阅读说明：本技术 一种快速检测样品中镉含量的试剂盒 (Kit for rapidly detecting cadmium 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镉含量的检测试剂盒,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检测仪器成本低,操作简便,快速,可常温储存运输,实现单人份包装且稳定性好的优点。(The invention relates to a detection kit for rapidly detecting cadmium 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 low cost of detection instruments, simple and convenient operation, quickness, capability of being stored and transported at normal temperature, single-person packaging and good stability.)

1. A kit for rapidly detecting the content of cadmium 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 a cadmium specific antibody and a fluorescent microsphere and a conjugate of a 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 cadmium 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 phosphate buffer, TRIS hydrochloric acid buffer, and glycine buffer.

3. The kit of claim 1, wherein the active proteins comprise one or more of bovine serum albumin, casein and ovalbumin.

4. The kit according to claim 1, wherein the surfactant comprises one or more of tween20, laureth Brij35, Triton-100.

5. The kit according to claim 1, wherein the mixture volume ratio of the cadmium specific antibody to the fluorescent microsphere conjugate to the chicken IgY antibody to the fluorescent microsphere conjugate is (4-6): 1.

6. the kit according to claim 1 or 5, wherein the particle size of the fluorescent microspheres is 100-200 nm, and the Ex excitation wavelength Ex measured by a fluorescence spectrophotometer is 365 nm; the emission wavelength Em was 610 nm.

7. The kit according to claim 1 or 5, wherein the cadmium specific antibody in the conjugate of the cadmium specific antibody and the fluorescent microsphere is a cadmium 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 2-3 h, and drying for 1-2 h to obtain a pretreated glass cellulose membrane; respectively dissolving the conjugate of the cadmium 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 carbonate buffer solution with the pH value of 8.5-9.6 and the pH value of 50-200 mM, TRIS hydrochloric acid buffer solution and glycine buffer solution as basic buffer solution, and also comprises 0.1-1.0 g/L of sucrose, 0.1-4 g/L of mannitol and 0.1% of SDS (sodium dodecyl sulfate) by mass percentage.

9. The kit of claim 1, wherein the quality control material is a buffer solution containing cadmium, and the buffer solution comprises the following components: 0.5mmol/L nitric acid buffer solution, 0.1 percent of Tween20, 0.5 percent of bovine serum albumin, 0.1 percent of Proclin300, water as a solvent and 5.2 of pH value.

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 takes a nitrate buffer solution with a pH value of 5.2-5.4 as a basic buffer solution, and further comprises 3-8 g/L of sucrose, 5-20 g/L of 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 cadmium in a sample.

Background

Cadmium is a heavy metal which is discovered by stromcyer in 1817 and has great harm to human bodies, and is mainly enriched in the bodies through food chains. The long-term accumulation of cadmium in human body can cause renal tubular injury, affect the absorption of phosphorus, calcium and vitamin D, cause osteoporosis, osteomalacia and fracture, change the morphology of the central system of brain, interfere the normal metabolism of iron in the body, induce cancer, damage the male reproductive system and affect fertility. With the rapid development of industry and agriculture in recent years, heavy metal pollution in the environment is becoming more and more serious. And the quality and speed requirements for heavy metal detection and analysis are higher and higher.

At present, a tungsten boat lead-cadmium element analyzer is widely used for measuring blood cadmium in clinical tests of 30 provincial and municipal medical institutions in China, and the tungsten boat is used as an atomizer (Beijing Bohui Innovative photoelectric technology, Inc.) of the instrument, and the tungsten boat is specially used for an atomic absorption spectrometer for detecting the blood cadmium. 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 finished. The atomization pool lens glass cover can cause pollution due to smoke adsorption, and the pollutants can fall into the tungsten boat to cause errors due to vibration of the electromagnetic valve, so that the atomization pool lens glass cover is stopped to be cleaned at any time according to conditions during large-scale continuous detection, particularly when a large number of samples with high cadmium content are measured. The tungsten boat becomes thinner gradually with the increase of the using times of the tungsten boat, and the temperature is too high and too fast in work, so that the sample can splash possibly to cause errors of a detection result, therefore, when the tungsten boat is used for more than 10 times, the temperature of the tungsten boat needs to be readjusted or replaced by a new tungsten boat, and 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 too many 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 samples with high flux, frequent replacement of element lamps, high detection cost and complex operation.

