CN119023970A - Purpose and use of combined ELISA kit - Google Patents
Purpose and use of combined ELISA kit Download PDFInfo
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- CN119023970A CN119023970A CN202410964791.3A CN202410964791A CN119023970A CN 119023970 A CN119023970 A CN 119023970A CN 202410964791 A CN202410964791 A CN 202410964791A CN 119023970 A CN119023970 A CN 119023970A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract
本发明涉及免疫分析技术领域，提供组合式ELISA试剂盒的用途及使用方法，组合式ELISA试剂盒用于作为阳性对照品或阴性对照品，以及定量检测人外周血中的SASP衰老相关分泌表型经典因子。本发明所制备的SASP经典因子抗体具有高特异性、高纯度和高稳定性，确保了对目标SASP因子的准确检测，本发明提供了一种直接而有效的非侵入性检测方法，能够定量检测人外周血中的SASP经典因子，从而评估衰老程度。组合式ELISA试剂盒作来阳性和阴性对照品提高了实验的准确性和可靠性，有助于识别实验误差。
The present invention relates to the field of immunoassay technology, and provides the purpose and use method of a combined ELISA kit, wherein the combined ELISA kit is used as a positive control or a negative control, and is used to quantitatively detect SASP aging-related secretory phenotype classical factors in human peripheral blood. The SASP classical factor antibodies prepared by the present invention have high specificity, high purity and high stability, ensuring accurate detection of the target SASP factor. The present invention provides a direct and effective non-invasive detection method, which can quantitatively detect the SASP classical factors in human peripheral blood, thereby evaluating the degree of aging. The combined ELISA kit as a positive and negative control improves the accuracy and reliability of the experiment, and helps to identify experimental errors.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a combined ELISA kit for quantitatively detecting human peripheral blood SASP classical factors and a use method thereof.
Background
The time sequence age-increasing causes common degeneration of the tissue structure and functions of each part of the organism, and the risk of the organism suffering from various chronic diseases in the senile stage is increased to a great extent. For many years, comprehensive studies conducted with mice and the like as model organisms revealed molecular changes that occur across multiple organs during aging, defining unique aging trajectories and corresponding time nodes. Specific organs, such as brain, heart, lung, liver and kidney, have significant differences in susceptibility and resistance to aging disorders in the human population. However, little is known to date about how the human organ changes with age at the molecular level. Understanding human organ aging at the molecular level is critical to solving the global increasing aging diseases and the social and medical burden that they pose, and it is possible to completely change the modes of patient care, preventive medicine and drug development and paradigms of medical intervention under clinical conditions.
Cell aging is an essentially irreversible state of cell cycle arrest, initially thought to be a tumor suppression program, which promotes tissue homeostasis by recognizing damaged cells for immune-mediated clearance. Senescence-associated secretory phenotypes (SENESCENCE-Associated Secretory Phenotype, SASP) are typical features of senescent cells, and can lead to chronic inflammation in a variety of tissues and organs, the extent of which is generally dependent on the specific microenvironment composition in which the senescent cells are located and their pathophysiological properties. SASP canonical factors refer to a series of specific, critical, representative molecules secreted by senescent cells during cellular senescence. These factors have a variety of functions, including affecting surrounding cells, promoting inflammatory responses, affecting tissue repair and regeneration, and the like.
The current general detection method of human blood markers in the medical and health field is widely applied to Enzyme-linked immunosorbent assay (ELISA for short) of Enzyme-Linked Immunosorbent Assay. The development of a diagnosis technology of SASP factors which can be used for monitoring the circulatory state in human peripheral blood, in particular an ELISA kit with high sensitivity, high specificity and high selectivity, is a senescence evaluation means which fully utilizes the international front edge of the SASP research field to promote the clinical detection target of the serum of elderly individuals or elderly related disease patients, and has great significance.
Disclosure of Invention
In order to solve the problems in the background technology, the invention provides the application of the combined ELISA kit as a positive control or a negative control, wherein the combined ELISA kit is pre-packaged with a micro-pore plate, a buffer solution, a sealing solution, a sample diluent, an enzyme diluent, a SASP factor standard substance, a SASP classical factor antibody, a biological enzyme marked SASP classical factor antibody and a luminescent substrate, wherein the SASP classical factor antibody is a specific polyclonal antibody of a human cell SASP classical exocrine factor.
The use method of the combined ELISA kit for the positive control or the negative control comprises the following steps:
S1, coating a microplate: mixing SASP classical exofactor antibody of a combined ELISA kit with buffer solution, adding into a micro-pore plate for coating, and then sealing the micro-pore plate by using sealing liquid to form a coating plate;
S2, preparing standard products and samples: mixing a SASP classical exocrine factor standard with a sample diluent to prepare an antigen standard; meanwhile, mixing the clinical blood supernatant to be tested with a sample diluent to prepare a solution to be tested;
S3, incubation and incubation: respectively adding a solution to be detected, an antigen standard substance and a biological enzyme marked SASP classical exocrine factor antibody solution into a coating plate for incubation, and finally adding a luminescent substrate for incubation;
s4, detecting a luminous value: detecting the luminescence value of each hole by a luminescence instrument;
S5, data analysis: calculating the content of SASP classical exocrine factors in a sample to be detected according to a standard curve;
s6, using a reference substance: adding a sample known to contain a target SASP classical factor to the assay to verify the detection capacity of the kit; the positive reference substance generates a signal to confirm that the experimental system can detect the target molecule; or:
sample dilutions without the classical factor of the target SASP were used as negative controls to ensure that no non-specific binding or background signal was present in the experiment.
