CN111458522B - 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 PDFInfo
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- CN111458522B CN111458522B CN202010312663.2A CN202010312663A CN111458522B CN 111458522 B CN111458522 B CN 111458522B CN 202010312663 A CN202010312663 A CN 202010312663A CN 111458522 B CN111458522 B CN 111458522B
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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 a detection reagent for detecting a natural antibody (Natural antibody for interleukin-6, NAIL6) of plasma interleukin6, a kit and application thereof. The invention utilizes linear antigen polypeptide highly complementary with target NAIL6 to realize qualitative and relative quantitative detection of NAIL6 in blood plasma, and can be used for treating and researching novel inflammatory related diseases such as pneumonia (Novel Coronavirus Pneumonia) infected by coronavirus, type II diabetes, cardiovascular and cerebrovascular infarction and the like.
Description
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 antibodies (Natural antibody for Interleukin, NAIL 6) of plasma Interleukin6 (IL 6 ) and an application method thereof.
Background
Inflammation is a defensive immune response in humans and animals that may be accompanied by tissue and cell damage in the body leading to the development of a variety of common diseases.
The presence of "inflammatory cytokine storm" characterized by elevated IL6 in patients with significant lung lesions in the 2019 new coronavirus pneumonia can lead to dramatic exacerbation and even death of the patient. Meanwhile, the influence of inflammatory cytokines in diseases such as diabetes, atherosclerosis, obesity, tumors, rheumatoid arthritis and the like is hidden, but the theory of the aging is not definite. Many inflammatory cytokines are currently being studied, mainly including interleukin 1 (Interleukin 1. Alpha. And. Beta., IL 1-. Alpha. And IL 1-. Beta.), interleukin6 (Interleukin 6, IL 6), interleukin 8 (Interleukin 8, IL 8) and tumor necrosis factor (tumor necrosis factor. Alpha., TNF-. Alpha.). The academy recognizes that inflammatory cytokines are involved in almost all inflammatory reaction processes and are involved in the occurrence and development of a variety of diseases. Numerous studies have reported that related inflammatory cytokines in the population also increase continuously with age or long-term sub-health, but a significant proportion of the occurrence and progression of cardiovascular infarction and type ii diabetes have not been demonstrated to be causally related. If the natural antibody level increase and decrease of inflammatory cytokines and the change of symptoms can be monitored in a targeted manner, further research on how to purposefully control and influence certain links is possible, and the method is a correct research direction for solving the damage of inflammatory response to the body.
In the clinical application of anti-inflammatory cytokine technology, in the novel coronavirus pneumonia diagnosis and treatment scheme (trial seventh edition) immunotherapy method, for heavy patients whose laboratory detection IL6 level is elevated, tolizumab therapy is considered. In the prior art, monoclonal antibody medicines are also commonly used for treating rheumatoid arthritis, such as monoclonal antibodies against tumor necrosis factor-Adalimumab (Adalimumab or Humira) and Infliximab (Infiniumab or Remica), anti-interleukin-1 beta monoclonal antibodies-kanamab (Canakiumab or Ilasis) and anti-interleukin-6 receptor monoclonal antibodies-tolizumab (actera or Tocilizumab), but the general toxic and side effects are large, and the antibodies are easy to generate and have drug resistance, so that the medicine is not suitable for long-term continuous use.
Currently, no stable, accurate, inexpensive and easy-to-use IL6 detection means exists clinically, which leads to the fact that the "laboratory detection of heavy patients with elevated IL6 levels" to which the novel coronavirus pneumonia diagnosis and treatment protocol is directed is virtually uncertain. This situation based on uncertain preconditions is also present in type ii diabetes, where patients generally have to undergo external intervention injuries with insulin for a long period of time in exchange for a slowing down of the progression of the disease, but where it is the blocking inflammatory factors that are the correct treatment for some patients to restore systemic function. In addition, the patients with high risk of cardiovascular and cerebrovascular infarction are overadministered for a long term with or without a vector.
Disclosure of Invention
The inventors found that natural antibodies (Natural antibody for Interleukin, NAIL 6) to Interleukin6 (Interleukin 6, il6) are present in human plasma, and that natural antibodies may be important factors in controlling the physiological levels of inflammatory cytokine IL6 in the body, and detecting NAIL6 levels in plasma predicts the risk of inflammation-related diseases. At present, no stable, accurate, low-cost and easy-to-use NAIL6 detection means exists clinically. The invention adopts a means for relatively quantitatively detecting the rising or falling level of natural IgG antibody (Natural antibody for Interleukin, NAIL 6) of Interleukin6 (IL 6 ), and can accurately detect the NAIL6 level, because the biological half-life of the IgG antibody in blood plasma is 3-5 weeks, and the NAIL6 level is stable in the corresponding detection period (such as 2-3 weeks), thereby being capable of specifically defining the IL6 condition.
