CN117554620B - Kit for detecting four soluble cytokine receptors, and preparation method and application thereof - Google Patents
Kit for detecting four soluble cytokine receptors, and preparation method and application thereof Download PDFInfo
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- CN117554620B CN117554620B CN202311217464.3A CN202311217464A CN117554620B CN 117554620 B CN117554620 B CN 117554620B CN 202311217464 A CN202311217464 A CN 202311217464A CN 117554620 B CN117554620 B CN 117554620B
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Abstract
The invention provides a kit for detecting four soluble cytokine receptors, a preparation method and application thereof, wherein coding nucleotide sequences of sCD25, sCD40L, sCD and sTREM-1 are subjected to codon optimization, an optimized signal peptide sequence is adopted, a pcDNA3.4 vector is used, a eukaryotic cell expression system is used for expression preparation of recombinant protein antigen, and then a recombinant protein antigen is used for immunization of animals to prepare sCD25 antibody, sCD40L antibody, sCD130 antibody and sTREM-1 antibody, and the four soluble cytokine receptors are simultaneously detected by a flow cytometer, so that the detection accuracy and sensitivity can be improved, the difference between batches can be controlled, the comprehensive evaluation of the immune function condition of a patient is facilitated, the immune state of the organism is comprehensively judged, and important references are provided for auxiliary diagnosis, guiding drug administration, supervision curative effect and prognosis of diseases.
Description
The application claims a prior application in China, application number: 202211144691.3, priority of 20, 9, 2022, the description, claims, abstract and drawings of this application are incorporated by reference as if set forth in full herein.
Technical Field
The invention relates to the technical field of flow cytometry, in particular to a kit for detecting four soluble cytokine receptors, a preparation method and application thereof.
Background
Soluble Cytokine Receptors (SCR) are circulating proteins that lack the extracellular portion of the receptor in the transmembrane region. SCRs are one of the normal body fluid components that regulate cytokine and lymphoid movements. The expression level of four soluble cytokine receptors of sCD25, sCD40L, sCD and sTREM-1 in human serum or plasma reflects whether the state of the body immune system is abnormal, and can provide the basis of auxiliary diagnosis for doctors.
The first SCR defined was the soluble interleukin 2receptor (sIL-2R, sCD 25) which regulates the cytokine activity of the IL-2 family. The soluble interleukin-2receptor (solubleinterleukin-2 receptor, scd 25/sIL-2R) is a soluble cytokine receptor that is expressed in small amounts in normal humans, mainly from activated lymphocytes (including B lymphocytes and T lymphocytes). In a pathological condition of the body, too many activated lymphocytes or tumor cells release sCD25 in large amounts. Serum sCD25 levels are markedly elevated in the clinical course of a variety of diseases, such as malignant tumors, autoimmune diseases, infectious diseases, and certain infectious diseases. Excessive activated macrophages and T lymphocytes in patients with hemophagocytic syndrome (HLH) lead to a significant increase in serum sCD25 levels, so that the provision of serum sCD 25.gtoreq.2400U/mL in the HLH-2004 guideline is one of the important diagnostic criteria for HLH. Previous studies focused on the value of sCD25 in HLH diagnostics. The level of sIL-2Rs in serum of patients suffering from immune activation related diseases such as autoimmune diseases, infections and tumors is significantly increased.
CD40 is a type I transmembrane protein and belongs to a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily. CD40L is a ligand for CD40, and belongs to type II transmembrane proteins and TNF family cytokines. CD40L also exists in soluble molecular form (sCD 40L), with about 95% of sCD40L in the blood circulatory system coming from activated platelets. Over-expression of CD40, CD40L in the lesion of Atherosclerosis (AS), the interaction of CD40-CD40L is involved in the processes of AS occurrence, development and plaque rupture, thrombosis. The CD40L/CD40 axis is an important co-stimulatory molecule pair in the immune response in vivo, involved in both the humoral and cellular immune responses of the body. CD40L is predominantly expressed in cd4+ T cells, cd8+ T cells and B cells, CD40 is predominantly expressed in B cells, activated monocytes/macrophages, dendritic Cells (DCs), epithelial cells, and the like. The CD40L/CD40 axis plays a very important role in B cell activation, proliferation, cytokine secretion and immunoglobulin class switching. The CD40L/CD40 interaction also plays an important role in the activation of cytotoxic T cell precursors, maintaining the homeostasis of regulatory T cells, and inducing differentiation of helper T cells. The CD40L/CD40 axis is also involved in the pathogenic processes of various immune-related diseases, such as autoimmune diseases, hepatitis, etc., and plays an important role in anti-tumor immunity.
SCD130 represents a CD130 molecule that is soluble in serum. CD130, also known as gp130, is a 130kD transmembrane glycoprotein, which is the receptor and signal transducer common to interleukin 6 (IL-6), LIF, oncoinhibin (OSM), ciliary neurotrophic factor (CNTF), IL-11 and myocardial trophic factor-l (CT-1), and therefore also belongs to the IL-6 family of cytokines, which are important for the signal transduction involved in cytokines. Studies have shown that sCD130 levels in serum from diabetic retinopathy patients are significantly elevated compared to normal tissues and are significantly positively correlated with IL-6 and sIL-6R levels.
STREM-1 is a soluble form of TREM-1, and exists in body fluids such as blood plasma, gastric juice, urine, etc. The molecular mass of sTREM-1 varies depending on the cell source, and is 17.5kDa from bone marrow cells and monocytes and 15kDa from neutrophils. There are two views of the origin of sTREM-1, one being considered to be encoded by a splice variant of TREM-1mRNA (mRNA lacking both transmembrane and intracellular coding), and the other being considered to be the product of a matrix metalloproteinase which hydrolyses and releases the extracellular domain of TREM-1. The expression of sTREM-1 in body fluid is consistent with that of TREM-1 in tissue. In the pancreatitis rat model, the expression of serum and ascites sTREM-1 is obviously increased in pancreatic, liver and kidney tissues, and the expression of TREM-1 and the expression are consistent. But the regulation of inflammation is not the same. TREM-1 amplifies inflammatory responses, while sTREM-1 attenuates inflammatory responses. Baruah et al used TREM-1 agonists to treat human neutrophils in vitro and exogenous sTREM-1 to intervene, and found a decrease in the release of the downstream inflammatory factor IL-1 beta.
Currently, the main methodologies for quantitatively detecting soluble cytokine receptors in the market are enzyme-linked immunosorbent assay and chemiluminescent immunoassay. The ELISA method is common, but the manual operation steps are complicated, the required serum amount is large, and the result repeatability is poor. The chemiluminescent immunoassay method has simple operation and high sensitivity, but the chemiluminescent substance is unstable and easy to hydrolyze. At present, the detection technology of soluble cytokine receptors sCD25, sCD40L, sCD, sTREM-1 is generally single-index detection, and has the defects of poor reagent sensitivity, precision and the like, long detection time and complex operation, so that a reagent with excellent performance capable of simultaneously detecting the four markers is needed.
Therefore, there is a need to provide a novel kit for detecting a soluble cytokine receptor and a preparation method thereof to solve the above-mentioned problems in the prior art.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a kit for detecting four soluble cytokine receptors, a preparation method and application thereof, wherein coding nucleotide sequences of sCD25, sCD40L, sCD and sTREM-1 are subjected to codon optimization, an optimized signal peptide sequence is adopted, a pcDNA3.4 vector is used, recombinant proteins are prepared through expression of a eukaryotic cell expression system, and then a recombinant protein immune animal is used for preparing sCD25 antibodies, sCD40L antibodies, sCD130 antibodies and sTREM-1 antibodies, so that simultaneous detection of the four soluble cytokine receptors is realized through a flow cytometry, the detection accuracy and sensitivity can be improved, the difference between batches is controlled, the comprehensive evaluation of the immune function condition of a patient is facilitated, the comprehensive judgment of the immune state of the organism is realized, and important references are provided for auxiliary diagnosis, instruction, supervision and treatment effects of diseases.
In one aspect, the invention provides a kit for simultaneous detection of four soluble cytokine receptors based on a flow cytometer, the kit comprising a microsphere cocktail of conjugated antibodies, a biotin conjugated antibody cocktail, and a sample diluent, the antibodies comprising any one or more of a sCD25 antibody, a sCD40L antibody, a sCD130 antibody, and a sTREM-1 antibody, the sCD25 antigen having a sequence as set ID NO:1, the sCD40L antigen has an amino acid sequence as set forth in Seq ID NO:2, the sCD130 antigen has an amino acid sequence as shown in Seq ID NO:3, the sTREM-1 antigen has an amino acid sequence as shown in Seq ID NO:4 to 6; the four soluble cytokine receptors are sCD25, sCD40L, sCD, and sTREM-1, respectively.
When a microsphere mixed solution of a coupling antibody and a biotin coupling antibody mixed solution prepared from a commercially available sCD25 antibody, a sCD40L antibody, a sCD130 antibody and a sTREM-1 antibody are used for detecting four soluble cytokine receptors by a flow cytometer, the detection accuracy and sensitivity are usually found to be low, serious batch-to-batch difference problems exist, and the detection result is unstable.
In order to solve the problem, the invention tries to adopt self-made sCD25, sCD40L, sCD and sTREM-1 recombinant proteins and carry out codon optimization on coding nucleotide sequences, and the prepared antibody obtained by immunizing animals with sCD25, sCD40L, sCD and sTREM-1 recombinant protein antigens can realize simultaneous and precise detection of four soluble cytokine receptors of sCD25, sCD40L, sCD and sTREM-1, can effectively control batch-to-batch difference and improve detection accuracy and sensitivity.
Further, the sTREM-1 antigen has a sequence as set forth in Seq ID NO:4, and a polypeptide having the amino acid sequence shown in (a) and (b).
The sTREM-1 antibody is easy to cause cross influence in the detection process, the reasons are probably that the sTREM-1 forms a polymer, the spatial configuration is changed so that part of antigen epitopes can not be exposed, the coding nucleotide sequence of the sTREM-1 is repeatedly regulated for a plurality of times, the most preferable signal peptide sequence of the sTREM-1 is obtained, and when the prepared sTREM-1 antibody is used for detection by a flow cytometer, the accuracy and the sensitivity of simultaneously detecting four soluble cytokine receptors can be improved, and batch-to-batch differences are eliminated.
Further, the sCD25 antibody, the sCD40L antibody and the sCD130 antibody are rabbit monoclonal antibodies, and the sTREM-1 antibody is a mouse monoclonal antibody.
