CN116449013A - Test paper strip, kit and detection method for measuring interleukin-6 in serum, plasma and whole blood - Google Patents
Test paper strip, kit and detection method for measuring interleukin-6 in serum, plasma and whole blood Download PDFInfo
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Classifications
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- 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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- 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
- G01N33/6869—Interleukin
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/548—Carbohydrates, e.g. dextran
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
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- 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
- G01N33/577—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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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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
- G01N33/58—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
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- 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
- G01N2333/52—Assays involving cytokines
- G01N2333/54—Interleukins [IL]
- G01N2333/5412—IL-6
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a test strip, a kit and a detection method for measuring interleukin-6 in human serum, plasma and whole blood samples, wherein the kit comprises the test strip, the test strip comprises a PVC bottom plate, a sample pad, a bonding pad, a nitrocellulose membrane and absorbent paper, the PVC bottom plate is of a strip-shaped structure, the sample pad, the bonding pad, the nitrocellulose membrane and the absorbent paper are sequentially arranged on one surface of the PVC bottom plate along the length direction of the PVC bottom plate, two ends of the nitrocellulose membrane are respectively overlapped and overlapped with one ends of the bonding pad and the absorbent paper, and the other ends of the bonding pad are overlapped and overlapped with one end of the sample pad. The kit has the characteristics of higher detection sensitivity, wider detection range, low price, rapidness, simplicity and convenience in operation and wide application prospect.
Description
Technical Field
The invention relates to the field of biomedical detection, in particular to a test strip, a kit and a detection method for measuring interleukin-6 in serum, plasma and whole blood.
Background
Interleukin 6 (IL-6) is mainly produced by cells such as macrophages, monocytes, fibroblasts, lymphocytes, T cells and the like, is a cytokine which belongs to the interleukins, has more complex and diverse physiological functions, and is one of important members of the cytokines in the human body. IL-6 has wide in vivo effects, has regulating effects on immune inflammatory reaction, acute protein synthesis of liver, hematopoiesis and bone metabolism, and has the main effects of promoting B cell secretion of antibodies and proliferation and differentiation, and also has wide effects on nerve block, hematopoiesis system, liver cells and T cells in vivo. The acute production of IL-6 can lead to acute inflammatory responses, and is also associated with injury, infection, stress, brain death, and other conditions.
Interleukin 6, which is a multifunctional cytokine and an inflammation marker, participates in various reactions in the body, plays an important role in the health of human bodies, and is an important means for early diagnosis and screening of infectious diseases. At present, various interleukin 6 detection methods are studied, and immunological methods are mainly adopted according to detection principles and different methods. The immunological method also comprises an enzyme-linked immunosorbent assay, an immune colloidal gold method, a radioimmunoassay and a chemiluminescent immunoassay; the respective merits are as follows.
1) The enzyme-linked immunosorbent assay (ELISA) is a relatively common technology, and is characterized in that a solid antigen or antibody is combined with an antigen and an antibody after enzyme labeling, and a common combination mode is a double antibody sandwich method and an indirect method. The ELISA method adopts a double antibody sandwich method to detect, and the coated antibody is adsorbed on the surface of a solid phase carrier and is used for combining with a specific antigen, so that when the specific antigen is contained in an object to be detected, an antigen-antibody complex is formed with the object to be detected. Another enzyme-labeled antibody is added as a detection antibody, which can retain the enzyme activity and the immunological activity of the antibody and is used for improving the sensitivity of the ELISA method. The antibody after enzyme labeling can be combined with different epitopes of antigen in the to-be-detected object to form a double antibody sandwich compound of antibody-antigen-antibody, the formed compound is also combined on a solid phase carrier, after a substrate for enzyme reaction is added, the substrate is catalyzed into a colored product by the enzyme, and the concentration of the antigen in the to-be-detected object can be judged by the color depth of the colored product, so that qualitative or quantitative experiments can be carried out. The method has the advantages of long detection time and narrow detection range.
