CN101196521A - The detection method of cadmium ion indirect competition ELISA - Google Patents

Abstract

The invention discloses an indirect competitive ELISA method for detecting cadmium ions. In the method, fish and shellfish sample extracts and cadmium ion standard solutions of serial concentrations are respectively added to the micropores of antigen pre-coated strips, and then anti-cadmium ions are added. Add the monoclonal antibody of the monoclonal antibody to each microwell, mix and incubate, add HRP-labeled goat anti-mouse IgG antibody to each microwell, and incubate; stop the reaction after the substrate color develops, and measure each microwell with a microplate reader The absorbance is compared with the curve regression equation obtained by the standard substance to calculate the content of cadmium ions in the sample. The present invention utilizes the obtained positive cells capable of secreting anti-cadmium ion monoclonal antibody to produce a large amount of anti-cadmium ion monoclonal antibody, and the established method is low in cost, and can quickly, easily and sensitively measure the content of cadmium ion in a sample. The invention can quickly and sensitively detect the residual heavy metal cadmium in marine fish and shellfish food on a large scale.

Description

The detection method of cadmium ion indirect competition ELISA

technical field

The invention belongs to the field of biotechnology, and relates to a rapid diagnosis method for heavy metal residues in aquatic meat products, in particular to an indirect competitive ELISA method for detecting cadmium ions using monoclonal antibodies against heavy metal cadmium ions.

Background technique

Cadmium (Cdmium) atomic number 48 is one of the heavy metals that seriously endanger human health, mainly from cadmium mines and cadmium smelters. Because cadmium and zinc are in the same family and often coexist with zinc, there must be ZnO and CdO in the emissions of zinc smelting. They are highly volatile and can spread thousands of meters away from the pollution source. With the rapid development of industry and agriculture in our country, the pollution of heavy metals in the environment is becoming more and more serious. The poison caused by heavy metal pollution persists and will enter the food chain with the recirculation of soil and water, posing a threat to food safety and endangering human life and health. Cadmium poisoning can damage kidney function, cadmium entering the respiratory tract can cause pneumonia, and emphysema acting on the digestive system can cause gastroenteritis. People with cadmium poisoning are often accompanied by anemia. Excessive cadmium accumulation in bones will soften, deform, fracture, atrophy, and cause cancer. my country's national health standards for heavy metals in various foods, and the safety and quality of agricultural products and the safety requirements for pollution-free aquatic products (2001) all limit the content of cadmium to ≤0.1mg/kg. The European Union, the United States, Japan, South Korea and other major exporting countries of my country's aquatic products have increasingly strict restrictions on the heavy metal content of aquatic products, and the regulations and standards have become more and more stringent.

The current detection methods for heavy metal cadmium mainly include:

Physical and chemical methods: atomic absorption spectrophotometry (AAS), inductively coupled plasma-atomic emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry (ICP-MS), atomic fluorescence spectrometry (AFS), etc. . These methods are characterized by sensitivity and accuracy, but require complex instruments and equipment, specialized analytical technicians, expensive standard products, troublesome pre-treatment, and cannot detect a large number of samples at the same time, so they are not suitable for on-site and large-scale promotion and application.

Immunological detection technology: At present, enzyme-linked immunosorbent assay (ELISA) is more widely used in the detection of heavy metal cadmium. The cadmium ions in the standard compete with the cadmium ions in the standard to bind to the antibody and use this to perform qualitative or quantitative analysis of the cadmium in the sample. The method is simple and fast, and can analyze a large number of samples at the same time.

At present, heavy metal immunological detection technology has been carried out abroad, and specific heavy metal antibodies such as In(III), Pb(II), Ni(II), Cd(II) and Hg(II) have been successfully prepared, and some antibodies have been used in For environmental testing, an enzyme-linked detection kit for heavy metals has been developed. There is no report on the immunological detection of heavy metals in China, and the reports in foreign literature are insufficient, and artificial antigens cannot be repeatedly synthesized based on it. Therefore, it is of great significance to develop an enzyme-linked detection method for heavy metals with independent intellectual property rights.

Contents of the invention

In order to solve the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a kind of indirect competitive ELISA method that utilizes heavy metal cadmium monoclonal antibody to detect cadmium ions, which can detect residual heavy metals in marine fish and shellfish foods on a large scale. Cadmium is quickly and sensitively detected to ensure the safety of seafood in our country and safeguard our country's interests in related international trade fields.

