阅读说明：本技术 一种快速检测样品中铅含量的试剂盒 (Kit for rapidly detecting lead content in sample ) 是由 周建平 王艳新 周裕军 于 2019-09-26 设计创作，主要内容包括：本发明涉及一种快速检测样品中铅含量的检测试剂盒,属于医学体外免疫检测技术领域。本发明所述试剂盒包括检测卡及质控品；所述检测卡包括底板及位于底板表面的,从加样端起顺序排列的样品垫、玻璃纤维膜、硝酸纤维素膜和吸水纸。本发明所述试剂盒具有前处理简单、无需消解、经济、快速、便捷等优势,能实现常温保存、快速、高通量、低仪器成本、操作简便、单人份随时检测,大大提高临床使用简便性。(The invention relates to a detection kit for rapidly detecting lead content in a sample, and belongs to the technical field of medical in-vitro immunoassay. The kit comprises a detection card and a quality control product; the detection card comprises a bottom plate, and a sample pad, a glass fiber membrane, a nitrocellulose membrane and absorbent paper which are arranged on the surface of the bottom plate in sequence from a sample adding end. The kit has the advantages of simple pretreatment, no need of digestion, economy, quickness, convenience and the like, can be stored at normal temperature, is quick and high in flux, is low in instrument cost, is simple and convenient to operate, can be detected by a single person at any time, and greatly improves the clinical use simplicity.)

1. A kit for rapidly detecting the content of lead in a sample comprises a detection card and a quality control product; the detection card comprises a bottom plate, and a sample pad, a glass fiber membrane, a nitrocellulose membrane and absorbent paper which are arranged on the surface of the bottom plate in sequence from a sample adding end;

the sample pad is soaked by a sample pad treatment buffer solution, and the sample pad treatment buffer solution comprises active protein and a surfactant;

the glass cellulose membrane is coated with: a conjugate of the lead specific antibody and the fluorescent microsphere and a conjugate of the chicken IgY antibody and the fluorescent microsphere;

the nitrocellulose membrane is marked with a detection limit and a quality control line, the detection line is coated with lead coupling hapten, and the quality control line is coated with goat anti-chicken IgY antibody.

2. The kit of claim 1, wherein the sample pad treatment buffer is based on one or more of triethanolamine buffer, borax borate buffer, and glycine buffer.

3. The kit of claim 1, wherein the active protein comprises one or more of fetal bovine serum, horse serum albumin, and bovine serum albumin.

4. The kit of claim 1, wherein the surfactant comprises one or more of S9, S13, and Tween 80.

5. The kit according to claim 1, wherein the mixing volume ratio of the conjugate of the lead-specific antibody and the fluorescent microsphere to the conjugate of the chicken IgY antibody and the fluorescent microsphere is (4-7): 1.

6. the kit according to claim 1 or 5, wherein the fluorescent microspheres have a particle size of 80-220 nm.

7. The kit according to claim 1 or 5, wherein the lead-specific antibody in the conjugate of the lead-specific antibody and the fluorescent microsphere is a lead-specific murine monoclonal antibody.

8. The kit according to claim 1, wherein the method for preparing the glass cellulose membrane comprises: soaking the glass cellulose membrane in a heavy suspension buffer solution for 1-2 h, and drying for 1-2 h to obtain a pretreated glass cellulose membrane; respectively dissolving the conjugate of the lead specific antibody and the fluorescent microsphere and the conjugate of the chicken IgY antibody and the fluorescent microsphere in a heavy suspension buffer solution, spraying the solution on a pretreated glass cellulose membrane, and drying; the heavy suspension buffer solution takes one or more of phosphate buffer solution, boric acid buffer solution and glycine buffer solution with the pH value of 5.5-6.8 and 50-200 mM as basic buffer solution, and also comprises 0.5-3 g/L of cane sugar and 0.1% of S9 by mass percentage.

9. The kit of claim 1, wherein the quality control substance is a lead-containing buffer solution comprising the following components: 0.5mM nitric acid buffer solution, 0.1% Tween20 by mass-volume ratio, 0.5% bovine serum albumin by mass, 0.1% Proclin300 by mass, water as solvent, and pH 4.8-5.0.

