CN117288966A - Kit for combined detection of IGF-1 and IGFBP-3 and lysate and buffer solution used by kit - Google Patents

Kit for combined detection of IGF-1 and IGFBP-3 and lysate and buffer solution used by kit Download PDF

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CN117288966A
CN117288966A CN202311590135.3A CN202311590135A CN117288966A CN 117288966 A CN117288966 A CN 117288966A CN 202311590135 A CN202311590135 A CN 202311590135A CN 117288966 A CN117288966 A CN 117288966A
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igf
mass
igfbp
antibody
kit
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CN117288966B (en
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杨帆
郑雯雯
杨金红
朱琦琦
杨致亭
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Shandong Kanghua Biomedical Technology Co ltd
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Shandong Kanghua Biomedical Technology Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4745—Insulin-like growth factor binding protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/65—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/38—Pediatrics

Abstract

The invention relates to a kit for jointly detecting IGF-1 and IGFBP-3 and a lysate and a buffer solution used by the kit, belonging to the field of immunofluorescence chromatography detection, and the kit for jointly detecting IGF-1 and IGFBP-3 comprises a test strip, wherein the test strip comprises a PVC plate, the PVC plate sequentially comprises water absorbing paper, a nitrocellulose membrane and a sample pad from left to right, one end of the water absorbing paper is lapped on one end of the nitrocellulose membrane, and one end of the sample pad is lapped on the other end of the nitrocellulose membrane.

Description

Kit for combined detection of IGF-1 and IGFBP-3 and lysate and buffer solution used by kit
Technical Field
The invention belongs to the field of immunofluorescence chromatography detection, and particularly relates to a kit for jointly detecting IGF-1 and IGFBP-3, and a lysate and a buffer solution used by the kit.
Background
Growth hormone deficiency is one of the most common reasons for infants suffering from short and small diseases, early and accurate diagnosis is a key point for determining the later treatment effect and the final height development condition, and a classical laboratory diagnosis method is to use the level after an excitation test as a gold standard, but the excitation test is extremely complicated, patients need to take blood for many times, have great expense and certain adverse reactions, are not easily accepted by parents, cause more infants to lose the optimal treatment opportunity, and the accuracy and repeatability of the result are greatly influenced by age, sex and sex hormone level.
Insulin-like growth factor IGF-1 is a main substance for mediating growth hormone GH to promote growth, and serum IGF-1 and insulin growth factor binding protein IGFBP-3 form a complex, so that the serum concentration is relatively stable, no obvious pulse secretion and circadian rhythm change exist, and therefore, the measurement value of IGF-1 can better reflect the GH secretion state of an individual. IGFBP-3 has diagnostic and therapeutic value for infants suffering from deficiency type dwarfism in terms of bioavailability of circulating IGF-1, making IGFBP-3 another marker for diagnosis of growth disorders, particularly during childhood and adolescence, while detecting IGF-1 and IGFBP-3 levels.
Pretreatment of the sample is necessary because IGF-1 binds to IGFBP-3 and the acid labile subunit ALS, which complicates the measurement of IGF-1, and for co-detection, ensures that lysates and buffers have essentially no effect on IGFBP-3 detection, which results in accuracy in detecting IGF-1 levels and in the breakthrough of co-detection.
The existing IGF-1 pretreatment reagent has the technical defects of an organic solvent extraction method, a strong acid method and a displacement method. After the organic solvent is extracted and cracked, the organic solvent needs to be centrifuged, so that the automatic operation is inconvenient to realize. The strong acid method relies on too low a pH to denature or change the conformation of IGFBP-3 and acid labile proteins, releasing IGF-1, but IGF-1 solution is a low pH solution, which is detrimental to the next step of immunoreaction of IGF-1 with IGF-1 antibodies. Displacement methods use IGF-1 analogs to competitively replace IGF-1, displacing IGF-1 from the bound protein. The disadvantage of this method is that the displacement reaction is a reversible reaction, and the addition of IGF-1 analogues does not allow complete displacement, resulting in inaccurate results.
Thus, it would be a great need for a person skilled in the art to provide a combined quantitative detection of IGF-1 and IGFBP-3 by a lysing agent and buffer treatment of a sample.
Disclosure of Invention
The invention aims to provide a kit for jointly detecting IGF-1 and IGFBP-3, and a lysate and a buffer solution used by the kit, which are convenient to use, simple to operate, high in detection sensitivity and good in specificity, can realize bidirectional joint detection, ensure that IGF-1 is split and decomposed, ensure complete and unchanged protein conformation of IGFBP-3, and ensure detection sensitivity and specificity of the methodology, and the stability of a resolving agent and the buffer solution is good.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a lysate for a kit for the combined detection of IGF-1 and IGFBP-3, comprising the following components: disodium hydrogen phosphate-citrate buffer, bovine serum albumin, urea, guanidine hydrochloride, proClin300; wherein the concentration of the disodium hydrogen phosphate-citric acid buffer solution is 10-200mM, and the molar ratio of the disodium hydrogen phosphate to the citric acid is 2:1, pH range 2.2-3.5; the mass percentage of bovine serum albumin is 0.2-1.5%, the mass percentage of urea is 10-15%, guanidine hydrochloride is 15-20%, and the mass percentage of ProClin300 is 0.03-0.05%.