The invention designs and synthesizes 2 linear antigen polypeptides highly complementary with target NAIL6 by utilizing advanced sequence analysis means and epitope drawing technology, wherein each polypeptide contains more than 10 high-specificity overlapped epitopes, and an epitope-specific enzyme-linked immunosorbent assay (ELISA) method is established on the basis, so that qualitative and relative quantitative detection of NAIL6 in blood plasma is realized, and an experimental basis is laid for exploring low-toxic side effect to assist in treating novel coronavirus-infected pneumonia (Novel Coronavirus Pneumonia), and long-term accurate treatment of type II diabetes, cardiovascular and cerebrovascular infarction and other inflammation-related diseases. Specifically, the technical scheme of the invention is to provide a linear antigen polypeptide for detecting the concentration of natural antibodies (Natural antibody for Interleukin, NAIL 6) of Interleukin6 (IL 6 ) in human blood plasma, wherein the linear antigen polypeptide can be a single polypeptide or a composite polypeptide, and a kit for detecting NAIL6 in human blood plasma can be prepared. The antigen polypeptide is an epitope of IL6 inflammatory cytokines. These antigen binding sites are capable of specifically binding to autoantibodies to IL6 in human plasma.
In the present invention, "natural antibody" is used interchangeably with "autoantibody" and refers to a mixture of antibodies (monoclonal and/or polyclonal) naturally occurring in the body that recognize an epitope of a substance of interest (e.g., a protein of interest, such as an inflammatory cytokine). The "natural interleukin6 antibody" or "NAIL6" as defined in the present invention is a mixture of a plurality of antibodies (monoclonal and/or polyclonal) which are naturally present in a human body and which recognize an epitope of interleukin6, and may be, for example, a natural IgG antibody which recognizes an epitope of interleukin 6. In some embodiments of the invention, the "natural interleukin6 antibody" or "NAIL6" refers to a mixture of antibodies (monoclonal and/or polyclonal) that recognize one or both polypeptide sequences selected from SEQ ID No. 1 and SEQ ID No. 2. The NAIL 6-enriched plasma may have the effect of treating or preventing an IL 6-associated disorder (i.e., a disorder characterized by an increase in IL 6). Furthermore, since circulating antibody levels are relatively stable, detection of NAIL6 levels in plasma may predict the risk of IL 6-related disease, and lower NAIL6 levels in plasma or negative may be at a higher risk of IL 6-related disease. Whereas those with low or negative NAIL6 levels in plasma can prevent IL 6-related diseases by periodic administration of NAIL 6-enriched plasma.
It has been recognized that antigen-antibody binding occurs primarily between an epitope (i.e., an epitope) and an antibody binding site. Therefore, the closer the two are to complete complementation in space structure and configuration, the more stable the binding of antigen-antibody, the stronger the specificity and the higher the binding efficiency, 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 properties of the whole protein antigen and antibody.
The inventor uses immunoinformatics method and computer software to carry out epitope mapping of human leukocyte class II antigen (Human leukocyte antigen II, HLA-II) on IL6 protein sequence, and screens sequences with high affinity, thereby designing HLA-II restriction epitopes and linear antigen polypeptides which can be recognized by most human antigen presenting cells. According to the biological characteristics of IL6 protein, the invention carries out immunoinformatics prediction and simulation on a plurality of epitopes of the protein, and respectively designs two linear 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 sequence is shown in table 1.
