CN109633163B - procalcitonin/C reactive protein two-in-one detection kit - Google Patents
procalcitonin/C reactive protein two-in-one detection kit Download PDFInfo
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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
- G01N33/68—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
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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
- G01N33/53—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
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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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
- G01N33/53—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
- 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
- G01N33/577—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
Abstract
The invention provides a procalcitonin/C-reactive protein two-in-one detection kit, which belongs to the technical field of fluorescence immunochromatography in medical immunology, and comprises (i) a detection card, wherein the detection card comprises: (a) respectively labeling a PCT monoclonal antibody and a CRP monoclonal antibody by using rare earth fluorescent microspheres, (b) coating a carrier coated with the PCT monoclonal antibody and the CRP monoclonal antibody and a carrier coated with a goat anti-mouse IgG antibody; (ii) an IC card for calibration; and (iii) a diluent for diluting the collected whole blood sample. The test sample of the kit is a human serum, plasma or whole blood sample, and the kit has good detection specificity, higher sensitivity, simplicity and convenience in operation and stable fluorescent marker, so that the detection accuracy is ensured.
Description
Technical Field
The invention belongs to the technical field of fluorescence immunochromatography in medical immunology, and particularly relates to a procalcitonin/C-reactive protein two-in-one detection kit.
Background
C-reactive protein (CRP) refers to a number of proteins that rise sharply in the blood (acute proteins) when the body is infected or tissue damaged. CRP can activate complement and enhance phagocytosis of phagocyte to play an opsonizing role, thereby eliminating pathogenic microorganism and damaged, necrotic and apoptotic histiocyte invading the body, and playing an important protective role in the natural immune process of the body. The C-reactive protein is an extremely sensitive index of acute phase reaction, and the CRP concentration in blood is rapidly and obviously increased in acute myocardial infarction, trauma, infection, inflammation and surgical operation, and can reach 2000 times of the normal level. The method is mainly used for differential diagnosis of bacterial and viral infection, prognosis of cardiovascular diseases and forecast of gynecological diseases in clinic. Procalcitonin (PCT) is a glycoprotein, a hormone-inactive calcitonin precursor, with a relative molecular weight of 13 kD. Normally, PCT is produced and secreted mainly in the thyroid parafollicular cells (C cells) and is broken down into calcitonin by intracellular specific proteases. PCT is stable in vivo and in vitro, and the half-life period is 25-30 h. The blood PCT concentration is lower than 0.5ng/ml in healthy adults, chronic inflammatory reaction and autoimmune diseases, virus infection or mild to moderate local bacterial infection patients, and the value has definite correlation with the severity of the systemic infection condition, so the detection of the blood PCT value has important clinical significance in the aspects of disease diagnosis, treatment and prognosis evaluation of the severely infected patients. The PCT/CRP combined detection has certain clinical value for comprehensive judgment of inflammation.
The principle of fluorescence quantitative immunochromatography is similar to that of ELISA, and the method is based on the principle of antigen-antibody specific reaction and achieves the purpose of detection by labeling an antibody or an antigen and forming an immune complex. Unlike the ELISA method, the immunoreaction process of the fluorescence quantitative immunochromatography technique occurs on a chromatographic membrane and advances the chromatography by capillary action. In recent years, chromatographic techniques including fluorescence labeling immunochromatography are widely applied to detection of low-concentration samples, and the detection method has certain advantages in the aspects of sensitivity, operability and accuracy, so that the method has wide application prospects in various subject fields including clinical chemistry, biological analysis and environmental analysis. The kit based on the fluorescence quantitative immunochromatography can be used for detecting mass samples, but cannot replace the existing instrument analysis technology, can be used as an important supplementary technology, and should be used together with other analysis technologies or mutually verified, so that the detection sensitivity is increased, the cross reaction is reduced, and more accurate quantification can be carried out. Therefore, the fluorescence quantitative immunochromatography has wide application prospect in field monitoring, detection of a simple basic laboratory and even primary screening of a large laboratory. Therefore, the method has important significance for establishing an effective procalcitonin/C-reactive protein detection two-in-one fluorescence quantitative immunochromatography method and developing a high-performance procalcitonin/C-reactive protein two-in-one rapid detection kit through research stages such as synthesis of hapten and complete antigen, pairing of antigen and antibody by a fluorescence quantitative immunochromatography platform, optimization of the fluorescence quantitative immunochromatography platform, performance evaluation of the kit, clinical application and the like.
