CN114236139A - Antibody detection kit for TNF-alpha biological agent and preparation method thereof - Google Patents

Antibody detection kit for TNF-alpha biological agent and preparation method thereof Download PDF

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CN114236139A
CN114236139A CN202111646130.9A CN202111646130A CN114236139A CN 114236139 A CN114236139 A CN 114236139A CN 202111646130 A CN202111646130 A CN 202111646130A CN 114236139 A CN114236139 A CN 114236139A
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cdr
tnf
antibody
variable region
detection kit
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陈菲
王莹
霍如松
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Hunan Herui Biotechnology Co ltd
Suzhou Herui Biotechnology Co ltd
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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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    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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    • G01N33/548—Carbohydrates, e.g. dextran
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    • G01—MEASURING; TESTING
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    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/525—Tumor necrosis factor [TNF]

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Abstract

The invention discloses an antibody detection kit of a TNF-alpha biological agent and a preparation method thereof, and relates to the technical field of immunodetection. The kit comprises an antibody capture device and an antibody detection device, wherein the antibody capture device comprises a solid phase carrier coated with a TNF-alpha biological agent, and the antibody detection device is a cellulose membrane for fixing a functional fragment or a conjugate coupled with the functional fragment; the functional fragment is a variable region, a heavy chain variable region, or a complementarity determining region of a TNF-alpha biologic of the TNF-alpha biologic. The invention overcomes the defects of false positive and false negative in the prior art. The inventor sets that the variable region, the heavy chain variable region or the complementary determining region of the detection antigen with small molecular weight, such as TNF-alpha biological preparation, has the advantages of small steric hindrance and easy combination with Fab of an anti-antibody, so that the detection sensitivity is higher, and the purpose of the concomitant diagnosis of the TNF-alpha biological preparation medicament is achieved. Thereby avoiding the occurrence of false negatives.

Description

Antibody detection kit for TNF-alpha biological agent and preparation method thereof
Technical Field
The invention relates to the technical field of immunodetection, in particular to an antibody detection kit of a TNF-alpha biological agent and a preparation method thereof.
Background
Tumor necrosis factor alpha (TNF α) is a cytokine produced mainly by inflammatory cells such as monocytes and macrophages, and was originally named based on its ability to induce necrosis of certain tumors in mice. TNF α was later discovered to be involved in the pathophysiology of a variety of other human diseases and conditions, including shock, sepsis, infection, autoimmune diseases, rheumatoid arthritis, crohn's disease, transplant rejection, and graft versus host disease.
Biological agents designed to target TNF α include TNF α mabs (e.g., Remicade (infliximab, human-mouse chimeric), Humira (humanized adalimumab), Cimzia (certolizumab ozoga)). Among them, Cimzia (certolizumab ozogamicin, pegylated Fab fragment) is FDA approved for the treatment of rheumatoid arthritis, Inflammatory Bowel Disease (IBD), psoriasis, and ankylosing spondylitis, etc. Although clinical practice has shown that such biological agents are effective in treating the above-mentioned diseases, not all patients are effective, some are ineffective from the outset (primary ineffective), and more are effective from the outset, but develop resistance after multiple uses and are ineffective (secondary ineffective). Further developments found that secondary nullimers were associated with the in vivo production of antibodies against the anti-biologic drug, and therefore, the detection of anti-biologic antibodies in the blood, which is recommended by clinical guidelines, is an important companion diagnostic.
The detection method of the biological agent antibody of the anti-TNF alpha is mainly carried out through immunological reaction, and comprises an indirect method or a double-antigen sandwich method. 201180042537.9 patent uses Fab of therapeutic monoclonal antibody to coat solid phase carrier to capture immune complex formed by target antibody, and uses labeling method to detect the content of immune complex, and this method belongs to indirect method. Patent 201180060851.X uses therapeutic antibody to capture the anti-antibody in the sample, and the molecular exclusion method distinguishes specific anti-antibody, and the principle is high performance liquid detection after the capture of full-length monoclonal antibody, belonging to indirect method and liquid phase method. For detection schemes with washing steps such as ELISA, tubular chemiluminescence, etc., indirect methods are highly efficient and effective detection methods. However, in the case of a detection scheme lacking a washing step in the image chromatography detection, the indirect method is interfered by a large amount of extraneous antibodies in the sample, and thus detection is distorted, such as false positive. The double-antigen sandwich method can effectively avoid the interference of irrelevant antibodies in a sample. For example, in the determination of anti-drug antibodies disclosed in patent 200780002957.8, a double antigen bridging immunoassay is mentioned, in which a mixture of drug antibodies is used as a capture antigen and a mixture of drug antibodies is used as a detection antigen. In patent 201510788470.3, biotinylated TNF α mab was used as capture antigen and AP-labeled TNF α mab was used as detection antibody, and the principle was that two full-length mabs were used to form a double antigen sandwich. However, the existing double-antigen sandwich method patents have the problem of false negative.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide an antibody detection kit of a TNF-alpha biological agent and a preparation method thereof so as to solve the technical problems.
The inventor creatively discovers that the root of the double-antigen sandwich method technical scheme, which causes the false negative result, is that: the problem of the binding efficiency between the immune complex formed after capture and the detection antigen is not fully considered, so that even if the target antibody in the positive sample is bound with the capture antigen to form the immune complex, the target antibody cannot be bound with the detection antigen with high sensitivity, and the detection result of the positive sample is misjudged (false negative is caused) in the subsequent detection.
The invention is realized by the following steps:
the invention provides an antibody detection kit of a TNF-alpha biological agent, which comprises an antibody capture device and an antibody detection device, wherein the antibody capture device comprises a solid phase carrier, and the antibody detection device is a cellulose membrane or a functional fragment with a detectable marker; the solid phase carrier is coated with TNF-alpha biological agent, and the cellulose membrane is fixed with functional fragments without markers; the functional fragment is a variable region, a heavy chain variable region or a complementarity determining region of the TNF-alpha biological agent.
The invention overcomes the defects of false positive and false negative in the prior art. The TNF alpha biological agent is used as the capture antigen, and the spatial contact surface is large, so that the anti-TNF-alpha biological agent antibody (namely the anti-antibody) in the sample can be captured more easily, and the advantage of high capture efficiency is achieved. By coating the TNF α biological agent on the capture device, the occurrence of false positives is avoided.
The antigen-antibody complex formed after capture has a large steric hindrance itself, and if the biological agent is coated on the antibody detection device, it is difficult for the biological agent coated on the detection device to bind to the other Fab of the antibody against the anti-TNF- α biological agent in the complex formed after capture because the biological agent (i.e., the detection antigen) has a large molecular weight. Therefore, the inventor sets that the variable region, the heavy chain variable region or the complementarity determining region of the detection antigen with small molecular weight, such as the TNF-alpha biological preparation, has the advantages of small steric hindrance and easy combination with Fab of an anti-antibody, so that the detection sensitivity is higher, and the purpose of the concomitant diagnosis of the TNF-alpha biological preparation drug is achieved. Therefore, the subsequent misjudgment of the sample detection result is avoided, and the phenomenon of false negative is avoided.
In an alternative embodiment, the functional fragment will generally have the same binding specificity as the antibody from which it is derived. The detection antigens for the above variable region (Fv) were: fv (variable region) in which the variable regions of the heavy chain and the variable regions of the light chain are linked by a linker peptide or a disulfide bond to maintain the stability of the Fv. The complementarity determining regions include CDR1, CDR2 and CDR3 in the heavy chain complementarity determining region and CDR1, CDR2 and CDR3 in the light chain complementarity determining region. It will be readily understood by those skilled in the art from the disclosure of the present invention that functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction cleavage of disulfide bonds. Based on the disclosure of the structure of the intact antibody, the above-described functional fragments are readily available to those skilled in the art.
Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art (e.g., recombinant expression by means of gene recombination) or by synthesis, for example, by an automated peptide synthesizer such as those sold by Applied BioSystems and the like.
In a preferred embodiment of the present invention, the TNF- α biological agent is selected from the group consisting of monoclonal antibody drugs of TNF- α.
In an alternative embodiment, the TNF- α mab drug is selected from Infliximab (Infliximab), Adalimumab (Adalimumab), Certolizumab Pegol (Certolizumab Pegol), or Golimumab (golimmab).
In a preferred embodiment of the present invention, the solid phase carrier is selected from fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, colloidal gold particles, quantum dots, microporous plates or microporous membranes. The colloidal gold is a colloidal solution obtained by reducing tetrachloroauric acid by a reducing agent. In other embodiments, the colloidal gold may be other colloidal substances, such as colloidal carbon, colloidal silver, or colloidal selenium.
In an alternative embodiment, the quantum dots are core-shell quantum dots, such as ZnS/CdSe or ZnS/CdTe quantum dots. According to the requirement, the quantum dots can be adjusted to quantum dots formed by a single compound, such as cadmium selenide (CdSe), zinc sulfide (ZnS), cadmium telluride (CdTe), cadmium sulfide (CdS), zinc selenide (ZnSe), indium phosphide (InP) or indium arsenide (InAs), or one of substances such as nanocrystals or semiconductor nanocrystals formed by wrapping a layer of ZnS or CdS on a CdSe core.
In an alternative embodiment, the TNF- α biologic coated on the solid support is modified with avidin or biotin. It should be noted that in an alternative embodiment, non-modified avidin or biotin may also be selected as desired on the TNF- α biologic.
In an alternative embodiment, the fluorescent microspheres are selected from time-resolved fluorescent microspheres and the magnetic microspheres are selected from magnetic beads.
In an alternative embodiment, the solid support is coated with the full length of the TNF- α biologic. The full length of the TNF alpha biological preparation is used as a capture antigen, and the anti-TNF-alpha biological preparation antibody (namely the anti-antibody) in a sample can be captured more easily due to the large space contact surface, so that the capture efficiency is high. By coating the full length of the TNF α biologic on the capture device, the occurrence of false positives can be better avoided.
In an alternative embodiment, the time-resolved fluorescent microspheres are selected from a combination of a time-resolved fluorescent substance and latex or nanoparticles, wherein the time-resolved fluorescent substance is one of lanthanide, a combination of lanthanide and latex, and a chelate of lanthanide; the lanthanide element can be any one of europium, terbium, samarium or dysprosium.
Detectable labels are substances having properties, such as luminescence, color development, radioactivity, etc., which can be observed directly by the naked eye or detected by an instrument, by which qualitative or quantitative detection of the respective target substance can be achieved.
In a preferred embodiment of the present invention, the detectable label includes, but is not limited to, a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioisotope, a chemiluminescent reagent, or a nanoparticle-based label.
In an alternative embodiment, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (e.g., including, but not limited to, Fluorescein Isothiocyanate (FITC) hydroxyphoton (FAM), tetrachloro-fluorescein (TET), etc. or analogs thereof), rhodamine-based dyes and derivatives thereof (e.g., including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine b (tritc), etc. or analogs thereof), Cy-series dyes and derivatives thereof (e.g., including, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or analogs thereof), Alexa-series dyes and derivatives thereof (e.g., including, but not limited to, Alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or analogs thereof), and protein-based dyes and derivatives thereof (e.g., including, but not limited to, Phycoerythrin (PE), Phycocyanin (PC), phycocyanin, phycoerythrin, Phycocyanin (PC), phycocyanin, and derivatives thereof), and derivatives thereof, Allophycocyanin (APC), polymethacrylic flavin-chlorophyll protein (precP), etc.).
In an alternative embodiment, enzymes that catalyze the development of a substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme.
In one optionIn embodiments of (1), the radioisotope includes, but is not limited to212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu and18F。
in an alternative embodiment, the chemiluminescent reagent comprises, without limitation, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridyl ruthenium and its derivatives, acridinium ester and its derivatives, dioxirane and its derivatives, lotucine and its derivatives, or peroxyoxalate and its derivatives.
In an alternative embodiment, the nanoparticle-based labels include, but are not limited to, nanoparticles and colloids.
In an alternative embodiment, colloids include, without limitation, colloidal metals, disperse dyes, dye-labeled microspheres, and latexes; in an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, colloidal carbon, or colloidal selenium.
In an alternative embodiment, the nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, or rare earth complex nanoparticles.
In a preferred embodiment of the present invention, the cellulose membrane further comprises a quality control antibody as a quality control T line, and the functional fragment and the quality control antibody are separately disposed.
In an alternative embodiment, the cellulose membrane includes, but is not limited to, a cellulose acetate membrane or a cellulose nitrate membrane.
In a preferred embodiment of the present invention, the variable regions of the heavy chain and the light chain of infliximab are shown in SEQ ID NO.1-2, respectively, and the variable regions (Fv) comprise the variable regions of the heavy chain and the variable regions of the light chain which are linked by disulfide bonds.
The complementarity determining regions of infliximab include: CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL 3;
CDR-VH1：NHWMN；CDR–VH2：EIRSKSINSATHYAESVKG；CDR–VH3：NYYGSTYDY；
CDR-VL1：RASQFVGSSIH；CDR-VL2：YASESMS；CDR-VL3：QQSHSWPFT。
the heavy chain variable region and the light chain variable region of the adalimumab are respectively shown in SEQ ID NO.3-4, and the variable region (Fv) thereof comprises the heavy chain variable region and the light chain variable region which are connected by a disulfide bond. The complementarity determining regions of adalimumab include: CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL 3;
CDR-VH1：DYAMH；CDR–VH2：AITWNSGHIDYADSVEG；CDR–VH3：VSYLSTASSLDY；
CDR-VL1：RASQGIRNYLA；CDR-VL2：AASTLQS；CDR-VL3：QRYNRAPYT。
the heavy chain variable region and the light chain variable region of the golimumab are respectively shown in SEQ ID NO.5-6, and the variable region (Fv) of the golimumab comprises the heavy chain variable region and the light chain variable region which are connected by disulfide bonds. The complementarity determining regions of golimumab include: CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL 3;
CDR-VH1：SYAMH；CDR–VH2：FMSYDGSNKKYADSVKG；CDR–VH3：DRGIAAGGNYYYYGMDV；
CDR-VL1：RASQSVYSYLA
CDR-VL2：DASNRAT
CDR-VL3：QQRSNWPPFT。
the variable regions of the heavy chain and the light chain of the certolizumab ozogamicin are respectively shown in SEQ ID NO.7-8, and the variable region (Fv) of the certolizumab ozogamicin comprises the variable regions of the heavy chain and the variable regions of the light chain which are connected by disulfide bonds. The complementarity determining regions of certolizumab ozogamicin include: CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL 3;
CDR-VH1：DYGMN；CDR–VH2：WINTYIGEPIYADSVKG；CDR–VH3：GYRSYAMDY。
CDR-VL1：KASQNVGTNVA；CDR-VL2：SASFLYS；CDR-VL3：QQYNIYPLT。
in other embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region of the TNF- α biologic provided by the invention can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the corresponding heavy chain variable region and light chain variable region described above.