The combined ELISA kit is used for detecting the content of SASP classical exocrine factors in a sample, and is pre-packaged with a microplate, a buffer solution, a blocking solution, a sample diluent, an enzyme diluent, a SASP factor standard, a SASP classical factor antibody, a biological enzyme-labeled SASP classical factor antibody and a luminescent substrate, wherein the SASP classical factor antibody is a specific polyclonal antibody of a human cell SASP classical exocrine factor.
The application of the combined ELISA kit as the detection of the SASP classical exocrine factor content in a sample is characterized in that: the preparation process of the SASP classical factor antibody comprises the following steps:
step one, molecular cloning 8 humanized SASP classical factors IL1a, IL1b, IL6, IL8, MMP1, MMP3, GM-CSF, SPINK1 in pET-28 fusion protein prokaryotic expression vector (5369 bp);
step two, designing a primer by using a T7 promoter sequence as a basis, determining an MCS section gene ID by sequencing, and sequentially transferring the vector into BL21DE3 competent escherichia coli strains;
Step three, after inducing the strain to express target proteins, obtaining high-purity recombinant proteins with N-terminal His labels through affinity chromatography;
Step four, sequentially injecting purified proteins into New Zealand white rabbits for immunization, and carrying out titer detection; the sample comprises gradient titer detection of preimmune negative blood control, first immunization, second immunization, third immunization and fourth immunization, and titer detection data is analyzed; the immunization period was 2 months;
Step five, animal serum collection and a small amount of antibody purification; after the immunization is finished, serum is collected in batches according to the immunization time for 7-12 days in a collection period; the volume of whole blood collected by each rabbit is not less than 50ml, and the volume of serum is not less than 30ml;
Step six, purifying the polyclonal antibody through a column; the antibody is purified and identified in small quantity, and the basic purity is determined according to SDS-PAGE gel diagram.
The use method of the combined ELISA kit for detecting the SASP classical exogen content in a sample comprises the following steps:
P1, mixing SASP classical exofactor antibodies with a buffer solution one by one, adding the obtained antibody solution into a micro-pore plate for coating, and then adding a sealing solution into the micro-pore plate for sealing to obtain a coated plate;
P2, mixing a sample to be detected with a sample diluent to prepare a solution to be detected and an antigen standard substance;
P3, mixing the SASP classical exocrine factor standard with a sample diluent to prepare an antigen standard;
p4, mixing the biological enzyme marked SASP classical exofactor antibody with enzyme diluent to obtain biological enzyme marked SASP classical exofactor antibody solution;
P5, respectively adding the solution to be detected and the antigen standard substance into a coating plate for incubation, then adding a biological enzyme marked SASP classical exofactor antibody solution for incubation, and finally adding a luminous substrate for incubation;
And P6, detecting a luminescence value, and obtaining the content of the SASP classical exocrine factor in the sample to be detected according to a standard curve.
The application method of the combined ELISA kit for detecting the SASP classical exofactor content in a sample is characterized in that the coating concentration of the SASP classical exofactor antibody is 4-10 mug/mL.
The application method of the combined ELISA kit for detecting the SASP classical exocrine factor content in a sample is characterized in that the volume ratio of the SASP classical exocrine factor antibody marked by biological enzyme to enzyme diluent in the SASP classical exocrine factor antibody solution marked by biological enzyme is 1 (2500-3500)
The preparation process of the SASP classical factor antibody comprises the following steps:
step one, molecular cloning 8 humanized SASP classical factors IL1a, IL1b, IL6, IL8, MMP1, MMP3, GM-CSF, SPINK1 in pET-28 fusion protein prokaryotic expression vector (5369 bp size); molecular cloning is a biotechnological method for replicating a specific DNA fragment in vitro and inserting it into a vector for replication or expression in an organism (e.g. a bacterial, yeast or mammalian cell). SASP factors are a group of proteins and other molecules released by senescent cells that can affect the cellular microenvironment, with important effects on cell fate, tissue homeostasis, and disease progression. The eight factors are respectively: IL1a (interleukin 1 a), IL1b (interleukin 1 β), IL6 (interleukin 6), IL8 (interleukin 8), MMP1 (matrix metalloproteinase 1), MMP3 (matrix metalloproteinase 3), GM-CSF (granulocyte-macrophage colony stimulating factor), and SPINK1 (trypsin inhibitor 1).
Prokaryotic expression vector of pET-28 fusion protein: pET-28 is a plasmid vector for expression of recombinant proteins in prokaryotes such as E.coli. By fusion protein prokaryotic expression vector is meant that the vector is designed to express a fusion protein, i.e., fusing the protein of interest to a tag or domain (e.g., an affinity tag) for subsequent purification or detection. "5369bp" means the linear size or length of this vector, i.e., it contains 5369 base pairs.
Step two, designing a primer by using a T7 promoter sequence as a basis, determining an MCS section gene ID by sequencing, and sequentially transferring the vector into BL21 competent escherichia coli strains; the primer was designed using the T7 promoter sequence as a basis:
The T7 promoter is a commonly used strong promoter, and is commonly used for efficiently expressing exogenous genes in Escherichia coli. Primers are usually designed to amplify specific DNA fragments by PCR (polymerase chain reaction). In this case, the primer is designed based on the T7 promoter sequence in order to amplify a DNA fragment containing the promoter, so that the T7 promoter can be used to drive efficient expression of the target gene in the subsequent transformation and expression processes.
Sequencing to determine MCS segment gene ID:
MCS (multiple cloning site) is a region on a vector that contains multiple different restriction sites for insertion of foreign DNA fragments. Dideoxy end termination sequencing is a method of determining the sequence of inserted DNA fragments in the MCS section so that the corresponding ID (identity or identifier of the gene) of the inserted gene can be determined. This helps to verify that the inserted DNA fragment is correct and to determine the protein information it encodes.
