CN114113636A - Heart, lung and kidney joint inspection test paper card, kit and preparation method - Google Patents
Heart, lung and kidney joint inspection test paper card, kit and preparation method Download PDFInfo
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Abstract
The invention relates to a heart, lung and kidney combined examination paper card, a kit and a preparation method, and belongs to the technical field of preparation of diagnostic kits. The test paper card is sequentially provided with a sample pad, a nitrocellulose membrane and a water absorption pad; four kinds of mouse anti-human monoclonal antibodies marked with fluorescence are respectively sprayed on different positions of the sample pad; the nitrocellulose membrane is respectively sprayed and scribed with a detection line and a quality control line, four mouse anti-human monoclonal antibodies are respectively sprayed and scribed on different positions of the detection line, and polyclonal antibodies are sprayed and scribed on the quality control line. The test paper card provided by the invention can be used for scratching the antibodies of the four biomarkers on the same test paper card, so that one-time sample adding is realized innovatively, the four biomarkers of BNP, cTnI, D-Dimer and NGAL in a sample are detected simultaneously, the detection time is short, the anti-interference performance is strong, the accuracy and the stability are high, the sample is not required to be pretreated, and the detection can be completed by directly utilizing EDTA (ethylene diamine tetraacetic acid) anticoagulated venous whole blood or plasma.
Description
Technical Field
The invention relates to the technical field of preparation of diagnostic kits, in particular to a heart, lung and kidney combined test paper card, a kit and a preparation method.
Background
The four items of heart, lung and kidney are the abbreviation of B-type natriuretic peptide (BNP), Neutrophil gelatinase-associated lipocalin (NGAL), D-Dimer (DD) and troponin I (Cardiac tropinin I, cTnI). BNP is a cardiac neuroendocrine hormone, can be specifically secreted by ventricular myocytes under the conditions of heart chamber volume overload, pressure overload and wall tension increase, has important clinical significance for quantitatively judging whether patients suffer from heart failure, and is closely related to the severity and prognosis of heart failure. cTn exists in myocardial cells in the form of a cTnI-C-T compound and free cTnI, and after being released into blood circulation during myocardial injury, the cTnI-C-T can be further decomposed into the cTnI-C compound and the free cTnI, and the cTnI is a 'gold index' for diagnosing acute myocardial infarction. Has important significance for the prognosis evaluation and risk stratification of acute coronary syndrome, the evaluation of myocardial ischemia injury area and the detection of myocardial injury degree caused by cardiac surgery. The D-Dimer is a specific degradation product generated by crosslinking a fibrinogen monomer with activated factor XIII and hydrolyzing with plasmin, is a specific fibrinolysis process marker, the generation or increase of the D-Dimer reflects the activation of a blood coagulation and fibrinolysis system, and the D-Dimer can be clinically used for diagnosing and evaluating deep vein thrombosis and pulmonary embolism and negatively eliminating the deep vein thrombosis and the pulmonary embolism and can be used as monitoring indexes of surgical operation and thrombolytic therapy and auxiliary diagnosis of cardiovascular diseases. NGAL is a member of lipocalin family, is remarkably increased within 2 hours after heart operation, has high prediction sensitivity and specificity on postoperative acute kidney injury, and is an early biomarker of postoperative acute kidney injury.
Clinically, acute chest pain is one of the common causes of adult emergency treatment. Chest pain has various causes, and the risk degree of chest pain needs to be immediately evaluated. Low-risk chest pain needs to be reasonably shunted, medium-risk chest pain needs to be dynamically evaluated and monitored, and fatal chest pain needs to immediately enter a rescue process. The common causes of chest pain include acute myocardial infarction, acute coronary syndrome, heart failure, pulmonary embolism and the like, the diseases are closely related to kidney injury and renal failure, the acute kidney injury and renal failure can be caused after heart operation, and the kidney injury and renal failure can be accompanied with various cardiovascular diseases. Meanwhile, patients with chest pain and abdominal pain are clinically detected whether the heart, lung and kidney function are disordered or not.
Therefore, the combined detection of BNP, cTnI, D-Dimer and NGAL can provide valuable guidance for clinical treatment of patients with heart failure, patients with acute myocardial infarction, acute coronary syndrome, pulmonary embolism, acute kidney injury and acute kidney failure, can be used for rapid diagnosis and identification of diseases, and can play a critical role in risk stratification, prognosis judgment and treatment decision.
At present, technologies such as ELLISA and chemiluminescence are mainly adopted for detecting corresponding biomarkers, but most of the existing detection methods and tools are single detection, and few of the existing detection methods and tools are combined detection, and a detection sample needs to be pretreated, so that the detection time is long, and the diagnosis and treatment time is delayed. In addition, the existing measuring method and tool have poor anti-interference performance, and various biological macromolecules in the blood sample to be detected can be non-specifically adsorbed with the measuring tool, such as a sample pad and a detection strip of a test paper card, so that the detection accuracy and stability are reduced, the measured value deviates from an actual value, and a diagnosis result obtained by a doctor according to the inaccurate measurement result is possibly wrong and treatment is delayed.
Disclosure of Invention
The invention aims to provide a combined test paper card for heart, lung and kidney, a kit and a preparation method. The test paper card provided by the invention can be used for scratching the antibodies of the four biomarkers on the same test paper card, so that one-time sample adding is realized innovatively, the four biomarkers of BNP, cTnI, D-Dimer and NGAL in a sample are detected simultaneously, the detection time is short, the anti-interference performance is strong, the accuracy and the stability are high, the sample is not required to be pretreated, and the detection can be completed by directly utilizing EDTA (ethylene diamine tetraacetic acid) anticoagulated venous whole blood or plasma.