TABLE 1 two linear epitope polypeptide sequences for detecting NAIL6 in human plasma
Full-length amino acid sequence (212 amino acids) of IL6 molecule and IL6a linear antigen epitope polypeptide region
Full-length amino acid sequence (212 amino acids) of IL6 molecule and IL6b linear antigen polypeptide region
The antigen polypeptide is synthesized by a solid-phase chemical method, and can be used for preparing ELISA antibody detection kits respectively or by mixing, and the NAIL6 concentration in human blood plasma can be detected according to set flow specifications. The detection reagent comprising one or two antigen polypeptides can be prepared into a simple and easy-to-use convenient kit in practical application, and can be vacuum-sealed and packaged by nonmetallic materials such as glass, medical plastic and the like, and can be stored for more than 6 months in a 4-DEG C (4 ℃) environment. Briefly, a 96-well microplate activated with Maleimide (Maleimide) is coated with one or a mixture of two antigen polypeptides, dried in a 45 degree (45 ℃) oven, and vacuum-sealed with a nonmetallic packaging material to make a kit. Preferably, the mixed solution of the two antigen polypeptides comprises the two antigen polypeptides with the mass volume concentration ratio of 1:1. Preferably, either or both of the antigenic polypeptides are preparations having a purity of > 95%.
Thus, according to one of the present invention there is provided a detection reagent useful for detecting human plasma NAIL6 comprising either or both of the following two antigenic polypeptides:
H-YLQNRFESSEEQARAVQMSTKVLICH-OH (SEQ ID NO: 1); and
H-DLTKLQAQNQWLQDMTTHLILRSFKC-OH(SEQ ID NO:2)。
in some embodiments, the detection reagent consists of any one or both of the two antigen polypeptides.
In some embodiments, either or both of the two antigenic polypeptides are high purity preparations, preferably chemical syntheses having a purity of > 95%.
In some embodiments, the two antigenic polypeptides may be used in a mixture, where the two antigenic polypeptides may be mixed in equal mass proportions. Thus, in some embodiments, the detection reagent is a mixture of the two antigen polypeptides, for example, may be a mixed solution. In some embodiments, the ratio of two antigen 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 detection reagent.
In some embodiments, the kit comprises a microplate, the detection reagent being coated within a well of the microplate.
In some embodiments, in the kit, the microplate coated with the above-described detection reagent is dried and then vacuum-sealed and packaged with a nonmetallic medical packaging material. In some embodiments, the microplate is a Maleimide (maleimid) activated 96-well microplate.
In some preferred embodiments, the nonmetallic medical packaging material is glass or medical grade 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 microplate. The positive control may also be referred to as a positive standard, e.g., a human anti-IL 6 antibody, and the negative control may be, e.g., any reagent that does not contain an IL6 antibody, e.g., an albumin solution.
As another aspect of the present invention, there is provided a method for detecting the concentration of NAIL6 in a sample to be tested using the above-described detection reagent or the above-described kit. In some embodiments, the method comprises detecting the level of NAIL6 in the test sample by an antigen-antibody binding reaction using the detection reagents described above. The method is preferably performed in vitro and the aim is to obtain test data per se without diagnostic purposes.
In some embodiments, the method comprises allowing an antigen-antibody binding reaction to occur between the detection reagent and NAIL6 in the test sample, and determining the level of NAIL6 in the test sample. Techniques for detecting or determining the level of antibodies in a sample to be tested by antigen-antibody binding reactions using antigen polypeptides are well known in the art, such as enzyme-linked immunosorbent assay (ELISA) methods. In a preferred embodiment, the NAIL6 level in a test sample is detected or determined by an enzyme-linked immunosorbent assay (ELISA) method in the above-described method.
In a more preferred embodiment, the enzyme-linked immunosorbent assay is a sandwich ELISA.
In some embodiments, the method comprises the steps of: (1) Mixing and incubating the detection reagent with a sample to be detected, a negative control and a positive control respectively under the condition suitable for antigen-antibody binding reaction between the detection reagent and NAIL6 in the sample to be detected, adding an enzyme-labeled secondary antibody for incubation, adding a color-developing agent, stopping the reaction after color development, and detecting an optical density value (OD); (2) determining the relative level of NAIL 6. The relative level refers to the relative level of NAIL6 in the test sample relative to the positive control.
In some embodiments, the relative levels of NAIL6 are determined using a Specific Binding Ratio (SBR). In some embodiments, the SBR calculation formula is:
SBR= [ OD value of sample to be measured-NC OD value ]/[ OD value of positive standard-NC OD value ]
Where NC is a negative control. The concentration of antibodies in a sample (e.g., plasma) is expressed as SBR mean and standard deviation.