The invention screens the antibody types of the coupled microspheres, and discovers that the microspheres using the rabbit anti-markers (the antibody coupled to the microspheres is a rabbit monoclonal antibody) have stronger specificity and higher detection sensitivity for sCD25 antibody, sCD40L antibody and sCD130 antibody, and the sTREM-1 antibody preferably adopts a mouse monoclonal antibody. After the rabbit antibody is replaced, the detection limit of sCD25 and sCD40L, sCD130,130 is reduced, and the detection sensitivity is obviously improved. The possible reasons for the above result are: rabbit antibodies are capable of recognizing epitopes on human antigens that are not immunogenic in rodents, increasing the total number of targetable epitopes; rabbit antibodies have a strong immune response to small molecules and haptens, which is not common in rodents; rabbits of inbred strains are more rare, while most mouse strains are inbred strains, so that the rabbits have more immune response diversity; rabbits use unique mechanisms to genetically produce and diversify antibodies with high affinity and specificity. However, the rabbit monoclonal antibody of sTREM-1 also causes cross-influence, so that the mouse monoclonal antibody is still used as the sTREM-1 antibody.
Further, the microsphere coupled with the sTREM-1 antibody is provided with a carboxyl modified functional group, and the microsphere coupled with the sCD25 antibody, the sCD40L antibody and the sCD130 antibody is provided with an amino modified functional group.
Further, the biotin-conjugated antibody mixture is a mixture of sCD25, sCD40L, sCD and sTREM-1 biotin-labeled antibody; the kit also comprises a fluorescent reagent, wherein the fluorescent reagent is a conjugate of streptavidin and phycoerythrin.
In another aspect, the present invention provides a method of preparing a kit for detecting a soluble cytokine receptor, the method comprising the steps of:
(1) Preparing recombinant proteins, wherein the recombinant proteins comprise any one or more of sCD25 recombinant proteins, sCD40L recombinant proteins, sCD130 recombinant proteins and sTREM-1 recombinant proteins; the coding nucleotide sequence of the sCD25 recombinant protein is as Seq ID NO:7, the coding nucleotide sequence of sCD40L recombinant protein is as shown in Seq ID NO:8, the coding nucleotide sequence of sCD130 recombinant protein is as shown in Seq ID NO:9, the coding nucleotide sequence of the sTREM-1 recombinant protein is shown as Seq ID NO: 10-Seq ID NO: 12.
(2) Immunizing an animal with the recombinant protein antigen prepared in the step (1) to obtain antibodies, including any one or more of sCD25 antibodies, sCD40L antibodies, sCD130 antibodies and sTREM-1 antibodies;
(3) Preparing a microsphere mixed solution of the conjugated antibody and a biotin conjugated antibody mixed solution by using the antibody prepared in the step (2).
Further, the coding nucleotide sequence of the sTREM-1 recombinant protein is as Seq ID NO: shown at 10.
Further, the preparation method of the recombinant protein in the step (1) comprises the following steps:
(a) Inserting the nucleotide for encoding the recombinant protein into an empty vector to obtain a recombinant vector;
(b) Transfecting the recombinant vector into a eukaryotic cell expression system;
(c) Collecting a culture medium of a eukaryotic cell expression system;
(d) Purifying by Ni-NTA agarose chromatographic column.
The traditional prokaryotic expression system has the advantages of high production speed, high yield and low cost. For example, the multiplication period of the genetically engineered bacteria is only 20-30 minutes, and the thallus can reach extremely high growth density within 16 hours. The yield of the recombinant protein obtained after purification can reach several grams per liter of culture medium. The culture condition of the escherichia coli is relatively simple, the cost of a culture medium is low, and the escherichia coli is generally suitable for producing recombinant proteins from small scale to large scale. However, prokaryotic expression systems are not suitable for producing a portion of recombinant proteins derived from eukaryotic cells. sCD40L, sCD, sTREM-1 and sCD130 are cell surface membrane proteins with complex glycosylation modifications. The traditional prokaryotic expression system cannot generate posttranslational modification and glycosylation which are the same as human cell membrane proteins, and does not have the same epitope and reactivity. The yield of 293T cell eukaryotic expression systems, although lower than prokaryotic expression systems, can reach tens of milligrams per liter of medium (calculated as reactive antigen).
Further, the empty vector is pcDNA3.4 empty vector; the eukaryotic cell expression system includes 293T cells; the culture medium is a high sugar culture medium of 10% fetal bovine serum and 90% DMEM.
In the invention, four recombinant proteins of sCD40L, sCD, sTREM-1 and sCD130 are all 293T cell eukaryotic expression systems, a plasmid vector is pcDNA3.4 vector provided by Kirsrui company, and a transfection technology based on cationic liposome transfection reagent is used. The 293T cell eukaryotic expression system can generate posttranslational modification and glycosylation consistent with natural cell membrane proteins, and can be well used as an antigen calibrator to meet the requirements of four inflammatory factor detection reagents of sCD40L, sCD, sTREM-1 and sCD 130. The pcDNA3.4 vector has a complete CMV promoter sequence and the yield of expressed recombinant protein is higher than that of a common incomplete CMV promoter. The gene sequence of each recombinant protein is optimized by codons, and an optimized signal peptide sequence is adopted, so that the recombinant protein yield of a eukaryotic expression system is improved to the maximum extent.
MRNA sequences of sCD40L, sCD, sTREM-1 and sCD130 are selected respectively, only the outer part of a cell membrane of protein is reserved, and then a deoxyribonucleic acid sequence which can be expressed as a His-Tag purification Tag is added. The recombinant protein DNA sequence is codon optimized to make it suitable for expression in human cell.
Based on pcDNA3.4 empty vector, the vector is a molecular biological vector capable of regulating and controlling the expression of exogenous genes. A multiple cloning site is additionally added to the pcDNA3.4 empty vector, and the ribonucleic acid molecule of the first aspect is connected to the pcDNA3.4 empty vector.
In some embodiments, the dna molecule of step (a) is inserted into the AflII and XbaI restriction endonuclease sites of the expression vector.
In some embodiments, the transfection method described in step (b) is a cationic liposome transfection reagent mediated transfection technique.
In some embodiments, the medium described in step (c) is 10% (v/v) of high-grade fetal bovine serum and 90% (v/v) of DMEM high-sugar medium.
In some embodiments, the purification of the Ni-NTA agarose column described in step (d) is a commonly used His-tag recombinant protein purification method. His-tag labels, namely 6-10 histidine residue side chains, have stronger binding force with nickel ions, and can be used for immobilized metal chelating affinity chromatography (IMAC) to separate and purify recombinant proteins.
The invention optimizes the use amount of the cationic liposome transfection reagent and the recombinant vector used in the transfection technology, and improves the yield of the recombinant protein to the maximum extent; the amino acid sequences in the four inflammatory factors are modified, the area crossing the cell membrane and the area in the cell are deleted, the extracellular area is reserved, and the recombinant protein is made into soluble protein and secreted to the outside of the cell; codon optimization is carried out on the original sequences of the four inflammatory factors, and the codon with higher occurrence frequency in a human cell expression system is adopted to optimize the proportion of GC base pairs, so that the yield of recombinant proteins is improved; the improved signal peptide sequence is adopted to replace the original signal peptide sequence of four inflammatory factors, the efficiency of producing secreted proteins is obviously improved, and meanwhile, the recombinant protein antigen is adopted to immunize animals to prepare antibodies, so that the antibodies are used for quantitative detection of sCD40L, sCD, sTREM-1 and sCD130, the inter-batch difference can be effectively controlled, and the accuracy and the sensitivity of detection are improved.
In a further aspect, the present invention provides the use of a set of recombinant proteins for the preparation of a reagent for increasing the detection sensitivity of a flow cytometer for simultaneous detection of four soluble cytokine receptors, characterized in that said recombinant proteins have the sequence as set forth in Seq ID NO: 1. seq ID NO: 2. seq ID NO: 3. seq ID NO:4, and the four soluble cytokine receptors are sCD25, sCD40L, sCD, 130 and sTREM-1, respectively.
The invention has the beneficial effects that:
1. The simultaneous detection of four soluble cytokine receptors of sCD25, sCD40L, sCD130 and sTREM-1 can be realized based on a flow cytometer;
2. The antibody obtained by immunizing animals with the prepared sCD25, sCD40L, sCD and sTREM-1 recombinant protein antigens can realize simultaneous and accurate detection of four soluble cytokine receptors of sCD25, sCD40L, sCD and sTREM-1, effectively control batch-to-batch difference and improve detection accuracy and sensitivity by adopting self-made sCD25, sCD40L, sCD and sTREM-1 recombinant protein and performing codon optimization on the coding nucleotide sequence;
3. The sCD25 antibody, the sCD40L antibody and the sCD130 antibody are labeled by using rabbit antibodies, and the sTREM-1 antibody is a mouse monoclonal antibody, so that cross influence is eliminated, and the detection accuracy is improved;
4. The detection sensitivity of sCD25 reaches 2.55pg/mL, the detection sensitivity of sCD40L reaches 5pg/mL, the detection sensitivity of sCD130 reaches 50.0pg/mL, and the detection sensitivity of sTREM-1 reaches 2.55pg/mL; the difference between batches is controlled within 7%.
Drawings
FIG. 1 is a schematic diagram of the detection principle of the flow cytometer based kit for simultaneously detecting four soluble cytokine receptors provided in example 1;
FIG. 2 shows the distribution of captured microspheres for sample detection provided in example 1;
FIG. 3 is a calibration curve for simultaneous detection of sCD25, sCD40L, sCD, 130 and sTREM-1 based on a flow cytometer as provided in example 1;
FIG. 4 is a graph showing the results of linear evaluation of sCD25 provided in example 1;
FIG. 5 is a graph showing the results of linear evaluation of sCD40L provided in example 1;
FIG. 6 is a graph showing the results of linear evaluation of sCD130 provided in example 1;
FIG. 7 is a linear evaluation result of sTREM-1 provided in example 1.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Unless otherwise defined, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. As used herein, the word "comprising" and the like means that elements or items preceding the word are included in the element or item listed after the word and equivalents thereof without precluding other elements or items. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
The culture medium comprises the following components: chinese fetal bovine serum (Top grade) was purchased from Minhai bioengineering Inc., lanzhou, DMEM high sugar medium was purchased from Zhejiang Ji Nuosai Biotech Inc., opti-MEM TM I reduced serum medium was purchased from Sieimer Feishier (China) Inc.
Biochemical reagent: lipofectamine TM transfection reagent, FASTDIGEST AFLII, FASTDIGEST XbaI, T4 DNA ligase was purchased from Simer Feier (China) Inc.
Cell culture consumable: t25 cell culture flasks were purchased from tin-free West Life technologies Co.
In the present invention, the recombinant protein is the antigen.