The invention provides a heart, lung and kidney combined examination paper card, which is sequentially provided with a sample pad, a nitrocellulose membrane and a water absorption pad;
the sample pad is a blood filtering membrane; fluorescent marked mouse-anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL are respectively sprayed on different positions of the sample pad;
the nitrocellulose membrane is respectively sprayed and scribed with a detection line and a quality control line, different positions of the detection line are respectively sprayed and scribed with mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL, and the quality control line is sprayed and scribed with polyclonal antibodies;
the fluorescence labeling condition of the fluorescence labeled mouse anti-human monoclonal antibody is as follows: the mouse anti-human monoclonal antibody of BNP and the mouse anti-human monoclonal antibody of cTnI are respectively marked with Eu-solid phase two-way chelate, and the mouse anti-human monoclonal antibody of D-Dimer and the mouse anti-human monoclonal antibody of NGAL are respectively marked with Sm-solid phase two-way chelate.
Preferably, the polyclonal antibody is capable of binding to fluorescently labeled murine anti-human monoclonal antibodies to BNP, cTnI, D-Dimer and NGAL.
Preferably, the sample pad is sprayed with fluorescently labeled murine anti-human monoclonal antibodies to NGAL, cTnI, D-Dimer and BNP at positions 17.5mm, 20mm, 22.5mm and 25mm from the bottom end of the sample pad, respectively.
Preferably, positions of the nitrocellulose membrane, which are sprayed with the mouse anti-human monoclonal antibodies of BNP, D-Dimer, cTnI and NGAL, are respectively 8mm, 15mm, 18mm and 25mm away from the bottom end of the nitrocellulose membrane; the polyclonal antibody is 28mm away from the bottom end of the nitrocellulose membrane.
Preferably, the material of the blood filtering membrane is a glass fiber membrane.
Preferably, the test paper card further comprises a housing.
The invention also provides a preparation method of the test paper card in the technical scheme, which comprises the following steps:
respectively labeling a mouse anti-human monoclonal antibody of BNP and a mouse anti-human monoclonal antibody of cTnI with Eu-solid phase two-way chelates, and labeling a mouse anti-human monoclonal antibody of D-Dimer and a mouse anti-human monoclonal antibody of NGAL with Sm-solid phase two-way chelates to obtain fluorescence labeled mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL;
respectively spraying mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL on a nitrocellulose membrane;
spraying and scratching the polyclonal antibody on a nitrocellulose membrane;
spraying fluorescence-labeled mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL on the sample pad;
and assembling the sample pad, the nitrocellulose membrane, the absorbent paper and the upper and lower shells to obtain the test paper card.
Preferably, the width of the nitrocellulose membrane is 5 ± 1mm, the width of the sample pad is 7 ± 1mm, and the width of the absorbent pad is 10 ± 1 mm.
Preferably, the working concentrations of the fluorescently labeled mouse anti-human monoclonal antibody, polyclonal antibody and mouse anti-human monoclonal antibody are 0.8mg/mL respectively before spraying.
The invention also provides a combined detection kit for heart, lung and kidney, which comprises the test paper card, the RFID card containing a standard curve, a thermal printing paper roll, a 200 mu L bag of straws and an instruction book.
The invention provides a combined test paper card for heart, lung and kidney (fluorescence immunoassay). The test paper card provided by the invention can be used for scratching the antibodies of the four biomarkers on the same test paper card, so that one-time sample adding is realized innovatively, the four biomarkers of BNP, cTnI, D-Dimer and NGAL in a sample are detected simultaneously, the detection time is short, the anti-interference performance is strong, the accuracy and the stability are high, the sample is not required to be pretreated, and the detection can be completed by directly utilizing EDTA (ethylene diamine tetraacetic acid) anticoagulated venous whole blood or plasma. The test paper card can realize one-time detection, differential diagnosis and prognosis evaluation of heart failure, myocardial infarction, myocardial injury, pulmonary embolism and renal injury renal failure, reasonably shunts patients with chest pain and chest pain combined with abdominal pain emergency treatment, and provides effective differential diagnosis for early lesions of organs after heart surgery or kidney surgery.
Drawings
FIG. 1 is a sequence diagram illustrating the assembly of a test card according to the present invention;
fig. 2 is a top view of the appearance of the test paper card provided by the present invention.
Detailed Description
The invention provides a heart, lung and kidney combined examination paper card based on a time-resolved immunofluorescence method, which is sequentially provided with a sample pad, a nitrocellulose membrane and a water absorption pad;
the sample pad is a blood filtering membrane; fluorescent marked mouse-anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL are respectively sprayed on different positions of the sample pad;
the nitrocellulose membrane is respectively sprayed and scribed with a detection line and a quality control line, different positions of the detection line are respectively sprayed and scribed with mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL, and the quality control line is sprayed and scribed with polyclonal antibodies;
the fluorescence labeling condition of the fluorescence labeled mouse anti-human monoclonal antibody is as follows: the mouse anti-human monoclonal antibody of BNP and the mouse anti-human monoclonal antibody of cTnI are respectively marked with Eu-solid phase two-way chelate, and the mouse anti-human monoclonal antibody of D-Dimer and the mouse anti-human monoclonal antibody of NGAL are respectively marked with Sm-solid phase two-way chelate.