10. The kit according to claim 1 or 9, wherein the quality control product is stored after being lyophilized, and the lyophilization buffer solution for lyophilization is a 0.5mM nitric acid buffer solution with a pH value of 4.5-4.8 as a basic buffer solution, and further comprises 5-10 g/L trehalose, 5-20 g/L mannitol and 0.1% by mass of Proclin 300.

Technical Field

The invention relates to the technical field of in-vitro detection of medical immunity, in particular to a detection kit for rapidly detecting the content of lead in a sample.

Background

Lead (Pb) is a heavy metal ion with accumulation and multi-affinity, and can cause damage to systems such as hematopoiesis, digestion, nerves and immunity of the human body. Lead, a heavy metal, is a hazardous substance with persistent toxicity and is difficult to degrade biologically, chemically, etc. in the natural environment. After heavy metal lead enters a human body through diet, the lead can be enriched in the human body to cause poisoning, and chronic poisoning of the lead of the human body is manifested by inappetence, anemia, diarrhea, emaciation, arthralgia and the like; lead injury is the first killer internationally recognized to harm development of the nervous system of children, and when the content of blood lead in the children exceeds 100 mug/L, the corresponding intelligence index is reduced by 10-20 minutes. Especially, after the lead is absorbed by infants, more than 30 percent of lead is kept in the body, which affects the growth and intelligence development of the infants, damages the brain functions of cognitive function, nerve behavior, learning, memory and the like, and causes dementia in serious patients. Heavy metal lead has been listed as a food contaminant necessity test item in the global environmental monitoring program (GEMS). Therefore, the establishment of an accurate and effective lead determination method has important practical significance and application value.

The traditional lead detection methods can be divided into three categories, namely a physical detection method, a chemical detection method and a biological detection method. The detection technology comprises spectrum detection, an electrophoresis apparatus, liquid chromatography detection, a dithizone contrast method and the like, and the detection methods have certain advantages in detection accuracy and extremely high accuracy, but the detection process is complex and high in cost, and is difficult to develop in the field of practical application. In the aspect of blood lead detection, a tungsten boat lead-cadmium element analyzer and a graphite furnace atomic absorption spectrophotometer are commonly used; the graphite furnace atomic absorption spectrophotometer is provided with an automatic sample injector, the sample measuring result is accurate, the precision is good, but the requirement on the laboratory conditions is strict, large current and high voltage are required, the graphite furnace input power is 4000VA, the input voltage is 380V, and a graphite tube is a consumable product which is easy to damage and is suitable for laboratories with better conditions.

At present, a tungsten boat lead element analyzer is widely used for measuring the blood lead in clinical examination of 30 provincial and municipal medical institutions in China, and the tungsten boat is taken as an atomizer (Beijing Bohui Innovative photoelectric technology, limited corporation) and is specially used for detecting the atomic absorption spectrometer of the blood lead. The tested sample in the tungsten boat is atomized through electric heating to generate a large amount of ground state free atoms, so that the characteristic spectral line of the tested element emitted by the hollow cathode lamp is absorbed, and the measurement process is completed. The atomization pool lens glass cover can cause pollution due to smoke adsorption, errors can be caused by the fact that the pollutants fall off a tungsten boat due to vibration of an electromagnetic valve, and therefore the pollutants can be cleaned at any time according to conditions during large-scale continuous detection, particularly when samples with high lead content are measured in a large quantity, the tungsten boat becomes thinner gradually along with increase of using times of the tungsten boat, splashing of the samples can be caused due to overhigh temperature during work, errors of detection results are caused, and therefore when the tungsten boat is used for more than 10 times, the temperature of the tungsten boat can be readjusted or replaced by a new tungsten boat, and meanwhile a standard curve is redone; with the use of the element lamp, the luminous intensity of the element lamp is gradually reduced, and the stability of the element lamp is possibly reduced, so that the element lamp is not suitable for being continuously used for a plurality of times after a standard curve is made, national standard substances are added every 10 persons to monitor the sensitivity of the element lamp, and if the sensitivity is reduced or the energy value is unstable, the standard curve is remade or the element lamp is replaced. The method has the defects of incapability of detecting a sample at high flux, frequent replacement of an element lamp, high detection cost and complex operation.