Example 1: preparation, use and effect evaluation of kit
1. Preparation of the kit
The specific composition is shown in table 1:
TABLE 1 composition of kit
The preparation method comprises the following steps:
1. Preparation of recombinant proteins
1.1 Design and Synthesis of recombinant protein coding Gene
1.1.1 Design and Synthesis of recombinant protein coding Gene of sCD25
The mRNA sequence of the sCD25 gene disclosed in NCBI (NCBI Reference Sequence accession number: NM-000417.3) is used as an optimization target, and Glu22-Cys213 sequences such as the sequence of Seq ID NO:13, at the amino terminus to:
ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGT to the carboxy terminus of the signal peptide sequence encoded by CACCACCACCACCACCACCACCACCACCACTAG. According to codon preference of a eukaryotic expression system of human cells, selecting a codon with highest use frequency, and redesigning a coding sequence to obtain a sequence like Seq ID NO:7, and performing artificial synthesis.
1.1.2SCD40L recombinant protein coding Gene design and Synthesis
The mRNA sequence of the sCD40L gene disclosed by NCBI (NCBI Reference Sequence accession number: NM-000074.3) is used as an optimization object, and Glu108-Leu261 sequences such as Seq ID NO:14, at the amino terminus to:
ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACA AACAGTCACCATCACCACCACCACCACCACCACGGATCTGGC and His-Tag sequence. According to codon preference of a eukaryotic expression system of human cells, selecting a codon with highest use frequency, and redesigning a coding sequence to obtain a sequence like Seq ID NO:8, and performing artificial synthesis.
1.1.3SCD130 recombinant protein coding Gene design and Synthesis
The mRNA sequence of the sCD130 gene disclosed by NCBI (NCBI Reference Sequence accession number: NM-002184.4) is used as an optimization object, and Glu23-Ile618 sequences such as the sequence of Seq ID NO:15, at the amino terminus to:
ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACA AACAGT to the carboxy terminus of the signal peptide sequence encoded by CACCACCACCACCACCACCACCACCACCACTAG. According to codon preference of a eukaryotic expression system of human cells, selecting a codon with highest use frequency, and redesigning a coding sequence to obtain a sequence like Seq ID NO:9, and performing artificial synthesis.
1.1.4 Design and Synthesis of coding Gene for sTREM-1 recombinant protein
Taking the mRNA sequence of the sTREM-1 gene disclosed by NCBI (NCBI Reference Sequence accession number: NM_ 018643.5) as an optimization object, selecting an Ala20-Asn205 sequence such as a Seq ID NO:16, at the amino terminus to:
ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACA AACAGT the signal peptide sequence encoded by CACCACCACCACCACCACCACCACCACCACTAG the His-Tag sequence was added at the carboxy terminus, requiring further sequence modifications. According to codon preference of a eukaryotic expression system of human cells, selecting a codon with highest use frequency, and redesigning a coding sequence to obtain a sequence like Seq ID NO:10, and carrying out artificial synthesis.
1.2 Construction of vectors Using AflII and XbaI restriction Endonuclease sites
(1) Treating the gene fragment of the recombinant protein and pcdna3.4 vector with FASTDIGEST AFLII and FASTDIGEST XBAI restriction enzymes;
(2) The gene fragments of pcDNA3.4 vector and recombinant protein treated with restriction enzymes were electrophoresed on a 1% agarose gel;
(3) Cleaving the DNA fragment of the correct molecular weight from the gel and purifying;
(4) Constructing a recombinant vector by using T4 DNA ligase;
(5) E.coli is transformed by the recombinant vector, and the plasmid is extracted and then sequenced for verification.
1.3 Transient transfection of 293T cells with the four recombinant vectors described above
(1) Approximately one hundred and a half million cells were seeded into T25 cell culture flasks one day before the experiment, and after one day the cells were grown to confluency of 80% or more;
(2) 6. Mu.g of the recombinant plasmid and 12. Mu.L of Lipofectamine TM transfection reagent were diluted with two 0.5mL of Opti-MEM TM I serum-reduced medium, respectively, and the two media were mixed and incubated for 30 minutes at ambient temperature;
(3) Replacing the original culture medium of the cells with 4mL of DMEM high-sugar culture medium, and adding 1mL of the mixed culture medium;
(4) Culturing at 37deg.C under 5% carbon dioxide for 4 hr, and changing the culture medium to 8mL complete culture medium containing 10% (v/v) of high-quality fetal bovine serum and 90% (v/v) of DMEM high-sugar culture medium;
(5) Culturing at 37deg.C under 5% carbon dioxide for 48-72 hr;
(6) Centrifuging for 5min to collect culture medium, wherein the centrifuging condition is 4 ℃ and 2000G;
(7) The culture medium was filtered through a 0.45. Mu.M filter membrane and purified by a Ni-NTA agarose column to obtain recombinant protein. .
2. Preparation of antibodies
The rabbit anti-human sCD25 monoclonal antibody, the rabbit anti-human sCD40L monoclonal antibody and the rabbit anti-human sCD130 monoclonal antibody are prepared by respectively immunizing rabbits (purchased from the Kohler rabbit industry) by adopting the prepared sCD25 and sCD40L, sCD recombinant proteins, and the specific steps are as follows: expressing sCD25 and sCD40L, sCD130,130 recombinant proteins by using 293F, immunizing New Zealand white rabbits by taking the purified proteins as immunogens, separating and culturing B cells, screening antigen-specific B cells by Elisa/WB, separating and sequencing single cells, preparing recombinant antibodies, and screening the antibodies after affinity chromatography purification by using a flow method.
The mouse (purchased from the Kong rabbit industry) is immunized by the prepared sTREM-1 recombinant protein to prepare the mouse anti-human sTREM-1 monoclonal antibody, and the specific steps are as follows: the 293F is used for expressing sTREM-1 recombinant protein, the purified protein is used as an immunogen to immunize Balb/c female mice, subcloned cell strains are obtained through a limiting dilution method, and all positive cell strains are screened by an indirect enzyme-linked immunosorbent assay method. Then, after the subcloned cell line was cultured, it was injected into the abdominal cavity of the mice (one week in advance of incomplete adjuvant injection). Taking the ascites of the mice after 10-15 days, and purifying the monoclonal antibody by a composite purification technology of ammonium sulfate precipitation and an affinity chromatographic column. The purified antibodies were screened for antibody pairs using a flow-through method.
3. Microsphere mixed solution for preparing coupling antibody
Taking 0.1mL of first fluorescent microspheres (amino groups) with the concentration of 5X 10 7/mL, adding PBST buffer solution for cleaning for 2 times, adding 100 mug of EDC and 50 mug of NHS into the first fluorescent microspheres after cleaning, standing for 30 minutes to activate the microspheres, adding 100 mug of rabbit anti-human sCD25 monoclonal antibody for rotary reaction at room temperature for 5 hours, cleaning the first fluorescent microspheres to remove excessive antibodies, adding skimmed milk powder with the mass fraction of 5% for sealing for 30 minutes, adding Tris buffer solution with the pH of 7.2 for preservation after removing skimmed milk powder, and obtaining 0.5mL of polystyrene microsphere solution coated with rabbit anti-human sCD25 monoclonal antibody;
Taking 0.1mL of second fluorescent microspheres (amino groups) with the concentration of 5X 10 7/mL, adding PBST buffer solution for cleaning for 1-3 times, adding 100 mug of EDC and 50 mug of NHS into the cleaned second fluorescent microspheres, standing for 30 minutes to activate the microspheres, adding 100 mug of rabbit anti-human sCD40L monoclonal antibody for rotary reaction at room temperature for 5 hours, adding 5% of skim milk powder for sealing for 30 minutes after the second fluorescent microspheres are cleaned to remove the superfluous antibody, adding Tris buffer solution with the pH of 7.2 for preservation after the skim milk powder is removed, and obtaining 0.5mL of polystyrene microsphere solution coated with rabbit anti-human sCD40L monoclonal antibody;
Taking 0.1mL of third fluorescent microspheres (amino groups) with the concentration of 5X 10 7/mL, adding PBST buffer solution for cleaning for 1-3 times, adding 100 mug of EDC and 50 mug of NHS into the third fluorescent microspheres after cleaning, standing for 30 minutes to activate the microspheres, adding 100 mug of rabbit anti-human sCD130 monoclonal antibody for rotary reaction at room temperature for 5 hours, adding 5% of skim milk powder with the mass fraction after cleaning the third fluorescent microspheres to remove the superfluous antibody, sealing for 30 minutes, adding Tris buffer solution with the pH of 7.2 after removing the skim milk powder, and preserving to prepare 0.5mL of polystyrene microsphere solution coated with rabbit anti-human sCD130 monoclonal antibody;
Taking 0.1mL of fourth fluorescent microspheres (carboxyl groups) with the concentration of 5X 10 7/mL, adding PBST buffer solution for cleaning for 1-3 times, adding 100 mug of EDC and 50 mug of NHS into the fourth fluorescent microspheres after cleaning, standing for 30 minutes to activate the microspheres, adding 100 mug of mouse anti-human sTREM-1 monoclonal antibody for rotary reaction at room temperature for 5 hours, adding 5% of skim milk powder for sealing for 30 minutes after cleaning the fourth fluorescent microspheres to remove the superfluous antibody, adding Tris buffer solution with the pH of 7.2 for preservation after removing the skim milk powder, and obtaining 0.5mL of polystyrene microsphere solution coated with the mouse anti-human sTREM-1 monoclonal antibody;
Table 2 shows the manufacturer and the source of the raw materials in the preparation of the antibody-coated microspheres.
TABLE 2 manufacturer and source of product number for each raw material in antibody-coated microspheres
| Microsphere(s) | Manufacturer' s | Goods number |
| First fluorescent microsphere | Spherotech | APAK-3567 |
| Second fluorescent microsphere | Spherotech | APAK-3567 |
| Third fluorescent microsphere | Spherotech | APAK-3567 |
| Fourth fluorescent microsphere | Bangslsbs | 238 |
98ML of Tris buffer solution with the pH of 7.2 is taken, and 0.5mL of polystyrene microsphere coated with the rabbit anti-human sCD25 monoclonal antibody, polystyrene microsphere coated with the rabbit anti-human sCD40L monoclonal antibody, polystyrene microsphere coated with the rabbit anti-human sCD130 monoclonal antibody and polystyrene microsphere coated with the mouse anti-human sTREM-1 monoclonal antibody are respectively added to prepare microsphere mixed solution of the coupled antibody.