In some embodiments, a NAIL6 positive sample threshold may be set. The results of sample testing of the normal population were used and the threshold for NAIL6 positive samples was determined by percentile method. The sample detection result is negative when being lower than the threshold value, which means that the NAIL6 content in the sample is lower; a positive sample detection result above the threshold value indicates that the NAIL6 content in the sample is higher. In some embodiments, the normal population may be normal adults. Normal populations may also be referred to as healthy populations, meaning populations that have not had diabetic patients and other severe inflammation-related disorders as determined by clinical physical examination. The percentile method may be, for example, the 95 th percentile method, the 90 th percentile method, or the like. The NAIL6 positive sample threshold is determined to screen out samples with relatively high NAIL6 content, and thus is not limited to a particular normal population, nor to a particular percentile when determining the threshold. For example, the detection may be performed using existing normal population detection results, or using a new set of samples of normal population. The number of individuals in the normal population may be arbitrarily selected as long as the detection result thereof has a statistical significance, and for example, it may be not less than 50, not less than 100, not less than 200, or the like. In different cases, different thresholds may be set according to different needs for different purposes. For example, when it is desired to screen a sample for a lower level of NAIL6 (e.g., a lower level of NAIL6 in a plasma sample, and possibly a higher risk of developing IL 6-related disease), a lower percentile may be used to determine the threshold, e.g., the 5 th percentile, the 10 th percentile, etc., where a negative sample is a lower level of NAIL6 in the sample; when a sample enriched in NAIL6 (i.e., a sample in which the NAIL6 content of the sample is relatively high in the population) is to be screened for treatment or prevention of an IL6 related disorder, a threshold can be determined using a higher percentile, e.g., 95 th percentile, 90 th percentile, etc., where the positive sample is NAIL6 enriched plasma.
The person skilled in the art will readily be aware of conditions suitable for an antigen-antibody binding reaction between the detection reagent and the NAIL6 in the sample to be tested, for example from conventional antigen-antibody reaction conditions and/or from routine experimentation.
In some embodiments, the mixed incubation of step (1) above comprises coating the detection reagent described 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' -Tetramethylbenzidine (TMB). In some embodiments, the reaction is terminated using a sulfuric acid solution, preferably at 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 630nm.
In some embodiments, the sample is human plasma. In some embodiments, the sample is healthy or normal human plasma. In some embodiments, the sample is more preferably a single individual's plasma. The healthy or normal person may be a person who has not had a diabetic patient and other severe inflammation-related disorders as determined by clinical physical examination. In some embodiments, the individual's plasma is plasma from a single healthy individual or a single normal individual.
In a more preferred embodiment, the step (1) includes:
dissolving two antigen polypeptides listed in Table 1 with 67% acetic acid to obtain 5 mg/ml stock solution, respectively, and storing in-20deg.C refrigerator, or mixing two stock solutions at equal volume;
diluting the independent 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 Maleimide (Maleimide) activated 96-well microplate with a working solution, and after overnight incubation at 4 ℃, washing the plate 3 times with a wash solution of 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, wherein the pH value is between 7.0 and 7.4;
the plasma sample to be tested is provided with double-compound holes, and 2 Negative Control (NC) holes (which are negative control liquids without anti-IL 6 antibodies, such as bovine serum albumin, thereby reflecting experimental index values of two polypeptide antigens in a NAIL6 negative reaction system as shown in table 1) and 2 Positive Control (PC) holes (which are a mixture of human anti-IL 6 (SEQ NO: 1) antibodies and human anti-IL 6 (SEQ NO: 2) antibodies), thereby reflecting experimental index values of two polypeptide antigens in a NAIL6 positive reaction system as shown in table 1);
diluting a plasma sample to be detected by using an analysis solution in a ratio of 1:100-200, 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 between 7.0 and 7.4, and 100 mu l of the solution is added into each hole; incubating for 1-2 hours at 20-25 ℃, and then washing the plate for 3 times;
diluting horseradish peroxidase-labeled goat anti-human IgG antibody (for verifying whether the detected substances in the blood plasma are specific antibodies) with an analysis solution (namely 0.1M phosphate buffer solution containing 0.15M sodium chloride and 10mM EDTA, wherein the pH value is between 7.0 and 7.4), wherein the dilution ratio of the antibody is 1:10000-1:50000, adding 100 μl of the antibody into each well, and incubating at 20-25 ℃ for 1-2 hours;
washing the plate 3 times with washing solution (0.1M phosphate buffer solution containing 0.15M sodium chloride and 0.1% TWEEN-20, pH value between 7.0-7.4), adding 100 μl of mixed solution of 3,3', 5' -Tetramethylbenzidine (TMB) and peroxidase into each well, and keeping away from light at room temperature for 20-30 minutes;
mu.l of a 12% sulfuric acid solution (12% H) of stop solution was added to each well 2 SO 4 ) The Optical Density (OD) value was then measured with a microplate reader at a wavelength of 450nm and a reference wavelength of 630nm, and the assay was completed within 10 minutes after the addition of the stop solution, thereby relatively quantifying NAIL6 levels in the plasma of different individuals.
