CN210514336U

CN210514336U - Combined quantitative detection kit for five cardiac markers

Info

Publication number: CN210514336U
Application number: CN201920575973.6U
Authority: CN
Inventors: 刘冰; 章燕; 周伟; 李亚楠; 刘凤鸣
Original assignee: Changzhou Bowendi Pharmaceutical Co ltd
Current assignee: Changzhou Bowendi Pharmaceutical Co ltd
Priority date: 2019-04-25
Filing date: 2019-04-25
Publication date: 2020-05-12
Anticipated expiration: 2029-04-25

Abstract

The utility model discloses a joint quantitative determination five heart marker kits, including drier, sealing bag and detection card. The detection card comprises a quintuplet buckle card and a reagent strip; the reagent strip comprises a sample pad, a combination pad, a blood filtering membrane, a nitrocellulose membrane, absorbent paper and a PVC bottom plate, wherein the combination pad is coated with an antibody of hypersensitive C-reactive protein (Hs-CRP), an antibody of Myeloperoxidase (MPO), an antibody of lipoprotein phospholipase A2(Lp-PLA2), an antibody of oxidized low-density lipoprotein (ox-LDL) and an antibody of F2-isoprostane (F2-iso); the antibody is one or more pairing combination of monoclonal antibody, polyclonal antibody, antibody fragment or chimeric antibody. The utility model discloses the method has specific, sensitive, the productivity is high, quick, simple and convenient, good reproducibility, easy automation, false positive have reached the minimizing, detection efficiency is high, detect outstanding advantage such as with low costs, have important practical meaning.

Description

Combined quantitative detection kit for five cardiac markers

Technical Field

The utility model relates to a clinical medicine field of examining, specifically speaking relates to a combined quantitative determination hypersensitivity C reaction protein, myeloperoxidase lipoprotein phospholipase A2, oxidation low density lipoprotein and F2-isoprostaglandin fluorescence immunochromatography detect reagent box.

Background

Cardiovascular diseases are clinically common critical diseases, high fatality rate and serious illness, and seriously threaten the health and life of human beings. There are data indicating that the basic pathological change of cardiovascular disease is atherosclerosis, and recent studies show that inflammatory responses underlie atherosclerosis and cardiovascular disease, and chronic inflammation in particular is thought to increase the risk of arterial injury, predispose atherosclerotic plaque to rupture, and play a key role in the formation of atherosclerosis. Arterial injury is a chronic inflammatory response of the cardiovascular system throughout the body. However, due to the lack of obvious clinical manifestations in the early stage of arterial injury, the diagnosis is often in the middle and late stages of the disease, resulting in poor prognosis and excessive medical costs for the patient. Therefore, the method has important clinical significance for searching the cardiovascular and cerebrovascular markers for effectively monitoring early arterial lesions and predicting the risk of serious cardiovascular and cerebrovascular diseases.

Hs-CRP is a sensitive marker of inflammatory lesions and can accurately detect low-concentration C-reactive protein. Clinical tests show that the serum Hs-CRP level is in a progressive rising trend along with the coronary disease. Hs-CRP is a sensitive and accurate index reflecting inflammatory process, the increase of Hs-CRP can be caused by slight inflammation in vivo, and epidemiological studies show that the increased Hs-CRP can indicate the CHD generation within 10 years. Lp-PLA2 is a calcium ion independent secreted protein with a molecular weight of 45 KDa. Lp-PLA2 is produced by macrophages, is a marker of vascular endothelial inflammation, and is not affected by other inflammatory diseases. Can specifically detect the occurrence of early vascular inflammation. Multiple clinical trial studies have shown that higher levels of basal Lp-PLA2 are followed by a significant increase in the proportion of cardiovascular events compared to lower levels of basal Lp-PLA 2. MPO is a neutral lysosomal enzyme produced by activated neutrophils, monocytes and macrophages and involved in a variety of inflammatory reactions and oxidative stress processes in humans. An increase in MPO suggests that the patient may have coronary inflammation, but the blood vessels are not completely occluded and do not cause myocardial necrosis, and can be used to predict the likelihood of an early adverse cardiovascular event. The ox-LDL level in the plasma of coronary patients was 2 times higher than that of normal controls. The level of F2-iso in blood is closely related to the extent and severity of the coronary heart disease. The utility model relates to a joint quantitative determination Hs-CRP, MPO, Lp-PLA2, ox-LDL and F2-iso fluorescence immunochromatography detect reagent box combine above five, through joint detection patient's blood Hs-CRP, MPO, Lp-PLA2, ox-LDL and F2-iso level, can more accurately effectively predict the possibility of patient's serious cardiovascular and cerebrovascular disease risk, realize serious cardiovascular event early diagnosis, risk prediction and severity aassessment.