4. Preparing biotin coupling antibody mixed solution
Taking a rabbit anti-human sCD25 monoclonal antibody, a rabbit anti-human sCD40L monoclonal antibody, a rabbit anti-human sCD130 monoclonal antibody and a mouse anti-human sTREM-1 monoclonal antibody, respectively adding biotin in a molar ratio of the antibody to biotin of 1:30, vibrating and incubating for 5 hours at room temperature, and then adding PBS solution with the concentration of 0.01mol/L into a 50K ultrafiltration tube for cleaning for 3 times to remove excessive unbound biotin, so as to obtain a biotin-coupled rabbit anti-human sCD25 monoclonal antibody, a biotin-coupled rabbit anti-human sCD40L monoclonal antibody, a biotin-coupled rabbit anti-human sCD130 monoclonal antibody and a biotin-coupled mouse anti-human sTREM-1 monoclonal antibody, and respectively diluting the anti-human sCD25 monoclonal antibody, the biotin-coupled rabbit anti-human sCD40L monoclonal antibody, the biotin-coupled rabbit anti-human sCD130 monoclonal antibody and the biotin-coupled mouse anti-human sTREM-1 monoclonal antibody to 2 mug/mL by using PBS solution with the concentration of 0.01 mol/L;
The diluted sCD25, sCD40L, sCD and sTREM-1 antibodies were mixed in a concentration of 2. Mu.g/mL, and 25mL of the labeled biotin-conjugated antibody was prepared, whereby the concentration of each of the biotin-labeled antibodies was 0.5. Mu.g/mL.
5. SA-PE fluorescent reagent, purchased from Thermofisher, was diluted to 4. Mu.g/mL for use.
6. Preparation of sample dilutions
4.76G of hydroxyethylpiperazine ethylsulfuric acid (HEPES) was dissolved in 2000mL of pure water to prepare a HEPES solution having a concentration of 0.01mol/L, and 0.1g of mouse IgG, 0.2g of rabbit IgG, 0.02g of anti-HAMA polyclonal antibody, 16.0g of sodium chloride (NaCl) and a Proclin300 preservative having a mass concentration of 0.05% were added to adjust the pH to 7.4 for use.
7. Preparation of washing buffer (10×)
2.4G of potassium dihydrogen phosphate (KH 2 PO 4), 36.32g of disodium hydrogen phosphate dodecahydrate (Na 2 HPO4.12H2O), 8g of NaCl and 2g of potassium chloride (KCl) are dissolved in 1000mL of pure water, and 25g of Bovine Serum Albumin (BSA), a Proclin300 preservative with a mass concentration of 1% and Tween 20 (Tween-20) with a mass concentration of 0.5% are added and mixed for later use.
8. Preparing a calibrator: the calibrator is diluted to sCD25, sCD40L, sCD, sTREM-1 recombinant protein solution with different concentrations by adopting a sample diluent, and is used for establishing a calibration curve during detection.
9. Preparing a quality control product: the quality control product is prepared from high-value and medium-value freeze-dried products of sCD25, sCD40L, sCD and sTREM-1 recombinant protein (prepared in the embodiment), and sample diluent is added for indoor quality control of the kit.
10. Matrix solution is prepared, which consists of 1% of bovine serum albumin, 20% of fetal bovine serum and 1% of glycine, and is used for simulating serum matrix and eliminating matrix effect.
2. Method for using kit
The kit prepared by the embodiment is used as follows:
(1) Adding 25 mu L of matrix solution into the calibration curve tube, and respectively adding 25 mu L of sample diluent into the sample tube and the quality control tube;
(2) Adding 25 mu L of a sample into a sample tube, adding 25 mu L of a calibrator into a calibration curve tube, and adding 25 mu L of a quality control product into a quality control tube;
(3) Adding 25 mu L of microsphere mixed solution of the coupling antibody into the sample tube, the calibration curve tube and the quality control tube respectively, fully and uniformly mixing, and oscillating for more than 30 seconds by an oscillator;
(4) Respectively adding 25 mu L of biotin-conjugated antibody mixed solution into a sample tube, a calibration curve tube and a quality control tube, and vibrating at room temperature at 1000-2000rpm in a dark place for 2 hours;
(5) Respectively adding 25 mu L of SA-PE fluorescent reagent into a sample tube, a calibration curve tube and a quality control tube, and vibrating at room temperature in a dark place for 0.5h;
(6) Adding 1000 mu L of washing buffer solution (1X) into the sample tube, the calibration curve tube and the quality control tube respectively, re-suspending the microspheres by vortex, fully and uniformly mixing, oscillating for more than 30 seconds by an oscillator, centrifuging for 5 minutes at 300g, and discarding the supernatant;
(7) 150-300 mu L of washing buffer (1X) is respectively added into the sample tube, the calibration curve tube and the quality control tube, the microspheres are resuspended by vortex, fully and uniformly mixed, the oscillator oscillates for more than 30 seconds, the fluorescence type and the fluorescence signal intensity are detected on a BriCyte E flow cytometer manufactured by Michael company, and the specific detection process is a conventional technical means of a person skilled in the art and is not described herein.
The principle of detection by using the kit is shown in figure 1, wherein 1 is polystyrene microsphere, 2 is sCD25, sCD40L, sCD or sTREM-1 antibody coupled with microsphere, 3 is sample to be detected, 4 is sCD25, sCD40L, sCD or sTREM-1 antibody coupled with biotin, 5 is biotin, 6 is streptavidin, and 7 is phycoerythrin. When two beams of excitation light with different wavelengths emitted by the flow cytometer irradiate the immune complex, the type of detection index is determined by the fluorescence intensity of different fluorescent microspheres, and the content of each detection index is determined by the fluorescence intensity of phycoerythrin.
FIG. 2 shows the distribution of captured microspheres for sample detection. Referring to fig. 2, the side scattered light (sidescatter, SSC) is on the ordinate and Allophycocyanin (APC) is on the abscissa. The ordinate determines the location interval of microsphere complexity, the abscissa distinguishes the fluorescence intensities of APCs carried by different microspheres, and fig. 2 can distinguish four kinds of polystyrene microspheres combined with sCD25, sCD40L, sCD, and sTREM-1 antibodies, wherein P1 represents the SSC and APC distributions of the polystyrene microspheres coated with sCD25 antibodies of this example, P2 represents the SSC and APC distributions of the polystyrene microspheres coated with sCD40L antibodies of this example, P3 represents the SSC and APC distributions of the polystyrene microspheres coated with sCD130 antibodies of this example, and P4 represents the SSC and APC distributions of the polystyrene microspheres coated with sTREM-1 antibodies of this example.
3. Analysis of detection results
1. Calibration curve
The kit provided by the invention is used for establishing calibration curves of four cytokine receptors of sCD25, sCD40L, sCD and sTREM-1, and respectively detecting calibrator materials with different concentrations of sCD25, sCD40L, sCD and sTREM-1 on a flow cytometer to obtain the calibration curve of figure 3.
2. Linear, blank, detection limit evaluation
Linear evaluation: and (3) selecting high-concentration samples at the upper limit of the linear range, diluting the high-concentration samples into at least 7 samples with different concentrations (xi), testing each concentration 3 times, and respectively obtaining the average value (yi) of the detection result. And (3) taking the dilution concentration (xi) as an independent variable and taking the average value (yi) of the detection result as a dependent variable to solve a linear regression equation. And calculating a correlation coefficient (R) of the linear regression.
Fig. 4 is a linear evaluation result of sCD25 according to an embodiment of the present invention, fig. 5 is a linear evaluation result of sCD40L according to an embodiment of the present invention, fig. 6 is a linear evaluation result of sCD130 according to an embodiment of the present invention, and fig. 7 is a linear evaluation result of sTREM-1 according to an embodiment of the present invention.
Referring to fig. 4-7, the scd25 linear regression equation is y= 1.0119x-9.6422, r 2 =0.9993; sCD40L linear regression equation is y= 1.0429x-40.797, r 2 =0.9995; the sCD130 linear regression equation is y= 1.0519x-2005.9, r 2 = 0.9988; the sTREM-1 linear regression equation is y= 1.0211x-85.745, and r 2 = 0.9981. With the kit of the invention, the linear interval of sCD25 is not narrower than [9.77,40000] pg/mL, the linear interval of sCD40L is not narrower than [43.95,180000] pg/mL, the linear interval of sCD130 is not narrower than [219.73,450000 ] pg/mL, the linear interval of sTREM-1 is not narrower than [12.21,50000] pg/mL, and the linear correlation coefficient |R| is not smaller than 0.990.
The blank limit detection method comprises the following steps: and (3) detecting by using the zero-concentration calibrator as a sample, repeatedly measuring for 20 times, obtaining concentration values of 20 measurement results according to a curve equation of the calibrator used by the kit, and calculating an average value (M) and a Standard Deviation (SD) of the concentration values to obtain M+2SD, namely the blank limit value.
Detection limit detection method: and detecting 5 low-value samples with the concentration approximate detection limit (the approximate detection limit is estimated according to the obtained blank limit value and is slightly higher than the blank limit value), detecting 5 times for each sample, sorting detection results according to the sizes, and the number of detection results lower than the blank limit value is smaller than or equal to 3.
The blank and detection limit results obtained by the above method are shown in Table 3, and the unit in Table 3 is pg/mL.
TABLE 3 blank Limit, detection Limit results (pg/mL)
3. Recovery evaluation
Adding a high level of a known concentration of a test substance A (sCD 25, sCD40L, sCD, sTREM-1 calibrator) to the matrix solution, wherein the volume ratio between the added test substance A and the matrix solution is not more than 1:9, repeating the detection 3 times, and taking an average value. Table 4 shows the results of recovery tests (pg/mL, n=3), with recovery rates of sCD25, sCD40L, sCD, sTREM-1 ranging from 95% to 105% according to Table 4.
TABLE 4 recovery rate test results
| Object to be measured A | sCD25 | sCD40L | sCD130 | sTREM-1 |
| Theoretical concentration | 2000.00 | 9000.00 | 22500.00 | 2500.00 |
| Detecting concentration | 1973.40 | 9217.13 | 23226.75 | 2410.82 |
| Recovery rate | 99％ | 102％ | 103％ | 96％ |
4. Repeatability and batch to batch difference assessment
Repeatability: 2 copies of sCD25, sCD40L, sCD, sctem-1 calibrator at different concentration levels were tested in parallel 10 times to calculate the coefficient of variation CV. Table 5 shows the results of the repeatability test (pg/mL).
TABLE 5 repeatability test results
As can be seen from Table 5, the repeatability variation coefficients CV of the sCD25, sCD40L, sCD, sTREM-1 high and low calibrator are all within 5%.
Batch-to-batch difference: three batches of the kit were taken, 10 batches each were each repeatedly tested for 1 reference, the average and standard deviation SD of 30 measurements were calculated, and the coefficient of variation CV was calculated. The results of the batch-to-batch difference measurements are shown in Table 6.
TABLE 6 evaluation results of batch to batch differences (pg/mL)
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According to Table 6, the 3 lot-to-lot differences CV for CD25, sCD40L, sCD, sTREM-1 were all within 7%.