2) The immune colloidal gold method (ICA) is to label colloidal gold with high electron density as a label on an antibody or antigen, which generates macroscopic color when aggregation occurs at the corresponding ligand, and thus can be used in qualitative or semi-quantitative detection studies. With the development of colloidal gold research, certain modification can be performed on the surface of gold cores, mainly including sulfur-containing ligands, phosphide, phosphorus oxide, amino and carboxyl ligands. The method has low sensitivity, and can not carry out quantitative detection and meet the requirement of clinical diagnosis.
3) Radioimmunoassay (RIA) is a method of using isotopically labeled and unlabeled antigens to competitively inhibit reactions with antibodies. The method has the advantages of cross reaction, long operation time, high instrument price and certain damage to operators due to certain radiation.
4) Chemiluminescent immunoassay (CLIA) is a detection analysis technique for various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, drugs, etc., by combining a chemiluminescent assay technique with high sensitivity with a highly specific immune reaction. The method has low detection precision and high cost of instruments and equipment.
Disclosure of Invention
Based on the above problems, the invention aims to provide a test strip, a kit and a detection method which have low cost and simple operation and can rapidly detect interleukin-6 in serum, plasma and whole blood.
The invention designs a technical scheme of three aspects.
The first technical scheme of the invention is as follows:
the test strip for measuring interleukin-6 in serum, plasma and whole blood samples comprises a PVC bottom plate, a sample pad, a combining pad, a nitrocellulose membrane and absorbent paper, wherein the PVC bottom plate is of a strip-shaped structure, the sample pad, the combining pad, the nitrocellulose membrane and the absorbent paper are sequentially arranged on one surface of the PVC bottom plate along the length direction of the PVC bottom plate, two ends of the nitrocellulose membrane are respectively overlapped with one ends of the combining pad and the absorbent paper, and the other ends of the combining pad are overlapped with one ends of the sample pad; the combination pad is provided with a fluorescent-labeled IL-6 antibody and a fluorescent-labeled goat anti-chicken IgY antibody; the nitrocellulose membrane is provided with a detection line and a quality control line which are parallel to each other, the detection line IL-6 is coated with an antibody in a monoclonal mode, and the quality control line is coated with a chicken IgY antibody.
In one embodiment, the biological source of the IL-6 antibody in the test strip is murine monoclonal IgG.
In one embodiment, the biological source of the IL-6 monoclonal antibody in the test strip is murine monoclonal IgG.
In one embodiment, in the test strip, the biological source of the goat anti-chicken IgY antibody is a goat-derived antibody.
In one embodiment, the biological source of the coated chicken IgY antibody in the test strip is chicken-derived antibody.
In one embodiment, fluorescent microspheres are labeled on the IL-6 antibody and the goat anti-chicken IgY antibody in the test strip.
The second technical scheme of the invention is as follows:
a kit for measuring interleukin-6 in serum, plasma and whole blood samples comprises a sample diluent, an ID card and a detection card; the ID card stores reagent standard information; the detection card comprises a card shell and any one of the test strips; the appearance structure of the card shell is consistent with that of the test strip, and a sample adding hole and a detection result observation area are arranged on the card shell; after the test strip is adaptively arranged in the clamping shell, the sample pad of the test strip is correspondingly arranged at the position of the sample adding hole, and the position of the detection result observation area is correspondingly arranged at the position of the nitrocellulose membrane.
In one embodiment, the kit, the sample diluent is phosphate buffer.
The technical scheme III of the invention is as follows:
a method for detecting interleukin-6 using the above kit, comprising the steps of:
preparing whole blood, plasma and serum samples respectively;
respectively taking whole blood, plasma and serum samples, adding the whole blood, the plasma and the serum samples into three diluted solutions, respectively and uniformly mixing the three diluted solutions to respectively obtain whole blood, plasma and serum sample solutions;
respectively taking whole blood, plasma and serum sample solutions, and adding the whole blood, the plasma and the serum sample solutions into sample adding holes on detection cards of three kits;
and (3) inserting the three detection cards into a fluorescence quantitative detector, and selecting a sample type of plasma, serum or whole blood for detection to obtain detection results of interleukin-6 in the serum, plasma or whole blood samples respectively.