Disclosure of Invention

The invention aims to provide a detection kit for rapidly detecting the lead content in a sample. The kit has the advantages of simple pretreatment, no need of digestion, economy, quickness, convenience and the like, can realize normal-temperature storage, quickness, high flux, low instrument cost, simple and convenient operation, single-person detection at any time, has high stability, and greatly improves the clinical use simplicity.

The invention provides a kit for rapidly detecting the lead content in a sample, which comprises a detection card and a quality control product; the detection card comprises a bottom plate, and a sample pad, a glass fiber membrane, a nitrocellulose membrane and absorbent paper which are positioned on the surface of the bottom plate and are sequentially arranged from a sample adding end;

the sample pad is soaked by a sample pad treatment buffer solution, and the sample pad treatment buffer solution comprises active protein and a surfactant;

the glass cellulose membrane is coated with: a conjugate of the lead specific antibody and the fluorescent microsphere and a conjugate of the chicken IgY antibody and the fluorescent microsphere;

the nitrocellulose membrane is marked with a detection limit and a quality control line, the detection line is coated with lead coupling hapten, and the quality control line is coated with goat anti-chicken IgY antibody.

Preferably, the sample pad treatment buffer is based on one or more of triethanolamine buffer, borax borate buffer, and glycine buffer.

Preferably, the active protein comprises one or more of fetal bovine serum, horse serum albumin and bovine serum albumin.

Preferably, the surfactant comprises one or more of S9, S13 and Tween 80.

Preferably, the mixing volume ratio of the conjugate of the lead specific antibody and the fluorescent microsphere to the conjugate of the chicken IgY antibody and the fluorescent microsphere is (4-7): 1.

preferably, the particle size of the fluorescent microsphere is 80-220 nm.

Preferably, the lead-specific antibody in the conjugate of the lead-specific antibody and the fluorescent microsphere is a lead-specific murine monoclonal antibody.

Preferably, the method for preparing the glass cellulose membrane comprises: soaking the glass cellulose membrane in a heavy suspension buffer solution for 1-2 h, and drying for 1-2 h to obtain a pretreated glass cellulose membrane; respectively dissolving the conjugate of the lead specific antibody and the fluorescent microsphere and the conjugate of the chicken IgY antibody and the fluorescent microsphere in a heavy suspension buffer solution, spraying the solution on a pretreated glass cellulose membrane, and drying; the heavy suspension buffer solution takes one or more of phosphate buffer solution, boric acid buffer solution and glycine buffer solution with the pH value of 5.5-6.8 and 50-200 mM as basic buffer solution, and also comprises 0.5-3 g/L of cane sugar and 0.1% of S9 by mass percentage.

Preferably, the quality control material is a lead-containing buffer solution, and the buffer solution comprises the following components: 0.5mM nitric acid buffer solution, 0.1% Tween20 by mass-volume ratio, 0.5% bovine serum albumin by mass, 0.1% Proclin300 by mass, water as solvent, and pH 4.8-5.0.

Preferably, the quality control product is freeze-dried and then stored, and the freeze-drying buffer solution for freeze-drying takes a 0.5mM nitric acid buffer solution with the pH value of 4.5-4.8 as a basic buffer solution, and further comprises 5-10 g/L trehalose, 5-20 g/L mannitol and 0.1% of Proclin300 in percentage by mass.

The invention provides a detection kit for rapidly detecting the content of lead in a sample. The invention provides a detection kit for realizing the lead content in blood by a brand new immunological methodology, and the kit has the advantages of low cost of a detection instrument, simplicity and convenience in operation, quickness, capability of being stored and transported at normal temperature, single-person packaging realization and good stability. Comparative differential potential dissolution measurement method: the interference of the matrix is large, and the operation is complex; the kit has the advantages that the immunochromatography method is used for detecting the content of lead in blood, the cost of a detection instrument is low, the complex pretreatment process is avoided, the detection is rapid for 15min, the high-throughput detection can be realized, the kit is packaged by a single person, the stability is good, and the repeatability is high; compared with a graphite furnace atomic absorption spectrometry determination method, the matrix interference is large, the price is high, the requirements on experimental conditions are strict, a large current is required, a high-voltage graphite tube is a consumable and is easy to damage, the cost of the detection instrument of the kit is low, the operation is simple, and the reagent can be stored at normal temperature; compared with a method for detecting lead element in whole blood by tungsten boat atomic absorption spectrometry (Beijing Bohui Innovative photoelectric technology, Inc., No. CN101592597A), the principle is that a detected sample in a tungsten boat is atomized by electric heating to generate a large amount of ground state free atoms, so that a characteristic spectral line of the detected element emitted by a hollow cathode lamp is absorbed, and the measurement process is completed.