As shown by the repeatability and the inter-batch difference evaluation, the kit has better repeatability and inter-batch difference, and the variation Coefficient (CV) of the intra-batch experiment is not more than 5%, and the inter-batch difference CV is not more than 7%.
5. Clinical sample detection
Samples of healthy persons and hemophagocytic syndrome patients were each examined 1 case using the kit in this example, and subjected to comparison analysis. Table 7 shows the results of a sample comparison test (pg/mL) for healthy subjects and patients with hemophagocytic syndrome. Referring to Table 7, HLH patients had sCD25, sCD40L, sCD, sTREM-1, each higher than 1 healthy human sample tested, with sCD25 being most pronounced and greater than 6400pg/mL, consistent with the national expert consensus for diagnosis and treatment of hemophagocytic syndrome in 2018.
TABLE 7 clinical sample testing
| Healthy person | Patients with HLH | |
| sCD25 | 1367.24 | 8872.54 |
| sCD40L | 6030.58 | 8625.43 |
| sCD130 | 112526.78 | 156562.89 |
| sTREM-1 | 50.05 | 110.22 |
The above example shows that the kit for simultaneously detecting sCD25, sCD40L, sCD, sTREM-1 by using the flow fluorescence technology provided by the invention can be used for measuring the concentration of sCD25, sCD40L, sCD, sTREM-1 in different samples, thereby facilitating the clinical detection and application and improving the detection accuracy.
Example 2: influence of antibodies produced by different recombinant proteins on detection results
1. Effects of different recombinant protein yields and concentrations
In this example, a control experiment was performed using a full-length recombinant protein and the recombinant protein prepared by the method provided in example 1, wherein sCD25 was prepared using the full-length recombinant protein (nm_ 000417.3) and the optimized Seq ID NO:7, constructing a recombinant vector, and expressing the recombinant vector by 293T cells to prepare a recombinant protein; sCD40L used full-length sequence recombinant protein (nm_ 000074.3) and optimized Seq ID NO:8, constructing a recombinant vector, and expressing the recombinant vector by 293T cells to prepare a recombinant protein; sCD130 uses the full-length sequence recombinant protein (nm_ 002184.4) and the optimized Seq ID NO:9 constructing a recombinant vector, and expressing the recombinant vector by 293T cells to prepare a recombinant protein; sTREM-1 adopts full-length sequence recombinant protein (NM_ 018643.5) and optimized Seq ID NO:10 constructing a recombinant vector, and expressing the recombinant vector by 293T cells to prepare a recombinant protein; the yields and the concentrations after purification of the recombinant proteins produced are shown in Table 8.
TABLE 8 production and concentration of different recombinant proteins
As can be seen from Table 8, the recombinant vector prepared from the optimized nucleotide sequence can remarkably improve the yield of recombinant protein and the concentration in the culture medium through 293T cell expression, improve the efficiency of secreted protein and reduce the cost.
2. Influence of the prepared antibody on the detection result
Recombinant proteins prepared with different sequences and commercially available recombinant proteins were used, respectively, sCD25 recombinant protein (from Thermofisher, model RP-75591), sCD40L recombinant protein (from Thermofisher, model RP-8640), sCD130 recombinant protein (from Bio-tech, model 228-GP), sTREM-1 recombinant protein (from Abcam, model ab 132233), and antibodies were prepared by immunizing animals according to the method provided in example 1, thereby preparing a microsphere mixed solution and biotin-conjugated antibody mixed solution of conjugated antibodies, and commercially available conjugated microsphere sCD25 antibodies (from Thermofisher, model MA 5-36078), conjugated microsphere antibody sCD40L antibodies (from Abcam, ab 303610), conjugated microsphere antibody sCD130 antibodies (from Abcam, ab 259634), conjugated microsphere sTREM-1 antibodies (from Thermofisher, model 5-30968), and commercially available conjugated biotin sCD25 antibodies (from Bio-tec, 10200 b1, model MA 5-36078), conjugated biological cd antibodies (from 35, 35 were repeated according to the methods shown in examples of fig. 35, and the results of the methods of performing the conjugated biological assay.
TABLE 9 influence of antibodies prepared with different recombinant proteins on detection results
As can be seen from Table 9, the recombinant protein prepared from the optimized nucleotide sequence and the antibody obtained by immunizing animals can remarkably improve the yield of the recombinant protein and the concentration in the culture medium, improve the efficiency of secreting the protein and reduce the cost.
Example 3: screening of sCD40L recombinant protein coding sequence
The sequence of the antigen sCD40L prepared initially is: his-Tag sequence coded by CACCACCACCACCACCACCACCACCACCACTAG is added at the carboxyl end of extracellular region sequence (Glu 108-Leu 261) of CD40L gene, and the sequence Seq ID NO:17. recombinant vectors were constructed and recombinant proteins prepared by expression in 293T cells were detected to have little activity, presumably due to the interference of the antibody recognition site by His-tag present at the carboxy terminus of the extracellular region sequence of the CD40L gene (Glu 108-Leu 261). Therefore, the research team adds HHHHHHHHHGSG to the amino terminal of the extracellular region sequence of the CD40L gene (Glu 108-Leu261 sequence is shown as Seq ID NO: 14), deletes His-tag from the carboxyl terminal, and obtains the sequence Seq ID NO:8. the test results before and after the optimization are shown in Table 10, as tested in example 1.
TABLE 10 screening of different sequences of antigen sCD40L
| Dilution ratio \sCD40L coding sequence | Seq ID NO：17 | Seq ID NO：8 |
| Background value (MFI) | 785 | 812 |
| Dilution 1-fold (MFI) | 1346 | 267954 |
| Dilution by a factor of 2 (MFI) | 953 | 125730 |
| Dilution by a factor of 4 (MFI) | 651 | 63680 |
As can be seen from Table 10, the recombinant protein expressed by the optimized pre-sequence has a test value close to the background value and has almost no activity, while the recombinant protein expressed by the optimized pre-sequence has very high activity.
The optimized sequence Seq ID NO:8 preparing recombinant protein.
Example 4: screening of coding sequences of sTREM-1 recombinant proteins
Since the antibody prepared by the sTREM-1 recombinant protein prepared by the initial optimization easily causes cross influence of four soluble cytokine receptors of sCD25, sCD40L, sCD and sTREM-1 in the detection process, so that the detection result is inaccurate, the reason is probably that sTREM-1 forms a polymer, the spatial configuration is changed, and part of antigen epitopes cannot be exposed, the research team carries out repeated adjustment on the coding nucleotide sequence of sTREM-1 for a plurality of times, three more typical groups are exemplified herein, the antibody prepared by the sTREM-1 recombinant protein prepared by the three groups of coding nucleotide sequences, and the commercially available sTREM-1 antibody are used for detecting a sample to be detected, wherein the sample to be detected is prepared by adding a sample A with known concentration into a matrix solution, and the volume ratio between the sample A and the matrix solution is 1:9, each of the test was repeated 3 times, and the average was taken, and the test results are shown in Table 11.
Seq ID NO:10 adding an amino acid Ala at the N end of a complete extracellular region sequence (Ala 21-Asn 205) of the TREM-1 gene, deleting an amino acid VPVFN at the C end, and adding His-tag;
seq ID NO:11 adding a segment of alpha helix structure coded by amino acid AEAAAKEAAAKA at the C end of the complete extracellular region sequence (Ala 21-Asn 205) of the TREM-1 gene to rigidly connect the polypeptide and His-tag;
Seq ID NO:12 deleting the amino acid VPVFN at the C end of the complete extracellular region sequence (Ala 21-Asn 205) of the TREM-1 gene, and increasing His-tag;
TABLE 11 screening of coding sequences for sTREM-1 recombinant proteins
| STREM-1 coding sequence | sCD25(pg/mL) | sCD40L(pg/mL) | sCD130(pg/mL) | sTREM-1(pg/mL) |
| Theoretical concentration | 2000.00 | 9000.00 | 22500.00 | 2500.00 |
| Seq ID NO：10 | 1903.68 | 8581.30 | 22136.05 | 2395.48 |
| Seq ID NO：11 | 1625.08 | 6324.25 | 20213.27 | 2008.65 |
| Seq ID NO：12 | 1235.46 | 9665.94 | 19864.73 | 2137.59 |
| Commercially available | 1058.27 | 7035.18 | 19953.24 | 2017.39 |
As can be seen from Table 11, antibodies prepared using different coding nucleotide sequences of sTREM-1 have a certain effect on the detection results of all four soluble cytokines, and therefore it is most preferable to use the sequence of Seq ID NO:10 is used as the coding nucleotide sequence of sTREM-1, and the prepared antibody can obviously improve the sensitivity and accuracy when sCD25, sCD40L, sCD, sTREM-1 are detected simultaneously.
Example 5: screening of microspheres
In this example, microspheres coupled with different functional groups are selected to optimize detection sensitivity of sCD25, sCD40L, sCD and sTREM-1, the groups coupled with the sCD25, sCD40L, sCD and sTREM-1 microspheres are modified by carboxyl groups to amino groups, and calibration products with known sCD25 concentration of 20pg/mL, sCD40L concentration of 80pg/mL, sCD130 concentration of 400pg/mL and sTREM-1 concentration of 25pg/mL are used for detection, the fluorescence values of the detection results are calculated, and the signal to noise ratio is shown in table 12.
TABLE 12 detection sensitivity of different microspheres
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As can be seen from Table 12, the fluorescence values of sCD25, sCD40L, sCD, sTREM-1 were examined using microspheres coupled with different functional groups, and it can be seen that the signal to noise ratio was directly affected by the different microsphere modified functional groups. When the sCD25 is selected from amino modified microspheres, the signal to noise ratio is higher, and the sensitivity is higher; when the sCD40L is amino modified microsphere, the signal to noise ratio is higher, and the sensitivity is higher; the sCD130 amino modified microsphere has higher signal to noise ratio and higher sensitivity; the signal to noise ratio is higher and the sensitivity is higher when the sTREM-1 selects carboxyl modified microspheres.
The example again verifies that the microsphere coupled with sTREM-1 antibody has carboxyl modified functional groups, and the detection results when the microsphere coupled with sCD25 antibody, sCD40L antibody and sCD130 antibody has amino modified functional groups are compared with the detection results when the microsphere of sCD25, sCD40L, sCD and sTREM-1 antibody has all amino modified functional groups, and the results prove that the signal to noise ratio and the detection sensitivity can be obviously improved.
Therefore, the microsphere coupled with the sTREM-1 antibody has a carboxyl modified functional group, and the microsphere coupled with the sCD25 antibody, the sCD40L antibody and the sCD130 antibody has an amino modified functional group.