The Chinese invention patent CN201810746660.2 discloses a test paper for combined quantitative detection of inflammation markers serum amyloid A, hypersensitive C reactive protein and procalcitonin, which only relates to inflammation markers, is generally detected after verification, and risk assessment cannot be realized. At present, a single-index detection product for clinical application can only detect one index at the same time, five indexes can be completed by five detection reagents, and the product has the defects of low detection efficiency, high cost, waste of consumables and the like.

In recent years, cardiovascular events have become the first cause of death in our country, so early identification and diagnosis and risk classification of cardiovascular events have important clinical significance. Therefore, it is necessary to develop a detection kit for early diagnosis and risk classification of cardiovascular and cerebrovascular diseases with high accuracy. The utility model discloses jointly use cardiovascular marker Hs-CRP, MPO, Lp-PLA2, ox-LDL and F2-iso's fluorescence immunochromatography to predict and diagnose cardiovascular disease, compare with traditional index, Hs-CRP, MPO, Lp-PLA2, ox-LDL and F2-iso's joint application is higher in the aspect of serious cardiovascular event early diagnosis and risk prediction accuracy, and the advantage is obvious, can reduce 30% mortality in the prediction. And the utility model discloses a fluorescence immunochromatography has peculiar, sensitive, productivity height, quick, simple and convenient, good reproducibility, easy automation, positive detectable rate reach more than 90%, false positive has reached the minimizing, detection efficiency is high, detect outstanding advantage such as with low costs, have important practical meaning.

Disclosure of Invention

The utility model aims to solve the defects of the prior art and provide a combined quantitative detection Hs-CRP, MPO, Lp-PLA2, ox-LDL and F2-iso fluorescent immunochromatography detection kit with high sensitivity, strong accuracy and simple and convenient operation.

The utility model discloses the purpose accessible adopts following technical scheme to realize:

the fluorescent immunochromatography detection kit for combined quantitative detection of the hypersensitive C-reactive protein, the myeloperoxidase, the lipoprotein phospholipase A2, the oxidized low-density lipoprotein and the F2-isoprostane is characterized by comprising a detection card, a drying agent and a sealing bag, wherein the detection card comprises a buckle card and a reagent strip; the reagent strip comprises a sample pad, a combination pad, a nitrocellulose membrane, absorbent paper and a PVC bottom plate, wherein the combination pad is respectively coated with an Hs-CRP antibody, an MPO antibody, an Lp-PLA2 antibody, an ox-LDL antibody and an F2-iso antibody; the cellulose nitrate membrane is coated and fixed with anti-human Hs-CRP, MPO, Lp-PLA2, ox-LDL and F2-iso capture antibodies which have the pairing and combination characteristics with the detection antibodies on human Hs-CRP, MPO, Lp-PLA2, ox-LDL and F2-iso.

The detection cards are independent detection cards, the detection cards of the five detection items are arranged in parallel, and the arrangement sequence of the detection cards is not limited.

The kit is cuboid.

The utility model discloses positive effect that has:

(1) oxidized low density lipoprotein is a new-generation cardiac marker, once the heart is damaged, the oxidized low density lipoprotein is immediately shown and detected, the purpose of early diagnosis is achieved, and valuable treatment time is won for patients.

(2) The utility model discloses a fluorescence immunochromatography detection method preparation kit has solved traditional colloidal gold method, immunoturbidimetry, ELISA and has had the judgement degree of accuracy not high, the false positive that easily appears, detect long consuming time, the problem of operation complicacy. The project method has the outstanding advantages of specificity, sensitivity, rapidness, accuracy, good repeatability, easy automation, minimized false positive, high detection efficiency, low detection cost, simple and convenient operation and the like.