The four pairs of antibodies of BNP, cTnI, D-Dimer and NGAL, namely the mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL and the fluorescently-labeled mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL, are all commercial products, have stable supply channels and can realize mass production. In the above four pairs of antibodies of the present invention, the epitope distance of each pair of antibodies is preferably relatively long. Specifically, the present invention preferably selects an antibody pair recommended by an antibody manufacturer according to the principle of the antibody sandwich method. The polyclonal antibody can be combined with fluorescence-labeled mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL, and particularly, the polyclonal antibody is used for being combined with redundant fluorescence-labeled mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL. The source of the polyclonal antibody is not particularly limited in the present invention, and any polyclonal antibody known to those skilled in the art, such as a polyclonal antibody of G5518 type commercially available from Sigma, which binds to fluorescently labeled monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL, may be used. The Eu-solid phase bidirectional chelate and the Sm-solid phase bidirectional chelate are commercial products, and the manufacturer is Saimeifei.
In the present invention, the positions of the fluorescently labeled murine anti-human monoclonal antibody sprays of NGAL, cTnI, D-Dimer and BNP on the sample pad are preferably 17.5mm, 20mm, 22.5mm and 25mm from the bottom end of the sample pad, respectively.
In the present invention, the positions of the BNP, D-Dimer, cTnI and NGAL sprayed with the mouse anti-human monoclonal antibodies are preferably 8mm, 15mm, 18mm and 25mm from the bottom end of the nitrocellulose membrane, respectively; the polyclonal antibody is 28mm away from the bottom end of the nitrocellulose membrane. The distance setting of the invention can ensure that the fluorescence is not interfered as much as possible, and the detection with high accuracy is realized.
In the invention, the material of the blood filtering membrane is preferably a glass fiber membrane.
In the present invention, the test paper card preferably further comprises a housing, more preferably an upper housing and a lower housing. The material of the shell is preferably PVC.
The test paper card is designed by using a fluorescence immunochromatographic method, mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL are simultaneously scratched on one test paper card, and the test paper card is subsequently detected by using a time-resolved immunofluorescence analyzer, preferably a dry type immunofluorometric analyzer. Specifically, the analyzer scans excitation light of the test paper card and collects emission light signals with wavelengths of 613nm and 600nm respectively to obtain fluorescence values on drawn lines of mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL and fluorescence values on a C line respectively.
The invention also provides a preparation method of the test paper card in the technical scheme, which comprises the following steps:
respectively labeling a mouse anti-human monoclonal antibody of BNP and a mouse anti-human monoclonal antibody of cTnI with Eu-solid phase two-way chelates, and labeling a mouse anti-human monoclonal antibody of D-Dimer and a mouse anti-human monoclonal antibody of NGAL with Sm-solid phase two-way chelates to obtain fluorescence labeled mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL;
respectively spraying mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL on a nitrocellulose membrane;
spraying and scratching the polyclonal antibody on a nitrocellulose membrane;
spraying fluorescence-labeled mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL on the sample pad;
and assembling the sample pad, the nitrocellulose membrane, the absorbent paper, the upper shell and the lower shell to obtain the test paper card.
In the invention, the width of the nitrocellulose membrane is 5 +/-1 mm, the width of the sample pad is 7 +/-1 mm, and the width of the absorbent pad is 10 +/-1 mm.
In the invention, before spraying, the working concentrations of the fluorescence-labeled mouse anti-human monoclonal antibody, polyclonal antibody and mouse anti-human monoclonal antibody are respectively 0.8 mg/mL.
The invention also provides a heart, lung and kidney combined examination kit based on the time-resolved immunofluorescence method, which comprises the test paper card, the RFID card containing the standard curve, the thermal printing paper roll, a 200 mu L straw bag and an instruction book.
When the joint inspection test is carried out, according to the industry standard, preferably, a standard substance containing four antigens of BNP, cTnI, D-Dimer and NGAL is firstly used for preparing reference substances with different concentrations, the test paper card of the application is used for detection, and a standard curve is drawn. In the present invention, the test paper card is preferably used for detection on a dry fluoroimmunoassay analyzer after loading.
The four-item combined test paper card for heart, lung and kidney, the kit and the preparation method of the invention are further described in detail with reference to the following specific examples, and the technical scheme of the invention includes but is not limited to the following examples.
Example 1
Selecting an antibody: the conventional commercially available antibody pair suitable for time resolution was selected according to the principle of the antibody sandwich method.