Drawings

FIG. 1 is a calibration graph provided in example 1 of the present invention;

FIG. 2 is a graph showing the correlation between reagents provided in comparative example 1 of the present invention;

FIG. 3 is a schematic structural diagram of a detection card in the kit provided by the present invention.

Detailed Description

The invention provides a kit for rapidly detecting the lead content in a sample, which comprises a detection card and a quality control product; the detection card comprises a bottom plate, and a sample pad, a glass fiber membrane, a nitrocellulose membrane and absorbent paper which are positioned on the surface of the bottom plate and are sequentially arranged from a sample adding end. The detection card of the invention is shown in figure 3, wherein 1 is a nitrocellulose membrane, in particular a coating NC membrane; 2 is a bottom plate, in particular a PVC plate; 3 is a glass fiber membrane, in particular a microsphere pad; 4 is a sample pad; 5 is absorbent paper.

In the present invention, the sample pad is soaked with a sample pad treatment buffer, which includes an active protein and a surfactant. After the sample pad is soaked in the sample pad treatment buffer solution, the adsorption of the sample pad on the sample can be reduced. In the present invention, the sample pad treatment buffer is preferably a buffer based on one or more of a triethanolamine buffer, a borax borate buffer, and a glycine buffer. The present invention uses these buffers to provide buffering capacity that corrects for differences in pH between individual samples. In the present invention, the active protein preferably includes one or more of fetal bovine serum, horse serum albumin and bovine serum albumin. The active protein can seal the active sites on the sample pad, and ensure that target detection objects flow away and fully participate in reaction. In the invention, the surfactant comprises one or more of S9(Tetronic1307), S13(TRITON X-45N 10.4426) and Tween 80. The surfactant provided by the invention can improve the detection limit and sensitivity of metal ion determination, and can also improve the dispersibility of a sample on a glass fiber membrane. In the invention, the soaking time is preferably 1-2 h, and more preferably 1 h. After the soaking treatment of the present invention, the sample pad is preferably subjected to a drying treatment, preferably at 37 ℃. The source of each component in the sample pad treatment buffer in the present invention is not particularly limited, and a conventional commercially available product well known to those skilled in the art may be used.

In the present invention, the glass cellulose film is coated with: a conjugate of the lead specific antibody and the fluorescent microsphere and a conjugate of the chicken IgY antibody and the fluorescent microsphere. In the invention, the conjugate of the lead specific antibody and the fluorescent microsphere has a specific recognition effect on heavy metal lead in a sample, forms an immune complex with the lead, chromatographs to a detection area (T) along a nitrocellulose membrane, is combined with a pre-coated lead coupling hapten, and has the fluorescence intensity inversely proportional to the pb content in the sample. The conjugate of the chicken IgY antibody and the fluorescent microsphere is chromatographed to a quality control area (C) and combined with the precoated goat anti-chicken IgY. In the invention, the mixing volume ratio of the conjugate of the lead specific antibody and the fluorescent microsphere and the conjugate of the chicken IgY antibody and the fluorescent microsphere is preferably 4-7: 1, more preferably 6: 1. The conjugate of the lead specific antibody and the fluorescent microsphere and the conjugate of the chicken IgY antibody and the fluorescent microsphere are preferably adjusted to have a total mass concentration of 0.2 percent before being sprayed on a glass cellulose membrane. In the invention, the particle size of the fluorescent microsphere is preferably 80-220 nm, and more preferably 120 nm. In the invention, the fluorescent microsphere has the advantages of large difference between excitation wavelength and receiving wavelength, small interference, high detection sensitivity and good repeatability. The fluorescent microspheres of the present invention are preferably conventional commercially available products. In the invention, the lead specific antibody in the conjugate of the lead specific antibody and the fluorescent microsphere is a lead specific mouse monoclonal antibody. In the present invention, the method for preparing the lead-specific murine monoclonal antibody preferably comprises the following steps: 1. injecting antigen protein (preferably lead coupled bovine serum albumin) into the mouse to enable the mouse to have immune response; 2. obtaining corresponding B lymphocyte; 3. fusing mouse myeloma cells and B lymphocytes, and then screening by using selection culture media (HAT and HT); 4. the screened cells are monoclonal cells, and can be propagated in a large quantity and can generate specific antibodies; 5. performing monoclonal culture and antibody detection on the hybridoma cells, and screening for multiple times to obtain cells stably secreting monoclonal antibodies; and 6, culturing the hybridoma cells in vitro in a large scale or injecting the hybridoma cells into the abdominal cavity of the mouse for proliferation to generate ascites, and purifying to obtain a large amount of lead-specific mouse monoclonal antibodies.