In some embodiments, the level of NAIL6 in plasma can be measured in vitro for type II diabetics, and in the case of a population random sampling assay, the data obtained for each individual measurement can be analyzed.
In another aspect of the invention, there is provided the use of the above detection reagent or kit for detecting Interleukin6 (il 6 ) natural antibodies (Natural antibody for Interleukin6, NAIL 6) in a sample. In some embodiments, the use is for in vitro, non-diagnostic purposes.
In another aspect of the invention, there is also provided the use of the above detection reagent in the preparation of a reagent for detecting Interleukin6 (Interleukin 6, il 6) natural antibody (Natural antibody for Interleukin6, nail 6) in a sample.
In a preferred embodiment, the sample is human plasma, more preferably plasma of a single individual.
In a more preferred embodiment, the individual's plasma is plasma from a healthy individual.
In another aspect of the invention there is also provided the use of the above detection reagent or kit for screening human plasma enriched in IL6 natural antibodies (Natural antibody for interleukin, NAIL 6).
Based on the scheme, the invention provides a NAIL6 detection technology with high precision, simple operation and moderate cost, and further provides a relative quantitative analysis and application scheme for NAIL6 on the basis of the NAIL6 detection technology, thereby laying an important foundation for predicting the onset risk of inflammation related diseases and developing a brand-new immunotherapy strategy with low side effect.
The detection system provided by the invention is an accurate relative quantitative method, can detect NAIL6 in blood plasma with reasonable cost, and plays a key role in the application of NAIL 6-enriched blood plasma and NAIL 6-enriched gamma globulin in the anti-inflammatory field with low toxic and side effects.
In summary, the invention discovers a group of brand-new antigen polypeptides and designs a practical kit, so that an effective and convenient human plasma NAIL6 detection means is provided, and the method can be used for auxiliary qualitative and relative quantitative detection of inflammatory cytokine autoantibodies such as NAIL6, auxiliary quantitative detection of NAIL6 levels of different individual plasmas and differentiation of the plasmas rich in NAIL6 (strong positive) and free of NAIL6 (negative). It is inferred that NAIL 6-enriched plasma may have a prophylactic or therapeutic effect on inflammation-related disorders. Therefore, the invention provides an important tool for the biological pharmaceutical company to develop new products such as tolizumab (actera or Tocilizumab) and new measures for preventing and treating inflammation related diseases in clinical medicine development. Because the antigen polypeptide synthesis means of the invention is relatively simple and has moderate cost, an important foundation is laid for the next clinical practice of applying NAIL 6-enriched plasma to prevent and treat inflammation-related diseases and evaluating the risk of inflammation-related diseases of individuals with negative plasma NAIL 6. In addition, whether the plasma NAIL6 negative individual detected by the product has higher risk of inflammation related diseases or not can be subjected to clinical follow-up and tracking, so that a positive conclusion can be obtained, and the aim of early implementation and control is fulfilled.
Detailed description of the preferred embodiments
In specific embodiments, the following is given in table 1 for 1 of two antigen polypeptides: the technical scheme of the invention is specifically illustrated by taking the mixed solution as a detection reagent. If a detection reagent containing only one antigen polypeptide is used, the mixed solution of two antigen polypeptides is changed into a 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 manipulation, each antigen was dissolved in 67% acetic acid to 5 mg/ml stock, then mixed in equal volumes and stored in a-20℃ (error within.+ -. 2℃) refrigerator.
2. At the beginning of the procedure, the mixture of the 2 antigen polypeptides listed in Table 1 was first diluted to 10-50. Mu.g/ml with a coating solution of 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA at a pH of between 7.0 and 7.4.
3. A96-well microplate (Corning, USA) activated with Maleimide (Maleimide) was coated with working solution, and after overnight 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 at a pH between 7.0 and 7.4.