In one embodiment, the diluent comprises 0.01M PB,1% PVP, 1% sodium chloride, and 1% Tween.
The invention is based on the principle of immunology and chromatography technology, uses immunofluorescence microsphere as a marker, uses a fluorescence detector to detect fluorescence signal intensity, and quantitatively determines the kit of interleukin-6 (IL-6) in human serum, plasma and whole blood samples; compared with the traditional rapid detection technology, the kit has the characteristics of higher detection sensitivity, wider detection range, low price, rapidness, simplicity and convenience in operation and wide application prospect.
Drawings
FIG. 1 is a schematic diagram of a test strip according to the present invention;
FIG. 2 is a schematic diagram of a shell structure according to the present invention;
FIG. 3 is a graph showing the correlation between the kit and the clinical whole blood sample values in example 5;
FIG. 4 is a graph showing the correlation between the clinical blood plasma sample values and the kit in example 5.
Detailed Description
The preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
As shown in FIG. 1, a test strip 10 for measuring interleukin-6 in human serum, plasma and whole blood samples comprises a PVC bottom plate 5, a sample pad 1, a bonding pad 2, a nitrocellulose membrane 3 and absorbent paper 4, wherein the PVC bottom plate 5 is of a strip-shaped structure, the sample pad 1, the bonding pad 2, the nitrocellulose membrane 3 and the absorbent paper 4 are sequentially arranged on one surface of the PVC bottom plate 5 along the length direction of the PVC bottom plate 5, two ends of the nitrocellulose membrane 3 are respectively overlapped and overlapped with one ends of the bonding pad 2 and the absorbent paper 4, and the other end of the bonding pad 2 is overlapped and overlapped with one end of the sample pad 1; the combination pad 2 is provided with a fluorescent-marked IL-6 antibody and a fluorescent-marked goat anti-chicken IgY antibody; the nitrocellulose membrane 3 is provided with a detection line 6 (T line) and a quality control line 7 (C line) which are parallel to each other, the detection line 6IL-6 monoclonal coats the antibody, and the quality control line 7 coats the chicken IgY antibody. The components and compositions of the test strips are shown in Table 1.
Table 1 Assembly of Interleukin-6 test strips and Main ingredients contained therein
| Numbering device | Component (A) | Main component of the composition |
| 1 | Sample pad | —— |
| 2 | Bonding pad | Fluorescent-labeled IL-6 antibody and fluorescent-labeled goat anti-chicken IgY |
| 3 | Nitrocellulose membrane | IL-6 monoclonal coated antibody, quality control line coated chicken IgY antibody |
| 4 | Water absorbing paper | —— |
| 5 | PVC bottom plate | ——- |
In one embodiment, the labeled antibody of the IL-6 antibody bound to the pad 2, i.e., the fluorescent-labeled IL-6 antibody biological source is shown as Ab1, the goat anti-chicken IgY antibody bound to the pad, i.e., the goat anti-chicken IgY labeled antibody biological source is shown as Ab3, the coated antibody for T-line detection, i.e., the IL-6 monoclonal coated antibody biological source is shown as Ab2, and the coated antibody for C-line quality control, i.e., the coated chicken IgY antibody biological source is shown as Ab 4.
Further, the Ab1 antibody is marked with fluorescent microspheres, and the Ab3 antibody is marked with fluorescent microspheres; the fluorescent microsphere is convenient to combine with a buffer solution to obtain the time-resolved fluorescence-antibody complex.