The invention also provides a preparation method of the antibody detection kit of the TNF-alpha biological agent, which comprises the following steps: the TNF-alpha biological agent is fixed on a solid phase carrier to be used as an antibody capture device, and the functional fragment is combined or fixed on a cellulose membrane or labeled by a label to be used as an antibody detection device.
In a preferred embodiment of the application of the present invention, when the solid-phase carrier is selected from a microplate, before the TNF- α biological agent is immobilized on the microplate, streptavidin is coated on the microplate, and then the TNF- α biological agent modified with biotin is incubated with the microplate coated with streptavidin.
In a preferred embodiment of the present invention, the antibody detection kit for TNF- α biological agents is a quantitative detection kit or a qualitative detection kit.
In a preferred embodiment of the present invention, the antibody detection kit for TNF- α biological agents is a time-resolved fluorescence immunochromatography detection kit, a colloidal gold immunochromatography detection kit, a quantum dot fluorescence immunochromatography detection kit, an enzyme-linked immunosorbent assay kit or a chemiluminescence detection kit.
The invention has the following beneficial effects:
the invention overcomes the defects of false positive and false negative in the prior art. The TNF alpha biological agent is used as the capture antigen, and the spatial contact surface is large, so that the anti-TNF-alpha biological agent antibody (namely the anti-antibody) in the sample can be captured more easily, and the advantage of high capture efficiency is achieved. By coating the TNF α biological agent on the capture device, the occurrence of false positives is avoided.
The antigen-antibody complex formed after capture has a large steric hindrance itself, and if the biological agent is coated on the antibody detection device, it is difficult for the biological agent coated on the detection device to bind to the other Fab of the antibody against the anti-TNF- α biological agent in the complex formed after capture because the biological agent (i.e., the detection antigen) has a large molecular weight. Therefore, the inventor sets that the variable region, the heavy chain variable region or the complementarity determining region of the detection antigen with small molecular weight, such as the TNF-alpha biological preparation, has the advantages of small steric hindrance and easy combination with Fab of an anti-antibody, so that the detection sensitivity is higher, and the purpose of the concomitant diagnosis of the TNF-alpha biological preparation drug is achieved. Therefore, the subsequent misjudgment of the sample detection result is avoided, and the phenomenon of false negative is avoided.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a diagram of the apparatus of the anti-inflixb antibody time-resolved fluorescence immunochromatographic test paper of the present invention;
FIG. 2 is a diagram of the device of the anti-adalimus antibody colloidal gold immunochromatographic test strip of the present invention;
FIG. 3 is a diagram of the apparatus of the anti-Golomicron anti-quantum dot fluorescence immunochromatographic test paper of the present invention;
FIG. 4 is a schematic diagram of the detection of the anti-certolizumab ozogamicin enzyme-linked immunoassay kit of the present invention;
FIG. 5 is a schematic diagram of the detection of the chemiluminescence detection kit for agolimumab of the invention;
FIG. 6 is a regression plot of the calibration curve of the anti-inflixb antibody time-resolved fluorescence immunochromatographic kit of the present invention;
FIG. 7 is a regression plot of the calibration curve of the anti-adalimus antibody colloidal gold immunochromatographic kit of the present invention;
FIG. 8 is a regression graph of a calibration curve of the anti-golimumab quantum dot fluorescence immunochromatographic kit of the present invention;
FIG. 9 is a regression plot of the calibration curve of the enzyme-linked immunoassay kit for anti-trastuzumab of the present invention;
fig. 10 is a regression graph of the calibration curve of the chemical luminescence assay kit for agouti mab of the present invention.
Reference numerals: 1-sample pad; 2-a conjugate pad; 3-nitrocellulose membrane; 4-detection line; 5-quality control line; 6-water absorbing film; 7-backing the base plate.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention has the working principle that 1) a solid phase carrier is fixed by a TNF-alpha biological preparation and is used for capturing an anti-antibody in a sample to be detected, the molecular space action area of the TNF-alpha biological preparation is large, and the anti-antibody in the sample can be captured efficiently to form an antigen-antibody compound; 2) the variable region, heavy chain variable region or complementarity determining region of the TNF-alpha biological agent is used as a detection antigen, and can effectively overcome steric hindrance due to small molecules, and can be combined with another Fab of an antibody in an antigen-antibody complex to form an antigen-antibody-antigen complex, and the antigen-antibody-antigen complex can be quantitatively or qualitatively detected by a well-known color development method.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a preparation method of an anti-inflixb antibody time-resolved fluorescence immunochromatography quantitative detection kit.
The embodiment provides an anti-inflixb antibody immunochromatographic test paper, as shown in fig. 1, the test paper is provided with a nitrocellulose membrane 3, a sample pad 1, a combination pad 2 and a water absorption membrane 6 on a backing substrate 7, the sample pad 1 is laminated on one end of the combination pad 2, and the other end of the combination pad 2 and the water absorption membrane 6 are respectively laminated on two ends of the nitrocellulose membrane 3; the combination pad 2 is coated with infliximab marked by time-resolved fluorescent microspheres and goat anti-chicken IgY antibody 2 marked by time-resolved fluorescent microspheres; the nitrocellulose membrane 3 is provided with a detection line 4(T line) and a quality control line 5(C line), the detection line 4(T line) is coated with a heavy chain variable region of inflixb, and the quality control line 5(C line) is coated with a chicken IgY antibody.
The variable heavy chain region of inflixb has the following sequence: evkleesggglvpgsmklscvasgfifsnhwmnwmwrwrwsrqspekglewvaeirsksinshyaesvkgrfsrtfisrddsksavylqmmtdlrttgvycrycsrnygstydysqgttlvss.
The preparation method of the immunochromatographic test paper comprises the following steps:
1) time-resolved labeling of fluorescent microspheres
500. mu.L (300nm) of time-resolved fluorescent microspheres (1% stock solution) are centrifuged at 13000rpm at 4 ℃ for 10min, and the supernatant is discarded. Adding 10mg/ml EDC, and reacting for 15 min; centrifuging, removing supernatant, adding 1000 μ L borate buffer (20mM, pH8.0), and mixing by ultrasonic for 10 s; adding 120 mu g (the labeling concentration is 120 mu g/mL) of labeled antibody (infliximab or goat anti-chicken IgY), and reacting for 2h at room temperature; centrifuging, removing supernatant, adding confining liquid, and reacting for 1 h; centrifuging, removing supernatant, adding 1000. mu.L borate buffer solution, ultrasonically mixing, labeling, and storing at 4 deg.C.
2) Sample pad, conjugate pad treatment
The polyester film was pre-blocked by soaking in a buffer solution containing a surfactant (formulation: 20mM pH8.0 BB, containing 2% BSA, 1% casein, 2% Tween-20, 1% S9 and 5% trehalose), and dried overnight at 37 ℃ to prepare a conjugate pad; and (3) mixing the infliximab marked with the time-resolved fluorescent microspheres and the goat anti-chicken IgY antibody by using a gold-labeled HM3035 gold spraying and membrane scratching instrument, spraying the mixture onto a pretreated binding pad with the width of 1cm at the rate of 5 mu L/cm, and drying at 37 ℃ for at least 3 hours to prepare the specific binding pad.
The glass cellulose membrane was pre-blocked by soaking in a surfactant-containing buffer (formulation: 100mM pH7.4 PB containing 2% NaCl, 2% BSA, 0.5% casein, 0.1% Tween-20, 0.5% S9 and 5% sucrose), dried overnight at 37 ℃ and cut into 30X 1.5cm to prepare a sample pad.