A buffer for a kit for the combined detection of IGF-1 and IGFBP-3 comprising the following components: tris (2-formylethyl) phosphine hydrochloride, tween 20, S9, EDTA-2NA and ProClin300, wherein the concentration of the tris (2-formylethyl) phosphine hydrochloride, the concentration of the tris (2-formylethyl) phosphine hydrochloride and the concentration of the tris (2-formylethyl) phosphine hydrochloride are respectively 20-100mM, 0.01-0.1% of the tris (2-formylethyl) phosphine hydrochloride, 0.05-0.15% of the tween 20, 0.8-1.2% of the S9, 0.1-0.15% of the EDTA-2NA and 0.03-0.05% of the ProClin 300.
A kit for jointly detecting IGF-1 and IGFBP-3 comprises a test strip, wherein the test strip comprises a PVC plate, the PVC plate sequentially comprises water absorbing paper, a nitrocellulose membrane and a sample pad from left to right, one end of the water absorbing paper is lapped on one end of the nitrocellulose membrane, and one end of the sample pad is lapped on the other end of the nitrocellulose membrane.
The preparation method of the test strip comprises the following steps:
(1) Scribing film
a. Coating a quality control line C and two detection lines T on a nitrocellulose membrane in sequence, wherein the formula of the membrane drawing liquid of the quality control line C and the two detection lines T is as follows: 0.1M PBS, 1% trehalose by mass;
b. labeling fluorescent dye IGFBP-3 fluorescent microsphere IGF-1 antibody, and sequentially coating the fluorescent dye IGFBP-3 fluorescent microsphere IGF-1 antibody on a nitrocellulose membrane, wherein the fluorescent membrane-drawing liquid comprises the following formula: 0.1M PBS, 0.5% trehalose by mass, 0.5% sucrose by mass, 0.9% NaCl by mass and 0.5% casein by mass.
(2) Preparation of sample pad
The sample pad treatment fluid is solidified on glass fiber, and after drying for 8 hours, the preparation of the sample pad is completed, and the sample pad treatment fluid comprises the following components: 0.2M pH8.0 Tris, 1% by mass of BSA, 0.1% by mass of PVA, 0.1% by mass of Tween 20, 0.1% by mass of ProClin300, 0.5% by mass of sucrose, 0.5% by mass of trehalose;
(3) Assembly of test strips
And (3) sequentially assembling the absorbent paper, the nitrocellulose membrane, the sample pad and the PVC base plate, and then cutting the assembled absorbent paper, the nitrocellulose membrane, the sample pad and the PVC base plate into test strips.
The fluorescent dye IGFBP-3 antibody labeling method comprises the following steps:
(1) Take out IThe GFBP-3 antibody was equilibrated at room temperature for 20min, the fluorochrome was dissolved to 1mol/L with PBS for use, 10. Mu.L fluorochrome and 4mg/mL IGFBP-3 antibody were added to 25. Mu.L per 50. Mu.L system, and the remaining volume was incubated with NaHCO at pH8.0, 1mol/L 3 Supplementing, mixing, and vibrating and reacting for 3 hours at room temperature by a shaking table;
(2) And (3) dialysis: and (3) labeling the antibody, dialyzing in a buffer solution for 36 hours, and replacing the dialyzate every 4 hours to obtain the labeled fluorescent dye IGFBP-3 antibody.
The labeling method of the fluorescent microsphere IGF-1 antibody comprises the following steps: (1) preparing microsphere suspension: preparing 50mM pH6.0 MES buffer solution, adding 1% fluorescent microsphere to prepare microsphere suspension with concentration of 1 w/v%;
(2) Activating: dissolving EDC in 50mM MES buffer solution with pH of 6.0, adding dissolved EDC into each mL of microsphere suspension to make its concentration 20mg/mL, incubating at room temperature for 20min, centrifuging or ultrafiltering, washing the microspheres twice with equal volume of 50mM MES buffer solution with pH of 6.0, and making into activated microsphere suspension, and continuously stirring;
(3) Labeling the antibody: rapidly adding IGF-1 antibody into the stirred activated microsphere suspension to ensure that the final concentration of IGF-1 antibody is 1mg/mL, continuously stirring, and incubating at room temperature for 2.5 hours to obtain microsphere suspension of labeled antibody;
(4) Closing: adding 2.5 mu L of ethanolamine into each mL of microsphere suspension of the labeled antibody, continuously stirring, incubating at room temperature for 10min, centrifuging or ultrafiltering;
(5) And (3) storing: suspending the microsphere with a storage buffer solution, repeating the steps twice, and removing unbound IGF-1 antibody and ethanolamine to obtain labeled fluorescent microsphere IGF-1 antibody;
the storage buffer formulation is: 0.2M Tris pH8.0, trehalose 0.5% by mass, sucrose 0.5% by mass, casein 0.2% by mass, BSA 2% by mass and sodium azide 0.1% by mass.