Drawings

Fig. 1 is a schematic diagram of the structure of the reagent strip of the present invention.

Fig. 2 is a schematic view of a five-link buckle.

Detailed Description

The following examples are further illustrative of the present invention with reference to the accompanying drawings, but the present invention is not limited to the following description.

As shown in fig. 1 and fig. 2, the detection kit of the present invention, in addition to the drying agent and the sealing bag, has a main structure of a detection card comprising a quintuplet card 7 and a reagent strip 1, wherein the reagent strip 1 comprises a sample pad 5, a binding pad 3, a nitrocellulose membrane 2, absorbent paper 4 and a PVC base plate 6; quintuplet detains card 7, detains the card and all includes application of sample hole 8 and observation window 9, and reagent strip 1 settles in quintuplet detains card 7.

Example (b):

(1) sample pad

And (2) dissolving BSA and NaCl by using a pH8.0 sodium bicarbonate buffer solution until the final concentration of the BSA is 2% and the final concentration of the NaCl is 0.1M, then adding a surfactant Tween20 until the final concentration is 0.5%, adjusting the pH to 8.0, uniformly spreading the buffer solution on a glass cellulose membrane according to the water absorption capacity of the glass cellulose membrane of 60uL/cm2, and placing the glass cellulose membrane at 37 ℃ for drying for 8 hours to obtain the sample pad. Storing at 4 deg.C for use.

(2) Combined pad

And (2) uniformly paving the monoclonal antibody marked by the fluorescent microspheres on the sample pad prepared in the step (1), carrying out vacuum freeze drying, sealing, and storing at room temperature for later use. The preparation process of the monoclonal antibody marked by the fluorescent microspheres comprises the following steps:

a. fluorescent microsphere labeled Hs-CRP monoclonal antibody

Taking 0.5mL of fluorescent microsphere solution with the particle size of 300nm, washing with 2mL of 50mM MES buffer solution with pH6.0 to remove a surfactant in the microsphere solution, then dissolving the microspheres to 2mL by using 50mM MES buffer solution with pH6.0, then activating with 500uL10mg/mL activator EDC and 500uL 50mg/mL stabilizer NHS, then washing with 50mM MES with pH6.0 to remove the rest activator, re-dissolving the microspheres by using 10mM PBS, mixing the activated fluorescent microspheres with Hs-CRP monoclonal antibody, wherein the final concentration of the antibody is 120ug/mL, the final volume is 2mL, fully stirring and reacting for 2h at room temperature, centrifuging to remove the supernatant, then carrying out a blocking reaction with 2mL of 1% casein solution for 1 hour for constant volume, then washing with the buffer solution, and dissolving to 5mL to obtain the Hs-CRP monoclonal antibody marked by the fluorescent microspheres.

b. Fluorescent microsphere marked MPO monoclonal antibody

Taking 0.5mL of fluorescent microsphere solution with the particle size of 300nm, washing with 2mL of 50mM MES buffer solution with pH6.0 to remove a surfactant in the microsphere solution, then dissolving the microspheres to 2mL by using 50mM MES buffer solution with pH6.0, then activating with 500uL10mg/mL activator EDC and 500uL 50mg/mL stabilizer NHS, then washing with 50mM MES with pH6.0 to remove the rest activator, re-dissolving the microspheres by using 10mM PBS, mixing the activated fluorescent microspheres with MPO monoclonal antibody, wherein the final concentration of the antibody is 120ug/mL, the final volume is 2mL, fully stirring and reacting for 2h at room temperature, centrifuging to remove the supernatant, then carrying out blocking reaction with 2mL of 1% casein solution for 1 hour, then washing with buffer solution, and dissolving to a constant volume of 5mL to obtain the MPO monoclonal antibody marked by the fluorescent microspheres.