In the present invention, the method for producing a glass cellulose film preferably comprises: soaking the glass cellulose membrane in a heavy suspension buffer solution for 1-2 h, and drying for 1-2 h to obtain a pretreated glass cellulose membrane; respectively dissolving the conjugate of the lead specific antibody and the fluorescent microsphere and the conjugate of the chicken IgY antibody and the fluorescent microsphere in a heavy suspension buffer solution, spraying the solution on a pretreated glass cellulose membrane, and drying; the heavy suspension buffer solution takes one or more of phosphate buffer solution, boric acid buffer solution and glycine buffer solution with the pH value of 5.5-6.8 and 50-200 mM as basic buffer solution, and also comprises 0.5-3 g/L of cane sugar and 0.1% of S9 by mass percentage. In the invention, the heavy suspension buffer solution has the beneficial effects that the unconjugated protein is removed, the heavy suspension buffer solution provides a proper pH and ionic strength environment, and the activity of the final antibody microsphere conjugate and the antibody are ensured to be not easy to fall off. In the invention, the drying is preferably carried out at the temperature of 45-65 ℃ for 2-6 h.

In the invention, the nitrocellulose membrane is marked with a detection limit and a quality control line, the detection line is coated with lead coupling hapten, and the quality control line is coated with goat anti-chicken IgY antibody. In the invention, the detection limit is positioned at one side close to the sample adding section, and the quality control line is positioned at one side far away from the sample adding end. In the present invention, the lead-coupled hapten is preferably lead-coupled bovine serum albumin, or lead-coupled ovalbumin. In the invention, the concentration of the lead coupling hapten is preferably 0.5-5 ug/mL, and more preferably 1 ug/mL. The source of the lead-coupled hapten is not particularly limited in the invention, and the lead-coupled hapten can be a conventional commercial product, such as a product purchased from Beijing Deolping Biotechnology Co. In the invention, the concentration of the goat anti-chicken IgY antibody is preferably 0.5-2 ug/mL, and more preferably 1.5 ug/mL. According to the invention, the lead coupling hapten and the goat anti-chicken IgY antibody are respectively scratched on a nitrocellulose membrane at a detection line position and a quality control line position, and after scratching is finished, the membrane is preferably dried for 2-6 hours at the temperature of 45-65 ℃.

In the invention, the quality control material is a lead-containing buffer solution which can ensure the stability of lead nitrate and avoid precipitation, and preferably comprises the following components: 0.5mM nitric acid buffer solution, 0.1% Tween20 by mass-volume ratio, 0.5% bovine serum albumin by mass, 0.1% Proclin300 by mass (used as a preservative), water as a solvent, and pH 4.8-5.0. The selection of the pH value can stabilize quality control products and inhibit the hydrolysis of lead nitrate. In the invention, the quality control product is preferably freeze-dried and then stored, and the freeze-drying buffer solution for freeze-drying takes a nitrate buffer solution with the pH value of 4.5-4.8 as a basic buffer solution, and also comprises 5-10 g/L of trehalose, 5-20 g/L of mannitol and 0.1% of Proclin300 by mass percentage. The freeze-drying buffer solution can ensure the stability of a freeze-dried finished product. In the invention, the freeze-drying is preferably vacuum freeze-drying, and the vacuum freeze-drying time is preferably 12-18 h. The method of adjusting pH in the present invention is not particularly limited, and the pH can be adjusted by using a conventional pH adjuster known in the art. The prepared detection card is preferably used for carrying out value determination on the quality control products, and freeze-drying is carried out after value assignment to prepare the quality control products with different concentrations, so that the subsequent detection of samples is facilitated. Specifically, the invention preferably selects a calibrator for tracing and assigning according to national standard substances, performs concentration setting on quality control substances, prepares freeze-drying buffer solutions with different concentrations, and performs vacuum freeze-drying for 12-18 hours to obtain the quality control substances.