A kit for jointly detecting IGF-1 and IGFBP-3 comprises a detection card, wherein the detection card comprises a card cover and a card body which are mutually buckled, and a test strip is arranged between the card cover and the card body.
The application of the kit for jointly detecting IGF-1 and IGFBP-3 comprises the steps of taking 40 mu L of lysate and 10 mu L of serum sample, fully mixing, adding 200 mu L of buffer solution, mixing, taking 75 mu L of buffer solution, adding the buffer solution into a sample adding hole of a detection card, inserting the detection card into a sample groove of a fluorescence analyzer, and outputting a result by a 15-min instrument.
The invention adopts the technical proposal and has the following advantages: the method has the advantages of convenient use, simple operation, high detection sensitivity and good specificity, can realize bidirectional joint inspection, ensures that IGF-1 is cracked by filling and decomposing, ensures complete and unchanged protein conformation of IGFBP-3, simultaneously ensures the detection sensitivity and specificity of the method, has good stability of a dissolving agent and a buffer solution, can be stored at normal temperature, can detect two items at the same time, has short detection time and can obtain a result after 15 minutes.
The invention is further described below with reference to the drawings and the detailed description.
Drawings
FIG. 1 is a graph showing the correlation between IGF-1 detection results and Siemens IGF-1 detection results in example 1 of the present invention;
FIG. 2 is a graph showing the correlation between IGFBP-3 detection results and Siemens IGFBP-3 detection results, respectively, in example 1 of the present invention;
FIG. 3 is a graph showing the correlation between IGF-1 detection results and Siemens IGF-1 detection results in example 2 of the present invention;
FIG. 4 is a graph showing the correlation between IGFBP-3 detection results and Siemens IGFBP-3 detection results, respectively, in example 2 of the invention;
FIG. 5 is a graph showing the correlation between IGF-1 detection results and Siemens IGF-1 detection results in example 3 of the present invention;
FIG. 6 is a graph showing the correlation between IGFBP-3 detection results and Siemens IGFBP-3 detection results, respectively, in example 3 of the invention.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples, in which specific conditions are not noted, according to conventional conditions or manufacturer's recommended conditions. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The described embodiments are intended to be illustrative of only some, but not all embodiments of the invention, and all other embodiments that may be made by one of ordinary skill in the art without inventive faculty are intended to be within the scope of the invention.
Example 1
The kit for jointly detecting IGF-1 and IGFBP-3 comprises a test strip, a lysate and a buffer solution, wherein the test strip comprises a PVC plate, the PVC plate sequentially comprises water absorbing paper, a nitrocellulose membrane and a sample pad from left to right, one end of the water absorbing paper is lapped on one end of the nitrocellulose membrane, and one end of the sample pad is lapped on the other end of the nitrocellulose membrane.
The lysate comprises the following components: disodium hydrogen phosphate-citrate buffer, bovine Serum Albumin (BSA), urea, guanidine hydrochloride, proClin300;
wherein, the concentration of the disodium hydrogen phosphate-citric acid buffer solution is 10mM, and the molar ratio of the disodium hydrogen phosphate to the citric acid is 2:1, PH2.2; the mass percentage of bovine serum albumin is 0.2%, the mass percentage of urea is 10%, the mass percentage of guanidine hydrochloride is 15%, and the mass percentage of ProClin300 is 0.03%.
The buffer comprises the following components: tris-hydrochloric acid buffer (Tris-HCl), tris (2-formylethyl) phosphine hydrochloride (TCEP), tween 20 (Tween-20), S9, EDTA-2NA, proClin300;
wherein, the concentration of the Tris-HCl buffer (Tris-HCl) is 20mM, the mass percentage of Tris (2-formylethyl) phosphine hydrochloride (TCEP) is 0.01%, the mass percentage of Tween 20 (Tween-20) is 0.05%, the mass percentage of S9 is 0.8%, the mass percentage of EDTA-2NA is 0.1%, and the mass percentage of ProClin300 is 0.03%.