Disclosure of Invention
The invention aims to provide a procalcitonin/C-reactive protein two-in-one detection kit, wherein a test sample is a human serum, plasma or whole blood sample, and the kit has the advantages of good detection specificity, higher sensitivity, simplicity and convenience in operation and guarantee of detection accuracy by a stable fluorescent marker.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a procalcitonin/C-reactive protein two-in-one detection kit comprises,
(i) a test card, said test card comprising:
(a) respectively using rare earth fluorescent microsphere labeled PCT monoclonal antibody and CRP monoclonal antibody,
(b) a carrier coated with a PCT monoclonal antibody and a CRP monoclonal antibody and a carrier coated with a goat anti-mouse IgG antibody;
(ii) an IC card for calibration; and
(iii) a diluent for diluting the collected whole blood sample.
Preferably, the PCT monoclonal antibody is directed against an epitope comprised in the sequence spanning amino acid residues 4 to 18 of procalcitonin. S of PCT protein4-18Amino acid sequence PFRSALESSPADPAT-NH of2The peptides of which the PCT monoclonal antibodies of the invention are specific for their epitopes in PCT and do not exhibit significant cross-reactivity for other epitopes in the peptides, particularly non-overlapping epitopes, use a peptide as an antigen to prepare antibodies that can be used to qualitatively or quantitatively determine human PCT proteins conveniently, with high sensitivity and specificity.
Preferably, the PCT monoclonal antibody is obtained by immunizing human PCT; the CRP monoclonal antibody is obtained by immunizing human CRP.
Preferably, the detection card comprises a sample pad, a chromatographic membrane, a combination pad, a lining plate and absorbent paper; a detection area of the chromatographic membrane is coated with a PCT monoclonal antibody and a CRP monoclonal antibody, and a quality control area is coated with a goat anti-mouse IgG antibody; the binding pad is sprayed with a PCT monoclonal antibody and a CRP monoclonal antibody marked by rare earth fluorescent microspheres.
More preferably, the chromatographic membrane is a nitrocellulose membrane.
More preferably, the rare earth fluorescent microsphere labeled PCT monoclonal antibody and CRP monoclonal antibody comprises the following steps:
1) washing 4.8-5.3mg of rare earth fluorescent microspheres with 20mM carbonate buffer solution with pH of 9.5, then suspending in 100ul of the carbonate buffer solution containing 1.3-1.7ul of monoethanolamine thioglycolate, adding 500ul of aldehyde-converted dextran and 0.19-0.22 mu g of 2-aminoethanesulfonic acid, mixing uniformly, carrying out dark reaction at room temperature for 3-5 hours, washing by a centrifugal method, then suspending in 100ul of the carbonate buffer solution, and placing at 4 ℃ for later use; the existence of the thioglycolic acid monoethanolamine and the 2-aminoethanesulfonic acid can improve the hydroformylation degree of the rare earth fluorescent microsphere, provide a coupling site for covalent combination of the anti-PG I monoclonal antibody and the PG II monoclonal antibody, improve the binding force of the rare earth fluorescent microsphere with the anti-PG I monoclonal antibody and the PG II monoclonal antibody, and increase the water solubility due to the existence of free aldehyde groups, so that the collision chance between the rare earth fluorescent microsphere and the anti-PG I monoclonal antibody and the PG II monoclonal antibody is increased, on the other hand, the cross-linking between the anti-PG I monoclonal antibody and the PG II monoclonal antibody can be reduced, the aggregation of the antibody is reduced, the immune activity of the antibody is relatively higher while the marking rate and the fluorescence intensity are improved to the maximum extent, and finally, the stability and the detection sensitivity of the kit are improved;
2) dialyzing 2-2.2mg of PCT monoclonal antibody and CRP monoclonal antibody with the carbonate buffer solution at 4 ℃ overnight according to the mass ratio of 1:1, mixing with the aldehyde-based rare earth fluorescent microspheres, and adding sodium borohydride for reaction; adding an equal volume of blocking solution, sealing overnight, and washing.
Preferably, the test sample of the kit is human serum, plasma or whole blood. The kit is only used for detecting human serum, plasma or whole blood samples, other body fluid samples are not applicable, the serum, plasma and whole blood samples are collected according to a conventional method, sodium citrate, EDTA or heparin anticoagulant plasma can be used as an anticoagulant, the serum or plasma is required to be detected within 4 hours after being separated, the serum or plasma can be stored for 5 days at the temperature of 2-8 ℃, the serum or plasma is required to be stored for more than 5 days at-20 ℃, the detection is finished within 1 hour after the sample is restored to the room temperature before the detection, and repeated freeze thawing is not needed; the whole blood sample can be stored for 3 days at the temperature of 2-8 ℃ without being frozen.