The object of the present invention is achieved through the following technical solutions: a detection method of cadmium ion indirect competition ELISA, comprising the steps of: adding the cadmium ion (Cd ²⁺ ) standard solution of fish and shellfish sample extract and serial concentrations to the antigen respectively Add anti-cadmium ion monoclonal antibody to each microwell of the pre-coated strip, mix and incubate, add HRP-labeled goat anti-mouse IgG antibody to each microwell, and incubate; The reaction was terminated after color development, and the absorbance (OD) of each well was measured with a microplate reader, and the content of cadmium ions (Cd ²⁺ ) in the sample was calculated according to the curve regression equation obtained from the standard substance.

The detection method of above-mentioned cadmium ion indirect competition ELISA comprises the steps:

(1) Add 10 μl of 100 mM EDTA (ethylenediammonium tetraacetic acid) to each microwell of the antigen pre-coated strip, and then add 80 μL of fish and shellfish sample extract and 80 μl of Cd ²⁺ standard solutions of serial concentrations, mix well; then add 10 μL 1:32000 times diluted anti-cadmium ion monoclonal antibody to each microwell; mix well, incubate at 37°C for 0.5~2h; then wash the microwell with washing solution ;

(2) Add 100 μL of HRP-labeled goat anti-mouse IgG antibody diluted with diluent 1:4000 to each microwell, incubate at 37°C for 0.5-1 h; then wash the microwell with washing solution;

(3) Add 100 μL chromogenic solution TMB (3,3′,5,5′-tetramethylbenzidine) substrate working solution to each microwell, and react for 10-20 minutes;

(4) Add 50 μL of 2M H ₂ SO ₄ (stop solution) to each microwell to stop the reaction, and vortex to mix;

(5) Measure the OD (absorbance) value at a wavelength of 450nm with an enzyme-linked reader;

(6) divide gained standard solution and sample absorbance value by the absorbance value of 0 standard (the standard solution of 0 concentration (ng/mL)) and multiply by 100%, with this standard solution calculated value is ordinate, cadmium ion concentration ( The logarithm of μg/kg) is the abscissa to draw the standard curve; read the corresponding cadmium ion concentration from the standard curve according to the B/B0 value of each sample, and then multiply it by the corresponding dilution factor to calculate the actual cadmium in the sample ion concentration. The diluent and washing liquid are both PBS-T, that is, 0.01M phosphate buffer saline containing 0.05% Tween-20.

In order to better realize the present invention, the fish and shellfish sample extract is pretreated according to the following method: every gram of fish meat or shellfish meat sample is added with 2mL ethyl acetate, fully smashed and homogenized; 4000rpm/min centrifugal 10min, get the supernatant in Dry the sample under nitrogen flow in a water bath at 50°C; mix and extract with equal volumes of cyclohexane and distilled water and vortex for 1 min, take the cyclohexane layer, add 12.5 μL of 1M HCl per gram of sample, vortex for 1 min, and let stand for 30 min; add 2 Distilled water twice the volume of HCl, 8000rpm/min, discard the upper layer of cyclohexane, absorb the lower layer, adjust the pH to 7.0-8.0 with 1N NaOH, and dilute 10 times with the diluent to obtain the fish and shellfish sample extract .

The antigen pre-coated strip is prepared as follows:

(1) Coating: Dilute Cd-iEDTA-BSA to 0.2 μg/mL with coating buffer, i.e. 0.05 M sodium carbonate buffer solution with pH 9.6, add 100 μL per microwell into the wells of polystyrene microwell plate, Coating overnight at 4°C or 2-3 hours at 37°C; the Cd-iEDTA-BSA is obtained by coupling cadmium ions to the carrier protein BSA with iEDTA;

(2) Washing: After pouring out the coating buffer in the wells of the microplate, add 200 μl of washing solution into each microwell; pour out the liquid in the wells again after 3 to 5 minutes, and completely remove the liquid in the microwells; The washing solution is 0.01M phosphate buffer solution containing 0.05% (mass percentage) Tween-20 (KH ₂ PO ₄ 0.2g, Na ₂ PO ₄ ·12H ₂ O 2.9g, NaCl 8g, dilute to 1000ml, pH7.4, plus 0.5ml Tween-20);

(3) Blocking: Add 150-200 μL of 0.01M PBS (phosphate buffer saline, pH 7.4) containing 0.5%-3% (W/V) BSA (bovine serum albumin) to each microwell of the microwell plate, 37 ℃ closed 0.5h ~ 1.5h;

(4) Wash the micropores 3 to 5 times according to step (2);

(5) Add 20% (W/V) sucrose phosphate buffer solution to protect at room temperature for 3 hours;

(6) Drying: After the microwell plate is dried, the antigen pre-coated strips are obtained.