The detection card also comprises absorbent paper, and the absorbent paper is not particularly limited, and can be commercially available absorbent paper for conventional detection cards.

After obtaining the sample pad, the glass fiber membrane, the nitrocellulose membrane and the absorbent paper, the present invention preferably performs the preparation of the test card according to a conventional method. If the sample pad, the dried glass fiber membrane, the dried nitrocellulose membrane and the absorbent paper are stuck on the bottom plate in sequence; and then, cutting the reagent large plate, then putting the reagent large plate into a reagent card, adding a drying agent and an aluminum foil bag, and sealing to obtain the detection card.

In the invention, the detection card preferably further comprises a card shell, the card shell further comprises a back card and an upper cover, the back card is provided with a detection card slot, the detection card is embedded in the detection card slot, the upper cover is provided with a test window and a sample adding hole, the position of the test window is matched with the positions of the detection line and the quality control line, and the position of the sample adding hole is matched with the position of the sample pad. In the present invention, the card case is preferably a plastic card case.

In the present invention, the samples that the kit can detect include blood samples and urine samples. (the urine sample is preferably added with a diluent for standing at room temperature for 5min for treatment, the diluent is preferably glycine 50-200 mmol buffer solution, 0.9% sodium chloride, 0.1% S13 and 0.05% preservative proclin 300. since different types of samples have different detection environments during detection, and complex protein components in the samples can be combined with antibodies and fluorescent markers coated on a test strip to different degrees, the detection result can be interfered, and a non-specific and very serious false signal, namely false positive, appears on a detection line, the invention preferably performs pretreatment operation on the sample, when the sample is blood, the kit preferably further comprises a blood filter membrane, the blood sample comprises whole blood, serum, plasma and the function of the blood filter membrane is that the whole blood can be added, and the function of blood cells can be removed; the invention has no special limitation on the source of the blood filter membrane, commercially available products of conventional blood filtration membranes well known to those skilled in the art may be used. Specifically, the detection method of the kit of the present invention preferably comprises the following steps: taking 20ul of serum sample by a sampling pipettor, adding the serum sample into the buffer solution, fully and uniformly mixing, standing for 5min at room temperature, and taking the supernatant. Sample adding: the test card was removed from the package and 80ul of the diluted sample was pipetted into the well of the test card. And (6) testing. And standing the sample at room temperature for 15min after sample addition, and putting the test strip into a dry type fluorescence immunoassay quantitative analyzer to read data. Please strictly control the time for 15 min. The dry type fluorescence immunoassay quantitative analyzer measures and analyzes the optical signal, and quantitatively obtains the concentration of the measured substance.

The kit disclosed by the invention applies the principle of competitive immunochromatographic detection, preferably is matched with an immune quantitative analyzer for use, lead in a sample is combined with a lead specific antibody coated on glass fiber and a coupling substance of fluorescent microspheres to form a fluorescent particle-antibody-antigen immune complex, the immune complex is chromatographed to a detection area (T) along a nitrocellulose membrane and is combined with a pre-coated lead coupling hapten, and the fluorescence intensity of the immune complex is inversely proportional to the lead content in a sample. And (3) carrying out chromatography on the conjugate of the chicken IgY antibody and the fluorescent microsphere to a quality control region (C) and combining the conjugate with the goat anti-chicken IgY antibody pre-coated on a quality control line. Therefore, as the concentration of the heavy metal lead residue in the sample increases, the detection line color development becomes gradually shallower due to the inhibition; because the quality control line contains the goat anti-chicken IgY antibody and recognizes and combines the goat anti-chicken IgY antibody, the control line can be colored no matter whether the sample contains heavy metal lead or not, so that the result of the detection product is effective.

The following describes a detection kit for rapidly detecting lead content in a sample in detail with reference to specific examples, and the technical solution of the present invention includes, but is not limited to, the following examples.