Sequentially transferring the vectors into BL21 competent escherichia coli strains:
BL21 is a commonly used E.coli strain, commonly used to express genes driven by the T7 promoter. Competent cells refer to cells that have been specifically treated to be able to readily take up and integrate exogenous DNA. The transfer of the vector (usually referred to as plasmid) into BL21 competent cells means that the plasmid containing the target gene is introduced into E.coli to express the target protein in the cells.
Step three, after inducing the strain to express target proteins, obtaining high-purity recombinant proteins (N-terminal His tags) through affinity chromatography; preliminary yield after purification of each protein was 5mg; inducing the strain to express target proteins:
after transferring the plasmid containing the target gene into BL21 E.coli, it is necessary to induce the expression of the target protein by specific conditions (e.g., addition of the inducer IPTG). This is because the T7 promoter is usually activated under specific conditions (e.g., IPTG induction) to drive transcription and translation of the target gene to produce the target protein.
Obtaining high-purity recombinant protein with His tag at N end through affinity chromatography:
Affinity chromatography is a method of separation and purification based on high affinity between proteins and specific ligands. In this step, the interaction between the His tag at the N-terminus of the recombinant protein and the metal ion on the affinity chromatography column is exploited. When the solution containing the recombinant protein flows through the chromatographic column, the His-tagged protein can be mutually combined with metal ions, and other impurities can not be combined with the metal ions or are combined with the metal ions weakly, so that the protein is eluted. Then, by setting elution conditions (e.g., determining the pH and/or ionic strength of an eluent in a certain range), the recombinant protein bound to the metal ions can be eluted, thereby obtaining the recombinant protein with high purity meeting the requirements of biochemical experiments or practical applications.
Step four, sequentially injecting purified proteins into New Zealand white rabbits for immunization, and carrying out titer detection; the sample comprises gradient titer detection of preimmune negative blood control, first immunization, second immunization, third immunization and fourth immunization, and titer detection data is analyzed; the immunization period was 2 months;
Step five, animal serum collection and a small amount of antibody purification; after the immunization is finished, serum is collected in batches according to the immunization time for 7-12 days in a collection period; the volume of whole blood collected by each rabbit is not less than 50ml, and the volume of serum is not less than 30ml;
Step six, purifying the polyclonal antibody through a column; the antibodies were purified and identified in small amounts, and the basic purity was determined from SDS-PAGE gel, and each batch of purification was provided with the corresponding gel identification as a reference.
Purifying by column: "passing through a column" generally refers to purification using a chromatography column (chromatography column). The column is packed with specific materials (e.g., gels, resins, etc.) that interact with the different components of the mixture in different ways to effect separation.
In antibody purification, the choice of chromatographic column and the operating conditions are determined according to the identity and desired purity of the antibody. When the antibody solution passes through the chromatographic column, impurities with weak binding force with the target antibody are eluted first, and the target antibody is collected in the subsequent step by changing the eluting condition.
Purifying and identifying: a small amount of antibody is typically purified and identified prior to large scale purification. This is to evaluate the effectiveness of the purification conditions and to ensure that the final purification results are as expected.
Purification and identification methods are various, but SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a common method. The separation of the antibody on the gel can be observed by SDS-PAGE, and the purity thereof can be primarily judged.
SDS-PAGE gel analysis: SDS-PAGE gel is a visual way to show the purity of antibodies. On the gel diagram, proteins of different molecular weights form different bands. The bands of the target antibody should be clear, single, and consistent with the expected molecular weight.
By analyzing the gel diagram, whether the purity of the antibody meets the requirement can be judged. The presence of multiple bands or unclear target bands on the pectin patterns may mean that the purity of the antibodies is not high enough and further adjustments of the purification conditions are required.
Each batch of purification provided the corresponding gum-plot identification as a reference: to ensure consistent high purity of each batch of purified antibody, SDS-PAGE analysis is typically performed after each purification and the corresponding gel map is provided as a reference. The purpose of this is to conveniently track and compare the purities of different batches of antibodies, and to ensure the stability and consistency of the product quality.
The beneficial effects achieved by the invention are as follows:
First, the detection process of the present invention is a direct and efficient non-invasive method that can detect senescence-associated proteins or metabolites to determine the senescence load in blood, can assist in future large-scale plasmaphorosis and machine learning, and can be used to non-invasively measure organ health and the degree of senescence in humans. The present invention uses a series of organ-specific proteins and fibrin, or protein molecules closely related to physiological age, to evaluate and monitor an individual. The technology has the potential of deconvolution of aging rates of different organs inside an individual by combining clinical diagnosis and intervention strategies of aging-promoting medicine in the future, so that aging is monitored and evaluated in aspects of organ level resolution and the like. Finally, through the determination of the luminescence value and the application of a standard curve, the content of the SASP classical factor in the sample can be accurately calculated, and quantitative data is provided for aging evaluation.
Secondly, the invention improves the accuracy of the experiment by using the positive and negative reference substances, which not only verifies the performance of the experiment operation and the kit, but also ensures the accuracy of the experiment result. Meanwhile, the use of the reference substance enhances the reliability of the experiment, and is helpful for identifying errors, such as cross contamination or misoperation, possibly occurring in the experimental process. In addition, by adding a certain concentration of the SASP classical factor antibody as a neutralizing antibody to the sample dilution, the detection range is enlarged, thereby enabling a more comprehensive assessment of the SASP classical factor level in the sample. The ELISA kit provides a direct and effective non-invasive detection method, and is particularly suitable for rapid detection under clinical conditions. In clinical application, the technology can monitor SASP factors in human peripheral blood, and has important potential value for evaluating the aging degree. In addition, quantitative detection of the SASP classical factor helps to drive the development of intervention strategies in the field of aging medicine.