Example 6: screening assays for microsphere antibodies
The biological activity and immunological properties of the antibodies coupled to the microspheres have an important influence on the detection sensitivity, and the detection limit of the antibodies according to this example was determined by screening different antibodies, the other steps being the same as those of example 1, and the results being shown in Table 13.
TABLE 13 screening of microsphere coated antibodies
| sCD25(pg/mL) | sCD40L(pg/mL) | sCD130(pg/mL) | sTREM-1(pg/mL) | |
| Mouse resistance | 5.75 | 15.25 | 165.50 | 2.5 |
| Rabbit antibody | 2.50 | 5.0 | 50.00 | 9.55 |
| Sheep anti | 12.50 | 30.55 | 350.00 | 12.5 |
The sources of the sheep antigens are shown in Table 14, and the sources of the mouse and rabbit antigens are shown in example 2:
Table 14, sheep anti-manufacturer and model information
| Raw materials | Manufacturer' s | Model number |
| Sheep anti-human sCD25 antibody | Bio-Techne | AF-223-NA |
| Sheep anti-human sCD40L antibody | Thermofisher | PA5-46964 |
| Sheep anti-human sCD130 antibody | Thermofisher | PA5-47650 |
| Sheep anti-human sTREM-1 antibody | Thermofisher | PA5-47112 |
The results show that: the murine antibody and rabbit antibody markers are obviously superior to sheep antibodies, and the sCD25 and sCD40L, sCD microspheres are preferably marked by rabbit antibodies, so that the detection sensitivity and accuracy are higher, and the reason is probably that the rabbits of the inbred line are more rare and have more immune response diversity; however, the sTREM-1 microsphere is preferably marked by a mouse antibody, and the reason is that the mouse antibody is selected for sTREM-1, so that the cross influence can be reduced, and the detection sensitivity can be improved.
The present example again verifies that the detection results when the sCD25, sCD40L, sCD, 130 microspheres are preferably labeled with rabbit antibody and the sctrem-1 microspheres are preferably labeled with mouse antibody, and indeed the detection sensitivity can be significantly improved compared with the detection results when all the sCD25, sCD40L, sCD, 130 and sctrem-1 microspheres are labeled with rabbit antibody or mouse antibody.
Therefore, the microsphere-coupled sCD25 antibody, sCD40L antibody, and sCD130 antibody are preferably rabbit monoclonal antibodies, and the sTREM-1 antibody is preferably murine monoclonal antibodies.
Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.
Sequence listing
Seq ID NO：1
Amino acid sequence of sCD25 recombinant protein:
MYRMQLLSCIALSLALVTNSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCHHHHHHHHHH-
Seq ID NO：2
amino acid sequence of sCD40L recombinant protein:
MYRMQLLSCIALSLALVTNSHHHHHHHHHGSGENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL-
Seq ID NO：3
amino acid sequence of sCD130 recombinant protein:
MYRMQLLSCIALSLALVTNSELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIISGLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADCKAKRDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGITYEDRPSKAPSFWYKIDPSHTQGYRTVQLVWKTLPPFEANGKILDYEVTLTRWKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACDFQATHPVMDLKAFPKDNMLWVEWTTPRESVKKYILEWCVLSDKAPCITDWQQEDGTVHRTYLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGNETAVNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIHHHHHHHHHH-
Seq ID NO：4
Amino acid sequence 1 of sTREM-1 recombinant protein:
MYRMQLLSCIALSLALVTNSAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDIIRHHHHHH-
Seq ID NO：5
Amino acid sequence 2 of sTREM-1 recombinant protein:
MYRMQLLSCIALSLALVTNSATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDIIRVPVFNAEAAAKEAAAKAHHHHHHHH-
Seq ID NO：6
amino acid sequence 3 of sTREM-1 recombinant protein:
MYRMQLLSCIALSLALVTNSATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDIIRHHHHHH-
Seq ID NO：7
coding nucleotide sequence of sCD25 recombinant protein:
ATGTATAGAATGCAACTGCTGAGCTGTATCGCCCTGTCTCTGGCCCTGGTGACAAACTCTGAGCTGTGCGACGATGATCCTCCAGAAATTCCCCACGCCACATTTAAGGCTATGGCCTACAAGGAAGGAACCATGCTCAACTGCGAGTGCAAGCGGGGCTTCCGGAGGATCAAGTCCGGCAGCCTGTACATGCTGTGTACCGGCAACAGCAGCCACTCTAGCTGGGACAACCAGTGCCAGTGTACAAGCTCCGCCACCAGAAATACCACCAAGCAGGTGACCCCTCAGCCTGAAGAACAGAAAGAGCGGAAGACAACCGAGATGCAGAGCCCTATGCAGCCTGTGGACCAGGCCAGCCTGCCTGGCCACTGCAGAGAGCCTCCACCTTGGGAGAACGAGGCCACAGAGAGAATCTACCACTTCGTGGTGGGCCAGATGGTCTACTACCAGTGCGTGCAGGGCTACCGGGCCCTGCACAGAGGACCTGCCGAGTCCGTGTGTAAAATGACCCACGGCAAGACCAGATGGACCCAGCCCCAGCTGATCTGCACCGGAGAAATGGAAACCAGCCAGTTCCCCGGCGAGGAAAAGCCCCAAGCTAGCCCCGAAGGCAGACCTGAGAGCGAGACAAGCTGCCACCACCACCACCACCACCACCATCATCACTGA
Seq ID NO：8
coding nucleotide sequence of sCD40L recombinant protein:
ATGTATAGAATGCAGCTGCTGTCTTGTATCGCTCTGAGCCTGGCCCTGGTGACCAACTCCCACCATCACCACCACCACCACCACCACGGATCTGGCGAGAACAGCTTTGAGATGCAGAAAGGCGACCAGAACCCCCAGATTGCCGCCCACGTGATCAGCGAGGCCTCTAGCAAGACCACAAGCGTGCTGCAGTGGGCCGAGAAGGGCTACTACACCATGAGCAACAACCTGGTCACCCTGGAAAACGGCAAGCAGCTGACCGTGAAGCGGCAGGGCCTGTACTACATCTACGCCCAGGTTACATTCTGCAGCAATCGGGAAGCTTCTAGCCAGGCCCCATTCATCGCCAGCCTGTGCCTGAAGAGCCCCGGCAGATTCGAAAGAATCCTGCTGAGAGCCGCTAATACCCACAGCTCCGCCAAGCCTTGCGGCCAGCAGAGCATCCACCTGGGAGGCGTGTTCGAGCTGCAACCTGGAGCCAGCGTGTTCGTGAACGTGACAGATCCTAGCCAAGTGTCCCACGGTACAGGCTTCACCTCTTTTGGCCTGCTCAAACTGTGA
Seq ID NO：9
Coding nucleotide sequence of sCD130 recombinant protein:
ATGTATCGGATGCAGCTGCTGTCTTGCATCGCCCTGTCTCTGGCTCTGGTCACAAACAGCGAGCTGCTGGATCCTTGTGGATACATCAGCCCCGAATCACCTGTTGTGCAGCTGCACAGCAACTTCACCGCCGTGTGTGTGCTGAAGGAAAAGTGCATGGATTACTTCCACGTGAACGCTAACTACATCGTGTGGAAGACCAATCACTTCACCATCCCGAAGGAACAGTACACAATCATCAATAGAACCGCCTCTTCTGTGACATTCACAGACATCGCCAGCCTGAACATCCAGCTGACCTGCAACATCCTGACATTTGGCCAGCTGGAACAGAACGTGTACGGCATCACCATCATTTCTGGACTGCCCCCTGAGAAGCCTAAGAACCTCAGCTGTATCGTGAACGAGGGCAAAAAGATGAGATGCGAGTGGGACGGCGGAAGAGAGACCCACCTGGAAACCAACTTTACCCTGAAGAGTGAGTGGGCCACCCACAAGTTCGCCGACTGTAAGGCCAAGCGGGACACACCCACATCTTGCACCGTTGACTACTCCACTGTGTACTTCGTGAACATCGAGGTGTGGGTGGAGGCTGAAAACGCCCTGGGCAAGGTGACCAGCGATCACATCAACTTTGACCCCGTGTACAAGGTGAAGCCCAACCCCCCACACAACCTGTCTGTGATTAACTCTGAAGAACTGAGCTCCATCCTCAAGCTGACCTGGACAAACCCCAGCATTAAGAGCGTGATCATCCTGAAGTACAACATCCAGTACAGAACCAAGGACGCCTCTACCTGGAGCCAGATCCCTCCTGAGGACACCGCCTCCACCAGAAGCAGCTTTACGGTCCAGGACCTGAAGCCTTTCACCGAATACGTGTTCCGGATCAGATGCATGAAAGAGGATGGCAAAGGCTACTGGAGCGACTGGTCCGAAGAGGCCAGCGGCATCACATACGAGGACCGGCCTAGCAAGGCTCCTAGCTTCTGGTACAAGATCGACCCCAGCCACACCCAAGGCTACAGAACCGTTCAACTGGTGTGGAAGACACTGCCTCCCTTTGAAGCCAATGGCAAGATCTTAGATTACGAAGTGACACTGACAAGATGGAAAAGCCACTTGCAGAATTATACCGTGAACGCCACCAAGCTGACCGTGAACCTTACAAACGACAGATACCTGGCCACCCTGACAGTGCGGAACCTGGTCGGAAAATCTGATGCCGCTGTGCTGACCATCCCTGCCTGCGACTTCCAGGCCACCCACCCTGTGATGGACCTGAAAGCCTTCCCTAAAGATAACATGCTGTGGGTCGAGTGGACCACCCCAAGAGAGAGCGTGAAGAAGTACATTCTGGAATGGTGCGTGCTGAGCGACAAGGCTCCTTGCATCACCGATTGGCAGCAGGAGGACGGCACAGTGCACAGAACCTATCTGAGAGGCAATCTGGCCGAGAGCAAGTGCTATCTGATCACAGTGACCCCCGTTTACGCCGACGGCCCTGGCAGCCCAGAGAGCATCAAGGCGTACCTGAAACAGGCCCCTCCTAGCAAAGGCCCTACCGTGCGGACCAAGAAGGTAGGAAAGAATGAGGCCGTGCTGGAATGGGATCAGCTGCCTGTGGATGTGCAAAACGGCTTCATCAGAAACTACACTATCTTCTACCGCACCATCATCGGAAACGAGACAGCCGTGAATGTGGACAGCTCCCACACAGAGTACACCCTGTCCAGCCTGACAAGCGACACTCTGTACATGGTGAGGATGGCCGCATACACAGACGAGGGCGGCAAGGACGGCCCCGAATTCACCTTCACAACCCCAAAGTTCGCCCAGGGCGAGATACACCACCATCACCATCACCACCACCACCATTGA