Disclosure of Invention

The invention aims to provide a detection kit for rapidly detecting the cadmium content in a sample. The kit has the advantages of low cost of detection instruments, high detection speed, simple and convenient operation, rapidness, high throughput, normal-temperature storage and transportation, single-person packaging and good stability.

The invention provides a kit for rapidly detecting cadmium 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 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 a cadmium specific antibody and a fluorescent microsphere and a conjugate of a 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 cadmium coupling hapten, and the quality control line is coated with goat anti-chicken IgY antibody.

Preferably, the sample pad treatment buffer is a base buffer based on one or more of a phosphate buffer, a TRIS hydrochloric acid buffer, and a glycine buffer.

Preferably, the active protein comprises one or more of bovine serum albumin, casein and ovalbumin.

Preferably, the surfactant comprises one or more of tween20, lauryl ether Brij35 and Txiton-100.

Preferably, the mixing volume ratio of the cadmium specific antibody to the fluorescent microsphere conjugate to the chicken IgY antibody to the fluorescent microsphere conjugate is (4-6): 1.

preferably, the particle size of the fluorescent microsphere is 100-200 nm, and the Ex excitation wavelength Ex measured by a fluorescence spectrophotometer is 365 nm; the emission wavelength Em was 610 nm.

Preferably, the cadmium specific antibody in the conjugate of the cadmium specific antibody and the fluorescent microsphere is a cadmium 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 2-3 h, and drying for 1-2 h to obtain a pretreated glass cellulose membrane; respectively dissolving the conjugate of the cadmium 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 carbonate buffer solution with the pH value of 8.5-9.6 and the pH value of 50-200 mM, TRIS hydrochloric acid buffer solution and glycine buffer solution as basic buffer solution, and also comprises 0.1-1.0 g/L of sucrose, 0.1-4 g/L of mannitol and 0.1% of SDS (sodium dodecyl sulfate) by mass percentage.

Preferably, the quality control material is a buffer solution containing cadmium, and the buffer solution comprises the following components: 0.5mmol/L nitric acid buffer solution, 0.1 percent of Tween20, 0.5 percent of bovine serum albumin, 0.1 percent of Proclin300, water as a solvent and 5.2 of pH value.

Preferably, the quality control product is freeze-dried and then stored, and the freeze-drying buffer solution for freeze-drying takes a nitrate buffer solution with the pH value of 5.2-5.4 as a basic buffer solution, and also comprises 3-8 g/L of sucrose, 5-20 g/L of mannitol and 0.1% of Proclin300 by mass percentage.