Labeling the fluorescent microspheres: respectively labeling a Eu-solid phase bidirectional chelate with a BNP mouse anti-human monoclonal antibody and a cTnI mouse anti-human monoclonal antibody, labeling an Sm-solid phase bidirectional chelate with a D-Dimer mouse anti-human monoclonal antibody and an NGAL mouse anti-human monoclonal antibody to obtain a fluorescent mouse anti-human monoclonal antibody, and labeling for later use;
configuring fluorescent monoclonal antibodies with specific concentrations: restoring the fluorescent mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL to room temperature, and configuring the working concentrations of the monoclonal antibodies by using an antibody buffer solution to be 0.8 mg/mL;
monoclonal antibodies were prepared at specific concentrations: restoring the mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL to room temperature, and configuring the working concentrations of the monoclonal antibodies by using an antibody buffer solution to be 0.8 mg/mL;
preparing polyclonal antibody with specific concentration: the polyclonal antibody is restored to room temperature, and the working concentration of the prepared polyclonal antibody by using an antibody buffer solution is 0.8 mg/mL;
scribing an NC film at a specific position: respectively scribing mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL and goat anti-mouse polyclonal antibodies at specific positions of an NC membrane by using different scribing heads of a scribing instrument, wherein the mouse anti-human monoclonal antibodies of BNP are scribed at positions 8mm away from the bottom end of the NC membrane, the mouse anti-human monoclonal antibodies of D-Dimer are scribed at positions 15mm away from the bottom end of the NC membrane, the mouse anti-human monoclonal antibodies of cTnI are scribed at positions 18mm away from the bottom end of the NC membrane, the mouse anti-human monoclonal antibodies of NGAL are scribed at positions 25mm away from the bottom end of the NC membrane, and the goat anti-mouse polyclonal antibodies are scribed at positions 28mm away from the bottom end of the NC membrane, and drying for later use;
draw filter membrane (sample pad) at specific location: spraying and scratching the prepared fluorescence-labeled mouse anti-human fluorescent monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL at a specific position on a hemofiltration membrane by using different scratching heads of a hemofiltration instrument, paying attention to avoid the position of a sample adding hole, scratching the fluorescence-labeled mouse anti-human monoclonal antibody of NGAL at a position 17.5mm away from the bottom end of the hemofiltration membrane, scratching the fluorescence-labeled mouse anti-human monoclonal antibody of cTnI at a position 20mm away from the bottom end of the hemofiltration membrane, scratching the fluorescence-labeled mouse anti-human monoclonal antibody of D-Dimer at a position 22.5mm away from the bottom end of the hemofiltration membrane, scratching the fluorescence-labeled mouse anti-human monoclonal antibody of BNP at a position 25mm away from the bottom end of the hemofiltration membrane, and drying for later use;
slitting: cutting the NC membrane, the blood filtration membrane and the water absorption pad into required sizes, wherein the width of the NC membrane is 5mm, the width of the filtration pad is 7mm, and the width of the water absorption pad is 10 mm;
assembling: as shown in fig. 1, the prepared blood filtering membrane, NC membrane and water absorption pad are placed on the plastic lower shell according to the operation sequence, and the plastic upper shell is buckled and assembled together;
welding and coding: welding the assembled test paper cards together, and printing the batch number information into a two-dimensional code on the test paper cards by using a coding machine;
generating a standard curve: the method comprises the steps of configuring an internal reference substance by using BNP, cTnI, D-Dimer and NGAL antigens, setting 8 gradient concentrations, testing the fluorescence concentration on a dry-type fluorescence immunoassay analyzer by using four joint test paper cards of heart, lung and kidney, collecting data, generating a standard curve which can be recorded into the analyzer by using software, and recording the standard curve into a corresponding time-resolved fluorescence immunoassay analyzer with multiple wavelengths and multiple excitation lights;
adding 200 mu L of sample into the test plate, reacting for 15min in the dark, placing into an analyzer for scanning, and displaying the concentration of each marker to be detected on the screen of the analyzer.
Example 2
Selecting 20 blood samples of acute chest pain patients in emergency department, using a 2mL purple cap tube to extract the blood samples, and reversing the blood samples up and down and mixing the blood samples for later use;
using a pipette, 200. mu.L of each blood sample was pipetted into four joint test paper cards for heart, lung and kidney. The test paper card is selected from a commercially available BNP detection kit, a cTnI detection kit, a D-Dimer detection kit and an NGAL detection kit which are produced by Hebei Tewent Biotechnology development Limited. After the test paper card is added into a sample, the sample reacts for 15min, and the reaction paper card is put into an analyzer to read the test result, and the record is shown in table 1:
TABLE 1 clinical specimen examination comparison results
The test data show that the four combined test paper cards for heart, lung and kidney can detect the concentrations of four biomarkers of BNP, D-Dimer, cTnI and NGAL at the same time, so that the time for patients to see a doctor can be saved, and the rapid shunting of emergency treatment can be realized.
Example 3
Linear detection
The B-type natriuretic peptide (BNP) is linear in the range of 50pg/mL to 4000pg/mL, and r is more than or equal to 0.95. Troponin I (cTnI) is linear within the range of 0.05 ng/mL-30 ng/mL, and r is more than or equal to 0.95. The D-Dimer (D-Dimer) is linear in the range of 100 ng/mL-5000 ng/mL, and r is more than or equal to 0.95. The Neutrophil Gelatinase Associated Lipocalin (NGAL) is linear in the range of 60 ng/mL-1300 ng/mL, and r is more than or equal to 0.95. Taking a standard solution, diluting the standard solution with calf serum to obtain a BNP concentration of 4000pg/mL, 2500pg/mL, 1000pg/mL, 100pg/mL, 50pg/mL, a cTnI concentration of 30ng/mL, 20ng/mL, 10ng/mL, 0.4ng/mL, 0.05ng/mL, a D-Dimer concentration of 5000ng/mL, 3000ng/mL, 1000ng/mL, 600ng/mL, 100ng/mL, an NGAL concentration of 1300ng/mL, 600ng/mL, 300ng/mL, 150ng/mL, 60ng/mL, and repeating the test sample measuring program for 5 concentrations of 3 times to obtain an average value (yi). The linear correlation coefficient r is calculated according to the formula (1) with the sample concentration (xi) as an independent variable and the measured mean value (yi) as a dependent variable.
In the formula: r is linear correlation coefficient
x: concentration of sample
Mean value of sample concentration
y: all measurements areValue of
Average of the mean values
The results of the linear measurements are shown in Table 2:
TABLE 2 Linear determination results of combined detection kit for heart, lung and kidney (fluoroimmunoassay)
The data in table 2 show that the linear measurement results of BNP, cTnI, D-Dimer and NGAL of the heart, lung and kidney combined test kit (fluorescence immunoassay) are all above 0.995, which are far higher than the basic requirements of the industry standard and can completely meet the design requirements.
Secondly, detecting the accuracy
The samples with high (BNP: 1000pg/mL, cTnI: 20ng/mL, D-Dimer: 2000ng/mL, NGAL: 1300ng/mL) and low (BNP: 100pg/mL, cTnI: 0.4ng/mL, D-Dimer: 200ng/mL, NGAL: 150ng/mL) concentrations were measured using the same lot of test paper cards, and the samples were separately tested on the same fluorescence immunoassay analyzer, and the measurement was repeated 3 times to obtain the average
The relative deviation (B%) is calculated according to equation (2) and should not be greater than 20%.