The kit disclosed by the invention is used for quantitatively detecting the content of Procalcitonin (PCT) and C-reactive protein (CRP) in a human serum, plasma or whole blood sample by adopting an immunofluorescence double antibody sandwich method. After a sample is dripped into a sample adding hole of the detection card, PCT and CRP in the sample are combined with anti-PCT monoclonal antibody and CRP monoclonal antibody marked by fluorescent substance in the combination pad to form a reaction compound, the reaction compound moves forwards along the nitrocellulose membrane along with the chromatography action and is captured by the corresponding monoclonal antibody on a detection line of the nitrocellulose membrane, the PCT and CRP capture amount in the sample is positively correlated with the signal intensity of the detection area, and the content of PCT and CRP in the sample is quantitatively detected by a fluorescence analyzer.
The invention also discloses the application of the procalcitonin/C-reactive protein two-in-one detection kit, which is characterized in that: the application of the kit in vitro quantitative detection of the content of procalcitonin and C-reactive protein in human serum, plasma or whole blood samples.
Preferably, the use of the procalcitonin/C-reactive protein two-in-one test kit is for the stratification of the use of a diagnostic, prognostic, risk stratification, therapy monitoring, therapy guidance or therapeutic measures for a disease or condition associated with elevated procalcitonin and C-reactive protein levels.
The invention also discloses a using method of the procalcitonin/C-reactive protein two-in-one detection kit, which comprises the steps of sucking 150 mu L of whole blood, adding a drop of diluent or serum and 100 mu L of plasma into a sample adding hole of a test card, inserting the test card into a card slot of an applicable instrument after 10 minutes, and carrying out quantitative interpretation on a result; the normal reference interval is: PCT: less than 0.5 ng/mL; CRP: less than 10 mg/L.
Compared with the prior art, the invention has the beneficial effects that: 1) the test sample of the kit is a human serum, plasma or whole blood sample, and the kit has good detection specificity, higher sensitivity, simplicity and convenience in operation and stable fluorescent marker, so that the detection accuracy is ensured; 2) the kit can simultaneously quantitatively detect the content of procalcitonin and C-reactive protein in a sample, can be used for diagnosis, prognosis, risk stratification, therapy monitoring, therapy guidance or application stratification of therapeutic measures of diseases or symptoms related to the rising of the levels of the procalcitonin and the C-reactive protein, and has the advantages of rapidness, effectiveness, accuracy and the like; 3) the two monoclonal antibody-microsphere coupled compounds prepared by the method have the advantages of large binding force, long fluorescence life, large Stokes shift, no overlapping of excitation spectrum and emission spectrum and no mutual interference phenomenon.
The procalcitonin/C-reactive protein two-in-one detection kit provided by the invention adopts the technical scheme, makes up for the defects of the prior art, and is reasonable in design and convenient to operate.
Detailed Description
The following further describes embodiments of the present invention with reference to specific examples. The following examples are illustrative only and are not to be construed as limiting the invention.
The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. In addition, the term "coating" in the present invention is a term in the field of immunization, and includes adsorption and immobilization.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1:
a procalcitonin/C-reactive protein two-in-one detection kit comprises,
(i) a test card, said test card comprising:
(a) respectively using rare earth fluorescent microsphere labeled PCT monoclonal antibody and CRP monoclonal antibody,
(b) a carrier coated with a PCT monoclonal antibody and a CRP monoclonal antibody and a carrier coated with a goat anti-mouse IgG antibody;
(ii) an IC card for calibration; and
(iii) a diluent for diluting the collected whole blood sample.
The above-described PCT monoclonal antibody is directed to an epitope contained in a sequence spanning amino acid residues 4 to 18 of procalcitonin. S of PCT protein4-18Amino acid sequence PFRSALESSPADPAT-NH of2The peptides of which the PCT monoclonal antibodies of the invention are specific for their epitopes in PCT and do not exhibit significant cross-reactivity for other epitopes in the peptides, particularly non-overlapping epitopes, use a peptide as an antigen to prepare antibodies that can be used to qualitatively or quantitatively determine human PCT proteins conveniently, with high sensitivity and specificity.
The PCT monoclonal antibody is obtained by immunizing human PCT; the CRP monoclonal antibody is obtained by immunizing human CRP.