3) Coating of nitrocellulose membranes (NC membranes)
The amino acid synthesis method synthesizes the heavy chain variable region of infliximab, and the synthetic polypeptide is verified to coat the ELISA plate and can react with the anti-infliximab antibody.
The NC film was attached to the designated position of the backing substrate, and the heavy chain variable region (Hv) of inflixb was diluted to 1.5mg/mL with 50mM of pH7.4 phosphate buffer for the preparation of T-line; diluting chicken IgY antibody to 0.5mg/mL by 50mM phosphate buffer solution with pH7.4 for preparing C line; uniformly scratching the two diluted antibodies on an NC film by a gold mark HM3035 gold spraying and film scratching instrument according to the scratching amount of 1 mu L/cm to prepare a T line and a C line; the scribed NC films were placed in a 37 ℃ dry box to dry overnight.
4) Assembly
Laminating the combination pad obtained in the step 2) on one end of the nitrocellulose membrane obtained in the step 3), fixing and laminating a water absorption membrane on the other end of the nitrocellulose membrane, finally laminating the sample pad obtained in the step 2) on the other end of the combination pad, cutting the sample pad according to the width of 4mm of each pad by using a membrane cutting instrument, and filling the sample pad into a chromatography strip shell to obtain a finished product.
5) Detection of
Diluting the serum by 20 times, adding 60 mu L of the diluted serum into a sample adding hole, reacting for 10min, and detecting by a fluorescence reader. And comparing the value with the value of a standard substance or a reference substance, and judging the content of the anti-antibody in the sample to be detected.
In this embodiment, the time-resolved fluorescent substance latex microspheres are europium-containing latex microspheres, i.e., the time-resolved fluorescent substance is a combination of europium lanthanide and latex. In other embodiments, the time-resolved fluorescent substance may be adjusted to one of lanthanide, a combination of lanthanide and latex, and a chelate complex of lanthanide, as needed; the lanthanide may be one of europium, terbium, samarium or dysprosium.
Example 2
The embodiment provides an anti-adalimus antibody colloidal gold immunochromatographic assay kit and a preparation method thereof.
The immunochromatographic test paper for anti-adalimus antibody provided in this embodiment is shown in fig. 2, in which a backing substrate 7 is provided with a nitrocellulose membrane 3, a sample pad 1, a combination pad 2 and a water-absorbing membrane 6, the sample pad 1 is laminated on one end of the combination pad 2, and the other end of the combination pad 2 and the water-absorbing membrane 6 are respectively laminated on two ends of the nitrocellulose membrane 3; the binding pad 2 is coated with a biotinylated adalimumab and goat anti-chicken IgY antibody 2 marked by colloidal gold; the sample pad 1 contains streptavidin; the nitrocellulose membrane 3 is provided with a detection line 4(T line) and a quality control line 5(C line), the detection line 4(T line) is coated with a variable region (Fv) of adalimus, and the quality control line 5(C line) is coated with a chicken IgY antibody.
The adalimumab variable region (Fv) comprises an adalimumab heavy chain variable region and an adalimumab light chain variable region linked by a disulfide bond, wherein the adalimumab heavy chain variable region has the following sequence:
evqlvesggglvqpgrslrlscaasgftfddyamhwvrqapgkglewvsaitwnsghidyadsvegrftisrdnaknslylqmnslraedtavyycakvsylstassldywgqgtlvtvss；
the adalimumab light chain variable region sequence is as follows:
diqmtqspsslsasvgdrvtitcrasqgirnylawyqqkpgkapklliyaastlqsgvpsrfsgsgsgtdftltisslqpedvatyycqrynrapytfgqgtkveik。
the preparation method comprises the following steps:
1) preparation of colloidal gold
Cleaning a round-bottom flask and a glass reagent bottle used for preparing the colloidal gold, soaking in a weak acid washing solution, thoroughly cleaning and drying. A clean round-bottom flask was taken, 100mL of ultrapure water was added thereto, 1mL of 1% chloroauric acid was added thereto, and the mixture was boiled. 2mL of 1% sodium citrate was added thereto quickly, and after the color became purple red, the reaction was continued for 10min, and the heating was stopped. After cooling, ultrapure water was added to 100mL and 4 degrees was used.
2) Biotinylation of labeled antibodies
1mg of adalimumab was placed in a 5mL centrifuge tube, 50. mu.L of 10mg/mL activated biotin (Sulfo-NHS-LC-Bintin) was added thereto, 0.02M PBS was added thereto to a volume of 1mL, the mixture was reacted at room temperature for 1 hour, and the reaction mixture was taken out and dialyzed overnight against 0.02M PBS. The biotinylated adalimumab antibody was removed and preserved by addition of 0.03% Proclin300 for 4 degrees.
3) Labeling of colloidal gold
10mL of prepared colloidal gold is added with 300 mu L of 0.2M potassium carbonate and mixed evenly, 150 mu g of biotinylated adalimumab or goat anti-chicken IgY is added to react for 30min at room temperature, and 1% BSA is added to seal for 30 min. After centrifugation at 12000rpm at 4 ℃ for 30min, the pellet was dissolved in 1mL of a resuspension solution (formulation: 20mM, pH8.0 BB, containing 2% BSA, 2% Tween-20, and 5% sucrose) and kept at 4 degrees.
3) Sample pad, conjugate pad treatment
And mixing the total length of biotinylated adalimus labeled with colloidal gold and the non-biotinylated goat anti-chicken IgY antibody by using a gold-labeled HM3035 gold spraying and membrane drawing instrument, spraying the mixture onto a glass fiber pad with the width of 1cm x 30cm at the rate of 4 mu L/cm, and drying the glass fiber pad for 3 hours at the temperature of 37 ℃ to prepare the colloidal gold binding pad.
The glass cellulose membrane was pre-blocked by soaking in a streptavidin-containing buffer (formulation: 100mM pH7.4 PB containing 1% streptavidin, 1% BSA, 0.5% casein, 0.5% S9, and 2% sucrose), dried overnight at 37 ℃, and cut at 30X 1.5cm to prepare a sample pad.
4) Coating of nitrocellulose membranes (NC membranes)
The amino acid synthesis method synthesizes the variable region of the adalimumab antibody, and the synthesized polypeptide is proved to be capable of reacting with the anti-adalimumab antibody by coating the ELISA plate.
The NC film was attached to the designated position of the backing substrate, and adalimus variable domain (Fv) was diluted to 1.5mg/mL with 50mM of pH7.4 phosphate buffer for the preparation of T-line; diluting chicken IgY antibody to 0.5mg/mL by 50mM phosphate buffer solution with pH7.4 for preparing C line; uniformly scratching the two diluted antibodies on an NC membrane by a gold spraying and membrane scratching instrument according to the scratching amount of 1 mu L/cm to prepare a T line and a C line; the scribed NC films were placed in a 37 ℃ dry box to dry overnight.
5) Assembly
Laminating the combination pad obtained in the step 2) on one end of the nitrocellulose membrane obtained in the step 3), fixing and laminating a water absorption membrane on the other end of the nitrocellulose membrane, finally laminating the sample pad obtained in the step 2) on the other end of the combination pad, cutting the sample pad according to the width of 4mm of each pad by using a membrane cutting instrument, and filling the sample pad into a chromatography strip shell to obtain a finished product.
6) Detection of
Diluting the serum by 20 times, adding 60 μ L into the sample adding hole, reacting for 10min, and detecting with a colloidal gold reader. And comparing the value with the value of a standard substance or a reference substance, and judging the content of the anti-antibody in the sample to be detected.