The preparation method of the test strip comprises the following steps:
(1) Scribing film
a. Coating a quality control line C and two detection lines T on a nitrocellulose membrane in sequence, wherein the formula of the membrane drawing liquid of the quality control line C and the two detection lines T is as follows: 0.1M PBS, 1% trehalose by mass;
b. labeling fluorescent dye IGFBP-3 fluorescent microsphere IGF-1 antibody, and sequentially coating the fluorescent dye IGFBP-3 and the fluorescent microsphere IGF-1 antibody on a nitrocellulose membrane, wherein the fluorescent membrane-drawing liquid comprises the following formula: 0.1M PBS, 0.5% trehalose by mass, 0.5% sucrose by mass, 0.9% NaCl by mass and 0.5% casein by mass;
(2) Preparation of sample pad
The sample pad treatment fluid is solidified on glass fiber, and after drying for 8 hours, the preparation of the sample pad is completed, and the sample pad treatment fluid comprises the following components: 0.2M pH8.0 Tris, 1% by mass of BSA, 0.1% by mass of PVA, 0.1% by mass of Tween 20, 0.1% by mass of ProClin300, 0.5% by mass of sucrose, 0.5% by mass of trehalose;
(3) Assembly of test strips
And (3) sequentially assembling the absorbent paper, the nitrocellulose membrane, the sample pad and the PVC base plate, and then cutting the assembled absorbent paper, the nitrocellulose membrane, the sample pad and the PVC base plate into test strips.
Wherein, in the step (1), the fluorescent dye IGFBP-3 antibody labeling method comprises the following steps:
(1) Taking out IGFBP-3 antibody, standing at room temperature for 20min, dissolving fluorochrome to 1mol/L with PBS for use, adding 10 μL fluorochrome and 4mg/mL IGFBP-3 antibody 25 μL into 50 μL system, and mixing the rest volume with pH8.0 and 1mol/L NaHCO 3 Supplementing, mixing, and vibrating and reacting for 3 hours at room temperature by a shaking table;
(2) And (3) dialysis: and (3) labeling the antibody, dialyzing in a buffer solution for 36 hours, and replacing the dialyzate every 4 hours to obtain the labeled fluorescent dye IGFBP-3 antibody.
The labeling method of the fluorescent microsphere IGF-1 antibody comprises the following steps: (1) preparing microsphere suspension: preparing 50mM pH6.0 MES buffer solution, adding 1% fluorescent microsphere to prepare microsphere suspension with concentration of 1 w/v%;
(2) Activating: dissolving EDC in 50mM MES buffer solution with pH of 6.0, adding dissolved EDC into each mL of microsphere suspension to make its concentration 20mg/mL, incubating at room temperature for 20min, centrifuging or ultrafiltering, washing the microspheres twice with equal volume of 50mM MES buffer solution with pH of 6.0, and making into activated microsphere suspension, and continuously stirring;
(3) Labeling the antibody: rapidly adding IGF-1 antibody into the stirred activated microsphere suspension to ensure that the final concentration of IGF-1 antibody is 1mg/mL, continuously stirring, and incubating at room temperature for 2.5 hours to obtain microsphere suspension of labeled antibody;
(4) Closing: adding 2.5 mu L of ethanolamine into each mL of microsphere suspension of the labeled antibody, continuously stirring, incubating at room temperature for 10min, centrifuging or ultrafiltering;
(5) And (3) storing: suspending the microsphere with a storage buffer solution, repeating the steps twice, and removing unbound IGF-1 antibody and ethanolamine to obtain labeled fluorescent microsphere IGF-1 antibody;
the storage buffer formulation is: 0.2M Tris pH8.0, trehalose 0.5% by mass, sucrose 0.5% by mass, casein 0.2% by mass, BSA 2% by mass and sodium azide 0.1% by mass.
The kit for jointly detecting IGF-1 and IGFBP-3 comprises a detection card, wherein the detection card comprises a card cover and a card body which are mutually buckled, the test strip is arranged between the card cover and the card body, and a sample adding hole for adding samples and an observation window for observing results are formed in the detection card.
Example 2
The kit for jointly detecting IGF-1 and IGFBP-3 comprises a test strip, a lysate and a buffer solution, wherein the test strip comprises a PVC plate, the PVC plate sequentially comprises water absorbing paper, a nitrocellulose membrane and a sample pad from left to right, one end of the water absorbing paper is lapped on one end of the nitrocellulose membrane, and one end of the sample pad is lapped on the other end of the nitrocellulose membrane.
The lysate comprises the following components: disodium hydrogen phosphate-citrate buffer, bovine Serum Albumin (BSA), urea, guanidine hydrochloride, proClin300;
wherein, the concentration of the disodium hydrogen phosphate-citric acid buffer solution is 100mM, and the molar ratio of the disodium hydrogen phosphate to the citric acid is 2:1, PH3; the mass percentage of bovine serum albumin is 1%, the mass percentage of urea is 12%, the mass percentage of guanidine hydrochloride is 18%, and the mass percentage of ProClin300 is 0.04%.