Seq ID NO：10
coding nucleotide sequence 1 of sTREM-1 recombinant protein:
ATGTATAGAATGCAGCTGCTCTCCTGCATCGCCCTGAGCCTGGCTCTGGTGACCAATAGCGCCGCCACAAAGCTGACCGAAGAGAAGTACGAGCTGAAGGAGGGACAGACACTGGACGTGAAGTGCGACTACACACTGGAAAAGTTCGCCAGCAGCCAGAAAGCCTGGCAGATCATCCGGGACGGCGAGATGCCTAAGACCCTGGCCTGCACCGAGCGGCCTAGCAAGAACAGCCACCCCGTCCAGGTGGGAAGAATCATCCTGGAAGATTACCACGACCACGGCCTGCTGAGAGTGCGGATGGTGAACCTGCAGGTTGAAGATTCTGGCCTGTACCAGTGCGTGATCTACCAACCTCCAAAAGAGCCTCACATGCTGTTCGACAGAATCAGACTGGTCGTGACCAAAGGCTTTAGCGGCACCCCTGGCAGCAACGAGAACTCCACCCAGAACGTGTACAAGATCCCCCCCACCACCACAAAGGCTCTGTGTCCTCTGTACACCAGCCCTAGAACCGTGACCCAAGCCCCTCCAAAGTCTACCGCCGACGTGTCTACACCCGACAGCGAGATCAACCTGACAAATGTGACTGATATCATTAGGCACCACCACCATCACCACTGA
Seq ID NO：11
coding nucleotide sequence 2 of sTREM-1 recombinant protein:
ATGTATAGAATGCAGCTGCTCTCCTGCATCGCCCTGAGCCTGGCTCTGGTGACCAATAGCGCCACAAAGCTGACCGAAGAGAAGTACGAGCTGAAGGAGGGACAGACACTGGACGTGAAGTGCGACTACACACTGGAAAAGTTCGCCAGCAGCCAGAAAGCCTGGCAGATCATCCGGGACGGCGAGATGCCTAAGACCCTGGCCTGCACCGAGCGGCCTAGCAAGAACAGCCACCCCGTCCAGGTGGGAAGAATCATCCTGGAAGATTACCACGACCACGGCCTGCTGAGAGTGCGGATGGTGAACCTGCAGGTTGAAGATTCTGGCCTGTACCAGTGCGTGATCTACCAACCTCCAAAAGAGCCTCACATGCTGTTCGACAGAATCAGACTGGTCGTGACCAAAGGCTTTAGCGGCACCCCTGGCAGCAACGAGAACTCCACCCAGAACGTGTACAAGATCCCCCCCACCACCACAAAGGCTCTGTGTCCTCTGTACACCAGCCCTAGAACCGTGACCCAAGCCCCTCCAAAGTCTACCGCCGACGTGTCTACACCCGACAGCGAGATCAACCTGACAAATGTGACTGATATCATTAGGGTGCCCGTGTTCAACGCCGAGGCCGCTGCCAAGGAAGCCGCCGCTAAGGCCCACCATCACCACCACCATCACCACTGA
Seq ID NO：12
coding nucleotide sequence 3 of sTREM-1 recombinant protein:
ATGTATAGAATGCAGCTGCTCTCCTGCATCGCCCTGAGCCTGGCTCTGGTGACCAATAGCGCCACAAAGCTGACCGAAGAGAAGTACGAGCTGAAGGAGGGACAGACACTGGACGTGAAGTGCGACTACACACTGGAAAAGTTCGCCAGCAGCCAGAAAGCCTGGCAGATCATCCGGGACGGCGAGATGCCTAAGACCCTGGCCTGCACCGAGCGGCCTAGCAAGAACAGCCACCCCGTCCAGGTGGGAAGAATCATCCTGGAAGATTACCACGACCACGGCCTGCTGAGAGTGCGGATGGTGAACCTGCAGGTTGAAGATTCTGGCCTGTACCAGTGCGTGATCTACCAACCTCCAAAAGAGCCTCACATGCTGTTCGACAGAATCAGACTGGTCGTGACCAAAGGCTTTAGCGGCACCCCTGGCAGCAACGAGAACTCCACCCAGAACGTGTACAAGATCCCCCCCACCACCACAAAGGCTCTGTGTCCTCTGTACACCAGCCCTAGAACCGTGACCCAAGCCCCTCCAAAGTCTACCGCCGACGTGTCTACACCCGACAGCGAGATCAACCTGACAAATGTGACTGATATCATTAGGCACCACCACCATCACCACTGA
Seq ID NO：13
Glu22-Cys213 sequence:
GAGCTCTGTGACGATGACCCGCCAGAGATCCCACACGCCACATTCAAAGCCATGGCCTACAAGGAAGGAACCATGTTGAACTGTGAATGCAAGAGAGGTTTCCGCAGAATAAAAAGCGGGTCACTCTATATGCTCTGTACAGGAAACTCTAGCCACTCGTCCTGGGACAACCAATGTCAATGCACAAGCTCTGCCACTCGGAACACAACGAAACAAGTGACACCTCAACCTGAAGAACAGAAAGAAAGGAAAACCACAGAAATGCAAAGTCCAATGCAGCCAGTGGACCAAGCGAGCCTTCCAGGTCACTGCAGGGAACCTCCACCATGGGAAAATGAAGCCACAGAGAGAATTTATCATTTCGTGGTGGGGCAGATGGTTTATTATCAGTGCGTCCAGGGATACAGGGCTCTACACAGAGGTCCTGCTGAGAGCGTCTGCAAAATGACCCACGGGAAGACAAGGTGGACCCAGCCCCAGCTCATATGCACAGGTGAAATGGAGACCAGTCAGTTTCCAGGTGAAGAGAAGCCTCAGGCAAGCCCCGAAGGCCGTCCTGAGAGTGAGACTTCCTGC
Seq ID NO：14
Glu108-Leu261 sequence:
GAAAACAGCTTTGAAATGCAAAAAGGTGATCAGAATCCTCAAATTGCGGCACATGTCATAAGTGAGGCCAGCAGTAAAACAACATCTGTGTTACAGTGGGCTGAAAAAGGATACTACACCATGAGCAACAACTTGGTAACCCTGGAAAATGGGAAACAGCTGACCGTTAAAAGACAAGGACTCTATTATATCTATGCCCAAGTCACCTTCTGTTCCAATCGGGAAGCTTCGAGTCAAGCTCCATTTATAGCCAGCCTCTGCCTAAAGTCCCCCGGTAGATTCGAGAGAATCTTACTCAGAGCTGCAAATACCCACAGTTCCGCCAAACCTTGCGGGCAACAATCCATTCACTTGGGAGGAGTATTTGAATTGCAACCAGGTGCTTCGGTGTTTGTCAATGTGACTGATCCAAGCCAAGTGAGCCATGGCACTGGCTTCACGTCCTTTGGCTTACTCAAACTC
Seq ID NO：15
glu23-Ile618 sequence:
GAACTTCTAGATCCATGTGGTTATATCAGTCCTGAATCTCCAGTTGTACAACTTCATTCTAATTTCACTGCAGTTTGTGTGCTAAAGGAAAAATGTATGGATTATTTTCATGTAAATGCTAATTACATTGTCTGGAAAACAAACCATTTTACTATTCCTAAGGAGCAATATACTATCATAAACAGAACAGCATCCAGTGTCACCTTTACAGATATAGCTTCATTAAATATTCAGCTCACTTGCAACATTCTTACATTCGGACAGCTTGAACAGAATGTTTATGGAATCACAATAATTTCAGGCTTGCCTCCAGAAAAACCTAAAAATTTGAGTTGCATTGTGAACGAGGGGAAGAAAATGAGGTGTGAGTGGGATGGTGGAAGGGAAACACACTTGGAGACAAACTTCACTTTAAAATCTGAATGGGCAACACACAAGTTTGCTGATTGCAAAGCAAAACGTGACACCCCCACCTCATGCACTGTTGATTATTCTACTGTGTATTTTGTCAACATTGAAGTCTGGGTAGAAGCAGAGAATGCCCTTGGGAAGGTTACATCAGATCATATCAATTTTGATCCTGTATATAAAGTGAAGCCCAATCCGCCACATAATTTATCAGTGATCAACTCAGAGGAACTGTCTAGTATCTTAAAATTGACATGGACCAACCCAAGTATTAAGAGTGTTATAATACTAAAATATAACATTCAATATAGGACCAAAGATGCCTCAACTTGGAGCCAGATTCCTCCTGAAGACACAGCATCCACCCGATCTTCATTCACTGTCCAAGACCTTAAACCTTTTACAGAATATGTGTTTAGGATTCGCTGTATGAAGGAAGATGGTAAGGGATACTGGAGTGACTGGAGTGAAGAAGCAAGTGGGATCACCTATGAAGATAGACCATCTAAAGCACCAAGTTTCTGGTATAAAATAGATCCATCCCATACTCAAGGCTACAGAACTGTACAACTCGTGTGGAAGACATTGCCTCCTTTTGAAGCCAATGGAAAAATCTTGGATTATGAAGTGACTCTCACAAGATGGAAATCACATTTACAAAATTACACAGTTAATGCCACAAAACTGACAGTAAATCTCACAAATGATCGCTATCTAGCAACCCTAACAGTAAGAAATCTTGTTGGCAAATCAGATGCAGCTGTTTTAACTATCCCTGCCTGTGACTTTCAAGCTACTCACCCTGTAATGGATCTTAAAGCATTCCCCAAAGATAACATGCTTTGGGTGGAATGGACTACTCCAAGGGAATCTGTAAAGAAATATATACTTGAGTGGTGTGTGTTATCAGATAAAGCACCCTGTATCACAGACTGGCAACAAGAAGATGGTACCGTGCATCGCACCTATTTAAGAGGGAACTTAGCAGAGAGCAAATGCTATTTGATAACAGTTACTCCAGTATATGCTGATGGACCAGGAAGCCCTGAATCCATAAAGGCATACCTTAAACAAGCTCCACCTTCCAAAGGACCTACTGTTCGGACAAAAAAAGTAGGGAAAAACGAAGCTGTCTTAGAGTGGGACCAACTTCCTGTTGATGTTCAGAATGGATTTATCAGAAATTATACTATATTTTATAGAACCATCATTGGAAATGAAACTGCTGTGAATGTGGATTCTTCCCACACAGAATATACATTGTCCTCTTTGACTAGTGACACATTGTACATGGTACGAATGGCAGCATACACAGATGAAGGTGGGAAGGATGGTCCAGAATTCACTTTTACTACCCCAAAGTTTGCTCAAGGAGAAATT
Seq ID NO：16
ala20-Asn205 sequence:
GCCGCAACTAAATTAACTGAGGAAAAGTATGAACTGAAAGAGGGGCAGACCCTGGATGTGAAATGTGACTACACGCTAGAGAAGTTTGCCAGCAGCCAGAAAGCTTGGCAGATAATAAGGGACGGAGAGATGCCCAAGACCCTGGCATGCACAGAGAGGCCTTCAAAGAATTCCCATCCAGTCCAAGTGGGGAGGATCATACTAGAAGACTACCATGATCATGGTTTACTGCGCGTCCGAATGGTCAACCTTCAAGTGGAAGATTCTGGACTGTATCAGTGTGTGATCTACCAGCCTCCCAAGGAGCCTCACATGCTGTTCGATCGCATCCGCTTGGTGGTGACCAAGGGTTTTTCAGGGACCCCTGGCTCCAATGAGAATTCTACCCAGAATGTGTATAAGATTCCTCCTACCACCACTAAGGCCTTGTGCCCACTCTATACCAGCCCCAGAACTGTGACCCAAGCTCCACCCAAGTCAACTGCCGATGTCTCCACTCCTGACTCTGAAATCAACCTTACAAATGTGACAGATATCATCAGGGTTCCGGTGTTCAAC
Seq ID NO：17
coding nucleotide sequence of sCD40L recombinant protein:
ATGTATAGAATGCAGCTGCTGTCTTGTATCGCTCTGAGCCTGGCCCTGGTGACCAACTCCGAGAACAGCTTTGAGATGCAGAAAGGCGACCAGAACCCCCAGATTGCCGCCCACGTGATCAGCGAGGCCTCTAGCAAGACCACAAGCGTGCTGCAGTGGGCCGAGAAGGGCTACTACACCATGAGCAACAACCTGGTCACCCTGGAAAACGGCAAGCAGCTGACCGTGAAGCGGCAGGGCCTGTACTACATCTACGCCCAGGTTACATTCTGCAGCAATCGGGAAGCTTCTAGCCAGGCCCCATTCATCGCCAGCCTGTGCCTGAAGAGCCCCGGCAGATTCGAAAGAATCCTGCTGAGAGCCGCTAATACCCACAGCTCCGCCAAGCCTTGCGGCCAGCAGAGCATCCACCTGGGAGGCGTGTTCGAGCTGCAACCTGGAGCCAGCGTGTTCGTGAACGTGACAGATCCTAGCCAAGTGTCCCACGGTACAGGCTTCACCTCTTTTGGCCTGCTCAAACTGCACCACCACCACCACCACCACCACCATCATTGA.