In this embodiment, the colloidal gold, i.e., a colloidal solution obtained by reducing tetrachloroauric acid with a reducing agent; the labeling substance may be any one of colloidal carbon, colloidal silver, and colloidal selenium, as required.
Example 3
The embodiment provides a preparation method of an anti-golimumab quantum dot fluorescence immunochromatography assay kit.
An anti-golimumab immune chromatography test paper is shown in figure 3, wherein a backing bottom plate 7 of the test paper is provided with a nitrocellulose membrane 3, a sample pad 1, a combination pad 2 and a water absorption membrane 6, the sample pad 1 is laminated at one end of the combination pad 2, and the other end of the combination pad 2 and the water absorption membrane 6 are respectively laminated at two ends of the nitrocellulose membrane 3; the combination pad 2 is coated with the golimumab marked by quantum dots and the goat anti-chicken IgY antibody; the nitrocellulose membrane 3 is provided with a detection line 4(T line) and a quality control line 5(C line), the detection line 4(T line) is coated with a Golimmumab heavy chain variable region, and the quality control line 5(C line) is coated with a chicken IgY antibody.
The sequence of the heavy chain variable region of golimumab is as follows: sklqvlqlvesgvvvprslrlscraasgsaasgfifssyamwvrqapgnglawvafmsdngsnkksnkkatvskdntlylglqmmnslraedtavyardachardgiaggnygyvgmdngvmtgvtvss.
The preparation method comprises the following steps:
1) labeling of quantum dots
Taking 100 mu L of commercially available quantum dots, centrifuging at 13000rpm at 4 ℃ for 10min, centrifuging, discarding the supernatant, adding 1000 mu L of MES buffer, ultrasonically mixing, weighing 20mg of EDC and 20mg of NHS, slowly adding into the microsphere mixed solution, ultrasonically mixing in time, and reacting for 15 min; centrifuging, removing supernatant, adding borate buffer solution (20mM, pH8.0), and ultrasonically mixing; adding 100 mu g of labeled antibody (golimumab or goat anti-chicken IgY) (labeled concentration is 100 mu g/mL), and reacting for 2h at room temperature; centrifuging, removing supernatant, adding confining liquid, ultrasonically mixing with ice water, and reacting at room temperature for 1 h; centrifuging, discarding supernatant, adding 1000 μ L borate buffer (20mM pH8.0), mixing with ultrasound, labeling, and storing at 4 deg.C.
2) Sample pad, conjugate pad treatment
The polyester film was pre-blocked by soaking in a buffer solution containing a surfactant (formulation: 20mM pH8.0 BB, containing 2% BSA, 2% Tween-20, 1% S9 and 5% trehalose), and dried overnight at 37 ℃ to prepare a conjugate pad; and (3) mixing the prepared binding pad with Golimumab labeled with quantum dots and goat anti-chicken IgY antibody by a gold-labeled HM3035 gold-spraying membrane-scratching instrument, spraying the mixture onto the binding pad with the width of 1cm, which is pretreated, at 4 mu L/cm, drying at 37 ℃ for at least 3 hours, and thus obtaining the specific binding pad.
The glass cellulose membrane was pre-blocked by soaking in a buffer containing a surfactant (formulation: 100mM pH7.4 PB containing 1% BSA, 0.5% casein, 0.1% Tween-20, 0.5% S9 and 2% sucrose), dried overnight at 37 ℃ and cut at 30X 1.5cm to prepare a sample pad.
3) Coating of nitrocellulose membranes (NC membranes)
The amino acid synthesis method is used for synthesizing a heavy chain variable region of the golimumab, and the synthetic polypeptide is verified to coat the ELISA plate and can react with the anti-golimumab.
Attaching an NC film to a designated position of a backing substrate, and diluting a heavy chain variable region (Hv) of golimumab to 1.5mg/mL with 50mM of a pH7.4 phosphate buffer solution for preparing a T-line; diluting chicken IgY antibody to 0.5mg/mL by 50mM phosphate buffer solution with pH7.4 for preparing C line; uniformly scratching the two diluted antibodies on an NC film by a gold mark HM3035 gold spraying and film scratching instrument according to the scratching amount of 1 mu L/cm to prepare a T line and a C line; the scribed NC films were placed in a 37 ℃ dry box to dry overnight.
4) Assembly
Laminating the combination pad obtained in the step 2) on one end of the nitrocellulose membrane obtained in the step 3), fixing and laminating a water absorption membrane on the other end of the nitrocellulose membrane, finally laminating the sample pad obtained in the step 2) on the other end of the combination pad, cutting the sample pad according to the width of 4mm of each pad by using a membrane cutting instrument, and filling the sample pad into a chromatography strip shell to obtain a finished product.
5) Detection of
Diluting the serum by 20 times, adding 60 mu L of the diluted serum into a sample adding hole, reacting for 10min, and detecting by a fluorescence reader. And comparing the value with the value of a standard substance or a reference substance, and judging the content of the anti-antibody in the sample to be detected.
In this embodiment, the quantum dots are core-shell quantum dots, ZnS/CdSe or ZnS/CdTe quantum dots. The quantum dots may be adjusted to be quantum dots formed of a single compound, such as any one of cadmium selenide (CdSe), zinc sulfide (ZnS), cadmium telluride (CdTe)/cadmium sulfide (CdS), etc., as needed.
Example 4
The embodiment provides a preparation method of an enzyme-linked immunoassay kit for anti-certolizumab ozogamicin.
The enzyme-linked immunoassay kit for anti-cetuzumab comprises an enzyme label plate, biotinylation cetuzumab, an anti-cetuzumab antibody calibrator, an anti-cetuzumab antibody quality control product, an enzyme conjugate, a cleaning solution and a stop solution. The enzyme conjugate is horseradish peroxidase (HRP) labeled cercis bead Complementarity Determining Regions (CDRs). The microplate is coated with streptavidin.
The cenosphere Complementarity Determining Regions (CDRs) include a heavy chain complementarity determining region and a light chain complementarity determining region. Wherein in the heavy chain complementarity determining region: CDR-VH 1: DYGMN; CDR-VH 2: WINTYIGEPIYADSVKG, respectively; CDR-VH 3: GYRSYAMDY are provided.
Light chain complementarity determining regions: CDR-VL 1: KASQNVGTNVA, respectively; CDR-VL 2: the compound of SaSFLYS; CDR-VL 3: QQYNIYPLT are provided. The complementarity determining regions are connected by a connecting peptide selected from GGGGSGGGGSGGGGS.
The preparation method and the detection method are shown in fig. 4, and the specific preparation is as follows:
1) biotinylation of Cytuzumab ozogamicin
1mg of certolizumab ozogamicin was put in a 5mL centrifuge tube, 50. mu.L of 10mg/mL activated biotin (Sulfo-NHS-LC-Bintin) was added thereto, 0.02M PBS was added thereto to a volume of 1mL, the mixture was reacted at room temperature for 1 hour, and the reaction mixture was taken out and dialyzed overnight with 0.02M PBS. The biotinylated certolizumab ozogamicin was removed and preserved by adding 0.03% Proclin300 for 4 degrees.
2) HRP-labeled Sautou bead Complementarity Determining Regions (CDRs)
The amino acid synthesis method synthesizes the complementary determining area of the certolizumab ozogamicin, and the synthetic polypeptide is used for coating the enzyme label plate after verification and can react with the anti-certolizumab ozogamicin antibody.