The detection card comprises a sample pad, a chromatographic membrane, a combination pad, a lining plate and absorbent paper; a detection area of the chromatographic membrane is coated with a PCT monoclonal antibody and a CRP monoclonal antibody, and a quality control area is coated with a goat anti-mouse IgG antibody; the binding pad is sprayed with a PCT monoclonal antibody and a CRP monoclonal antibody marked by rare earth fluorescent microspheres. The detection card is stored at 4-30 ℃, the validity period is 18 months, and the detection card is valid within 2 hours after the reagent aluminum foil bag is opened.
Wherein, the chromatographic membrane is a nitrocellulose membrane.
The rare earth fluorescent microsphere labeled PCT monoclonal antibody and CRP monoclonal antibody comprises the following steps:
1) taking 5mg rare earth fluorescent microspheres, washing for 3 times by using 20mM carbonate buffer solution with pH of 9.5 by using a centrifugal method, wherein the centrifugal speed is 12000rpm, the time is 5 minutes, finally suspending the microspheres in 100ul of the carbonate buffer solution containing 1.5ul of monoethanolamine thioglycolate, adding 500ul of aldehyde dextran and 0.2 mu g of 2-aminoethanesulfonic acid, uniformly mixing, carrying out dark reaction for 4 hours at room temperature, washing and suspending the microspheres in 100ul of the carbonate buffer solution by using the same centrifugal method, and placing the microspheres at 4 ℃ for later use. The existence of the thioglycolic acid monoethanolamine and the 2-aminoethanesulfonic acid can improve the hydroformylation degree of the rare earth fluorescent microsphere, provide a coupling site for covalent combination of the anti-PG I monoclonal antibody and the PG II monoclonal antibody, improve the binding force of the rare earth fluorescent microsphere with the anti-PG I monoclonal antibody and the PG II monoclonal antibody, and increase the water solubility due to the existence of free aldehyde groups, so that the collision chance between the rare earth fluorescent microsphere and the anti-PG I monoclonal antibody and the PG II monoclonal antibody is increased, on the other hand, the cross-linking between the anti-PG I monoclonal antibody and the PG II monoclonal antibody can be reduced, the aggregation of the antibody is reduced, the immune activity of the antibody is relatively higher while the marking rate and the fluorescence intensity are improved to the maximum extent, and finally, the stability and the detection sensitivity of the kit are improved;
2) dialyzing 2mg of PCT monoclonal antibody and CRP monoclonal antibody with the carbonate buffer solution at 4 ℃ overnight according to the mass ratio of 1:1, mixing with the aldehyde-based rare earth fluorescent microspheres, and reacting at 4 ℃ overnight; then, sodium borohydride was added to a final concentration of 5mM, and the reaction was carried out at 4 ℃ for 4 hours; adding equal volume of blocking solution (50mM Tris-HCl, pH 7.4, containing 2.5% BSA, 5% sucrose), and blocking at 4 deg.C overnight; then, the cells were washed 3 times with 50mM Tris-HCl, pH 7.4 buffer by centrifugation, resuspended in 100. mu.l of 50mM Tris-HCl buffer (containing 1.2% NaCl, 0.5% BSA, 0.1% Tween 20), and stored at 4 ℃ in the dark for further use.
The test sample of the kit is human serum, plasma or whole blood. The kit is only used for detecting human serum, plasma or whole blood samples, other body fluid samples are not applicable, the serum, plasma and whole blood samples are collected according to a conventional method, sodium citrate, EDTA or heparin anticoagulant plasma can be used as an anticoagulant, the serum or plasma is required to be detected within 4 hours after being separated, the serum or plasma can be stored for 5 days at the temperature of 2-8 ℃, the serum or plasma is required to be stored for more than 5 days at-20 ℃, the detection is finished within 1 hour after the sample is restored to the room temperature before the detection, and repeated freeze thawing is not needed; the whole blood sample can be stored for 3 days at the temperature of 2-8 ℃ without being frozen.
Suitable instruments for the kit include: NP007 dry fluoroimmunoassay analyzer of wiener biotechnology limited, AFS1000 dry fluoroimmunoassay analyzer of cantonese blue brib biotechnology limited.