The anti-cadmium ion monoclonal antibody is prepared by the following method: iEDTA (i.e. 1-(4-isothiocyanate benzyl) vinyl diamine-N, N, N', N'-tetraacetic acid) Coupling cadmium ions to hemocyanin (KLH), mixing Freund's incomplete adjuvant to immunize BALB/c mice, taking splenocytes from immunized mice and SP2/0 myeloma cells to fuse, and screening out stable secretion of anti-cadmium ions The positive cell line of monoclonal antibody (MAb-Cd) was expanded and cultured, the cells were injected into mice to induce ascites, and the anti-cadmium ion monoclonal antibody was purified.

The above-mentioned indirect competitive ELISA method for detecting cadmium ions can be applied to the detection of marine fish and shellfish and the like.

In the present invention, Cd-iEDTA-BSA is coated on a 96-well enzyme label plate, cadmium ion standard solution (Cd-EDTA) and fish and shellfish sample extract (X-EDTA) to be tested are added, and then anti-cadmium ion monoclonal Antibody, coated antigen Cd-iEDTA-BSA competes with free Cd-EDTA anti-cadmium ion monoclonal antibody, washes free Cd-EDTA and MAb-Cd-EDTA complex, and coated antigen Cd-iEDTA-BSA The combined MAb was combined with the enzyme-labeled goat anti-mouse IgG antibody, and the reaction was terminated after the substrate was developed, and the absorbance (OD) of each well was measured with a microplate reader. The larger the OD value, the free Cd-EDTA in the sample The less the content is, the curve regression equation obtained from the standard substance is used to calculate the content of Cd ²⁺ in the sample.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

The present invention utilizes the obtained positive cells secreting anti-cadmium ion monoclonal antibody to produce a large amount of anti-cadmium ion monoclonal antibody, the established method is low in cost, and can quickly, easily and sensitively measure the content of Cd2 ⁺ in a sample. The entire detection process only takes about 2 hours, does not require large-scale equipment, and the sensitivity can reach the national detection standard of 100 μg/kg sample. This method can quickly and sensitively detect heavy metal cadmium residues in marine shellfish on a large scale, so as to ensure the safety of seafood in our country and safeguard our country's interests in related international trade fields.

Description of drawings

Figure 1 is a standard inhibition curve.

Figure 2 is a linear regression diagram of the inhibition curve.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Preparation of reagents:

Both the diluent and the washing solution are PBS-T, that is, 0.01M phosphate buffer solution containing 0.05% Tween-20 (KH ₂ PO ₄ 0.2g, Na ₂ PO ₄ ·12H ₂ O 2.9g, NaCl 8g, dilute to 1000ml, pH7.4, plus 0.5ml Tween-20); the chromogenic solution is TMB (3,3′,5,5′-tetramethylbenzidine) substrate working solution; the stop solution is 2M sulfuric acid solution Measure 111.2ml of concentrated sulfuric acid (18M) and dilute to 1000ml.

Example 1

Utilize anti-cadmium ion monoclonal antibody to detect the indirect competition ELISA method of cadmium ion, comprises the steps:

(A) Cadmium ions were coupled to hemocyanin (KLH) using iEDTA (i.e., 1-(4-isothiocyanobenzyl)ethylenediamine-N,N,N',N'-tetraacetic acid), Mix Freund's incomplete adjuvant to immunize BALB/c mice, take the splenocytes of the immunized mice and fuse them with SP2/0 myeloma cells, screen out positive cell lines that stably secrete anti-cadmium ion monoclonal antibody (MAb-Cd) and expand Cultured, injected cells into mice to induce ascites, and purified to obtain monoclonal antibodies against cadmium ions.

(B) Using iEDTA to couple cadmium ions to the carrier protein BSA to obtain the detection antigen Cd-iEDTA-BSA.