Weighing 5mg HRP, placing in a 5mL centrifuge tube, adding 1mL pure water for dissolving, adding 1mL NaIO4The solution (10mg/mL, ready to use) was reacted for 30min at 4 ℃ in the dark. 0.02mL of ethylene glycol was added and the reaction was carried out at room temperature for 15 min. Dialyzing the solution and taking out. Adding 1mg of Cetuo bead complementarity determining region into the activated solution, reacting at room temperature in a dark place for 2 hours, and adding 0.2mL of NaBH4The solution (5mg/mL, ready to use) was mixed well and dialyzed against 0.05M CB overnight. And (4) taking out the marked enzyme-labeled antibody, adding glycerol with the same volume, and storing at-20 ℃ for later use.
3) Coated enzyme label plate
Streptavidin was diluted to 1. mu.g/mL in CB buffer, coated in 100. mu.L wells in microtiter plates, for 2h at 37 ℃. The coating solution was aspirated off, incubated with 200. mu.L of blocking buffer containing 2% BSA per well for 2 hours at 37 ℃, the blocking solution was tapped off, and dried at 37 ℃ for future use.
4) Coating biotinylated certolizumab ozogamicin
Biotinylated certolizumab ozogamicin was diluted to 1. mu.g/mL with neutral phosphate buffer, 100. mu.L per well, 2h at 37 ℃. The plate was washed 3 times, incubated with 200. mu.L of blocking buffer containing 2% BSA per well for 2 hours at 37 ℃, blotted off and dried at 37 ℃ for use.
5) Diluting a serum sample to be detected by 100 times, adding 100 mu L of the serum sample into each hole of the sample, incubating at 37 ℃ for 2 hours, and washing the plate for 4 times.
6) HRP-labeled Saito bead Complementarity Determining Regions (CDRs) were added, incubated for 1 hour, and the plate was washed 4 times.
7) Adding a substrate TMB of HRP enzyme into the microplate, incubating for 15 minutes, adding a termination solution HCL solution to terminate the color reaction, detecting the OD value by using the wavelength of 450nm, and comparing with the OD value of a standard substance or a reference substance to judge the content of the anti-antibody in the sample to be detected.
Example 5
The embodiment provides a preparation method of an anti-golimumab chemiluminescence assay kit.
The chemiluminescence detection kit for the anti-golimumab antibody comprises biotinylation golimumab, an anti-golimumab antibody calibrator, an anti-golimumab antibody quality control product, an acridinium ester-labeled golimumab heavy chain variable region, a magnetic particle reagent, an excitation liquid and a cleaning liquid.
The sequence of the golimumab heavy chain variable region is as follows: sklqvlsoggvvqpgrslrlscaasgsfasgufsyahwrvrqapgnglwvafmsdngsnkkatvsnktsnkntlymqmnsnandlayratavycargiaggngngngygmvwg
The preparation and detection steps are as follows:
1) biotinylation of golimumab
1mg of golimumab is put into a 5mL centrifuge tube, 50 μ L of 10mg/mL activated biotin (Sulfo-NHS-LC-Bintin) is added, 0.02M PBS is added to the tube until the volume is 1mL, the reaction solution is reacted for 1 hour at room temperature, and the reaction solution is taken out and dialyzed with 0.02M PBS overnight. The biotinylated golimums full-length antibody was removed and preserved by adding 0.03% Proclin300 for 4 degrees.
2) Acridine ester labeled golian heavy chain variable region (Hv)
The amino acid synthesis method is used for synthesizing a heavy chain variable region of the golimumab, and the synthetic polypeptide is verified to coat the ELISA plate and can react with the anti-golimumab.
Taking 2mg of golimums heavy chain variable region (Hv), and according to the antibody: and adding the activated acridine ester in a molar ratio of 1: 10-50, and reacting at room temperature for 30 min. Dialyzing the reaction solution with 0.05M CB solution, taking out, adding glycerol with the same volume, and storing at-20 ℃ for later use.
3) Detection step
The method realizes the detection of the anti-single antibody by utilizing the acridinium ester chemiluminescence immunoassay technology-the combination of the biotin-avidin magnetic particle separation technology. The detection principle is as shown in FIG. 5: mixing a sample (diluted by 20 times), streptavidin magnetic particles (1) and biotinylated golimumab (2) to obtain a streptavidin magnetic bead-biotinylated golimumab-sample complex (3), washing, adding an acridinium ester labeled golimumab heavy chain variable region (Hv) for reaction, and forming the streptavidin magnetic bead-biotinylated golimumab-sample-acridinium ester labeled golimumab Hv complex. Adding luminous excitation liquid, measuring luminous intensity, comparing with the value of standard or reference, and judging the content of the anti-antibody in the sample to be detected.
Experimental example 1
This experimental example quantitatively determined the kit provided in example 1 to examine its performance.
1.1 Standard Curve preparation
1.1.1A set of anti-inflixb antibody calibrators was taken, and the specific values are shown in Table 1.
TABLE 1
Figure BDA0003445209930000161
1.1.2 detection method
Taking 60 mu L of a calibrator, and directly adding the calibrator into a sample window in the chromatographic strip; after 10 minutes, the signal values were quantitatively determined with a time-resolved fluorescence quantitative analyzer.
Each calibrator was tested 2 times and the T/C values averaged. The specific results are shown in Table 2.
TABLE 2
Figure BDA0003445209930000162
1.1.3 Standard Curve preparation
According to the detection result, taking the T/C value as an X axis and the concentration value as a Y axis, performing linear regression to obtain a linear equation: 77.666x-18.729, R20.9965, see fig. 6.
1.2 precision test:
1.2.1 preparing 10 chromatography strips, and preparing an anti-inflixb antibody working calibration product with the concentration (100 ng/mL);
1.2.2 directly adding 60 mu L of calibrator into a sample adding hole of the reagent strip;
1.2.3 after the calibrator is chromatographed for 10min, a time-resolved fluorescence quantitative analyzer is used for detecting, the results of 10 reagent strips are measured according to the linear equation, the results are shown in table 3, and the precision of the anti-inflixb antibody time-resolved fluorescence immunochromatography assay kit is good.
TABLE 3
Figure BDA0003445209930000171
1.3 compared to other methods:
marking microspheres with infliximab, and coating an NC membrane with the infliximab as a method 2; marking microspheres with infliximab, and coating NC membrane with infliximab as method 3. The conditions were the same for the three methods except that the labeled antibody and the coated antibody were different, and the results were shown in Table 4.
TABLE 4
This example Method 2 Method 3
Detection limit ＜4ng/ml ＜10ng/ml ＜20ng/ml
Detection range 4-500ng/ml 10-500ng/ml 20-400ng/ml
Linear R2 0.9965 0.9892 0.9902
Precision degree 5.53％ 17.31％ 14.62％
Non-specific background interference Is low in Height of Is low in
Experimental example 2
This example quantitatively measures the kit provided in example 2 to test its performance.
2.1 Standard Curve preparation
2.1.1 taking a set of anti-adalimumab antibody calibrator, the specific values are shown in Table 5.
TABLE 5
Figure BDA0003445209930000172
Figure BDA0003445209930000181
2.1.2 detection method
Taking 60 mu L of a calibrator, and directly adding the calibrator into a sample window in the chromatographic strip; after 10 minutes, the signal values were measured with a colloidal gold reader. Each calibrator was tested 2 times and the T/C values averaged. The specific results are shown in Table 6.