The invention provides a detection kit for rapidly detecting cadmium content in a sample. The invention provides a brand-new immunochromatography method for detecting heavy metal cadmium, and has the advantages of low cost of a detection instrument, simplicity and convenience in operation, rapidness, capability of being stored and transported at normal temperature, capability of realizing single-person packaging and good stability. Compared with a graphite furnace atomic absorption spectrometry method, the method has the advantages that the matrix interference is large, the price is high, the requirement on experimental conditions is strict, high current is required, a high-voltage graphite tube is a consumable and is easy to damage, the kit disclosed by the invention is applied to an immunochromatography method, the detection is quick and only needs 15min, the cost of a detection instrument is low, the operation is simple, and the reagent can be stored at normal temperature; compared with a biosensor method, the kit has the advantages that the cost of a detection instrument is low, the operation is simple and convenient, the rapid reagent can be stored at normal temperature and packaged by a single person, the sample types are serum and urine, the stability is good, and the sensitivity is high; compared with an indication biological method, the kit has the advantages of higher specificity and high sensitivity; compared with an enzyme analysis method, the kit has the advantages of higher specificity and selectivity; compared with an ELISA (enzyme-Linked immuno sorbent assay) immunoassay method, the kit has the advantages that the cost of a detection instrument is low, the operation is simple and convenient, the rapid reagent can be stored at normal temperature and packaged by a single person, the types of samples are serum and urine, the stability is good, and the sensitivity is high; compared with a method for detecting cadmium 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 characteristic spectral lines of the detected element emitted by a hollow cathode lamp are 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 cadmium 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 arranged on the surface of the bottom plate in sequence 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 solution including 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 to the sample can be reduced. In the present invention, the sample pad treatment buffer is a base buffer based on one or more of a phosphate buffer, a TRIS hydrochloric acid buffer, and a glycine buffer. The use of the sample pad treatment buffer solution can keep the sample pad at proper ionic strength, so as to correct the pH difference among individual samples in response to samples with higher ionic strength. In the present invention, the active protein includes one or more of bovine serum albumin, casein and ovalbumin. The active protein added in the invention can seal the active sites on the sample pad, and ensure that target detection objects all flow away to fully participate in the reaction. In the invention, the surfactant comprises one or more of Tween20, lauryl ether Brij35 and Txiton-100. The surfactant provided by the invention can improve the detection limit and sensitivity of metal ion determination and increase the specificity. In the invention, the soaking time is preferably 1-2 h, and more preferably 2 h. After the soaking treatment of the present invention, the sample pad is preferably dried, 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 a cadmium specific antibody and a fluorescent microsphere and a conjugate of a chicken IgY antibody and the fluorescent microsphere. In the invention, the conjugate of the cadmium specific antibody and the fluorescent microsphere has a specific recognition effect on heavy metal cadmium in a sample, and can form an immune complex with the cadmium, the immune complex is chromatographed to a detection area (T) along a nitrocellulose membrane and is combined with the pre-coated cadmium coupling hapten, and the fluorescence intensity of the immune complex is inversely proportional to the content of Cd in the sample. The conjugate of the chicken IgY antibody and the fluorescent microsphere is chromatographed to a quality control region (C) and combined with the precoated goat anti-chicken IgY. In the invention, the mixing volume ratio of the cadmium specific antibody to the fluorescent microsphere conjugate and the chicken IgY antibody to the fluorescent microsphere conjugate is preferably 4-6: 1, more preferably 5: 1. The conjugate of the cadmium 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 100-200 nm, more preferably 100nm, and the Ex excitation wavelength Ex measured by a fluorescence spectrophotometer is 365 nm; the emission wavelength Em was 610 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. The coupling preparation method of the conjugate of the cadmium specific antibody and the fluorescent microsphere and the conjugate of the chicken IgY antibody and the fluorescent microsphere is preferably a conventional method. Specifically, the preparation method of the conjugate of the cadmium specific antibody and the fluorescent microsphere and the conjugate of the chicken IgY antibody and the fluorescent microsphere comprises the following steps: adding 1% of fluorescent microspheres, EDC10mg/ml and cadmium specific antibody 2-10 ug/ml into two tubes, uniformly mixing and coupling for 2 hours, centrifuging for 30 minutes at a speed of 8000-14000 r/min, removing supernatant, repeating the operation twice, adding 1% BSA, and sealing for 1 hour; ② adding 1 percent of fluorescent microspheres and EDC10mg/ml, adding chicken IgY antibody 50ug, uniformly mixing, coupling for 2h, centrifuging for 30min at the speed of 8000-14000 r/min, removing supernatant, repeating the operation twice, adding 1 percent BSA, and sealing for 1 h. In the invention, the cadmium specific antibody in the conjugate of the cadmium specific antibody and the fluorescent microsphere is a cadmium specific mouse monoclonal antibody. In the present invention, the method for preparing the cadmium-specific murine monoclonal antibody preferably comprises the following steps: 1. injecting antigen protein (preferably cadmium-coupled bovine serum albumin) into the mice to enable the mice to have immune response; 2. obtaining corresponding B lymphocyte; 3. fusing mouse myeloma cells and B lymphocytes, and then screening by using selection 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; 6. culturing the hybridoma cells in vitro in a large scale or injecting the hybridoma cells into the abdominal cavity of a mouse for proliferation to generate ascites, and purifying to obtain a large amount of the cadmium specific mouse monoclonal antibody.