(C) Preparation of antigen pre-coated strips:

(1) Coating: Dilute Cd-iEDTA-BSA to 0.2 μg/mL with coating buffer (0.05M sodium carbonate buffer, pH9.6), add 100 μL per microwell into a polystyrene microwell plate, Coating overnight at 4°C.

(2) Washing: After pouring out the coating buffer in the wells of the microwell plate, turn the microwell plate upside down on absorbent paper and pat to ensure that the liquid in the wells is completely removed. Add 200 μl of wash solution to the wells using a wash bottle. After 3 minutes, pour out the liquid in the microwell again to completely remove the liquid in the microwell.

(3) Blocking: Add 160 μL of 0.01M PBS (Phosphate-Buffered Saline, phosphate-buffered saline, pH7.4) containing 2% (W/V) BSA (Bovine serum album, bovine serum albumin) to each microwell, 37 ℃ closed for 1.5h;

(4) Wash the micropores 3 to 5 times according to step (2);

(5) Add 20% (W/V) sucrose phosphate buffer solution to protect at room temperature for 3 hours;

(6) Drying: After drying in a drying room, the antigen-precoated strips are obtained, and stored in packaging bags containing desiccant.

(D) Sample pretreatment (fish sample extract)

Take the shredded fish sample and place it in a centrifuge tube, add 2 mL of ethyl acetate per gram of the sample to fully break up and homogenize; centrifuge at 4000 rpm/min for 10 min, take the supernatant and dry the sample in a nitrogen flow in a water bath at 50°C; etc. Mix the volume of cyclohexane and distilled water and extract by vortexing for 1 min, take the cyclohexane layer, add 12.5 μL of 1M HCl per gram of sample, vortex for 1 min, and let it stand for 30 min; add distilled water twice the volume of HCl, 8000 rpm/min, discard Remove the upper layer of cyclohexane, absorb the lower layer, adjust the pH to 7.0-8.0 with 1N NaOH, dilute 10 times with the diluent, and take 80 μL for analysis.

(E) Detection of heavy metal cadmium ions

(1) Add 10 μL of 100 mM EDTA solution to each microwell, and then add 80 μL of cadmium ion standard solution with serial concentrations (0, 10, 50, 125, 250, 500, 2000, 5000 ng/mL) and 80 μL of the above-mentioned processed fish Sample extraction solution into the microwell;

(2) Add 10 μL of the anti-cadmium ion monoclonal antibody diluted with diluent 1:32000 to the microwells of the existing standard solution and fish sample extract, mix gently, and incubate at 37°C for 2 hours;

(3) Wash the micropores with washing solution for 3 to 5 times;

(4) Add 100 μL of HRP-labeled goat anti-mouse IgG antibody diluted with diluent 1:4000 to each microwell, and incubate at 37°C for 1 h;

(5) Wash the micropores with washing solution for 3 to 5 times;

(6) Add 100 μL chromogenic solution TMB (3,3′,5,5′-tetramethylbenzidine) substrate working solution to each microwell, and react for 10-20 minutes;

(7) Add 50 μL of 2M H ₂ SO ₄ (stop solution) to each microwell to stop the reaction, and shake gently to mix;

(8) Measure the OD (absorbance) value at a wavelength of 450 nm with an enzyme-linked reader within 15 minutes. The standard readings were 1.785, 1.645, 1.431, 1.147, 0.958, 0.663, 0.362, 0.120, and the sample readings were 1.250.

(F) Result Judgment Criteria

The resulting standard solution and the sample absorbance value are divided by the absorbance value of the 0 standard (standard solution of 0 concentration) and then multiplied by 100%. The calculated value of this standard solution is the ordinate, and the logarithm of the cadmium ion concentration (μg/kg) is The abscissa draws the standard curve.

According to the B/B0 value of each sample, the corresponding cadmium ion concentration of 92.826ppb can be read from the above standard curve, and then multiplied by the corresponding dilution factor to finally calculate the actual cadmium ion concentration in the sample as 928.26μg/kg.

Example 2

Utilize anti-cadmium ion monoclonal antibody to detect the indirect competition ELISA method of cadmium ion, comprises the steps:

(A) Use iEDTA to couple cadmium ions to hemocyanin (KLH), mix Freund's incomplete adjuvant to immunize BALB/c mice, take splenocytes from immunized mice and fuse SP2/0 myeloma cells, and screen out Stable positive cell lines secreting anti-cadmium ion monoclonal antibody (MAb-Cd) were expanded and cultured, the cells were injected into mice to induce ascites, and the anti-cadmium ion monoclonal antibody was purified.