TABLE 6
Figure BDA0003445209930000182
2.1.3 Standard Curve preparation
According to the detection result, taking the T/C value as an X axis and the concentration value as a Y axis, performing linear regression to obtain a linear equation: y is 236.43x-23.977 and R2 is 0.996, as shown in fig. 7.
2.2 precision test:
2.2.1 preparing 10 chromatography strips, and preparing an anti-adalimus antibody work calibration product with the concentration (50 ng/mL);
2.2.2 directly adding 60 mu L of calibrator into the sample adding hole of the reagent strip;
2.2.3 after the calibrator is chromatographed for 10min, detecting by using a colloidal gold reading instrument, and measuring results of 10 reagent strips according to the linear equation, wherein the results are shown in Table 7, which indicates that the anti-adalimus antibody colloidal gold immunochromatography assay kit has good precision.
TABLE 7
Figure BDA0003445209930000183
Figure BDA0003445209930000191
2.3 compared to other methods:
using this example as a control, adalimumab labeled colloidal gold, and adalimumab coated NC membrane as method 2; colloidal gold is labeled with adalimumab, and NC membrane is coated with adalimumab as method 3. The results are shown in Table 8, except for the labeled antibody and the coated antibody, which are different under the same conditions.
TABLE 8
This example Method 2 Method 3
Detection limit ＜5ng/ml ＜10ng/ml ＜30ng/ml
Detection range 5-500ng/ml 10-500ng/ml 30-400ng/ml
Linear R2 0.996 0.9883 0.9891
Precision degree 5.57％ 16.77％ 13.36％
Non-specific background interference Is low in Height of Is low in
Experimental example 3
This example quantitatively measures the kit provided in example 3 to test its performance.
3.1 Standard Curve preparation
3.1.1A set of anti-golimumab calibrator is taken, and specific values are shown in Table 9.
TABLE 9
Figure BDA0003445209930000192
3.1.2 detection method
Taking 60 mu L of a calibrator, and directly adding the calibrator into a sample window in the chromatographic strip; after 10 minutes, the signal values were measured with a fluorescence quantitative analyzer. Each calibrator was tested 2 times and the T/C values averaged. The specific results are shown in Table 10.
Watch 10
Figure BDA0003445209930000193
Figure BDA0003445209930000201
3.1.3 Standard Curve preparation
According to the detection result, taking the T/C value as an X axis and the concentration value as a Y axis, carrying out curve fitting to obtain a curve equation: 160.29x2+72.168x-0.1583，R20.9993, see fig. 8.
3.2 precision test:
3.2.1 preparing 10 prepared chromatographic strips, and preparing an anti-Golomikon anti-antibody working calibration product with the concentration (50 ng/mL);
3.2.2 directly adding 60 mu L of calibrator into the sample adding hole of the reagent strip;
3.2.3 after the calibrator is chromatographed for 10min, detecting by using a fluorescence quantitative analyzer, and measuring results of 10 reagent strips according to the equation, wherein the results are shown in Table 11, which shows that the anti-golimumab quantum dot fluorescence immunochromatography assay kit has good precision.
TABLE 11
Figure BDA0003445209930000202
3.3 compared to other methods:
marking quantum dots with golimumab, and coating an NC film with golimumab as a method 2; and (3) marking the quantum dots with golimumab fab, and coating the NC film with golimumab fab as a method 3. The conditions were the same for the three methods except that the labeled antibody and the coated antibody were different, and the three results were shown in Table 12.
TABLE 12
This example Method 2 Method 3
Detection limit ＜5ng/ml ＜10ng/ml ＜20ng/ml
Detection range 5-500ng/ml 10-500ng/ml 20-200ng/ml
Linear R2 0.9993 0.9862 0.9911
Precision degree 5.91％ 15.67％ 12.76％
Non-specific background interference Is low in Height of Is low in
Experimental example 4
This example quantitatively tests the kit provided in example 4 to test its performance.
4.1 detection method
4.1.1A set of anti-Satuzumab antibody calibrators was taken, and the specific values are shown in Table 13.
Watch 13
Figure BDA0003445209930000211
4.1.2 detection step
1) Adding 100 mu L of calibrator and quality control material into the microporous plate per well, incubating at 37 ℃ for 2h, and washing the plate for 4 times.
2) Add the complementarity determining region of HRP-labeled certolizumab, incubate for 1h, and wash the plate 4 times.
3) Adding a substrate TMB of HRP enzyme into the microporous plate, incubating for 15min, adding a stop solution to stop the color reaction, detecting the OD value by using the wavelength of 450nm, and judging the performance of the kit. The specific results are shown in Table 14.
TABLE 14
Figure BDA0003445209930000212
The detection values are as follows:
Figure BDA0003445209930000213
Figure BDA0003445209930000221
4.1.3 Standard Curve
According to the detection result, curve fitting is carried out by taking the OD value as an X axis and the concentration value as a Y axis to obtain a curve equation: y 39.02x2+74.984x-6.4689，R20.9982, see fig. 9.
4.2 precision testing:
according to the OD value of the detected precision quality control product, a result value is calculated by a curve equation, and the result is shown in table 15, which shows that the anti-Saturuzu antibody enzyme-linked immunoassay kit has good precision.
Watch 15
Figure BDA0003445209930000222
4.3 compared to other methods:
labeling HRP with the certuzumab, and coating a microporous plate with the certuzumab as a method 2; using Satuo bead fab to mark HRP, and using Satuo bead fab to coat the microplate as method 3. The conditions were the same for the three methods except that the labeled antibody and the coated antibody were different, and the results were shown in Table 16.
TABLE 16
This example Method 2 Method 3
Detection limit ＜5ng/ml ＜20ng/ml ＜20ng/ml
Detection range 5-500ng/ml 20-500ng/ml 20-200ng/ml
Linear R2 0.9982 0.9833 0.9796
Precision degree 2.9％ 13.41％ 11.17％
Non-specific background interference Is low in Height of Is low in
Experimental example 5
This example quantitatively tests the kit provided in example 5 to test its performance.
5.1 detection method
5.1.1A set of anti-golimumab calibrator is taken, and specific values are shown in Table 17.
TABLE 17
Figure BDA0003445209930000223
5.1.2 detection step
1) And respectively adding 20 mu L of calibrator, 20 mu L of streptavidin magnetic particles and 50 mu L of biotinylation golimumab into the micropores, mixing, incubating at 37 ℃ for 15min, and cleaning for 3 times.
2) Adding acridinium ester to mark the Goligumu heavy chain variable region (Hv), incubating at 37 ℃ for 15min, and washing for 3 times.
3) Adding luminous excitation liquid, and measuring luminous intensity. The results are shown in Table 18.
Watch 18
Figure BDA0003445209930000231
5.1.3 Standard Curve
According to the detection result, taking the logarithm value of the concentration value as an X axis and the logarithm value of the luminous value as a Y axis, performing linear regression to obtain a linear equation: 0.9601x +2.9521, R2The curve is shown in fig. 10, 0.9959.
5.2 precision test:
adding 20 μ L of precision quality control product, 20 μ L of streptavidin magnetic particle, and 50 μ L of biotinylation golimumab into the micropore, mixing, incubating at 37 deg.C for 15min, and cleaning for 3 times; adding acridinium ester to mark a golian heavy chain variable region (Hv), incubating for 15min at 37 ℃, and cleaning for 3 times; adding luminous excitation liquid, and measuring luminous intensity. And calculating a result value according to the curve equation. The results are shown in table 15, which shows that the anti-golimums antibody chemiluminescence immunoassay kit of the method has good precision. The results are shown in Table 19.