4. Then the sample is added and analyzed step by step according to the following steps:
(1) The plasma sample to be tested was provided with double wells, and 2 Negative Control (NC) wells (reference was a negative control solution without anti-IL 6 antibody, such as bovine serum albumin (Sigma-Aldrich, inc.), thereby reflecting the experimental index values of the 2 polypeptide antigens in the NAIL6 negative reaction system described in Table 1) and 2 Positive Control (PC) wells (reference was a polyclonal antibody mixture of human anti-IL 6 full-length protein, thereby reflecting the experimental index values of the 2 polypeptide antigens in the NAIL6 positive reaction system described in Table 1).
(2) Diluting the plasma sample to be tested by using an analysis solution in a ratio of 1:100-200, wherein the analysis solution is the same as the antigen coating solution, namely 0.1M phosphate buffer solution containing 0.15M sodium chloride and 10mM EDTA, the pH value is between 7.0 and 7.4, 100 mu l of the solution is added to each hole, the solution is incubated for 1-2 hours at 20-25 ℃, and then the plate is washed for 3 times.
(3) The goat anti-human IgG antibody marked by horseradish peroxidase (used for verifying whether the detected substances in the blood plasma are specific antibodies) is diluted by an analysis solution (namely 0.1M phosphate buffer solution containing 0.15M sodium chloride and 10mM EDTA, wherein the pH value is between 7.0 and 7.4), the dilution ratio of the antibody is 1:10000-1:50000, 100 mu l of the antibody is added to each hole, and the mixture is incubated for 1-2 hours at 20-25 ℃.
(4) After washing the plates 3 times with wash 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 3,3', 5' -Tetramethylbenzidine (TMB) was added to each well and protected from light at room temperature for 20-30 minutes.
(5) Mu.l of a 12% sulfuric acid solution (12% H) of stop solution was added to each well 2 SO 4 ) The Optical Density (OD) value was then measured with an enzyme-labeled instrument at a wavelength of 450nm and a reference wavelength of 630nm. The assay procedure was completed within 10 minutes after the addition of the stop solution, thereby relatively quantitatively analyzing the NAIL6 levels in the plasma of different individuals.
(6) Definition of a threshold for positive IgG levels in plasma enriched with anti-IL 6 Natural antibodies
(7) When analyzing the data obtained by the detection, the specific binding ratio (Specific binding ratio, SBR) is adopted to judge the IgG level of the natural antibody rich in IL6 in the blood plasma, and the SBR has the following calculation formula:
SBR= [ OD value of sample to be measured-NC OD value ]/[ positive control OD value-NC OD value ]
NC is the negative control for each sample. The antibody concentration in plasma of all subjects was expressed as SBR mean and standard deviation. The SBR threshold (cut-off) of IL6 natural antibody IgG level positive samples 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-IL 6 natural antibody plasma, and samples with plasma IgG levels below this threshold were defined as negative samples of anti-IL 6 natural antibody plasma. The anti-IL 6-enriched natural antibody plasma can be packaged in standard storage bags (150-200 ml of plasma per bag) and stored in a negative 80 degree (-80 ℃) refrigerator for no more than 6 months.
(8) For in vitro detection of NAIL6 levels in plasma in type II diabetics, a population random sampling analysis may be performed on the data obtained from each individual detection. In some embodiments, the relative levels of NAIL6 in plasma are determined using specific binding ratios (Specific binding ratio, SBR). The SBR calculation method comprises the following steps:
sbr= [ OD value of sample to be measured-NC OD value ]/[ OD value of positive control standard-NC OD value ].
The 95 th percentile method was used to determine the SBR threshold (cut-off) of NAIL6 positive plasma in normal adult plasma samples, below which samples were NAIL6 free (negative) plasma.
Application example one screening of NAIL6 enriched plasma
1. 200 healthy human plasma samples were provided from a central blood station in the city, all of which 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 all synthesized by SEVERN BIOTECH, inc. In the united kingdom, with a purity of 95%, and the specific procedure was as follows:
(2) Before the operation, each antigen was dissolved in 67% acetic acid to 5.0mg/ml stock, the two antigens were mixed in equal volumes and stored in-20 ℃ (error within + -2 ℃) refrigerator.
(3) At the beginning of the procedure, 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 determined to be 7.3.
(4) A Maleimide (Maleimide) -activated 96-well microplate (Thermo Scientific, USA) was coated with the coating solution containing the 2 antigen mixtures obtained in the above step (2), and after incubation overnight (18 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 7.3.