Preferably, the final concentration of the IL-6 labeled antibody on the test strip is 0.5mg/mL, the final concentration of the IL-6 coated antibody on the test strip is 1mg/mL, the final concentration of the labeled antibody for C line quality control on the test strip is 0.5mg/mL, and the final concentration of the coated antibody for C line quality control on the test strip is 1mg/mL. The biological origin of the antibody for interleukin-6 (IL-6) was quantitatively determined as shown in Table 2.
TABLE 2 detection of the biological origins of antibodies and antigens to interleukin-6 (IL-6)
| Main raw materials | Biogenic origin |
| anti-IL-6 labeled antibody Ab1 | Murine monoclonal IgG |
| anti-IL-6 coated antibody Ab2 | Murine monoclonal IgG |
| Goat anti-chicken IgY labeled antibody Ab3 | Sheep-derived antibody |
| Antibody Ab4 coated with chicken IgY | Chicken-derived antibody |
Carrying out chromatography under capillary effect, combining IL-6 antigen in the combined pad sample with fluorescent labeled IL-6 antibody, diffusing to a test area, capturing by IL-6 monoclonal antibody coated by a detection line, and forming an antibody-antigen-fluorescent antibody complex; the IL-6 concentration in the sample is proportional to the fluorescence intensity of the complex, and the immunofluorescence detector converts the fluorescence signal value into the IL-6 concentration in the sample according to a set standard curve.
The preparation process of the test strip comprises the following steps:
step one, preparation of bond pad
IL-6 immunofluorescence activation: 100uL of fluorescent microspheres were added to a 800uL MES (2-morpholinoethanesulfonic acid) centrifuge tube and sonicated for 3min. 30uL of EDC (1-ethyl-carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) (1:1) activation solution were mixed with the centrifuge tube, sonicated until dispersed uniformly, and equilibrated for a period of time at room temperature. 12000r/min, centrifuging at 4deg.C for 15min, and removing supernatant.
Antibody coupling: 1mL MES was added to the centrifuge tube, vortexed, sonicated for 3min, and 0.5mg of IL-6 antibody and goat anti-chicken IgY antibody were added and the reaction was swirled for 2h.
Closing and storing: 100uL of 10% BSA (bovine serum albumin) solution was added to the centrifuge tube and blocked for 30min,12000r/min, and centrifuged at 4℃for 15min. The supernatant was removed, 1mL of Tris buffer (Tris) was added, sonicated for 3min, followed by 12000r/min, centrifugation at 4℃for 15min, and this step was repeated once. Adding 1mL of microsphere complex solution into the supernatant, performing ultrasonic treatment for 3min, centrifuging for 3min at 2000r/min, removing precipitate, and keeping at 4deg.C in dark place.
And (3) a bonding pad: uniformly spraying the prepared fluorescent particle solution on 1.5-1.8cm glass fiber at the speed of 6uL/cm, and drying at 55 ℃ for 3-4 hours.
Step two, preparation and treatment of sample pad
Preparing a treatment fluid: 0.1M BB,1% sucrose, 1% trehalose, 1% PVPK3O (polyvinylpyrrolidone), 0.1mg/mLRBC (human anti-erythrocyte antibody).
Sample pad treatment: uniformly coating the treating fluid on a sample pad of 0.8-1.0cm according to 3-4 mL/strip (width: 17-18, length: 30 cm), and drying at 55deg.C for 6-8 h.
Step three, preparation of nitrocellulose membrane (also called immunochromatography test strip)
Coating liquid preparation: 0.05M Tris (Tris), 1% sucrose.
Spraying pad: and sticking a nitrocellulose membrane (namely an NC membrane) on a PVC bottom plate, coating the antibody by using the coating liquid to ensure that the coating concentration of the IL-6 antibody on an NC membrane detection line is 1mg/mL, the coating concentration of the chicken IgY on a quality control line is 1mg/mL, and the drying time of the coating is selected to be 48 hours.
Step four, assembling test paper strips
And (3) sequentially attaching the treated sample pad, the bonding pad and the absorbent paper on a PVC plate, and cutting the assembled PVC plate into the immunochromatographic test strip with the width of 3.85-4.00mm by a slitter.