Claims (8)
1. A kit for simultaneously detecting four soluble cytokine receptors based on a flow cytometer, which is characterized by comprising a microsphere mixed solution of coupled antibodies, a biotin-coupled antibody mixed solution and a sample diluent, wherein the antibodies comprise a sCD25 antibody, a sCD40L antibody, a sCD130 antibody and a sTREM-1 antibody, and the antibodies are respectively obtained from recombinant proteins sCD25, sCD40L, sCD and sTREM-1 immunized animals; coding nucleotide sequences for sCD25 recombinant proteins such as Seq ID NO:7, the coding nucleotide sequence of sCD40L recombinant protein is as shown in Seq ID NO:8, the coding nucleotide sequence of sCD130 recombinant protein is as shown in Seq ID NO:9, the coding nucleotide sequence of the sTREM-1 recombinant protein is shown as Seq ID NO:10~ Seq ID NO:12, any one of the following; the four soluble cytokine receptors are sCD25, sCD40L, sCD and sTREM-1, respectively; the sCD25 antibody, the sCD40L antibody and the sCD130 antibody are rabbit monoclonal antibodies, and the sTREM-1 antibody is a mouse monoclonal antibody.
2. The kit of claim 1, wherein the microspheres coupled to the sTREM-1 antibody have a carboxyl-modified functionality and the microspheres coupled to the sCD25 antibody, the sCD40L antibody, and the sCD130 antibody have an amino-modified functionality.
3. The kit of claim 2, wherein the mixture of biotin-conjugated antibodies is a mixture of sCD25, sCD40L, sCD130,130, and sTREM-1 biotin-labeled antibodies; the kit also comprises a fluorescent reagent, wherein the fluorescent reagent is a conjugate of streptavidin and phycoerythrin.
4. A method of preparing a kit for detecting a soluble cytokine receptor, comprising the steps of:
(1) Preparing recombinant proteins, wherein the recombinant proteins comprise sCD25 recombinant proteins, sCD40L recombinant proteins, sCD130 recombinant proteins and sTREM-1 recombinant proteins; the coding nucleotide sequence of the sCD25 recombinant protein is as Seq ID NO:7, the coding nucleotide sequence of sCD40L recombinant protein is as shown in Seq ID NO:8, the coding nucleotide sequence of sCD130 recombinant protein is as shown in Seq ID NO:9, the coding nucleotide sequence of the sTREM-1 recombinant protein is shown as Seq ID NO:10~ Seq ID NO:12, any one of the following;
(2) Immunizing an animal with the recombinant protein prepared in step (1) to obtain antibodies including sCD25 antibody, sCD40L antibody, sCD130 antibody and sTREM-1 antibody; the sCD25 antibody, the sCD40L antibody and the sCD130 antibody are rabbit monoclonal antibodies, and the sTREM-1 antibody is a mouse monoclonal antibody;
(3) Preparing a microsphere mixed solution of the conjugated antibody and a biotin conjugated antibody mixed solution by using the antibody prepared in the step (2).
5. The method of claim 4, wherein the coding nucleotide sequence of the sTREM-1 recombinant protein is as set forth in Seq ID No: shown at 10.
6. The method of claim 5, wherein the method of producing a recombinant protein according to step (1) comprises the steps of:
(a) Inserting the nucleotide for encoding the recombinant protein into an empty vector to obtain a recombinant vector;
(b) Transfecting the recombinant vector into a eukaryotic cell expression system;
(c) Collecting a culture medium of a eukaryotic cell expression system;
(d) Purifying by Ni-NTA agarose chromatographic column.
7. The method of claim 6, wherein the empty vector is pcdna3.4 empty vector; the eukaryotic cell expression system includes 293T cells; the culture medium is a high sugar culture medium of 10% fetal bovine serum and 90% DMEM.
8. Use of a set of recombinant proteins for the preparation of a reagent for increasing the detection sensitivity of a flow cytometer for simultaneous detection of four soluble cytokine receptors, characterized in that the recombinant proteins are sCD25, sCD40L, sCD and sTREM-1; the coding nucleotide sequence of the sCD25 recombinant protein is as Seq ID NO:7, the coding nucleotide sequence of sCD40L recombinant protein is as shown in Seq ID NO:8, the coding nucleotide sequence of sCD130 recombinant protein is as shown in Seq ID NO:9, the coding nucleotide sequence of the sTREM-1 recombinant protein is shown as Seq ID NO:10~ Seq ID NO:12, any one of the following; the four soluble cytokine receptors are sCD25, sCD40L, sCD and sTREM-1, respectively; such agents include sCD25 antibodies, sCD40L antibodies, sCD130 antibodies, and sTREM-1 antibodies; the antibodies are obtained from animals immunized with recombinant proteins sCD25, sCD40L, sCD, 130 and sTREM-1, respectively; the sCD25 antibody, the sCD40L antibody and the sCD130 antibody are rabbit monoclonal antibodies, and the sTREM-1 antibody is a mouse monoclonal antibody.
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| CN202211144691.3A CN115586338A (en) | 2022-09-20 | 2022-09-20 | Kit for detecting soluble cytokine receptor and preparation method thereof |
| CN2022111446913 | 2022-09-20 |
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102964428A (en) * | 2012-11-23 | 2013-03-13 | 同昕生物技术（北京）有限公司 | Kit for detecting soluble leukocyte differentiation antigen 40 ligand and polypeptide ligand |
| CN104220456A (en) * | 2012-02-15 | 2014-12-17 | 诺和诺德A/S（股份有限公司） | Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1) |
| CN104744582A (en) * | 2015-04-03 | 2015-07-01 | 福州大学 | gp130 extracellular domain protein as well as production method and application thereof |
| CN107921128A (en) * | 2015-08-05 | 2018-04-17 | 詹森生物科技公司 | Anti- CD154 antibody and its application method |
| CN110049995A (en) * | 2017-11-17 | 2019-07-23 | 盖立复诊断解决方案公司 | Novel human immunodeficiency's virus envelope protein antigen of mammal expression |
| CN112430621A (en) * | 2019-08-09 | 2021-03-02 | 百奥赛图(北京)医药科技股份有限公司 | Construction method and application of IL2RA gene humanized non-human animal |
| CN112813080A (en) * | 2019-11-15 | 2021-05-18 | 复旦大学 | Swine fever virus E2 protein optimized by artificial codon and preparation method and application thereof |
| CN113214397A (en) * | 2014-07-17 | 2021-08-06 | 诺和诺德股份有限公司 | Site-directed mutagenesis of TREM-1 antibodies to reduce viscosity |
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Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104220456A (en) * | 2012-02-15 | 2014-12-17 | 诺和诺德A/S（股份有限公司） | Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1) |
| CN102964428A (en) * | 2012-11-23 | 2013-03-13 | 同昕生物技术（北京）有限公司 | Kit for detecting soluble leukocyte differentiation antigen 40 ligand and polypeptide ligand |
| CN113214397A (en) * | 2014-07-17 | 2021-08-06 | 诺和诺德股份有限公司 | Site-directed mutagenesis of TREM-1 antibodies to reduce viscosity |
| CN104744582A (en) * | 2015-04-03 | 2015-07-01 | 福州大学 | gp130 extracellular domain protein as well as production method and application thereof |
| CN107921128A (en) * | 2015-08-05 | 2018-04-17 | 詹森生物科技公司 | Anti- CD154 antibody and its application method |
| CN110049995A (en) * | 2017-11-17 | 2019-07-23 | 盖立复诊断解决方案公司 | Novel human immunodeficiency's virus envelope protein antigen of mammal expression |
| CN112430621A (en) * | 2019-08-09 | 2021-03-02 | 百奥赛图(北京)医药科技股份有限公司 | Construction method and application of IL2RA gene humanized non-human animal |
| CN112813080A (en) * | 2019-11-15 | 2021-05-18 | 复旦大学 | Swine fever virus E2 protein optimized by artificial codon and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
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| Biological variation of immunological blood biomarkers in healthy individuals and quality goals for biomarker tests;Najib Aziz等;《BMC Immunology》;20191231;第20卷(第33期);1-11页 * |
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