The buffer comprises the following components: tris-hydrochloric acid buffer (Tris-HCl), tris (2-formylethyl) phosphine hydrochloride (TCEP), tween 20 (Tween-20), S9, EDTA-2NA, proClin300;
wherein, the concentration of the Tris-HCl buffer (Tris-HCl) is 60mM, the mass percentage of Tris (2-formylethyl) phosphine hydrochloride (TCEP) is 0.06%, the mass percentage of Tween 20 (Tween-20) is 0.1%, the mass percentage of S9 is 1%, the mass percentage of EDTA-2NA is 0.12%, and the mass percentage of ProClin300 is 0.04%.
The preparation method of the test strip comprises the following steps:
(1) Scribing film
a. Coating a quality control line C and two detection lines T on a nitrocellulose membrane in sequence, wherein the formula of the membrane drawing liquid of the quality control line C and the two detection lines T is as follows: 0.1M PBS, 1% trehalose by mass;
b. labeling fluorescent dye IGFBP-3 fluorescent microsphere IGF-1 antibody, and sequentially coating the fluorescent dye IGFBP-3 and the fluorescent microsphere IGF-1 antibody on a nitrocellulose membrane, wherein the fluorescent membrane-drawing liquid comprises the following formula: 0.1M PBS, 0.5% trehalose by mass, 0.5% sucrose by mass, 0.9% NaCl by mass and 0.5% casein by mass;
(2) Preparation of sample pad
The sample pad treatment fluid is solidified on glass fiber, and after drying for 8 hours, the preparation of the sample pad is completed, and the sample pad treatment fluid comprises the following components: 0.2M pH8.0 Tris, 1% by mass of BSA, 0.1% by mass of PVA, 0.1% by mass of Tween 20, 0.1% by mass of ProClin300, 0.5% by mass of sucrose, 0.5% by mass of trehalose;
(3) Assembly of test strips
And (3) sequentially assembling the absorbent paper, the nitrocellulose membrane, the sample pad and the PVC base plate, and then cutting the assembled absorbent paper, the nitrocellulose membrane, the sample pad and the PVC base plate into test strips.
Wherein, in the step (1), the fluorescent dye IGFBP-3 antibody labeling method comprises the following steps:
(1) Taking out IGFBP-3 antibody, standing at room temperature for 20min, dissolving fluorochrome to 1mol/L with PBS for use, adding 10 μL fluorochrome and 4mg/mL IGFBP-3 antibody 25 μL into 50 μL system, and mixing the rest volume with pH8.0 and 1mol/L NaHCO 3 Complement, shake reaction 3 with room temperature shaking table after mixingh；
(2) And (3) dialysis: and (3) labeling the antibody, dialyzing in a buffer solution for 36 hours, and replacing the dialyzate every 4 hours to obtain the labeled fluorescent dye IGFBP-3 antibody.
Labeling of fluorescent microsphere IGF-1 antibodies: (1) preparing microsphere suspension: preparing 50mM pH6.0 MES buffer solution, adding 1% fluorescent microsphere to prepare microsphere suspension with concentration of 1 w/v%;
(2) Activating: dissolving EDC in 50mM MES buffer solution with pH of 6.0, adding dissolved EDC into each mL of microsphere suspension to make its concentration 20mg/mL, incubating at room temperature for 20min, centrifuging or ultrafiltering, washing the microspheres twice with equal volume of 50mM MES buffer solution with pH of 6.0, and making into activated microsphere suspension, and continuously stirring;
(3) Labeling the antibody: rapidly adding IGF-1 antibody into the stirred activated microsphere suspension to ensure that the final concentration of IGF-1 antibody is 1mg/mL, continuously stirring, and incubating at room temperature for 2.5 hours to obtain microsphere suspension of labeled antibody;
(4) Closing: adding 2.5 mu L of ethanolamine into each mL of microsphere suspension of the labeled antibody, continuously stirring, incubating at room temperature for 10min, centrifuging or ultrafiltering;
(5) And (3) storing: suspending the microsphere with a storage buffer solution, repeating the steps twice, and removing unbound IGF-1 antibody and ethanolamine to obtain labeled fluorescent microsphere IGF-1 antibody;
the storage buffer formulation is: 0.2M Tris pH8.0, trehalose 0.5% by mass, sucrose 0.5% by mass, casein 0.2% by mass, BSA 2% by mass and sodium azide 0.1% by mass.
The kit for jointly detecting IGF-1 and IGFBP-3 comprises a detection card, wherein the detection card comprises a card cover and a card body which are mutually buckled, the test strip is arranged between the card cover and the card body, and a sample adding hole for adding samples and an observation window for observing results are formed in the detection card.