In the formula: b%: relative deviation of
T: nominal value
The accuracy test results are shown in table 3:
TABLE 3 accuracy determination results of combined detection kit for heart, lung and kidney (fluoroimmunoassay)
The data in table 3 show that the relative deviation measurement results of BNP, cTnI, D-Dimer and NGAL of the heart, lung and kidney combined test kit (fluorescence immunoassay) are all below 10%, which are far higher than the basic requirements of the industry standard and can completely meet the design requirements.
Third, detecting the internal precision of the batch
Taking quality control substances with high concentration (BNP: 1000pg/mL, cTnI: 20ng/mL, D-Dimer: 2000ng/mL, NGAL: 1300ng/mL) and low concentration (BNP: 100pg/mL, cTnI: 0.4ng/mL, D-Dimer: 200ng/mL, NGAL: 150ng/mL) as samples, repeatedly measuring each level of quality control substance for 10 times by using a same batch of test paper cards, respectively calculating the average value of the measuring results
And Standard Deviation (SD), calculating relative standard deviation RSD (or coefficient of variation CV) according to formula (3), wherein the relative standard deviation RSD (or coefficient of variation CV) is less than or equal to 15%.
In the formula: RSD: relative Standard Deviation (SD)
SD: standard deviation of
The results of the in-batch precision measurements are shown in Table 4:
TABLE 4 measurement results of precision in batch of four-item combined detection kit (fluorescence immunoassay) for heart, lung and kidney
The data in table 4 show that the relative standard deviation measurement results of BNP, cTnI, D-Dimer and NGAL of the heart, lung and kidney combined test kit (fluorescence immunoassay) are all below 10%, which are far higher than the basic requirements of the industry standard and can completely meet the design requirements.
Four, detection of precision between batches
Using three kits with different batch numbers, taking 3 test paper cards for each batch number, taking 1000pg/mL of B-type natriuretic peptide (BNP), 15ng/mL of troponin (cTnI), 2000ng/mL of D-Dimer (D-Dimer) and 150ng/mL of neutrophil gelatinase-associated lipocalin (NGAL) for testing, and respectively calculating the average value of the detection results of the 3 batch number test paper cards
And calculating the total average value of the detection results of all three batches
Calculating the relative range R between batches according to the formula (4), wherein the relative range R between batches is not more than 20%.
In the formula:
maximum value of
The results of the batch-to-batch precision measurements are shown in Table 5:
TABLE 5 measurement results of precision between batches of four-item combined detection kit (fluorescence immunoassay) for heart, lung and kidney
The data in Table 5 show that the relative range difference measurement results among the BNP, cTnI, D-Dimer and NGAL of the heart, lung and kidney combined test kit (fluorescence immunoassay) are all below 10 percent, and the design requirements can be completely met.
Fifth, detection of the lowest detection Limit
Taking zero value plasma as a blank matrix sample, repeatedly testing for 20 times by using a test paper card with the same batch number, and calculating a measurement mean value
And Standard Deviation (SD), and the lowest detection limit (LoD) is calculated according to equation (5). The minimum detection limit of B-type natriuretic peptide (BNP) is not more than 50 pg/mL. The minimum detection limit of troponin (cTnI) is not more than 0.05 ng/mL. The lowest detection limit of the D-Dimer (D-Dimer) is not more than 100 ng/mL. The minimum detection limit of neutrophil gelatinase-associated lipocalin (NGAL) is not more than 60 ng/mL.
In the formula
Mean value of measurement
SD: standard deviation of
The results of the lowest detection limit are shown in Table 6:
TABLE 6 determination of the lowest detection limit of the combined detection kit (fluorescence immunoassay) for heart, lung and kidney
The data in table 6 show that the measurement results of the lowest detection limits of BNP, cTnI, D-Dimer and NGAL of the heart, lung and kidney combined test kit (fluorescence immunoassay) are far lower than the industrial standard, and can completely meet the design requirements.
Sixth, interference immunity detection
Taking quality control products of B-type natriuretic peptide (BNP)3000pg/mL, troponin (cTnI)15ng/mL, D-Dimer (D-Dimer)4000ng/mL and neutrophil gelatinase-associated lipocalin (NGAL)1300ng/mL, respectively adding the interferents so that the concentration of hemoglobin is 10g/L, the concentration of cholesterol is 100mg/mL, the concentration of triglyceride is 1000mg/mL, the concentration of bilirubin is 2mg/mL, the concentration of fibrinogen is 1mg/mL, testing each for 3 times, and respectively comparing with the quality control products without the interferents, wherein the relative deviation is not more than 5%.
The results of the anti-interference measurements are shown in table 7:
TABLE 7 anti-interference detection results of heart, lung and kidney combined detection kit (fluorescence immunoassay)
The data in Table 7 show that the anti-interference measurement results of BNP, cTnI, D-Dimer and NGAL of the heart, lung and kidney combined test kit (fluorescence immunoassay) can meet the design requirements.
Seventhly, stability detection
The long-term stability test result of the kit shows that the product can be stably stored for at least one year when being refrigerated at the temperature of 2-8 ℃. The evaluation of the analysis performance is carried out every 6 months, and the test results of the product at 6 months and 12 months are in accordance with the design requirements of the product through a plurality of tests. Therefore, stability evaluation is carried out every 6 months, and according to the design requirements of the product, three indexes of linearity, accuracy, batch precision and minimum detection limit are detected, so that the stability of the product can be effectively analyzed.