The performance indexes of the kit are as follows:
1. linear range:
PCT is in the range of 0.1 ng/mL-40.0 ng/mL, and the correlation coefficient r is more than or equal to 0.9900;
1.2 CRP is in the range of 0.5 mg/L-150.0 mg/L, and the correlation coefficient r is more than or equal to 0.9900.
2. Repeatability:
2.1. the PCT Coefficient of Variation (CV) is less than or equal to 15 percent;
2.2, CRP Coefficient of Variation (CV) is less than or equal to 15 percent.
3. Inter-batch difference:
3.1. the PCT relative range (R) is less than or equal to 20 percent;
3.2 CRP relative range (R) is less than or equal to 20%.
4. Accuracy:
4.1.PCT uses internal reference as detection sample, and the relative deviation of the detection result and the marked value is within the range of plus or minus 20 percent;
and 4.2, the CRP takes a reference product as a detection sample, and the deviation of the measurement result and the marked value is within the range of +/-15%.
5. The lowest detection limit is:
PCT: not higher than 0.1 ng/mL;
CRP: not higher than 0.5 mg/L.
1. The notes on the above kit:
2. the product is a disposable in vitro diagnostic reagent, and can be used repeatedly within the effective period.
3. The test card and assembly are only suitable for use with an associated fluorescence analyzer.
4. The product is not required to be opened before use, and the package is obviously damaged.
5. Reagent components of different batches cannot be used in a mixed mode, and the IC card and the detection card cannot be used in a mixed mode.
6. Appropriate protective measures should be taken during collection, disposal, storage, blending of the sample and detection.
7. The aluminum foil bag is filled with a drying agent, and the food cannot be eaten.
8. The test card stored at low temperature needs to be opened after being restored to room temperature so as not to absorb moisture.
9. The detection card and the fluorescence analyzer should avoid vibration and electromagnetic environment when in use; in normal use, the instrument generates vibration and is normal; the detection is performed without pulling out the IC card.
10. It is recommended to use a fresh sample, which if there is significant hemolysis or blood clotting in the sample will interfere with the test and lead to erroneous results, which are never used.
11. The detection result of the reagent is only used for clinical reference, and the clinical diagnosis and treatment of patients should be comprehensively considered by combining the symptoms, physical signs, medical history, other laboratory examinations, treatment response and other conditions.
12. All specimens, waste liquids, tubes and the like are treated in an infectious pollutant treatment mode. After detection, the used detection card is discarded in a corresponding biohazard container and is treated according to biohazard.
13. If there is a problem or suggestion in using the reagent, please contact the manufacturer.
The kit adopts an immunofluorescence double-antibody sandwich method to quantitatively detect the content of Procalcitonin (PCT) and C-reactive protein (CRP) in human serum, plasma or whole blood samples. After a sample is dripped into a sample adding hole of the detection card, PCT and CRP in the sample are combined with anti-PCT monoclonal antibody and CRP monoclonal antibody marked by fluorescent substance in the combination pad to form a reaction compound, the reaction compound moves forwards along the nitrocellulose membrane along with the chromatography action and is captured by the corresponding monoclonal antibody on a detection line of the nitrocellulose membrane, the PCT and CRP capture amount in the sample is positively correlated with the signal intensity of the detection area, and the content of PCT and CRP in the sample is quantitatively detected by a fluorescence analyzer.
The application of the procalcitonin/C-reactive protein two-in-one detection kit is the application of the procalcitonin/C-reactive protein two-in-one detection kit in-vitro quantitative detection of the content of procalcitonin and C-reactive protein in human serum, plasma or whole blood samples. For use in the diagnosis, prognosis, risk stratification, therapy monitoring, therapy guidance or stratification for the use of therapeutic measures of a disease or condition associated with elevated procalcitonin and C-reactive protein levels.
The using method of the procalcitonin/C-reactive protein two-in-one detection kit comprises the steps of sucking 150 mu L of whole blood, adding a drop of diluent or 100 mu L of serum and plasma samples into a sample adding hole of a test card, inserting the test card into a card slot of an applicable instrument after 10 minutes, and carrying out quantitative interpretation on the result; the normal reference interval is: PCT: less than 0.5 ng/mL; CRP: less than 10 mg/L.
Comparative example 1:
the rare earth fluorescent microsphere labeled PCT monoclonal antibody and CRP monoclonal antibody comprises the following steps:
taking 5mg rare earth fluorescent microspheres, washing for 3 times by using 20mM carbonate buffer solution with pH of 9.5 by adopting a centrifugal method, wherein the centrifugal speed is 12000rpm, the time is 5 minutes, finally suspending in 100ul of the carbonate buffer solution, adding 500ul of aldehyde-group-converted glucan, uniformly mixing, carrying out dark reaction for 4 hours at room temperature, washing and suspending in 100ul of the carbonate buffer solution by adopting the same centrifugal method, and placing at 4 ℃ for later use. The rest of the process was the same as in example 1.