Example 3
The kit for jointly detecting IGF-1 and IGFBP-3 comprises a test strip, a lysate and a buffer solution, wherein the test strip comprises a PVC plate, the PVC plate sequentially comprises water absorbing paper, a nitrocellulose membrane and a sample pad from left to right, one end of the water absorbing paper is lapped on one end of the nitrocellulose membrane, and one end of the sample pad is lapped on the other end of the nitrocellulose membrane.
The lysate comprises the following components: disodium hydrogen phosphate-citrate buffer, bovine Serum Albumin (BSA), urea, guanidine hydrochloride, proClin300;
wherein, the concentration of the disodium hydrogen phosphate-citric acid buffer solution is 200mM, and the molar ratio of the disodium hydrogen phosphate to the citric acid is 2:1, PH3.5; the mass percentage of bovine serum albumin is 1.5%, the mass percentage of urea is 15%, guanidine hydrochloride is 20%, and the mass percentage of ProClin300 is 0.05%.
The buffer comprises the following components: tris-hydrochloric acid buffer (Tris-HCl), tris (2-formylethyl) phosphine hydrochloride (TCEP), tween 20 (Tween-20), S9, EDTA-2NA, proClin300;
wherein, the concentration of the Tris-HCl buffer (Tris-HCl) is 100mM, the mass percentage of Tris (2-formylethyl) phosphine hydrochloride (TCEP) is 0.1%, the mass percentage of Tween 20 (Tween-20) is 0.15%, the mass percentage of S9 is 1.2%, the mass percentage of EDTA-2NA is 0.15%, and the mass percentage of ProClin300 is 0.05%.
The preparation method of the test strip comprises the following steps:
(1) Scribing film
a. Coating a quality control line C and two detection lines T on a nitrocellulose membrane in sequence, wherein the formula of the membrane drawing liquid of the quality control line C and the two detection lines T is as follows: 0.1M PBS, 1% trehalose by mass;
b. labeling fluorescent dye IGFBP-3 and fluorescent microsphere IGF-1 antibody, and coating the fluorescent dye IGFBP-3 and the fluorescent microsphere IGF-1 antibody on a nitrocellulose membrane in sequence, wherein the fluorescent membrane-drawing liquid comprises the following formula: 0.1M PBS, 0.5% trehalose by mass, 0.5% sucrose by mass, 0.9% NaCl by mass and 0.5% casein by mass;
(2) Preparation of sample pad
The sample pad treatment fluid is solidified on glass fiber, and after drying for 8 hours, the preparation of the sample pad is completed, and the sample pad treatment fluid comprises the following components: 0.2M pH8.0 Tris, 1% by mass of BSA, 0.1% by mass of PVA, 0.1% by mass of Tween 20, 0.1% by mass of ProClin300, 0.5% by mass of sucrose, 0.5% by mass of trehalose;
(3) Assembly of test strips
And (3) sequentially assembling the absorbent paper, the nitrocellulose membrane, the sample pad and the PVC base plate, and then cutting the assembled absorbent paper, the nitrocellulose membrane, the sample pad and the PVC base plate into test strips.
Wherein, in the step (1), the fluorescent dye IGFBP-3 antibody labeling method comprises the following steps:
(1) Taking out IGFBP-3 antibody, standing at room temperature for 20min, dissolving fluorochrome to 1mol/L with PBS for use, adding 10 μL fluorochrome and 4mg/mL IGFBP-3 antibody 25 μL into 50 μL system, and mixing the rest volume with pH8.0 and 1mol/L NaHCO 3 Supplementing, mixing, and vibrating and reacting for 3 hours at room temperature by a shaking table;
(2) And (3) dialysis: and (3) labeling the antibody, dialyzing in a buffer solution for 36 hours, and replacing the dialyzate every 4 hours to obtain the labeled fluorescent dye IGFBP-3 antibody.
Labeling of fluorescent microsphere IGF-1 antibodies: (1) preparing microsphere suspension: preparing 50mM pH6.0 MES buffer solution, adding 1% fluorescent microsphere to prepare microsphere suspension with concentration of 1 w/v%;
(2) Activating: dissolving EDC in 50mM MES buffer solution with pH of 6.0, adding dissolved EDC into each mL of microsphere suspension to make its concentration 20mg/mL, incubating at room temperature for 20min, centrifuging or ultrafiltering, washing the microspheres twice with equal volume of 50mM MES buffer solution with pH of 6.0, and making into activated microsphere suspension, and continuously stirring;
(3) Labeling the antibody: rapidly adding IGF-1 antibody into the stirred activated microsphere suspension to ensure that the final concentration of IGF-1 antibody is 1mg/mL, continuously stirring, and incubating at room temperature for 2.5 hours to obtain microsphere suspension of labeled antibody;
(4) Closing: adding 2.5 mu L of ethanolamine into each mL of microsphere suspension of the labeled antibody, continuously stirring, incubating at room temperature for 10min, centrifuging or ultrafiltering;
(5) And (3) storing: suspending the microsphere with a storage buffer solution, repeating the steps twice, and removing unbound IGF-1 antibody and ethanolamine to obtain labeled fluorescent microsphere IGF-1 antibody;
the storage buffer formulation is: 0.2M Tris pH8.0, trehalose 0.5% by mass, sucrose 0.5% by mass, casein 0.2% by mass, BSA 2% by mass and sodium azide 0.1% by mass.