(B) Using iEDTA to couple cadmium ions to the carrier protein BSA to obtain the detection antigen Cd-iEDTA-BSA.

(C) Preparation of antigen pre-coated strips:

(1) Coating: Dilute Cd-iEDTA-BSA to 0.2 μg/mL with coating buffer (0.05M sodium carbonate buffer, pH9.6), add 100 μL per microwell to the microwell of polystyrene microwell plate Wells were coated overnight at 4°C.

(2) Washing: After pouring out the coating buffer in the wells of the microwell plate, turn the microwell plate upside down on absorbent paper and pat to ensure that the liquid in the wells is completely removed. Add 200 µl of wash solution to the microwells with a multichannel pipette. After 5 minutes, pour out the liquid in the microwell again to completely remove the liquid in the microwell.

(3) Blocking: Add 150 μL of 0.01M PBS (Phosphate-Buffered Saline, phosphate-buffered saline, pH7.4) containing 3% (W/V) BSA (Bovine serum album, bovine serum albumin) to each microwell, 37 ℃ closed for 1h;

(4) Wash the micropores 5 times with washing liquid according to step (2);

(5) Add 20% (W/V) sucrose phosphate buffer solution to protect at room temperature for 3 hours;

(6) Drying: After drying in a drying room, the antigen-precoated strips are obtained, and stored in packaging bags containing desiccant.

(D) Sample pretreatment (shellfish sample extract)

Take the shredded shellfish sample and place it in a centrifuge tube, add 2 mL of ethyl acetate per gram of the sample to fully break up and homogenize; centrifuge at 4000 rpm/min for 10 minutes, take the supernatant and dry the sample in a nitrogen flow in a water bath at 50°C; Equal volumes of cyclohexane and distilled water were mixed and extracted, vortexed for 1 min, and the cyclohexane layer was taken, and 12.5 μL of 1M HCl was added per gram of sample, vortexed for 1 min, and allowed to stand for 30 min; distilled water with 2 times the volume of HCl was added, 8000 rpm/min, Discard the upper layer of cyclohexane, absorb the lower layer, adjust the pH to 7.0-8.0 with 1N NaOH, dilute 10 times with the diluent, and take 80 μL for analysis.

(E) Detection of heavy metal cadmium ions

(1) Add 10 μL of 100 mM EDTA solution to each microwell, and then add 80 μL of serial concentrations of cadmium ion standard solution and 80 μL of the treated shellfish sample extract to the microwells;

(2) Add 10 μL of 1:32000 diluted anti-cadmium ion monoclonal antibody to the microwells of the existing standard solution and shellfish sample extract, mix gently, and incubate at 37°C for 0.5h;

(3) Wash the micropores with washing solution for 3 to 5 times;

(4) Add 100 μL of 1:4000 diluted HRP-labeled goat anti-mouse IgG antibody to each microwell, and incubate at 37°C for 0.5h;

(5) Wash the micropores with washing solution for 3 to 5 times;

(6) Add 100 μL chromogenic solution TMB (3,3′,5,5′-tetramethylbenzidine) substrate working solution to each microwell, and react for 10-20 minutes;

(7) Add 50 μL of 2M H ₂ SO ₄ (stop solution) to each microwell to stop the reaction, and shake gently to mix;

(8) Measure the OD (absorbance) value at a wavelength of 450 nm with an enzyme-linked reader within 15 minutes. The standard solution readings were 1.757, 1.684, 1.419, 1.128, 0.955, 0.659, 0.344, 0.126, and the sample reading was 1.404.

(F) Result Judgment Criteria

The resulting standard solution and the sample absorbance value are divided by the absorbance value of the 0 standard (standard solution of 0 concentration) and then multiplied by 100%. The calculated value of this standard solution is the ordinate, and the logarithm of the cadmium ion concentration (μg/kg) is The abscissa draws the standard curve.

According to the B/B0 value of each sample, the corresponding cadmium ion concentration of 58.288ppb can be read from the above standard curve, and then multiplied by the corresponding dilution factor to finally calculate the actual cadmium ion concentration of 582.88μg/kg in the sample.