(5) The post step loading analysis was as follows:
a) The plasma samples to be tested were double-countersunk, 2 additional Negative Control (NC) wells (bovine serum albumin, supplied by Sigma-Aldrich) and 2 Positive Control (PC) wells (antibody mixture of human anti-IL 6 whole molecule, supplied by Sigma-Aldrich).
b) Plasma was diluted 1:150 with an assay solution, which was the same as the antigen-coated liquid, 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, pH 7.3, 100 μl per well, and incubated at 25deg.C for 1.5 hours.
c) After washing the plates 3 times with wash solution (i.e., 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, pH 7.3), horseradish peroxidase-labeled goat anti-human IgG antibody (supplied by Sigma-Aldrich Co., ltd.) was diluted with assay solution (0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, pH 7.3) at an antibody dilution ratio of 1:25000, 100. Mu.l was added to each well and incubated at 25℃for 1.5 hours.
d) After washing the plates 3 times with the above washing 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' -Tetramethylbenzidine (TMB) was added to each well and protected from light at room temperature for 25 minutes.
e) After adding 50. Mu.l of a 12% sulfuric acid solution (12% H2SO 4) to each well, the Optical Density (OD) was measured with an enzyme-labeled instrument at a wavelength of 450nm and a reference wavelength of 630nm, and after adding the stop solution, the measurement was completed within 10 minutes, and the subsequent steps were performed for each individual by the relative quantitative comparative analysis of NAIL6 according to the result.
(6) Analyzing the data obtained by the detection, and judging the IgG level of the natural antibody rich in IL6 in the blood plasma by adopting a specific binding ratio (Specific binding ratio, SBR), wherein the SBR has a calculation formula of: sbr= [ OD value of sample to be measured-NC OD value ]/[ OD value of positive control standard-NC OD value ], NC is a negative control for each sample. The concentration of antibodies in plasma was expressed as SBR mean and standard deviation. And determining the positive and strong positive thresholds of the plasma NAIL6 by applying a percentile method to the antibody concentration detection results of 200 healthy human plasma samples, wherein the definition of the plasma IgG antibody level lower than the positive threshold is negative samples, and the definition higher than the strong positive threshold is strong positive samples.
2. Experimental results
In 200 normal adult human plasma samples randomly drawn in this example, the average value of the NAIL6 concentration is sbr=0.78±0.42, and the coefficient of variation is 53.8%. Determining a plasma NAIL6 negative threshold of 0.25 using the 5 th percentile method; the 95 th percentile method was used to determine a strong positive threshold of 1.55 for plasma NAIL6 (shown in Table 1).
TABLE 1 detection of anti-IL 6 antibody concentration in 200 normal adult blood plasma samples at a blood station
As can be seen from Table 1, the number of NAIL6 strong positives was 12 (6%) in 200 normal adult human plasma samples tested.
Application example II type diabetes patient NAIL6 detection
1. Sample collection: 420 plasma samples were provided from a hospital, a blood station, and from both diabetics II and normal adults. The normal adult is an individual who has not had a diabetic patient and other severe inflammation-related disorders as determined by clinical physical examination. All plasma samples were stored at minus 80 degrees (-80 ℃) and thawed in a 4 degree (4 ℃) refrigerator 12 hours prior to use.
2. Sample detection: the 2 polypeptide antigens listed in table 1 used in this experiment were all synthesized by SEVERN BIOTECH, inc. In the united kingdom, with a purity of 95%, and the specific procedure was as follows:
(1) Before the operation, each antigen was dissolved in 67% acetic acid to 5.0mg/ml stock solution, and then mixed in equal volumes to form a coating solution and stored in a-20 ℃ (error within + -2 ℃) refrigerator.
(2) At the beginning of the procedure, 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 determined to be 7.3.
(3) Then, a Maleimide (maleimid) -activated 96-well microplate (Thermo Scientific, U.S.) obtained in the above step (2) was coated with a coating solution containing a mixture of 2 antigens, and after incubation at 4℃overnight (14-18 hours), the plate was washed 3 times with a washing solution of 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, and pH was measured to be 7.3.