The kit for jointly detecting IGF-1 and IGFBP-3 comprises a detection card, wherein the detection card comprises a card cover and a card body which are mutually buckled, the test strip is arranged between the card cover and the card body, and a sample adding hole for adding samples and an observation window for observing results are formed in the detection card.
Example 4
Use of a kit for the combined detection of IGF-1 and IGFBP-3: and (3) taking 40 mu L of lysate and 10 mu L of serum sample, mixing uniformly, adding 200 mu L of buffer solution, mixing uniformly, taking 75 mu L of buffer solution, adding the sample solution into a sample adding hole of a detection card, inserting the detection card into a sample groove of a fluorescence analyzer, and outputting a result by a 15-min instrument.
Experiment
Experiment 1:
30 serum samples were taken and assayed using the kit for the combined detection of IGF-1 and IGFBP-3 of examples 1-3, respectively, and IGF-1 and IGFBP-3 were assayed using the chemiluminescence inlet kit Siemens insulin-like growth factor-1 assay kit and the insulin-like growth factor binding protein-3 assay kit, respectively, and the results were compared as shown in Table 1 below:
TABLE 1
FIGS. 1-6 are correlation comparisons of the test results of examples 1-3 with Siemens test results, respectively.
As can be seen from the above results, the kit for combined detection of IGF-1 and IGFBP-3 according to the present invention is comparable to the detection results of higher-sensitivity platform chemiluminescence assay for quantitative detection of IGF-1 and IGFBP-3, and has a correlation R2 of 0.99, and can realize joint detection of both IGF-1 and IGFBP-3.
Experiment 2:
comparing the results of examples 1-3 and Siemens detection with the IGF-1 national standard, the results are shown in Table 2, which shows that the correlation with the national standard is higher.
TABLE 2
According to the results, the IGF-1 and IGFBP-3 are quantitatively detected by combining the sample treated by the lysate and the buffer solution, compared with a higher sensitivity platform chemiluminescence method, the quantitative detection results of IGF-1 and IGFBP-3 products are not greatly different, the correlation R2 is 0.99, the combined detection of the IGF-1 and the IGFBP-3 products can be realized, the IGF-1 has a national standard, the IGFBP-3 has no national standard, and compared with the national standard, the IGFBP-3 has higher correlation with the national standard.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the invention, but is merely illustrative of the embodiments, and all the details not described herein are common knowledge of a person skilled in the art, so that it is possible for a person skilled in the art to modify the technical solutions described in the foregoing embodiments or to make equivalent substitutions for some technical features thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (8)

1. A lysate for a kit for the combined detection of IGF-1 and IGFBP-3, characterized by: comprises the following components: disodium hydrogen phosphate-citrate buffer, bovine serum albumin, urea, guanidine hydrochloride, proClin300; wherein the concentration of the disodium hydrogen phosphate-citric acid buffer solution is 10-200mM, and the molar ratio of the disodium hydrogen phosphate to the citric acid is 2:1, pH range 2.2-3.5; the mass percentage of bovine serum albumin is 0.2-1.5%, the mass percentage of urea is 10-15%, guanidine hydrochloride is 15-20%, and the mass percentage of ProClin300 is 0.03-0.05%.
2. A buffer for a kit for the combined detection of IGF-1 and IGFBP-3, characterized in that: comprises the following components: tris (2-formylethyl) phosphine hydrochloride, tween 20, S9, EDTA-2NA and ProClin300, wherein the concentration of the tris (2-formylethyl) phosphine hydrochloride, the concentration of the tris (2-formylethyl) phosphine hydrochloride and the concentration of the tris (2-formylethyl) phosphine hydrochloride are respectively 20-100mM, 0.01-0.1% of the tris (2-formylethyl) phosphine hydrochloride, 0.05-0.15% of the tween 20, 0.8-1.2% of the S9, 0.1-0.15% of the EDTA-2NA and 0.03-0.05% of the ProClin 300.
3. A kit for the combined detection of IGF-1 and IGFBP-3, characterized in that: including the test paper, the test paper includes the PVC board, from left to right includes water absorption paper, nitrocellulose membrane, sample pad on the PVC board in proper order, and the one end overlap joint of water absorption paper is on the one end of nitrocellulose membrane, and the one end overlap joint of sample pad is on the other end of nitrocellulose membrane.