In the preparation process of the SASP classical factor antibody, the specific polyclonal antibody of the human cell SASP classical exocrine factor is utilized for specific recognition, so that the accurate detection of the target SASP factor is ensured. Then, the recombinant protein with high purity and His tag at the N end is obtained by an affinity chromatography technology, so that the quality and the titer of the antibody are ensured. By immunizing New Zealand white rabbits and performing potency detection, the prepared antibodies are ensured to have high immunoreactivity, which is critical for subsequent ELISA detection. In the antibody purification stage, the purity and quality of the antibody are further improved through animal serum collection and a small amount of purification and combination of column purification steps. The purified antibodies of each batch were identified by SDS-PAGE gel to ensure stability and consistency of product quality. This series of refined procedures together ensure high specificity, high purity and high stability of the SASP classical factor antibodies.
Drawings
FIG. 1 is a graph showing the result of linear regression curve fitting of the luminescence values of the standard of comparative example 1;
FIG. 2 is a graph showing the result of linear regression curve fitting of the luminescence values of the standard of example 1.
FIG. 3 shows a signal diagram of a nucleotide sequence of pET28-IL1a after DNA sequencing.
FIG. 4 shows a signal diagram of a nucleotide sequence of pET28-IL1b after DNA sequencing.
FIG. 5 shows a signal diagram of a nucleotide sequence of pET28-IL6 after DNA sequencing.
FIG. 6 shows a signal diagram of a nucleotide sequence of pET28-IL8 after DNA sequencing.
FIG. 7 shows a signal pattern of a nucleotide sequence after DNA sequencing of pET28-MMP1 in a molecular cloning step.
FIG. 8 shows a signal pattern of a nucleotide sequence after DNA sequencing of pET28-MMP3 in a molecular cloning step.
FIG. 9 shows a signal pattern of a nucleotide sequence after DNA sequencing of pET28-GM-CSF in the molecular cloning procedure.
FIG. 10 shows a signal diagram of a nucleotide sequence of pET28-SPINK1 after DNA sequencing.
Detailed Description
The technical scheme of the invention will be clearly, completely and accurately described below with reference to the accompanying drawings. The configurations of the structures described in the following embodiments are merely examples, and the present invention is not limited to the structures described in the following embodiments, but all other embodiments obtained by a person skilled in the art without making any creative effort are within the scope of protection of the present invention.
Referring to fig. 1-10, the invention discloses a combined ELISA kit for quantitatively detecting human peripheral blood SASP classical factors and a use method thereof, and a person skilled in the art can refer to the content of the specification to properly improve the process parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
English and Chinese contrast:
ADP: adenosine diphosphate;
casein: casein;
SASP classical factor: IL1a, IL1b, IL6, IL8, MMP1, MMP3, GM-CSF and SPINK1;
IL1a is interleukin 1 alpha, IL1b is interleukin 1 beta, IL6 is interleukin 6, IL8 is interleukin 8, MMP1 is matrix metalloproteinase 1, MMP3 is matrix metalloproteinase 3, GM-CSF is granulocyte-macrophage colony stimulating factor, and SPINK1 is pancreatic secretion trypsin inhibitor 1;
PBST: english full name phosphatebufferedsolution, namely phosphate buffer salt containing Tween-20.
The invention is suitable for instruments:
sample feeder, incubator, plate washer, vibration mixer and luminometer.
Sample requirements:
1) Before use, the sample is balanced for more than 30 minutes at room temperature, and the sample is uniformly mixed before the experiment of freezing and storing.
2) Samples containing suspended fibrin or aggregates, and severely hemolyzed cannot be detected.
3) The sample should be free of microorganism, and the aseptically separated sample can be stored at 2-8deg.C for one week, and stored at low temperature for long term to avoid repeated freezing and thawing.
Preparation before experiment:
1) 1 bag of the solid washing liquid was dissolved in 500ml of distilled water and used.
2) The kit and the sample to be tested were removed and all reagents and samples were returned to room temperature (18-25 ℃).
3) And (5) adjusting the temperature of the incubator or the water bath kettle to the reaction temperature.
4) The luminescent substrate A, B solution was mixed in equal proportion 30 minutes in advance to the volume required for this test.
The kit is stored at 2-8 ℃ and has a valid period of 6 months. The kit was equilibrated to room temperature (about 30 minutes) prior to use. The unused microplates must be stored with desiccant and its packaging sealed with a self-sealing bag at 2-8 ℃. The liquid reagent is gently mixed by shaking before the experiment, and is immediately sealed and stored at 2-8 ℃.
The reagent kit for quantitative detection of SASP classical factor and the reagent or instrument used in the detection method for clinical diagnosis purpose provided by the invention can be purchased from the market.
The invention provides a combined ELISA kit for quantitatively detecting a human peripheral blood SASP classical factor, which comprises a SASP classical factor antibody, wherein the SASP classical factor antibody is a specific polyclonal antibody of a human cell SASP classical exocrine factor. The combined ELISA kit for quantitatively detecting the human peripheral blood SASP classical factor is pre-packaged with a microplate, a buffer solution, a sealing solution, a sample diluent, an enzyme diluent, a SASP factor standard, a SASP classical factor antibody, a biological enzyme-labeled SASP classical factor antibody and a luminescent substrate.
The biological enzyme marked SASP classical exofactor antibody is horseradish peroxidase marked SASP classical exofactor antibody. The buffer solution is CB buffer solution with the molar concentration of 0.01-0.1M and the pH value of 90-100. The preservative is ProClin300, 300 TM antibacterial agent.