Watch 19
Figure BDA0003445209930000232
Figure BDA0003445209930000241
5.3 compared to other methods:
marking acridine ester by using golimumab, and marking biotin by using golimumab as a method 2; and (3) marking acridine ester by using golimab, and marking biotin by using golimab as a method 3. The three methods were performed under the same conditions except that the acridinium ester-labeled antibody and the biotin-labeled antibody were different, and the results were shown in Table 20.
Watch 20
This example Method 2 Method 3
Detection limit ＜5ng/ml ＜10ng/ml ＜10ng/ml
Detection range 5-500ng/ml 10-500ng/ml 10-200ng/ml
Linear R2 0.9959 0.9763 0.9896
Precision degree 4.35％ 11.61％ 10.16％
Non-specific background interference Is low in Height of Is low in
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Suzhou & Rui Biotechnology Ltd
<120> antibody detection kit for TNF-alpha biological agent and preparation method thereof
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Val Tyr Leu Gln Met Thr Asp Leu Arg Thr Glu Asp Thr Gly Val Tyr
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Gly Thr Thr Leu Thr Val Ser Ser
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Claims (10)

1. The antibody detection kit of the TNF-alpha biological agent is characterized by comprising an antibody capture device and an antibody detection device, wherein the antibody capture device comprises a solid phase carrier, and the antibody detection device is a cellulose membrane or a functional fragment with a detectable marker; and a TNF-alpha biological agent is coated on the solid phase carrier, and the functional fragment without the label is fixed on the cellulose membrane; the functional fragment TNF-alpha biological agent comprises a variable region, a heavy chain variable region or a complementarity determining region.
2. The antibody detection kit for a TNF-a biologic according to claim 1, wherein the TNF-a biologic is selected from the group consisting of a monoclonal antibody drug of TNF-a;
preferably, the TNF-alpha mab drug is selected from Infliximab (Infliximab), Adalimumab (Adalimumab), Certolizumab Pegol (Certolizumab Pegol), or Golimumab (golimab).
3. The antibody detection kit for TNF- α biologics of claim 1 or 2, wherein said solid support is selected from the group consisting of fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, colloidal gold particles, quantum dots, microwell plates, and microporous membranes;
preferably, the TNF-alpha biological agent coated on the solid phase carrier is modified with avidin or biotin;
preferably, the fluorescent microspheres are selected from time-resolved fluorescent microspheres, and the magnetic microspheres are selected from magnetic beads;
preferably, the solid support is coated with the full length of the TNF- α biological agent.
4. The antibody detection kit for TNF- α biologies of claims 1 or 2, wherein the detectable label is selected from the group consisting of a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioisotope, a chemiluminescent reagent, or a nanoparticle-based label;
preferably, the fluorescent dye is selected from fluorescein dyes and derivatives thereof, rhodamine dyes and derivatives thereof, Cy dyes and derivatives thereof, Alexa dyes and derivatives thereof, and protein dyes and derivatives thereof;
preferably, the enzyme catalyzing the color development of the substrate is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme;
preferably, the radioisotope is selected from212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu and18F；
preferably, the chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, lowhine and its derivatives or peroxyoxalate and its derivatives;
preferably, the nanoparticle-based label is selected from the group consisting of nanoparticles and colloids;
preferably, the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latexes; preferably, the colloidal metal is selected from colloidal gold, colloidal silver, colloidal carbon or colloidal selenium;
preferably, the nanoparticles are selected from organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles or rare earth complex nanoparticles.
5. The antibody detection kit for TNF-alpha biological agent according to claim 1 or 2, wherein a quality control antibody is further fixed on the cellulose membrane as a quality control T line, and the functional fragment and the quality control antibody are separately disposed at an interval;
preferably, the cellulose membrane is selected from a cellulose acetate membrane or a cellulose nitrate membrane.
6. The antibody detection kit for TNF-a biologicals according to claim 1 or 2, wherein the variable regions of the heavy and light chains of infliximab are respectively represented by SEQ ID No.1-2, and the variable region (Fv) thereof comprises the variable regions of the heavy and light chains linked by a linker peptide or disulfide bond;
the complementarity determining regions of infliximab include: CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL 3;
CDR-VH1：NHWMN；CDR–VH2：EIRSKSINSATHYAESVKG；CDR–VH3：NYYGSTYDY；
CDR-VL1：RASQFVGSSIH；CDR-VL2：YASESMS；CDR-VL3：QQSHSWPFT；
the adalimumab variable region (Fv) comprises a heavy chain variable region and a light chain variable region linked by a linker peptide or disulfide bond; the heavy chain variable region and the light chain variable region of the adalimumab are respectively shown in SEQ ID NO.3-4, and the complementarity determining region of the adalimumab comprises: CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL 3;
CDR-VH1：DYAMH；CDR–VH2：AITWNSGHIDYADSVEG；CDR–VH3：VSYLSTASSLDY；CDR-VL1：RASQGIRNYLA；CDR-VL2：AASTLQS；CDR-VL3：QRYNRAPYT；
the golimumps single anti-variable region (Fv) comprises a heavy chain variable region and a light chain variable region which are connected by a connecting peptide or disulfide bond; the heavy chain variable region and the light chain variable region of the golimumab are respectively shown in SEQ ID NO.5-6, and the complementarity determining region of the golimumab comprises: CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL 3;
CDR-VH1：SYAMH；CDR–VH2：FMSYDGSNKKYADSVKG；CDR–VH3：DRGIAAGGNYYYYGMDV；CDR-VL1：RASQSVYSYLA；CDR-VL2：DASNRAT；CDR-VL3：QQRSNWPPFT；
the certolizumab ozogamicin variable region (Fv) comprises a heavy chain variable region and a light chain variable region linked by a linker peptide or disulfide bond; the heavy chain variable region and the light chain variable region of the certolizumab ozogamicin are respectively shown in SEQ ID NO.7-8, and the complementarity determining region of the certolizumab ozogamicin comprises: CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL 3;
CDR-VH1：DYGMN；CDR–VH2：WINTYIGEPIYADSVKG；CDR–VH3：GYRSYAMDY；CDR-VL1：KASQNVGTNVA；CDR-VL2：SASFLYS；CDR-VL3：QQYNIYPLT。
7. the method for preparing an antibody detection kit for a TNF- α biological agent according to claim 1 or 2, wherein the antibody detection kit for a TNF- α biological agent is a quantitative detection kit or a qualitative detection kit.
8. The method for preparing an antibody detection kit for a TNF-alpha biological agent according to claim 1 or 2, wherein the antibody detection kit for the TNF-alpha biological agent is a time-resolved fluorescence immunochromatography detection kit, a colloidal gold immunochromatography detection kit, a quantum dot fluorescence immunochromatography detection kit, an enzyme-linked immunosorbent assay kit or a chemiluminescent detection kit.
9. A method of making a TNF- α biologic antibody detection kit according to any of claims 1 to 8, comprising: TNF-alpha biological agents are fixed on a solid phase carrier to be used as an antibody capture device, functional fragments without labels are combined or fixed on a cellulose membrane, or the functional fragments are labeled to be used as an antibody detection device.
10. The method of claim 9, wherein when the solid support is selected from a microplate, streptavidin is coated on the microplate before the TNF- α biological agent is immobilized on the microplate, and then the biotin-modified TNF- α biological agent is incubated with the streptavidin-coated microplate.
CN202111646130.9A 2021-12-30 2021-12-30 Antibody detection kit for TNF-alpha biological agent and preparation method thereof Pending CN114236139A (en)

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