Step five, sample inspection and result judgment
Sample diluent preparation: 0.01M PB,1% PVP (polyvinylpyrrolidone), 1% sodium chloride, 1% Tween.
Sample inspection: taking 20-30uL of serum, plasma or whole blood sample to be detected, adding the serum, the plasma or the whole blood sample into 100uL of the sample diluent, and dripping the mixture onto an immunochromatography reagent card after the mixture is uniformly mixed for carrying out an immunochromatography reaction; fluorescence detection is then performed under the fluorescence detector using the wavelength of the emitted light corresponding to the fluorescent particles. In the case of fluorescence detection under a fluorescence detector, the detection result can be confirmed to be effective only by detecting a signal on a quality control line.
The invention also provides a kit for measuring interleukin-6 in human serum, plasma and whole blood samples, which comprises sample diluent, an ID card and a detection card; the ID card stores reagent standard information, and the test card includes a card case 20 and a test strip 10. As shown in fig. 2, the cartridge 20 has a shape and structure identical to those of the test strip 10, and a sample loading hole 21 and a detection result observation area 22 are provided on the cartridge 20; after the test strip is adaptively accommodated in the cartridge 20, the sample pad 1 of the test strip 10 is arranged corresponding to the position of the sample adding hole 21, and the position of the detection result observation area 22 is arranged corresponding to the position of the nitrocellulose membrane 3; in use, the detection result observation area 22 is used for observing the fluorescent stripes of the C line and the T line to judge whether the detection is effective.
Preferably, in order to prevent the test card from being polluted and wet, the test card is also required to be sealed and packaged by tin foil or aluminum foil, and a drying agent is also contained in the tin foil or aluminum foil.
In one embodiment, the sample diluent of the kit is phosphate buffer.
The invention also provides a detection method for measuring interleukin-6 in human serum, plasma and whole blood samples by using the kit, which comprises the following steps:
1. preparing whole blood, plasma and serum samples respectively;
2. respectively sucking 100uL of whole blood, plasma and serum samples by a pipette, adding the whole blood, the plasma and the serum samples into three dilutions, and respectively and uniformly mixing the three dilutions to respectively obtain whole blood, plasma and serum sample solutions;
3. opening an aluminum foil bag, taking out three detection cards, and respectively horizontally placing the three detection cards on a tabletop;
4. respectively sucking 100uL of whole blood, plasma and serum sample solution by using a pipette, and adding the sample solution into sample adding holes on three detection cards;
5. opening the fluorescent quantitative detector, and inserting an ID chip which is the same as the reagent batch number;
6. selecting a sample type of "plasma, serum or whole blood" on a fluorescent quantitative detector;
7. and (3) testing in real time: after reacting for 15min at room temperature, placing the detection card into an instrument card slot, selecting an 'instant test' mode, and clicking a 'test'; standard test: placing the detection card into an instrument card slot, selecting a standard test mode, clicking a test mode, automatically timing the instrument, automatically testing and displaying a detection result after timing is finished;
8. clicking "print" to print the report of the detection result of interleukin-6 in serum, plasma or whole blood sample respectively.
Whole blood, plasma, serum samples were prepared as follows:
the invention uses a sample collection method, which is to collect venous blood of a tested person by using an anticoagulant tube containing heparin lithium, EDTA-K2 or sodium citrate anticoagulant for whole blood collection, and shake the collected blood sample for later use; collecting blood plasma by using an anticoagulant tube containing heparin lithium, EDTA-K2 or sodium citrate anticoagulant to collect venous blood of a tested person, and separating the blood plasma as soon as possible after blood collection so as to avoid hemolysis; serum collection serum tubes or rapid serum tubes containing coagulants collect venous blood from subjects and serum should be separated as soon as possible after collection to avoid hemolysis.
The sample types include: whole blood, plasma, serum.