The sample diluent contains:
SASP classical factor antibody with mass fraction of 5-15 mug/ml; the SASP classical factor antibody is a polyclonal antibody of any one of human cell SASP classical exofactors IL1a, IL1b, IL6, IL8, MMP1, MMP3, GM-CSF and SPINK 1;
PBST with mass fraction of 15-25mg/ml, wherein the PBST is phosphate Tween buffer; fetal bovine serum with the volume fraction of 13-17 vt%; preservative with volume fraction of 0.08-0.12 vt%; make up the water used for proportioning. In a preferred embodiment, the enzyme diluent comprises: PBST with mass fraction of 20g/l, wherein the PBST is phosphate Tween buffer; bovine serum albumin with mass fraction of 10 g/l; preservative with volume fraction of 0.1 vt%; make up the water used for proportioning.
The invention also provides the application of the combined ELISA kit for quantitatively detecting the SASP classical factor in the peripheral blood of the human body as a positive reference substance and/or a negative reference substance.
The application method of the combined ELISA kit for quantitatively detecting the SASP classical factor of the peripheral blood of the human comprises the following steps:
P1, mixing SASP classical exofactor antibodies with a buffer solution one by one, adding the obtained antibody solution into a micro-pore plate for coating, and then adding a sealing solution into the micro-pore plate for sealing to obtain a coated plate;
P2, mixing a sample to be detected with a sample diluent to prepare a solution to be detected and an antigen standard substance;
P3, mixing the SASP classical exocrine factor standard with a sample diluent to prepare an antigen standard;
p4, mixing the biological enzyme marked SASP classical exofactor antibody with enzyme diluent to obtain biological enzyme marked SASP classical exofactor antibody solution;
P5, respectively adding the solution to be detected and the antigen standard substance into a coating plate for incubation, then adding a biological enzyme marked SASP classical exofactor antibody solution for incubation, and finally adding a luminous substrate for incubation;
And P6, detecting a luminescence value, and obtaining the content of the SASP classical exocrine factor in the sample to be detected according to a standard curve.
The coating concentration of the SASP classical exofactor antibody is 4-10 mug/mL. In the biological enzyme marked SASP classical exocrine factor antibody solution, the volume ratio of the biological enzyme marked SASP classical exocrine factor antibody to the enzyme diluent is 1 (2500-3500).
The use method of the combined ELISA kit for the positive control or the negative control comprises the following steps:
S1, coating a microplate: mixing SASP classical exofactor antibody of a combined ELISA kit with buffer solution, adding into a micro-pore plate for coating, and then sealing the micro-pore plate by using sealing liquid to form a coating plate;
S2, preparing standard products and samples: mixing a SASP classical exocrine factor standard with a sample diluent to prepare an antigen standard; meanwhile, mixing the clinical blood supernatant to be tested with a sample diluent to prepare a solution to be tested;
S3, incubation and incubation: respectively adding a solution to be detected, an antigen standard substance and a biological enzyme marked SASP classical exocrine factor antibody solution into a coating plate for incubation, and finally adding a luminescent substrate for incubation;
s4, detecting a luminous value: detecting the luminescence value of each hole by a luminescence instrument;
S5, data analysis: calculating the content of SASP classical exocrine factors in a sample to be detected according to a standard curve;
s6, using a reference substance: adding a sample known to contain a target SASP classical factor to the assay to verify the detection capacity of the kit; the positive reference substance generates a signal to confirm that the experimental system can detect the target molecule; or:
sample dilutions without the classical factor of the target SASP were used as negative controls to ensure that no non-specific binding or background signal was present in the experiment.
The invention is further illustrated by the following examples:
Example 1 the kit of the invention and its preparation method,
1. The kit comprises the following components in parts by weight:
CB buffer formulations of Table 1.0.05M, pH 9.6:
| Article name | Raw material requirements | Standard quantity per liter |
| Purified water | Self-made and qualified quality inspection | 1000ml |
| Na2CO3 | Analytical grade | 1.99g |
| NaHCO3 | Analytical grade | 2.53g |
Table 2. Blocking solution formulation:
| Reagent name | Raw material requirements | Theoretical weighing |
| NaH2PO4·2H2O | Analytical grade | 2.25g |
| Na2HPO4·12H2O | Analytical grade | 25g |
| NaCl | Analytical grade | 35g |
| Casein | Analytical grade | 50g |
| ADP | / | 5g |
| P300 | / | 5ml |
| Sucrose | Analytical grade | 500g |
| Purified water | Homemade quality inspection compliance | 500ml |
Table 3. Sample dilution formulation (ph=7.4):
| Name of product | Raw material requirements | Theoretical amount of |
| Purified water | Homemade quality inspection compliance | 850ml |
| PBST | Quality inspection compliance | 20g |
| Fetal bovine serum | Quality inspection compliance | 150ml |
| P300 | Quality inspection compliance | 1ml |
| Rabbit anti-human SASP antibodies | Homemade quality inspection compliance | 5mgl |
Table 4. Enzyme dilution formulation (ph=7.4):
| Name of product | Raw material requirements | Theoretical amount of |
| Purified water | Homemade quality inspection compliance | 1000ml |
| PBST | Quality inspection compliance | 20g |
| Fetal bovine serum | Quality inspection compliance | 10g |
| P300 | Quality inspection compliance | 1ml |
A rabbit anti-human SASP classical factor antibody;
6 antigen standard (SDS-PAGE quantitative, more than 95% purity);
7 horseradish peroxidase labeled rabbit anti-human SASP classical factor antibody;
8 luminescent substrate: tobacco Confucius Biotechnology Co., ltd;
substrate luminescence solution a (light-protected preservation): luminol, tris buffer, etc.;
substrate luminescence liquid B: hydrogen peroxide, tris-buffer, etc.;
9 positive control: standards of recombinant human SASP classical factor antibodies (concentration 200. Mu.g/mL, 100. Mu.g/mL, 50. Mu.g/mL, 25g/mL, 1250. Mu.g/mL, 6250. Mu.g/mL, 31250. Mu.g/mL, 1560. Mu.g/mL) were diluted with sample dilutions containing neutralizing antibodies
10 Negative control: sample dilutions.
2. The corresponding preparation method of each reagent in the kit comprises the following steps:
Preparation procedure of 1CB buffer
1) The preparation volume of the coating buffer is determined according to the actual requirement.