(4) The step-wise loading analysis was then as follows:
a. the plasma samples to be tested were double-countersunk, 2 additional Negative Control (NC) wells (bovine serum albumin, supplied by Sigma-Aldrich) and 2 Positive Control (PC) wells (IgG antibody mixture of human anti-IL 6 whole molecule, supplied by Sigma-Aldrich).
b. Plasma was diluted 1:150 with an assay solution, which was the same as the antigen-coated liquid, 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, pH 7.3, 100 μl per well, and incubated at 25deg.C for 1.5 hours.
c. After washing the plates 3 times with wash solution (i.e., 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, pH 7.3), horseradish peroxidase-labeled goat anti-human IgG antibody (supplied by Sigma-Aldrich Co., ltd.) was diluted with assay solution (0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, pH 7.3) at an antibody dilution ratio of 1:25000, 100. Mu.l was added to each well and incubated at 25℃for 1.5 hours.
d. After washing the plates 3 times with the above washing 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' -Tetramethylbenzidine (TMB) (supplied by Life Technologies company) was added to each well, and the plate was protected from light at room temperature for 25 minutes.
e. Mu.l of a 12% sulfuric acid solution (12% H) of stop solution was added to each well 2 SO 4 And (d) and then detecting the Optical Density (OD) value by using an enzyme-labeled instrument, wherein the detection wavelength is 450nm, the reference wavelength is 630nm, and after the stop solution is added, the detection is completed within 10 minutes, and the subsequent steps are carried out relative quantitative comparison analysis of NAIL6 for each individual according to the result.
(5) Analyzing the data obtained by the detection, and judging the NAIL6 level in the blood plasma by adopting a specific binding ratio (Specific binding ratio, SBR) with a calculation formula of SBR: sbr= [ OD value of sample to be measured-NC OD value ]/[ OD value of positive control standard-NC OD value ]. The 95 th percentile method was used to determine the threshold (cut-off) of NAIL6 (negative) free plasma.
3. Experimental results: the average NAIL6 concentration in 220 normal adult human plasma samples randomly drawn in this example was sbr=1.07±0.35. The 5 th percentile method was used to determine that the plasma NAIL6 negative threshold was 0.25 and that the SBR was below 0.25 was negative for human NAIL6 detection. As shown in table 2, of the 200 diabetics tested, 20.5% were NAIL 6-negative; of 220 normal individuals, 4.5% were negative for NAIL6 detection; meridian-chi square (X) 2 ) The difference between the two groups was significant (X 2 ＝25.0，P<0.001)。
TABLE 2 analytical comparison of the results of NAIL6 negative detection rate for 420 type II diabetes mellitus and normal adult human plasma samples
* Represents Odds Ratio (OR)
4. Evaluation of results: the 2 linear antigen polypeptides can be specifically combined with NAIL6 in blood plasma. That is, the amino acid sequence of these 2 linear antigen polypeptides is fully complementary in spatial structure and configuration to the NAIL6 molecule.
The related embodiments and application examples are only some specific embodiments for illustrating the technical solution of the present invention, but the protection scope of the present invention is not limited thereto, and any changes or substitutions that are conceivable by those skilled in the art within the technical scope of the present invention are intended to be covered in the protection scope of the present invention.
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Claims (10)
1. A detection reagent for detecting natural antibodies to IL6, characterized in that: the detection reagent comprises any one or two antigen polypeptides selected from the following two antigen polypeptides:
H-YLQNRFESSEEQARAVQMSTKVLICH-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 is composed of any one or two antigen polypeptides selected from the two antigen polypeptides.
3. The detection reagent according to claim 1 or 2, wherein the detection reagent comprises the two antigen polypeptides, and the mass-to-volume concentration ratio of the two antigen polypeptides is 1:1.
4. A kit for detecting a natural antibody to IL6, characterized in that: the kit comprising the detection reagent of any one of claims 1-3.
5. The kit of claim 4, wherein the kit comprises a microplate, the detection reagent being coated within a well of the microplate.
6. A method for detecting IL6 natural antibodies of non-diagnostic interest in vitro comprising detecting IL6 natural antibodies 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) Mixing and incubating the detection reagent with a sample to be detected, a negative control and a positive control respectively under the condition suitable for antigen-antibody binding reaction between the detection reagent and IL6 natural antibodies in the sample to be detected, adding enzyme-labeled secondary antibodies for incubation, adding a color reagent, stopping the reaction after color development, and detecting an optical density value (OD);
(2) Determining the relative level of the IL6 natural 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 an IL6 natural antibody in a sample.
9. Use of the detection reagent of any one of claims 1-3 or the kit of claim 4 or 5 for in vitro detection of IL6 natural antibodies for non-diagnostic purposes.
10. Use of the detection reagent of any one of claims 1-3 or the kit of claim 4 or 5 for screening human plasma enriched in IL6 natural antibodies.
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