4. A kit for the combined detection of IGF-1 and IGFBP-3 according to claim 3, wherein: the preparation method of the test strip comprises the following steps:
(1) Scribing film
a. Coating a quality control line C and two detection lines T on a nitrocellulose membrane in sequence, wherein the formula of the membrane drawing liquid of the quality control line C and the two detection lines T is as follows: 0.1M PBS, 1% trehalose by mass;
b. labeling fluorescent dye IGFBP-3 fluorescent microsphere IGF-1 antibody, and sequentially coating the fluorescent dye IGFBP-3 fluorescent microsphere IGF-1 antibody on a nitrocellulose membrane, wherein the fluorescent membrane-drawing liquid comprises the following formula: 0.1M PBS, 0.5% trehalose by mass, 0.5% sucrose by mass, 0.9% NaCl by mass and 0.5% casein by mass;
(2) Preparation of sample pad
The sample pad treatment fluid is solidified on glass fiber, and after drying for 8 hours, the preparation of the sample pad is completed, and the sample pad treatment fluid comprises the following components: 0.2M pH8.0 Tris, 1% by mass of BSA, 0.1% by mass of PVA, 0.1% by mass of Tween 20, 0.1% by mass of ProClin300, 0.5% by mass of sucrose, 0.5% by mass of trehalose;
(3) Assembly of test strips
And (3) sequentially assembling the absorbent paper, the nitrocellulose membrane, the sample pad and the PVC base plate, and then cutting the assembled absorbent paper, the nitrocellulose membrane, the sample pad and the PVC base plate into test strips.
5. A kit for the combined detection of IGF-1 and IGFBP-3 according to claim 4, wherein: the fluorescent dye IGFBP-3 antibody labeling method comprises the following steps:
(1) Taking out IGFBP-3 antibody, standing at room temperature for 20min, dissolving fluorochrome to 1mol/L with PBS for use, adding 10 μL fluorochrome and 4mg/mL IGFBP-3 antibody 25 μL into 50 μL system, and mixing the rest volume with pH8.0 and 1mol/L NaHCO 3 Supplementing, mixing, and vibrating and reacting for 3 hours at room temperature by a shaking table;
(2) And (3) dialysis: and (3) labeling the antibody, dialyzing in a buffer solution for 36 hours, and replacing the dialyzate every 4 hours to obtain the labeled fluorescent dye IGFBP-3 antibody.
6. A kit for the combined detection of IGF-1 and IGFBP-3 according to claim 5, wherein: the labeling method of the fluorescent microsphere IGF-1 antibody comprises the following steps: (1) preparing microsphere suspension: preparing 50mM pH6.0 MES buffer solution, adding 1% fluorescent microsphere to prepare microsphere suspension with concentration of 1 w/v%;
(2) Activating: dissolving EDC in 50mM MES buffer solution with pH of 6.0, adding dissolved EDC into each mL of microsphere suspension to make its concentration 20mg/mL, incubating at room temperature for 20min, centrifuging or ultrafiltering, washing the microspheres twice with equal volume of 50mM MES buffer solution with pH of 6.0, and making into activated microsphere suspension, and continuously stirring;
(3) Labeling the antibody: rapidly adding IGF-1 antibody into the stirred activated microsphere suspension to ensure that the final concentration of IGF-1 antibody is 1mg/mL, continuously stirring, and incubating at room temperature for 2.5 hours to obtain microsphere suspension of labeled antibody;
(4) Closing: adding 2.5 mu L of ethanolamine into each mL of microsphere suspension of the labeled antibody, continuously stirring, incubating at room temperature for 10min, centrifuging or ultrafiltering;
(5) And (3) storing: suspending the microsphere with a storage buffer solution, repeating the steps twice, and removing unbound IGF-1 antibody and ethanolamine to obtain labeled fluorescent microsphere IGF-1 antibody;
the storage buffer formulation is: 0.2M Tris pH8.0, trehalose 0.5% by mass, sucrose 0.5% by mass, casein 0.2% by mass, BSA 2% by mass and sodium azide 0.1% by mass.
7. A kit for the combined detection of IGF-1 and IGFBP-3 according to any one of claims 3-6, characterized in that: the test card comprises a card cover and a card body which are buckled with each other, and the test paper strip in any one of claims 3-6 is arranged between the card cover and the card body.
8. The use of a kit for the combined detection of IGF-1 and IGFBP-3 according to claim 7, wherein: and (3) taking 40 mu L of lysate and 10 mu L of serum sample, mixing uniformly, adding 200 mu L of buffer solution, mixing uniformly, taking 75 mu L of buffer solution, adding the sample solution into a sample adding hole of a detection card, inserting the detection card into a sample groove of a fluorescence analyzer, and outputting a result by a 15-min instrument.
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