The stability test results are shown in table 8:
TABLE 8 stability test results of combined detection kit for heart, lung and kidney (fluorescence immunoassay)
The data in Table 8 show that the heart, lung and kidney combined test kit (fluorescence immunoassay) can keep the stability of one year when being refrigerated at 2-8 ℃ and can completely meet the design requirements.
Eighthly, comparing detection results with other types of kit
Selecting quality control products with high concentration (BNP: 1000pg/mL, cTnI: 20ng/mL, D-Dimer: 2000ng/mL, NGAL: 1300ng/mL) and low concentration (BNP: 100pg/mL, cTnI: 0.4ng/mL, D-Dimer: 200ng/mL, NGAL: 150ng/mL) as samples, randomly selecting a heart, kidney and lung combined test kit (fluorescence immunoassay), selecting a B-type natriuretic peptide test kit (fluorescence immunoassay) marketed by Hebei Tentt Biotech development Co., Ltd, a troponin I test kit (fluorescence immunoassay), and a D-Dimer test kit (fluorescence immunoassay)Epidemic method), neutrophilic granulocyte gelatinase-associated lipocalin detection kit (fluorescence immunoassay method) as control for detection, repeating the determination for 10 times for each level quality control, and calculating average value of determination results
And Standard Deviation (SD), calculating relative standard deviation RSD (or coefficient of variation CV) according to formula (3), and comparing the differences of detection results of different kits.
The results are shown in tables 9-1 and 9-2:
TABLE 9-1 detection results of the four-item Combined detection kit (fluorescence immunoassay) for heart, kidney, and lung
TABLE 9-2 test results of other types of kits
The data comparison results in tables 9-1 and 9-2 show that the precision of the heart-kidney-lung four-item joint inspection kit (fluorescence immunoassay) and other single kits are all in accordance with requirements, the consistency is good, the four biomarkers of BNP, cTnI, D-Dimer and NGAL can be detected simultaneously by one-time sample adding, and the purposes of clinical rapid identification and diagnosis of heart-kidney-lung organ diseases and prognosis evaluation thereof are facilitated.
Comparative example 1
And adopting a fluorescence immune time resolution technology to selectively use the Eu-bidirectional chelate and the Sm-bidirectional chelate labeled monoclonal antibody according to the excitation wavelength.
The marking method and the serial number are shown in table 10:
TABLE 10 test numbers and labeling methods
| Test number | BNP | cTnI | D-Dimer | NGAL |
| TY-1 | Eu-two-way chelate | Eu-two-way chelate | Eu-two-way chelate | Eu-two-way chelate |
| TY-2 | Sm-bidirectional chelate | Sm-bidirectional chelate | Sm-bidirectional chelate | Sm-bidirectional chelate |
| TY-3 | Eu-two-way chelate | Eu-two-way chelate | Sm-bidirectional chelate | Sm-bidirectional chelate |
| TY-4 | Sm-bidirectional chelate | Sm-bidirectional chelate | Eu-two-way chelate | Eu-two-way chelate |
| TY-5 | Eu-two-way chelate | Sm-bidirectional chelate | Eu-two-way chelate | Sm-bidirectional chelate |
| TY-6 | Sm-bidirectional chelate | Eu-two-way chelate | Sm-bidirectional chelate | Eu-two-way chelate |
According to the result of the previous data inquiry, the antibody with specific working concentration is prepared, and the concentration is 0.8 mg/mL.
And (3) scribing membranes, namely sequentially scribing mouse anti-human monoclonal antibodies of BNP, D-Dimer, cTnI and NGAL and goat anti-mouse polyclonal antibodies at the same intervals at different positions of the NC membrane by using different scribing heads of a membrane scribing instrument, and spraying and scribing the prepared mouse anti-human fluorescent monoclonal antibodies of BNP, D-Dimer, cTnI and NGAL on the hemofiltration membrane by using different scribing heads of the membrane scribing instrument, wherein the positions of the sample adding holes are carefully avoided.
And cutting and assembling to form a test board, and detecting by using a dual-wavelength time-resolved fluorescence immunoassay analyzer.
The test was carried out according to the formula (2) and the formula (3) for accuracy and in-batch precision, and the test results are shown in Table 11:
TABLE 11 test results
The comparison test result shows that both the TY-5 condition and the TY-6 condition meet the technical requirements of the products of enterprises, and the TY-5 condition is selected as the final condition.
Comparative example 2
According to the previous test data, a TY-5 marking method is selected, the marking position is further verified, and the test serial number and the marking position are shown in a table 12:
TABLE 12 verification of NC scribe line positions
And cutting and assembling to form a test board, and detecting by using a dual-wavelength time-resolved fluorescence immunoassay analyzer.
The test was carried out according to the formula (2) and the formula (3) for accuracy and in-batch precision, and the test results are shown in Table 13:
TABLE 13 test results
The comparison test result shows that the NC membrane scratching condition of TH-3, accuracy and precision effect are better by using a TY-5 fluorescent labeling method.
And (3) optimizing the membrane scratching position of the blood filtering membrane in consideration of the time-resolved fluorescence characteristic, wherein the membrane scratching position is as follows: different membrane scribing instruments are utilized to spray and scribe the configured BNP, cTnI, D-Dimer and NGAL mouse anti-human fluorescent monoclonal antibodies at specific positions on the blood filtering membrane, the positions of the sample adding holes are carefully avoided, the NGAL mouse anti-human monoclonal antibodies are scribed at a position 17.5mm away from the bottom end of the blood filtering membrane, the cTnI mouse anti-human monoclonal antibodies are scribed at a position 20mm away from the bottom end of the blood filtering membrane, the D-Dimer mouse anti-human monoclonal antibodies are scribed at a position 22.5mm away from the bottom end of the blood filtering membrane, and the BNP mouse anti-human monoclonal antibodies are scribed at a position 25mm away from the bottom end of the blood filtering membrane. The results of the optimization experiments are shown in Table 14.
