEQARAVQMSTKV L ICH-OH (SEQ ID NO:1), and
H-DLTKLQAQNQWLQDMTTHLILRSFKC-OH(SEQ ID NO:2)。
2. the detection reagent according to claim 1, wherein the detection reagent consists of any one or two antigenic polypeptides selected from the two antigenic polypeptides.
3. The detection reagent according to claim 1 or 2, wherein the detection reagent comprises the two antigenic polypeptides, and the ratio of the mass to volume concentrations of the two antigenic polypeptides is 1: 1.
4. A kit for detecting I L6 natural antibody, characterized in that the kit comprises the detection reagent according to any one of claims 1 to 3.
5. A kit according to claim 4, wherein the kit includes a microplate, the detection reagents being coated in wells of the microplate.
6. An in vitro method for the detection of I L6 natural antibody for non-diagnostic purposes comprising detecting I L6 natural antibody in a sample using the detection reagent of any one of claims 1-3 or the kit of claim 4 or 5.
7. The detection method according to claim 6, comprising the steps of:
(1) respectively mixing and incubating the detection reagent with a sample to be detected, a negative control and a positive control under the condition suitable for an antigen-antibody binding reaction between the detection reagent and a natural I L6 antibody in the sample to be detected, then adding an enzyme-labeled secondary antibody and incubating, then adding a color developing agent, stopping the reaction after color development and detecting the Optical Density (OD) value;
(2) determining the relative level of I L6 native antibody in the sample.
8. Use of the detection reagent of any one of claims 1-3 in the preparation of a reagent for detecting a native antibody to I L6 in a sample.
9. Use of a detection reagent according to any one of claims 1 to 3 or a kit according to claim 4 or 5 for the in vitro detection of natural antibodies to I L6 for non-diagnostic purposes.
10. Use of the detection reagent of any one of claims 1 to 3 or the kit of claim 4 or 5 for screening human plasma for native antibodies enriched in I L6.
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