Example 2:
stability test
Detecting the stability of the card: the detection card in the kit is usually stored in a dry and cool environment at room temperature. The test card is placed in a 50 ℃ oven for one month for accelerated destruction stability test, which is equivalent to the effectiveness of the test card stored for 1 year at normal temperature. And (3) placing the detection card in a 50 ℃ oven for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks and 8 weeks respectively, then taking out and placing the detection card into a drying room, comparing the detection card with a reagent card placed in the drying room at normal temperature, storing the sample diluent at 4 ℃, and detecting the stability of the card. The reaction curve of the test card of example 2 in the case of the standard test was substantially matched with the test card stored at room temperature after the test card was placed in an oven at 50 ℃ for accelerated destruction for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, and 7 weeks, indicating that the T/C value of the fluorescence signal at low, medium, and high concentrations did not change, indicating that the stability of the fluorescence quantitative immunochromatographic test card was good after accelerated destruction test and the card could be left at room temperature for 21 months. The coincidence degree of the reaction curve of the test card in the example 1 and the test card stored at normal temperature under the condition that the test card is placed in a 50 ℃ oven to be accelerated and damaged for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks and 8 weeks under the standard product test is poorer than that of the test card in the example 2, which shows that the stability of the test card in the example 2 is better than that of the test card in the example 1, and further shows that the existence of the monoethanolamine thioglycolate and the 2-aminoethanesulfonic acid can improve the binding force of the rare earth fluorescent microspheres and the anti-PG I monoclonal antibody and the PG II monoclonal antibody, and further improve the stability of the test card.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (2)
1. procalcitonin/C reactive protein two-in-one detection kit, which is characterized in that: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
(i) the detection card comprises a sample pad, a chromatographic membrane, a combination pad, a lining plate and absorbent paper; the detection area of the chromatographic membrane is coated with a PCT monoclonal antibody and a CRP monoclonal antibody, the quality control area is coated with a goat anti-mouse IgG antibody, and the chromatographic membrane is a nitrocellulose membrane; the binding pad is sprayed with a PCT monoclonal antibody and a CRP monoclonal antibody marked by rare earth fluorescent microspheres;
(ii) an IC card for calibration; and
(iii) a diluent for diluting the collected whole blood sample;
the PCT monoclonal antibody is directed to an epitope contained in a sequence spanning amino acid residues 4 to 18 of procalcitonin; the PCT monoclonal antibody is obtained by immunizing human PCT; the CRP monoclonal antibody is obtained by immunizing human CRP;
the rare earth fluorescent microsphere labeled PCT monoclonal antibody and CRP monoclonal antibody comprises the following steps:
1) washing 4.8-5.3mg of rare earth fluorescent microspheres with 20mM carbonate buffer solution with pH of 9.5, then suspending in 100ul of the carbonate buffer solution containing 1.3-1.7ul of monoethanolamine thioglycolate, adding 500ul of aldehyde-converted dextran and 0.19-0.22 mu g of 2-aminoethanesulfonic acid, mixing uniformly, carrying out dark reaction at room temperature for 3-5 hours, washing by a centrifugal method, then suspending in 100ul of the carbonate buffer solution, and placing at 4 ℃ for later use;
2) dialyzing 2-2.2mg of PCT monoclonal antibody and CRP monoclonal antibody with the carbonate buffer solution at 4 ℃ overnight according to the mass ratio of 1:1, mixing with the aldehyde-based rare earth fluorescent microspheres, and adding sodium borohydride for reaction; adding sealing liquid with the same volume for sealing overnight, and washing;
the normal reference interval of the quantitative interpretation result of the detection card is as follows: PCT: less than 0.5 ng/mL; CRP: less than 10 mg/L.
2. The procalcitonin/C-reactive protein two-in-one assay kit according to claim 1, characterized in that: the test sample of the kit is human serum, plasma or whole blood.
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| CN110568203B (en) * | 2019-09-12 | 2022-05-24 | 重庆科技学院 | Application method of multi-channel fluorescence immunochromatographic microfluidic chip |
| CN111537737B (en) * | 2020-05-18 | 2023-05-12 | 巴迪泰（广西）生物科技有限公司 | Procalcitonin and C-reactive protein combined detection kit |
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