TABLE 14 test results of optimization test
| BNP | D-Dimer | cTnI | NGAL | |
| Accuracy of | 6.96％ | 5.94％ | 7.41％ | 7.67％ |
| Precision degree | 4.37％ | 7.61％ | 4.91％ | 6.43％ |
Comparing the test results, the optimal fluorescence labeling method is that BNP, D-Dimer labeled Eu-solid phase two-way chelate, cTnI and NGAL labeled Sm-solid phase two-way chelate are respectively used as fluorescence monoclonal antibodies, and then the film scribing position is as follows: dividing mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL and goat anti-mouse polyclonal antibodies at specific positions of an NC membrane by using different membrane dividing heads, dividing the mouse anti-human monoclonal antibodies of BNP at a position 8mm away from the bottom end of the NC membrane, dividing the mouse anti-human monoclonal antibodies of D-Dimer at a position 15mm away from the bottom end of the NC membrane, dividing the mouse anti-human monoclonal antibodies of cTnI at a position 18mm away from the bottom end of the NC membrane, dividing the mouse anti-human monoclonal antibodies of NGAL at a position 25mm away from the bottom end of the NC membrane, and dividing the goat anti-mouse polyclonal antibodies at a position 28mm away from the bottom end of the NC membrane; different membrane scribing instruments are utilized to spray and scribe the configured BNP, cTnI, D-Dimer and NGAL mouse anti-human fluorescent monoclonal antibodies at specific positions on the blood filtering membrane, the positions of the sample adding holes are carefully avoided, the NGAL mouse anti-human monoclonal antibodies are scribed at a position 17.5mm away from the bottom end of the blood filtering membrane, the cTnI mouse anti-human monoclonal antibodies are scribed at a position 20mm away from the bottom end of the blood filtering membrane, the D-Dimer mouse anti-human monoclonal antibodies are scribed at a position 22.5mm away from the bottom end of the blood filtering membrane, and the BNP mouse anti-human monoclonal antibodies are scribed at a position 25mm away from the bottom end of the blood filtering membrane.
Comparative example 3
Validation of working concentration of antibody
According to the previous data, the working concentration of the antibody is determined to be 0.8mg/mL, the antibody marking method and the membrane scribing position are determined in the test, in order to ensure the optimal performance of the test condition, the accuracy and the precision of the test plate under different working concentrations of the antibody are verified, the accuracy and the batch precision are tested according to the formula (2) and the formula (3), and the test sequence number, the working concentration of each antibody and the test result are as follows.
3.1 validation of working concentration of fluorescent BNP mab, each antibody working concentration is as in table 15:
TABLE 15 working concentration (unit: mg/mL) of antibody and test number
The test results are given in table 16 below:
TABLE 16 test results
As can be seen from the comparison of the test results, the optimal working concentration of the fluorescent BNP monoclonal antibody is 0.8 mg/mL.
3.2 validation of working concentrations of fluorescent D-Dimer monoclonal antibody, each working concentration of antibody is as in Table 17:
TABLE 17 working concentration (unit: mg/mL) of antibody and test number
The test results are given in table 18 below:
TABLE 18 test results
As can be seen from the comparison of the test results, the optimal working concentration of the fluorescent D-Dimer monoclonal antibody is 0.8 mg/mL.
3.3 validation of working concentration of fluorescent cTnI monoclonal antibody, the working concentration of each antibody is shown in Table 19:
TABLE 19 working concentration (unit: mg/mL) and test number of antibody
The test results are shown in table 20 below:
TABLE 20 test results
As can be seen from the comparison of the test results, the optimal working concentration of the fluorescent cTnI monoclonal antibody is 0.8 mg/mL.
3.4 validation of working concentrations of fluorescent NGAL mab, each antibody working concentration is as in table 21:
TABLE 21 working concentration (unit: mg/mL) and test number of antibody
The test results are shown in table 22 below:
TABLE 22 test results
As can be seen from the comparison of the test results, the optimal working concentration of the fluorescent NGAL monoclonal antibody is 0.8 mg/mL.
3.5 validation of the working concentration of BNP monoclonal antibody, each working concentration of antibody is as shown in Table 23:
TABLE 23 working concentration (unit: mg/mL) and test number of antibody
The test results are given in table 24 below:
TABLE 24 test results
As can be seen from the comparison of the test results, the optimal working concentration of the BNP monoclonal antibody is 0.8 mg/mL.
3.6 validation of working concentrations of D-Dimer monoclonal antibody, each working concentration of antibody is as in Table 25:
TABLE 25 working concentration (unit: mg/mL) and test number of antibody
The test results are shown in table 26 below:
TABLE 26 test results
As can be seen from the comparison of the test results, the optimal working concentration of the D-Dimer monoclonal antibody is 0.8 mg/mL.
3.7 validation of working concentrations of cTnI monoclonal antibody, each working concentration of antibody is as in Table 27:
TABLE 27 working concentration (unit: mg/mL) of antibody and test number
The test results are given in table 28 below:
TABLE 28 test results
As can be seen from the comparison of the test results, the optimal working concentration of the cTnI monoclonal antibody is 0.8 mg/mL.
3.8 validation of working concentrations of NGAL mab, each working concentration of antibody is as in table 29:
TABLE 29 working concentration (unit: mg/mL) and test number of antibody
The test results are shown in table 30 below:
TABLE 30 test results
As can be seen from the comparison of the test results, the optimum working concentration of NGAL monoclonal antibody is 0.8 mg/mL.