c. Lp-PLA2 monoclonal antibody marked by fluorescent microsphere

Taking 0.5mL of fluorescent microsphere solution with the particle size of 300nm, washing with 2mL of 50mM MES buffer solution with pH6.0 to remove a surfactant in the microsphere solution, then dissolving the microspheres to 2mL by using 50mM MES buffer solution with pH6.0, then activating with 500uL10mg/mL activator EDC and 500uL 50mg/mL stabilizer NHS, then washing with 50mM MES with pH6.0 to remove the rest activator, re-dissolving the microspheres by using 10mM PBS, mixing the activated fluorescent microspheres with Lp-PLA2 monoclonal antibody, wherein the final concentration of the antibody is 120ug/mL, the final volume is 2mL, fully stirring and reacting for 2h at room temperature, centrifuging to remove supernatant, then carrying out a blocking reaction with 2mL of 1% casein solution for 1 hour, then washing with the buffer solution, and dissolving to 5mL to obtain the Lp-PLA2 monoclonal antibody marked by the fluorescent microspheres.

d. Fluorescent microsphere labeled ox-LDL monoclonal antibody

Taking 0.5mL of fluorescent microsphere solution with the particle size of 300nm, washing with 2mL of 50mM MES buffer solution with pH6.0 to remove a surfactant in the microsphere solution, then dissolving the microspheres to 2mL by using 50mM MES buffer solution with pH6.0, then activating with 500uL10mg/mL activator EDC and 500uL 50mg/mL stabilizer NHS, then washing with 50mM MES with pH6.0 to remove the rest activator, re-dissolving the microspheres by using 10mM PBS, mixing the activated fluorescent microspheres with an ox-LDL monoclonal antibody, wherein the final concentration of the antibody is 120ug/mL, the final volume is 2mL, fully stirring and reacting for 2h at room temperature, centrifuging to remove the supernatant, then carrying out a blocking reaction with 2mL of 1% casein solution for 1 hour for constant volume, then washing with the buffer solution, and dissolving to 5mL to obtain the ox-LDL monoclonal antibody marked by the fluorescent microsphere.

e. F2-iso monoclonal antibody marked by fluorescent microsphere

Taking 0.5mL of fluorescent microsphere solution with the particle size of 300nm, washing with 2mL of 50mM MES buffer solution with pH6.0 to remove a surfactant in the microsphere solution, then dissolving the microspheres to 2mL by using 50mM MES buffer solution with pH6.0, then activating with 500uL10mg/mL activator EDC and 500uL 50mg/mL stabilizer NHS, then washing with 50mM MES with pH6.0 to remove the rest activator, re-dissolving the microspheres by using 10mM PBS, mixing the activated fluorescent microspheres with F32-iso monoclonal antibody, wherein the final concentration of the antibody is 120ug/mL, the final volume is 2mL, fully stirring and reacting for 2h at room temperature, centrifuging to remove supernatant, then carrying out a blocking reaction with 2mL of 1% casein solution for 1 h, washing with the buffer solution to fix the volume for 1 mL, and dissolving 2-iso monoclonal antibody to obtain the F2-iso monoclonal antibody marked by the fluorescent microsphere.

(3) A blood filtering membrane: the hemofiltration membrane was cut into pieces of 10cm by 4 mm.

(4) Nitrocellulose membrane (NC membrane) coated with detection line and quality control line

a. Coating with Hs-CRP nitrocellulose membrane: diluting a goat anti-mouse IgG antibody to a concentration of 1.0mg/mL by using 50mM/L PB buffer solution with pH7.2, scribing at a scribing rate of 0.8ul/cm at a C line mark position at the upper end of an NC membrane to obtain a quality control line, diluting a goat anti-human Hs-CRP polyclonal antibody to a concentration of 1.0mg/mL by using a dilution coating solution, scribing at a scribing rate of 0.8ul/cm at a T line mark position on the NC membrane to obtain a detection line, wherein the interval between the detection line and the quality control line is 5mM, and drying at 37 ℃ for 8 hours after scribing to obtain the NC membrane coated with the detection line and the quality control line.

b. Coating MPO nitrocellulose membrane: diluting a goat anti-mouse IgG antibody to a concentration of 1.0mg/mL by using 50mM/L PB buffer solution with pH7.2, scribing a scribing rate of 0.8ul/cm at a C line marking position at the upper end of an NC membrane to obtain a quality control line, diluting a goat anti-human MPO polyclonal antibody to a concentration of 1.0mg/mL by using a dilution coating solution, scribing a T line marking position on the NC membrane at a scribing rate of 0.8ul/cm to obtain a detection line, wherein the space between the detection line and the quality control line is 5mM, and drying at 37 ℃ for 8 hours after scribing is finished to obtain the NC membrane coated with the detection line and the quality control line.