2) The amounts of the above reagents were calculated according to standard formulations.
3) Adding the accurately weighed reagents into a container one by one, adding the required amount of purified water, and fully stirring for dissolution. The pH value is measured and recorded, and may be theoretical ph±0.05.
4) Formulation was completed the day prior to the planned coating.
5) The prepared coating buffer solution is placed at 2-8 ℃ for preservation.
2 Process for preparing a sealing liquid
1) The preparation volume of the buffer solution is determined according to the requirement, and the dosage of various raw materials is calculated according to a standard formula.
2) The container is filled with accurately weighed amounts of each chemical reagent and the desired amount of purified water.
3) And (5) fully stirring and dissolving, and then split charging.
4) After the preparation, the name, batch number, preparer, preparation date and liquid distribution amount are noted, qualified state identification is made, and if long-term storage is needed, the product is required to be stored at-20 ℃.
3 Sample diluent preparation flow
1) And determining the preparation volume of the buffer solution according to the requirement, and calculating the corresponding dosage of each raw material according to a standard formula.
2) The required amount of solid wash was added to the vessel and 15% fetal bovine serum was added.
3) P300 ml was added per liter.
4) And (5) fully stirring and dissolving, and then split charging.
5) After the preparation, the names and batch numbers are noted, and if the product needs to be stored for a long time, the product needs to be stored at-20 ℃.
Preparation of 4SASP classical factor rabbit polyclonal antibody and horseradish peroxidase (HRP) labeled antibody.
Animal immunization and rabbit polyclonal antibody preparation: the immunogen and the detecting antigen are all human SASP classical factors, and the purity is more than 95 percent, and the immunogen and the detecting antigen are obtained from the tobacco stand kenolais biotechnology Co. The purified product of prokaryotic expression recombinant protein of human SASP classical factor is used as immune antigen.
The preparation process comprises the following steps:
(1) Molecular cloning 8 human SASP classical factors (IL 1a, IL1b, IL6, IL8, MMP1, MMP3, GM-CSF or SPINK 1) were cloned into pET-28 fusion protein prokaryotic expression vectors (5369 bp in size).
(2) The MCS section gene ID (FIGS. 3-10) was determined by sequencing using the T7 promoter sequence as a basis for designing primers, and the vector was transferred into BL21 competent E.coli strain in sequence.
(3) After the strain is induced to express each target protein, high-purity recombinant protein (N-terminal His tag) is obtained through affinity chromatography. The preliminary yield after purification of each protein was 5mg.
(4) The New Zealand white rabbits are sequentially injected with purified proteins for immunization and potency detection is carried out. The samples include gradient titer assays for preimmune negative blood (control), first immunization, second immunization, third immunization, and fourth immunization, and assay titer assay data.
(5) The general immunization period is 2 months, and if there are special cases where the immunization is boosted, the latter is immunized at most once.
(6) Animal serum was collected and antibodies were purified in small amounts. After the immunization is finished, serum is collected in batches according to the immunization time for 7-12 days in a collection period. The volume of whole blood collected by each rabbit is not less than 50ml, and the volume of serum is not less than 30ml.
(7) Purifying polyclonal antibody by column. The antibodies were purified and identified in small amounts, and the basic purity was determined from SDS-PAGE gel, and each batch of purification was provided with the corresponding gel identification as a reference.
(8) Polyclonal antibody preparation was 2mg antibody/animal. This stage provides a total of 4mg, and the subsequent stage obtains 1mg for development of a high quality SASP kit.
(9) ELISA kit conditions were determined. 5 96-well plates were used per SASP factor protein for the determination of conditions.
(10) The mode of 8-joint holes and longitudinal arrangement is selected to form 12 rows, and 12 8-joint holes can be arranged in each 96-well plate. The combined ELISA kit is formed and is specially used for combined detection of the peripheral blood SASP factors under clinical conditions.
Horseradish peroxidase (HRP) labeled antibodies
Dissolving horseradish peroxidase in triple distilled water, adding sodium periodate solution for activation, and adding glycol for reaction. Putting the reaction mixture into a dialysis bag, dialyzing with acetic acid buffer, adding the activated horseradish peroxidase into a to-be-labeled substance (rabbit anti-human SASP classical factor antibody) respectively, mixing uniformly, regulating pH to be alkaline with carbonic acid buffer, reacting at 4 ℃ for 20-24h, adding NaHB4 solution, reacting at 4 ℃ for 2h, putting into a dialysis bag, dialyzing with phosphate buffer, subpackaging, adding a protein protectant (containing glycerol with 50% of final concentration), and preserving at 20 ℃ for later use.
Preparation of 5 antigen standard
SASP classical factor antigen was subjected to the following gradient dilutions of 200. Mu.g/ml, 100. Mu.g/ml, 50. Mu.g/ml, 25g/ml, 12.50. Mu.g/ml, 6.250. Mu.g/ml, 3.1250. Mu.g/ml, 1.560. Mu.g/ml, with the dilutions as sample dilutions, to prepare standards.