Preferably, the different samples should be used as immediately as possible after collection. If the detection can not be carried out in time, the serum/plasma/whole blood can be stored for 3 hours at room temperature; the serum/plasma can be preserved for 7 days at 2-8deg.C, and the whole blood can be preserved for 3 days at 2-8deg.C; serum/plasma can be stored for 90 days at-20+ -5deg.C, and whole blood sample cannot be frozen. The refrigerated and frozen samples are fully dissolved and evenly mixed before detection, and can be tested after being restored to the room temperature without repeated freezing and thawing for later use.
In the above detection method, the diluent comprises 0.01M PB,1% PVP, 1% sodium chloride and 1% Tween.
The kit is applicable to human serum, plasma and whole blood samples.
The kit is used for judging the detection effectiveness according to the following criteria: and each time the fluorescent strip appears on the C line, the test paper card is not damaged, and the instrument is not in fault, so that the experimental result is effective.
Further details are provided below by way of specific examples.
Example 1 composition, package and quantity (25 parts/box) of a kit for rapid detection of interleukin-6 (IL-6) in human serum, plasma and whole blood samples are shown in table 3:
TABLE 3 composition, package and quantity of kit
Example 2 blank Performance evaluation experiment of kit
100uL of negative bovine serum is taken and added with 100uL of sample diluent, 100uL is taken and added into a sample adding hole after uniform mixing, and the detection is carried out by adopting a fluorescence detector after 15min, and the steps are repeated for 20 times. The relative T/C average (M) and Standard Deviation (SD) are used to obtain the value of M+2SD, which is put into a standard curve equation to obtain the corresponding concentration value, namely the blank limit, as shown in Table 4. The results show that the blank limit of the kit is not more than 3.00pg/mL.
TABLE 4 blank test results
Example 3 test for evaluation of Linear Performance of kit
The negative bovine serum is adopted as a diluent, interleukin-6 standard (NIBSC 89/548) is prepared into standard substance solutions with the concentration of 3pg/mL, 30pg/mL, 200pg/mL, 1000pg/mL and 4000pg/mL, 100uL of standard substance is taken and added into 100uL of sample diluent, 100uL of sample diluent is taken after uniform mixing, and the sample solution is added into a sample adding hole for 15min and then detected by adopting a fluorescence detector. The results show that the linear correlation coefficient r2 is greater than 0.9900, the detection limit is 3.00pg/mL, and the detection variation coefficient of each concentration is less than 10%, as shown in Table 5.
TABLE 5 results of test for different concentration standards
Example 4 accuracy Performance assessment experiment of kit
A certain amount of interleukin-6 standard substance is tested and added into negative calf serum, so that the final concentration of interleukin-6 in the serum is respectively 7pg/mL and 1000pg/mL, and 6 interleukin-6 samples are repeated. And (3) fully mixing 100uL of sample to be detected with sample diluent in a 100uL kit for 1min, then adding the 100uL of mixed solution into a sample adding hole of a reagent card, horizontally placing the reagent card for 15min, inserting the reagent card into a fluorescence quantitative detector, reading a fluorescence signal T/C value, and calculating corresponding sample concentration according to a standard curve, wherein the sample concentration is shown in Table 6. As can be seen from the test data in Table 6, the relative deviation of the 6 samples is not more than + -15%.
TABLE 6 accuracy test experiment results
Example 5 clinical application experiment
20 cases of whole blood and plasma samples are collected from a hospital, 100 mu L of sample diluent is taken in a small centrifuge tube, then 100 mu L of clinical sample is sucked in the small centrifuge tube by a liquid-transferring gun, after the mixed solution is uniformly shaken by a vortex oscillator for reaction for 1min, 100 mu L of mixed solution is sucked in a sample-adding hole of a reagent card by the liquid-transferring gun for sample adding (the sample-adding process is carried out with the attention that bubbles are not required to be injected into the sample-adding hole so as to prevent the bubbles from entering a chromatographic system to influence the measurement result). Each sample was tested in duplicate. The reagent card is horizontally placed for 15min for chromatographic reaction, and then inserted into a fluorescent quantitative detector for reading the T/C value. The test values were obtained by inverse calculation using the graphs of fig. 3 and 4, and compared with the clinical sample values, and the results are shown in table 7.