To verify the reliability of the effect of the present invention, carry out the following identification:

Take the sealed microtiter plate, add 10 μL 100mM EDTA solution to each microwell, add 80 μL prepared Cd2 ⁺ standard solution (0, 10, 50, 125, 250, 500, 2000, 5000ng/mL) and mix well as To compete with the antigen, add 10 μL of the best diluted monoclonal antibody working solution (1:32000 times), mix well, add 100 μL of washing solution to one well as a blank control well without monoclonal antibody; incubate at 37 ° C for 1.5 h, wash Plate) Add HRP enzyme-labeled goat anti-mouse secondary antibody diluted with diluent 1:4000, incubate at 37°C for 1 h, add 100 μL TMB substrate solution for color development for 10 minutes, stop with 50 μL 2M H ₂ SO ₄ , use a microplate reader Read at a wavelength of 450nm. The standard inhibition curve was drawn with the logarithm of Cd ²⁺ concentration as the abscissa and B/B ₀ (%) as the ordinate.

From Figure 1 and Figure 2, it can be seen that within the range of 0.050-2μg/ml (B/B ₀ ) has a good linear relationship with the logarithmic value of Cd ²⁺ concentration, and the correlation coefficient is r=0.9978. When the inhibition rate is 10%, Cd ² The concentration of ⁺ is the lowest detection limit, reaching 0.010μg/ml.

(2) Reproducibility of results

The precision test for Cd ²⁺ detection was repeated 5 times between batches, and the coefficient of variation (CV%) of the absorbance values of the standard solutions of different concentrations obtained is shown in Table 1, showing that the method of the present invention has high reproducibility.

Table 1 Coefficient of variation between batches of competition ELISA

Cd ²⁺ (ng/mL) 10 50 125 250 500 1000 2000 Coefficient of variation (%) 2.32 3.83 1.44 3.42 4.00 2.89 7.72

(3) Calculation of recovery rate

Take 10 g of blank fish sample and add Cd ²⁺ standard sample, after diluting the extract (diluted to about within the concentration range of the standard curve), carry out ELISA determination. According to the OD value and B/B0 value, the Cd ²⁺ content was obtained from the standard curve and then multiplied by the dilution factor to obtain the Cd ²⁺ content in the sample, and then the recovery rate was calculated, as shown in Table 2.

Table 2 Determination of the recovery rate of the sample

The amount of Cd2+ added to the sample (μg/g) The amount of Cd2+ measured Recovery rate 50 50.56±1.4  101.12±2.8 5 4.81±0.2 96.34±4.0 0.5 0.46±0.01 93.17±2.0

As can be seen from the table, the recovery rate is 93-101%.

(4) Result specificity

Competitive ELISA was used for the specific cross-reaction test for the following heavy metals, and the results are as follows:

Table 3 Cross-reactivity of other heavy metal cadmium

Metal ion _IC50 (ng/mL)   Cross-reactivity rate (%) Cd ²⁺ Hg ²⁺ Cu ²⁺ Mn ²⁺ Zn ²⁺ Fe ³⁺ Ca ²⁺   250200＞100000＞100000＞300000＞500000＞500000   100125<0.25<0.25<0.08<0.05<0.05

pb ²⁺ Ni ²⁺ Al ³⁺ Mg ²⁺  ＞5000000＞5000000＞5000000＞5000000  ＜0.005＜0.005＜0.005＜0.005

The results show that the antibody has obvious cross-reactivity to Hg ²⁺ except for Cd ²⁺ , and has little cross-reactivity to other metals. Therefore, this monoclonal antibody can be used to detect both Cd ²⁺ and Hg ²⁺ simultaneously. Heavy metals, expanding the scope of application.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (5)

1. the detection method of a cadmium ion indirect competitive ELISA, it is characterized in that comprising the steps: that respectively cadmium ion titer with fish and shellfish sample extracting solution and series concentration joins antigen and wraps in advance by in the micropore of bar, the monoclonal antibody that adds the Cadmium resistance ion again is to each micropore, mixing is hatched, in each micropore, add the goat anti-mouse igg antibody of HRP mark, hatch; Cessation reaction after substrate colour developing is measured the absorbance of each micropore, the Regression Equations of reference standards gained, the content of cadmium ion in the calculation sample with microplate reader.