Comparative example 1 control kit
The sample dilutions in the control kit were different from example 1, lacking rabbit anti-human SASP classical factor antibodies, and the other were identical to example 1. The sample dilutions (ph=7.4) in the control kit were formulated as follows:
table 5 sample dilution (ph=7.4) formulation in control kit
| Name of product | Raw material requirements | Theoretical amount of |
| Purified water | Homemade quality inspection compliance | 950ml |
| PBST | Quality inspection compliance | 20g |
| Fetal bovine serum | Quality inspection compliance | 150ml |
| P300 | Quality inspection compliance | 1.0ml |
Test example 1 Combined ELISA detection method of SASP classical factor
1 Test materials
Example 1 kit, comparative example 1 kit.
2 Test procedure
Preparation of coated plates
1) And (5) coating. Respectively diluting SASP classical factor antibody (rabbit polyclonal antibody) to 5 mug/ml by using CB buffer solution with the concentration of 0.05M and the pH of 9.6, respectively covering preservative films, marking a beaker body, placing the beaker body on a magnetic stirrer, stirring for more than 20 minutes, adding a luminescent micro-pore plate after shaking and mixing uniformly, adding 100 mu l/hole of sample volume, ensuring that the liquid is fully paved at the bottom of the hole, covering a plate, horizontally standing at the temperature of 4 ℃ for coating overnight (12-24 hours);
2) And (5) sealing. Spin-drying the coating liquid, washing the plate twice, beating the plate with absorbent paper, sealing with Casein sealing liquid at 150 μl/hole, and standing at 4deg.C in refrigerator for overnight (12-24 hr);
3) Harvesting. Throwing off the sealing liquid, using water absorbing paper to beat for later use, bagging with aluminum foil, adding desiccant, attaching labels (name of detection plate, lot number and expiration date), sealing with sealing machine, and storing at 4deg.C for later use. The preparation of the coated plate is completed.
The combined ELISA experimental operation steps are that,
1) Recovering the SASP classical factor coated plate to room temperature;
2) The SASP classical factor antigen is diluted to 200 mug/ml, 100 mug/ml, 50 mug/ml, 25 mug/ml, 12.50 mug/ml, 6.250 mug/ml, 3.1250 mug/ml and 1.560 mug/ml according to the following gradient, and the diluted liquid is a sample diluted liquid to prepare a standard substance;
diluting the clinical blood supernatant to be tested by adopting the sample diluent to obtain a sample to be tested;
3) Adding 50 μl/well of diluted standard and sample into each well, and incubating at 37deg.C for 30min;
4) Throwing off the supernatant, washing the plate 5 times by using solid plate washing liquid (PBST), and beating the plate with water-absorbing paper;
5) Respectively adding HRP-SASP classical factor 1:3000 enzyme-labeled antibody, and adding sample amount: 50 μl/well, incubated at 37deg.C for 30min;
6) Throwing off the supernatant, washing the plate 5 times by using solid plate washing liquid (PBST), and beating the plate with water-absorbing paper;
7) An equal volume of luminescent substrate A+substrate B100 μl/well was added to each well, incubated at 37deg.C for 5min in the absence of light, and then placed in a luminometer for reading.
8) The clinical blood supernatants were relatively quantified according to a standard curve and the classical factor content of each SASP was calculated.
3 Experimental results:
standard luminescence value without neutralizing antibody added:
TABLE 6 luminescence values for comparative example 1 Standard (e.g., IL 6)
The result is shown in fig. 1 by calculation software in combination with the relevant parameters.
Luminescence value after addition of neutralizing antibody:
TABLE 7 luminescence values for example 1 Standard
| Standard +5 mug/mlIL 6 neutralizing antibody | Luminescence value (OD 450) |
| 200 | 20.1342.3 |
| 100 | 110526.8 |
| 50 | 56043.0 |
| 25 | 39405.2 |
| 125 | 18685.8 |
| 625 | 11659.9 |
| 3125 | 5584.1 |
| 15625 | 2965.8 |
| 0 | 463.5 |
The result is shown in fig. 2 by calculating the relevant parameters.
Five experimental conclusions:
1, and then, through cross pairing, selecting a reactive and linear optimal pairing human SASP classical factor+HRP-human SASP classical factor enzyme-labeled antibody.
2 Screening coating conditions of ELISA luminescent plates, and finally determining the working concentration of the established human SASP classical factor quantitative determination kit: SASP classical factor coating concentration 5 mug/ml, SASP classical factor-HRP 1:3000;
3 adding 5 μg/ml rabbit anti-human SASP classical factor antibody, changing the correlation from R 2 = 0.99985 to R 2 = 0.99998, and detecting in 0-200 μg/ml.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (8)
1. The application of the combined ELISA kit as a positive reference substance or a negative reference substance is characterized in that: the combined ELISA kit is pre-packaged with a microplate, a buffer solution, a blocking solution, a sample diluent, an enzyme diluent, a SASP factor standard substance, a SASP classical factor antibody, a biological enzyme-labeled SASP classical factor antibody and a luminescent substrate, wherein the SASP classical factor antibody is a specific polyclonal antibody of a human cell SASP classical exocrine factor.