TABLE 7 detection results of 20 examples of whole blood and plasma samples
As can be seen from Table 7, the 20 clinical samples were 5 negative and 25 positive, and the results were the same when tested by the kit, and the correlation between the test value of the kit and the clinical sample value was found to be good. The results show that the quantitative detection of the interleukin 6 content in human whole blood and plasma samples by the kit is more reliable.
It is to be understood that the foregoing description of the preferred embodiments of the invention is not to be considered as limiting the scope of the invention, which is defined by the appended claims.
Claims (10)
1. The test strip for measuring interleukin-6 in serum, plasma and whole blood samples is characterized by comprising a PVC bottom plate, a sample pad, a combining pad, a nitrocellulose membrane and water absorbing paper, wherein the PVC bottom plate is of a strip-shaped structure, the sample pad, the combining pad, the nitrocellulose membrane and the water absorbing paper are sequentially arranged on one surface of the PVC bottom plate along the length direction of the PVC bottom plate, two ends of the nitrocellulose membrane are respectively overlapped with one end of the combining pad and one end of the water absorbing paper, and the other end of the combining pad is overlapped with one end of the sample pad; the combination pad is provided with a fluorescent-labeled IL-6 antibody and a fluorescent-labeled goat anti-chicken IgY antibody; the nitrocellulose membrane is provided with a detection line and a quality control line which are parallel to each other, the detection line IL-6 is coated with an antibody in a monoclonal mode, and the quality control line is coated with a chicken IgY antibody.
2. The test strip of claim 1, wherein the biological source of the IL-6 antibody is murine monoclonal IgG.
3. The test strip of claim 1, wherein the biological source of the IL-6 monoclonal coated antibody is murine monoclonal IgG.
4. The test strip of claim 1, wherein the biological source of the goat anti-chicken IgY antibody is a goat-derived antibody.
5. The test strip of claim 1, wherein the biological source of the coated chicken IgY antibodies is chicken-derived antibodies.
6. The test strip of claim 1, wherein the IL-6 antibody and the goat anti-chicken IgY antibody are labeled with fluorescent microspheres.
7. A kit for measuring interleukin-6 in serum, plasma and whole blood samples, which is characterized by comprising a sample diluent, an ID card and a detection card; the ID card stores reagent standard information; the detection card comprises a card shell and the test strip as claimed in any one of claims 1 to 5; the appearance structure of the card shell is consistent with that of the test strip, and a sample adding hole and a detection result observation area are arranged on the card shell; after the test strip is adaptively arranged in the clamping shell, the sample pad of the test strip is correspondingly arranged at the position of the sample adding hole, and the position of the detection result observation area is correspondingly arranged at the position of the nitrocellulose membrane.
8. The kit of claim 7, wherein the sample diluent is phosphate buffer.
9. A method for detecting interleukin-6, characterized in that the method uses the kit according to claim 7 or 8, said method comprising the steps of:
preparing whole blood, plasma and serum samples respectively;
respectively taking whole blood, plasma and serum samples, adding the whole blood, the plasma and the serum samples into three diluted solutions, respectively and uniformly mixing the three diluted solutions to respectively obtain whole blood, plasma and serum sample solutions;
respectively taking whole blood, plasma and serum sample solutions, and adding the whole blood, the plasma and the serum sample solutions into sample adding holes on detection cards of three kits;
and (3) inserting the three detection cards into a fluorescence quantitative detector, and selecting a sample type of plasma, serum or whole blood for detection to obtain detection results of interleukin-6 in the serum, plasma or whole blood samples respectively.
10. The method of claim 9, wherein the diluent comprises 0.01M PB,1% pvp, 1% sodium chloride, and 1% tween.
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