2. the detection method of a kind of cadmium ion indirect competitive ELISA according to claim 1 is characterized in that comprising the steps:

(1) in antigen wraps by each micropore of bar in advance, all adds the 100mM EDTA of 10 μ l respectively, in above-mentioned each micropore, add the Cd of 80 μ l fish and shellfish sample extracting solutions and 80 μ L series concentration then more respectively ²⁺Titer, mixing; Each micropore adds the monoclonal antibody of the Cadmium resistance ion of 1: 32000 times of dilution of 10 μ L then; Mixing is hatched 0.5～2h for 37 ℃; Wash micropore with cleansing solution then;

(2) each micropore adds the goat anti-mouse igg antibody of 100 μ L with the HRP mark of dilution dilution in 1: 4000, hatches 0.5～1h for 37 ℃; Wash micropore with cleansing solution then;

(3) each micropore adds 100 μ L colour developing liquid tmb substrate working fluid, reaction 10～20min;

(4) each micropore adds 50 μ L stop buffer 2M H ₂SO ₄Cessation reaction, and vibration mixing;

(5) be determined at absorbance under the 450nm wavelength with enzyme connection readout instrument;

(6) gained titer and sample absorbance being multiply by 100% again divided by the absorbance of 0 standard, is ordinate with this titer calculated value, and the logarithm of concentration of cadmium ions is a horizontal ordinate drawing standard curve; B/B according to each sample ₀Value is read corresponding concentration of cadmium ions from typical curve, multiply by corresponding extension rate again, calculates concentration of cadmium ions actual in the sample; Described dilution and cleansing solution are the 0.01M phosphate buffer that PBS-T promptly contains 0.05% Tween-20.

3. the detection method of a kind of cadmium ion indirect competitive ELISA according to claim 2 is characterized in that: the pre-service as follows of described fish and shellfish sample extracting solution: every gram flesh of fish or shellfish meat sample add the 2mL ethyl acetate, fully smash even matter; The centrifugal 10min of 4000rpm/min gets supernatant nitrogen stream under 50 ℃ of water-baths and dries up sample; Mix extraction vortex 1min with equal-volume cyclohexane and distilled water, get the cyclohexane layer, every gram sample adds 12.5 μ L 1M HCl, and vortex 1min leaves standstill 30min again; Add the distilled water of 2 times of HCl volumes, 8000rpm/min discards the upper strata cyclohexane, draws subnatant, adjusts to pH7.0～8.0 with 1N NaOH, after 10 times of diluted, promptly gets the fish and shellfish sample extracting solution.

4. the detection method of a kind of cadmium ion indirect competitive ELISA according to claim 2 is characterized in that: the pre-bag of described antigen is prepared as follows by bar:

(1) bag quilt: to be cushioned liquid be that the 0.05M sodium carbonate buffer of pH9.6 is diluted to 0.2 μ g/mL with Cd-iEDTA-BSA with wrapping, and every micropore 100 μ L join in the polystyrene micropore plate hole, 4 ℃ of bags spent the night or 37 ℃ of bags by 2～3h; Described Cd-iEDTA-BSA is coupled to carrier protein BSA with iEDTA with cadmium ion and goes up acquisition;

(2) washing: after pouring out in the micropore plate hole bag and being cushioned liquid, 200 μ l cleansing solutions are added in each micropore; Pour out the liquid in the hole after 3～5 minutes once more, remove the liquid in the micropore fully; Described cleansing solution is the 0.01M phosphate buffer that contains 0.05% Tween-20;

(3) sealing: add the 0.01MPBS that 150～200 μ L contain 0.5%～3%BSA at the every micropore of microwell plate, 37 ℃ of sealing 0.5h～1.5h;

(4) set by step (2) washing micropore is 3～5 times;

(5) add 20% sucrose phosphate buffer room temperature protection 3 hours;

(6) drying: after the microwell plate drying, promptly obtain antigen and wrap by bar in advance.

5. the detection method of a kind of cadmium ion indirect competitive ELISA according to claim 2, the monoclonal antibody that it is characterized in that described Cadmium resistance ion is prepared from as follows: with iEDTA cadmium ion is coupled on the hemocyanin, mix not formula Freund immunity BALB/c mouse, getting immune mouse spleen cell and SP2/0 myeloma cell merges, filter out the positive cell strain and the enlarged culture of stably excreting Cadmium resistance ion monoclonal antibody, the injection cell advances in the mouse body to induce ascites, and purifying obtains the monoclonal antibody of Cadmium resistance ion.

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