c. Cellulose nitrate membrane coated with Lp-PLA 2: diluting a goat anti-mouse IgG antibody to a concentration of 1.0mg/mL by using 50mM/L PB buffer solution with pH7.2, scribing at a scribing rate of 0.8ul/cm at a C line mark position at the upper end of an NC membrane to obtain a quality control line, diluting a goat anti-human Lp-PLA2 polyclonal antibody to a concentration of 1.0mg/mL by using a dilution coating solution, scribing at a scribing rate of 0.8ul/cm at a T line mark position on the NC membrane to obtain a detection line, wherein the interval between the detection line and the quality control line is 5mM, and drying at 37 ℃ for 8 hours after scribing is finished to obtain the NC membrane coated with the detection line and the quality control line.

d. ox-LDL nitrocellulose coating: diluting a goat anti-mouse IgG antibody to a concentration of 1.0mg/mL by using 50mM/L PB buffer solution with pH7.2, drawing a line at the marked position of a C line at the upper end of an NC membrane at a drawing rate of 0.8ul/cm to obtain a quality control line, diluting a goat anti-human ox-LDL polyclonal antibody to a concentration of 1.0mg/mL by using a dilution coating solution, drawing a line at the marked position of a T line on the NC membrane at a drawing rate of 0.8ul/cm to obtain a detection line, wherein the interval between the detection line and the quality control line is 5mM, and drying at 37 ℃ for 8 hours after the drawing is finished to obtain the NC membrane coated with the detection line and the quality control line.

e. Coating F2-iso nitrocellulose membrane: diluting a goat anti-mouse IgG antibody to a concentration of 1.0mg/mL by using 50mM/L PB buffer solution with pH7.2, drawing a line at the marked position of a C line at the upper end of an NC membrane at a drawing rate of 0.8ul/cm to obtain a quality control line, diluting a goat anti-human F2-iso polyclonal antibody to a concentration of 1.0mg/mL by using a dilution coating solution, drawing a line at the marked position of a T line on the NC membrane at a drawing rate of 0.8ul/cm to obtain a detection line, wherein the interval between the detection line and the quality control line is 5mM, and drying at 37 ℃ for 8 hours after finishing drawing to obtain the NC membrane coated with the detection line and the quality control line.

(5) Water absorption paper: the absorbent paper was cut into pieces of 30cm by 2.7 cm.

(6) Assembling: and (3) sequentially sticking the sample pad obtained in the step (1), the bonding pad obtained in the step (2), the blood filtering membrane obtained in the step (3), the NC membrane obtained in the step (4) and the absorbent paper obtained in the step (5) on a PVC (polyvinyl chloride) base plate, cutting the stuck sample pad, the NC membrane and the absorbent paper into test strips with the width of 4mm by using a slitter, placing the test strips into a quintuplet buckle, and adding a drying agent for packaging to obtain the test paper.

Claims

1. The kit for combined quantitative detection of five cardiac markers is characterized by comprising a detection card, a drying agent and a sealing bag, wherein the detection card comprises a buckle card and a reagent strip; the reagent strip comprises a sample pad, a combination pad, a nitrocellulose membrane, absorbent paper and a PVC bottom plate, wherein the combination pad is coated with a hypersensitivity C-reactive protein antibody, a myeloperoxidase antibody, a lipoprotein phospholipase A2 antibody, an oxidized low-density lipoprotein antibody and a F2-isoprostane antibody; the cellulose nitrate membrane is coated and fixed with anti-human hypersensitive C-reactive protein, myeloperoxidase, lipoprotein phospholipase A2, oxidized low density lipoprotein, F2-isoprostane pairing and binding characteristics, myeloperoxidase, lipoprotein phospholipase A2, oxidized low density lipoprotein and F2-isoprostane capture antibody.

2. The kit for combined quantitative detection of five cardiac markers according to claim 1, wherein the detection cards are independent detection cards, the detection cards for five detection items are arranged in parallel, and the arrangement sequence of the detection cards is not limited.

3. The kit for combined quantitative detection of five cardiac markers according to claim 1, wherein the kit is in a rectangular parallelepiped shape.