3.9 validation of working concentrations of polyclonal antibodies, each working concentration of antibody is as in Table 31:
TABLE 31 working concentration (unit: mg/mL) of antibody and test number
The test results are shown in table 32 below:
TABLE 32 test results
As can be seen from comparison of the test results, the working concentration of polyclonal antibody is optimally 0.8 mg/mL.
In summary, the optimal fluorescence labeling method is to use BNP, D-Dimer labeled Eu-solid phase two-way chelate, cTnI, NGAL labeled Sm-solid phase two-way chelate as fluorescence monoclonal antibody, and then the film-scribing position is as follows: dividing mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL and goat anti-mouse polyclonal antibodies at specific positions of an NC membrane by using different membrane dividing heads, dividing the mouse anti-human monoclonal antibodies of BNP at a position 8mm away from the bottom end of the NC membrane, dividing the mouse anti-human monoclonal antibodies of D-Dimer at a position 15mm away from the bottom end of the NC membrane, dividing the mouse anti-human monoclonal antibodies of cTnI at a position 18mm away from the bottom end of the NC membrane, dividing the mouse anti-human monoclonal antibodies of NGAL at a position 25mm away from the bottom end of the NC membrane, and dividing the goat anti-mouse polyclonal antibodies at a position 28mm away from the bottom end of the NC membrane; and spraying and scratching the configured mouse anti-human fluorescent monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL at a specific position on the blood filtering membrane by using different scratching heads of a film scratching instrument, paying attention to avoid the position of a sample adding hole, scratching the mouse anti-human monoclonal antibody of NGAL at a position 17.5mm away from the bottom end of the blood filtering membrane, scratching the mouse anti-human monoclonal antibody of cTnI at a position 20mm away from the bottom end of the blood filtering membrane, scratching the mouse anti-human monoclonal antibody of D-Dimer at a position 22.5mm away from the bottom end of the blood filtering membrane, and scratching the mouse anti-human monoclonal antibody of BNP at a position 25mm away from the bottom end of the blood filtering membrane. The optimal working concentration before all antibody puffs was 0.8 mg/mL.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A heart, lung and kidney combined examination test paper card is characterized in that the test paper card is sequentially provided with a sample pad, a nitrocellulose membrane and a water absorption pad;
the sample pad is a blood filtering membrane; fluorescent marked mouse-anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL are respectively sprayed on different positions of the sample pad;
the nitrocellulose membrane is respectively sprayed and scribed with a detection line and a quality control line, different positions of the detection line are respectively sprayed and scribed with mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL, and the quality control line is sprayed and scribed with polyclonal antibodies;
the fluorescence labeling condition of the fluorescence labeled mouse anti-human monoclonal antibody is as follows: the mouse anti-human monoclonal antibody of BNP and the mouse anti-human monoclonal antibody of cTnI are respectively marked with Eu-solid phase two-way chelate, and the mouse anti-human monoclonal antibody of D-Dimer and the mouse anti-human monoclonal antibody of NGAL are respectively marked with Sm-solid phase two-way chelate.
2. The test paper card of claim 1, wherein the polyclonal antibody is capable of binding to fluorescently labeled murine anti-human monoclonal antibodies to BNP, cTnI, D-Dimer, and NGAL.
3. The test paper card of claim 1, wherein the sample pad is sprayed with fluorescently labeled murine anti-human monoclonal antibodies to NGAL, cTnI, D-Dimer and BNP at positions 17.5mm, 20mm, 22.5mm and 25mm from the bottom end of the sample pad, respectively.
4. The test paper card according to claim 1, wherein BNP, D-Dimer, cTnI and NGAL murine anti-human monoclonal antibody are sprayed on the nitrocellulose membrane at positions 8mm, 15mm, 18mm and 25mm from the bottom end of the nitrocellulose membrane, respectively; the polyclonal antibody is 28mm away from the bottom end of the nitrocellulose membrane.
5. The test paper card of claim 1, wherein the blood filtering membrane is made of glass fiber membrane.
6. The test card of claim 5, further comprising a housing.
7. A method of making a test card as claimed in any one of claims 1 to 6, comprising the steps of:
respectively labeling a mouse anti-human monoclonal antibody of BNP and a mouse anti-human monoclonal antibody of cTnI with Eu-solid phase two-way chelates, and labeling a mouse anti-human monoclonal antibody of D-Dimer and a mouse anti-human monoclonal antibody of NGAL with Sm-solid phase two-way chelates to obtain fluorescence labeled mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL;
respectively spraying mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL on a nitrocellulose membrane;
spraying and scratching the polyclonal antibody on a nitrocellulose membrane;
spraying fluorescence-labeled mouse anti-human monoclonal antibodies of BNP, cTnI, D-Dimer and NGAL on the sample pad;
and assembling the sample pad, the nitrocellulose membrane, the absorbent paper and the upper and lower shells to obtain the test paper card.
8. The method according to claim 7, wherein the nitrocellulose membrane has a width of 5 ± 1mm, the sample pad has a width of 7 ± 1mm, and the absorbent pad has a width of 10 ± 1 mm.
9. The method of claim 7, wherein the working concentrations of the fluorescently labeled murine anti-human monoclonal antibody, polyclonal antibody, and murine anti-human monoclonal antibody are 0.8mg/mL, respectively, prior to the spraying.
10. A combined test kit for heart, lung and kidney, which comprises the test paper card of any one of claims 1 to 6, an RFID card containing a standard curve, a thermal printing paper roll, a 200 μ L bag of straws and instructions.
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