2. The use of a combination ELISA kit according to claim 1 as a positive or negative control, characterized in that: the preparation process of the SASP classical factor antibody comprises the following steps:
step one, molecular cloning 8 humanized SASP classical factors IL1a, IL1b, IL6, IL8, MMP1, MMP3, GM-CSF, SPINK1 in pET-28 fusion protein prokaryotic expression vector (5369 bp);
step two, designing a primer by using a T7 promoter sequence as a basis, determining an MCS section gene ID by sequencing, and sequentially transferring the vector into BL21DE3 competent escherichia coli strains;
Step three, after inducing the strain to express target proteins, obtaining high-purity recombinant proteins with N-terminal His labels through affinity chromatography;
Step four, sequentially injecting purified proteins into New Zealand white rabbits for immunization, and carrying out titer detection; the sample comprises gradient titer detection of preimmune negative blood control, first immunization, second immunization, third immunization and fourth immunization, and titer detection data is analyzed; the immunization period was 2 months;
Step five, animal serum collection and a small amount of antibody purification; after the immunization is finished, serum is collected in batches according to the immunization time for 7-12 days in a collection period; the volume of whole blood collected by each rabbit is not less than 50ml, and the volume of serum is not less than 30ml;
Step six, purifying the polyclonal antibody through a column; the antibody is purified and identified in small quantity, and the basic purity is determined according to SDS-PAGE gel diagram.
3. The method for using the combined ELISA kit for the positive control or the negative control is characterized in that the method for using the combined ELISA kit for the use as the positive control or the negative control as set forth in claim 1 comprises the following steps:
S1, coating a microplate: mixing SASP classical exofactor antibody of a combined ELISA kit with buffer solution, adding into a micro-pore plate for coating, and then sealing the micro-pore plate by using sealing liquid to form a coating plate;
S2, preparing standard products and samples: mixing a SASP classical exocrine factor standard with a sample diluent to prepare an antigen standard; meanwhile, mixing the clinical blood supernatant to be tested with a sample diluent to prepare a solution to be tested;
S3, incubation and incubation: respectively adding a solution to be detected, an antigen standard substance and a biological enzyme marked SASP classical exocrine factor antibody solution into a coating plate for incubation, and finally adding a luminescent substrate for incubation;
s4, detecting a luminous value: detecting the luminescence value of each hole by a luminescence instrument;
S5, data analysis: calculating the content of SASP classical exocrine factors in a sample to be detected according to a standard curve;
s6, using a reference substance: adding a sample known to contain a target SASP classical factor to the assay to verify the detection capacity of the kit; the positive reference substance generates a signal to confirm that the experimental system can detect the target molecule; or:
sample dilutions without the classical factor of the target SASP were used as negative controls to ensure that no non-specific binding or background signal was present in the experiment.
4. The application of the combined ELISA kit as the detection of the SASP classical exocrine factor content in a sample is characterized in that:
The combined ELISA kit is pre-packaged with a microplate, a buffer solution, a blocking solution, a sample diluent, an enzyme diluent, a SASP factor standard substance, a SASP classical factor antibody, a biological enzyme-labeled SASP classical factor antibody and a luminescent substrate, wherein the SASP classical factor antibody is a specific polyclonal antibody of a human cell SASP classical exocrine factor.
5. The use of a combination ELISA kit according to claim 4 as a detection of the classical secretion of SASP in a sample characterized in that: the preparation process of the SASP classical factor antibody comprises the following steps:
step one, molecular cloning 8 humanized SASP classical factors IL1a, IL1b, IL6, IL8, MMP1, MMP3, GM-CSF, SPINK1 in pET-28 fusion protein prokaryotic expression vector (5369 bp);
step two, designing a primer by using a T7 promoter sequence as a basis, determining an MCS section gene ID by sequencing, and sequentially transferring the vector into BL21DE3 competent escherichia coli strains;
Step three, after inducing the strain to express target proteins, obtaining high-purity recombinant proteins with N-terminal His labels through affinity chromatography;
Step four, sequentially injecting purified proteins into New Zealand white rabbits for immunization, and carrying out titer detection; the sample comprises gradient titer detection of preimmune negative blood control, first immunization, second immunization, third immunization and fourth immunization, and titer detection data is analyzed; the immunization period was 2 months;
Step five, animal serum collection and a small amount of antibody purification; after the immunization is finished, serum is collected in batches according to the immunization time for 7-12 days in a collection period; the volume of whole blood collected by each rabbit is not less than 50ml, and the volume of serum is not less than 30ml;
Step six, purifying the polyclonal antibody through a column; the antibody is purified and identified in small quantity, and the basic purity is determined according to SDS-PAGE gel diagram.
6. Use of a combination ELISA kit for detecting the classical factor content of SASP in a sample, characterized in that it uses a combination ELISA kit as defined in claim 4 for the detection of the classical factor content of SASP in a sample comprising the following steps:
P1, mixing SASP classical exofactor antibodies with a buffer solution one by one, adding the obtained antibody solution into a micro-pore plate for coating, and then adding a sealing solution into the micro-pore plate for sealing to obtain a coated plate;
P2, mixing a sample to be detected with a sample diluent to prepare a solution to be detected and an antigen standard substance;
P3, mixing the SASP classical exocrine factor standard with a sample diluent to prepare an antigen standard;
p4, mixing the biological enzyme marked SASP classical exofactor antibody with enzyme diluent to obtain biological enzyme marked SASP classical exofactor antibody solution;
P5, respectively adding the solution to be detected and the antigen standard substance into a coating plate for incubation, then adding a biological enzyme marked SASP classical exofactor antibody solution for incubation, and finally adding a luminous substrate for incubation;
And P6, detecting a luminescence value, and obtaining the content of the SASP classical exocrine factor in the sample to be detected according to a standard curve.
7. The method of using the combined ELISA kit of claim 6 for detecting SASP classical exocrine factor content in a sample, wherein the coating concentration of the SASP classical exofactor antibody is 4-10 mug/mL.
8. The method of claim 6, wherein the volume ratio of the biological enzyme-labeled SASP classical exocrine factor antibody to the enzyme dilution in the biological enzyme-labeled SASP classical exocrine factor antibody solution is 1: (2500-3500).
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