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 2-3 h, and drying for 1-2 h to obtain a pretreated glass cellulose membrane; respectively dissolving the conjugate of the cadmium 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 carbonate buffer solution with the pH value of 8.5-9.6 and the pH value of 50-200 mM, TRIS hydrochloric acid buffer solution and glycine buffer solution as basic buffer solution, and also comprises 0.1-1.0 g/L of sucrose, 0.1-4 g/L of mannitol and 0.1% of SDS (sodium dodecyl sulfate) by mass percentage. In the invention, the heavy suspension buffer can remove unconjugated protein, and provides a proper pH and ionic strength environment to ensure the activity of the final antibody microsphere conjugate and the difficulty in falling off of the antibody. In the invention, the drying is preferably carried out for 2-6 h at the temperature of 45-65 ℃.

In the invention, the nitrocellulose membrane is marked with a detection limit and a quality control line, the detection line is coated with a cadmium coupling hapten, and the quality control line is coated with a goat anti-chicken IgY antibody. In the invention, the detection limit is positioned at one side close to the sample adding end, and the quality control line is positioned at one side far away from the sample adding end. In the present invention, the cadmium-conjugated hapten is preferably cadmium-conjugated bovine serum albumin or cadmium-conjugated ovalbumin. In the invention, the concentration of the cadmium coupling hapten is preferably 1-4 mg/mL, and more preferably 2 mg/mL. The source of the cadmium-conjugated hapten is not particularly limited in the invention, and the cadmium-conjugated 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 mg/mL, and more preferably 1.5 mg/mL. The cadmium coupled hapten and the goat anti-chicken IgY antibody are scratched on a nitrocellulose membrane at a detection line position and a quality control line position respectively, and after scratching is finished, the membrane is dried for 2-6 hours at the temperature of 45-65 ℃.

In the invention, the quality control product is a buffer solution containing cadmium, the buffer solution can ensure the stability of cadmium nitrate to avoid precipitation, and the buffer solution preferably comprises the following components: 0.5mmol/L nitric acid buffer solution, 0.1% Tween20, 0.5% bovine serum albumin, 0.1% Proclin300 (preservative), water as solvent, and pH 5.2. The pH value of the invention is selected to stabilize quality control products and inhibit the hydrolysis of cadmium 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 5.2-5.4 as a basic buffer solution, and also comprises 3-8 g/L of sucrose, 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 product, 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 housing is preferably a plastic card housing.

In the present invention, the samples that the kit can detect include blood samples and urine samples. When the sample is blood, the kit preferably further comprises a blood filtration membrane. The source of the blood filtration membrane is not particularly limited in the present invention, and a conventional blood filtration membrane commercially available product well known to those skilled in the art may be used. The detection method of the kit preferably comprises the following steps: and taking 100ul of serum sample by a sampling pipette, adding the serum sample into the buffer solution, fully mixing, standing at room temperature for 5min, 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 immunochromatography detection, preferably is matched with an immune quantitative analyzer for use, cadmium in a sample is combined with a conjugate of a cadmium specific antibody coated on glass fiber and fluorescent microspheres to form an immune complex of fluorescent particles, an antibody and an antigen, the immune complex is chromatographed to a detection area (T) along a nitrocellulose membrane and is combined with a cadmium coupling hapten pre-coated on a detection line, and the fluorescence intensity of the immune complex is inversely proportional to the cadmium content in the 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. The immunoassay analyzer calculates a T/C signal value by collecting fluorescence signals of strips of a detection line (T) and a quality control line (C), and the T/C signal value is compared with a standard curve to obtain the content of the heavy metal cadmium in the detection sample. Therefore, as the concentration of the residual heavy metal cadmium in the sample increases, the color development of the detection line is gradually reduced due to 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 cadmium or not, so that the result of the detection product is effective.

The detection kit for rapidly detecting cadmium content in a sample according to the present invention is further described in detail with reference to the following specific examples, and the technical solutions of the present invention include, but are not limited to, the following examples.