CN109633159A - A kind of method for detecting content of melamine and its dedicated colloid gold test paper - Google Patents
A kind of method for detecting content of melamine and its dedicated colloid gold test paper Download PDFInfo
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Abstract
The method and its dedicated colloid gold test paper that the invention discloses a kind of for detecting content of melamine.In the colloid gold test paper, the colloidal gold of melamine antibody label is coated on quenching antibody bonding pad, Quality Control line position on reaction film is coated with the antibody of the melamine antibody, and detection line position is coated with the antigen of the melamine antibody, is coated with fluorescent material on backboard；The melamine antibody is the antibody prepared using the conjugate of formula (I) compound represented and carrier protein as immunogene.It is demonstrated experimentally that can quickly detect the content of melamine in milk sample using colloid gold test paper provided by the present invention, easy to operate, time-consuming short, accuracy rate is high, and high sensitivity, and minimum detection limit is up to 5 μ g/L.Colloid gold test paper provided by the present invention has important application value in quickly detection content of melamine.
Description
Technical field
The invention belongs to technical field of food safety detection, and in particular to a method of for detecting content of melamine
And its dedicated colloid gold test paper.
Background technique
Melamine is a kind of triazines nitrogen heterocyclic ring organic compound, important azacyclo- Organic Chemicals, referred to as
Triamine is commonly called as melamine or extract of protein, molecular formula C3N6H6, molecular weight 126.12.Due to nitrogenous in the molecular formula of melamine
Amount is 66% or so, and the average nitrogen content of protein is 16% or so, therefore, because dairy industry protein content test side
The defect of method, melamine is often used as food additives by illegal businessman, to promote the protein content index in food inspection.
Although the toxicity of melamine is humbleer, long-term intake still can not be ignored.International Chemical Safety Agency in 1994 and
" international chemicals security manual " world third Juan He chemicals that European Commission EC compiles in collaboration with safe card explanation, it is long-term or
Largely intake melamine may have an impact kidney and bladder repeatedly, cause to generate calculus.
Currently, the detection method of melamine residual mainly uses instrumental method and immunoassay in dairy products.Instrument
Analytic approach includes high performance liquid chromatography (HPLC) method, gas chromatography mass spectrometry (GC-MS) method and LC-MS (LC-MS/MS) method.HPLC method
It is quantitatively accurate to have many advantages, such as, but pre-treatment is relative complex when detecting, and it is low equal scarce to there is method sensitivity in practical applications
Point.GC-MS method can carry out confirmation detection and high sensitivity, but pretreatment process is complicated, need to perform the derivatization processing.LC-MS/MS
Method has many advantages, such as that high sensitivity, selectivity are good, qualitative, quantitative is accurate, strong antijamming capability, but there is also pre-treatments for this method
The disadvantages of process is complicated, time-consuming long, at high cost.Immunoassay includes enzyme linked immunosorbent assay analysis method and colloid gold immune
Chromatography has the advantages that quick and convenient, easy to operate, cost is relatively low, screening sample efficiencies are high, but enzyme linked immunosorbent assay
Antibody used and enzyme belong to bioactive substance, need cryo-conservation, stability is poor；Colloidal gold immunity chromatography detection sensitivity
It is low, it is mainly used for quickly screening analysis, and enzyme linked immunosorbent assay analysis method and colloidal gold immunity chromatography have non-specificity
Easily there is the problem of false positive in reaction, testing result.Therefore, it is badly in need of formulating not only easy to be quick but also energy accurate quantitative analysis detection side
Method improves status, fills up the missing of original rapid detection method, promoted and standardize entire fast inspection industry technical level and
Product quality.
Summary of the invention
The purpose of the present invention is quickly detect the content of melamine in solution to be measured.
The present invention protects a kind of for detecting the colloid gold test paper of content of melamine first, quenches on antibody bonding pad
It is coated with the colloidal gold of melamine antibody label, the Quality Control line position on reaction film is coated with the anti-of the melamine antibody
Body, detection line position are coated with the antigen of the melamine antibody, spray fluorescent material on backboard or under reaction film；
The melamine antibody is to be prepared using the conjugate of formula (I) compound represented and carrier protein as immunogene
Antibody；
In above-mentioned colloid gold test paper, the melamine antibody can be a1) or a2):
A1) conjugate of formula (I) compound represented and carrier protein is as miscellaneous through body cell after immunogen immune animal
Hand over the melamine monoclonal antibody obtained；
A2) conjugate of formula (I) compound represented and carrier protein obtains animal booster immunization as immunogene
Melamine polyclonal antibody.
The animal can be any one of mouse, rabbit, goat, sheep or horse.
In above-mentioned colloid gold test paper, the melamine monoclonal antibody is made of heavy chain and light chain.The heavy chain can
The amino acid sequence for becoming area can be as shown in the sequence 1 of sequence table.The amino acid sequence of the variable region of the light chain can be such as sequence table
Sequence 2 shown in.
In above-mentioned colloid gold test paper, the antibody of the melamine antibody can be the IgG antibody of sheep anti mouse.The sheep anti mouse
IgG antibody concretely U.S. Jackson ImmunoResearch company product.
In above-mentioned colloid gold test paper, the antigen of the melamine antibody can be formula (I) compound represented and carrier egg
White conjugate.
Any of the above-described carrier protein can be bovine serum albumin(BSA) or oralbumin.
In the above method, formula (I) compound represented and carrier protein are keyed by amide.The amido bond is formula
(I) carboxyl of compound represented is formed by the amino on active ester and carrier protein.
The conjugate can be melamine-bovine serum albumin(BSA) conjugate (compound shown in formula (II)) or melamine
Amine-oralbumin conjugate (compound shown in formula (III)).
In above-mentioned colloid gold test paper, the fluorescent material can be organic fluorescent dye (such as RB 200).
In above-mentioned colloid gold test paper, in addition to spraying fluorescent material on backboard or under reaction film, it can also be coated with ProClin 300
(as preservative).
The specific steps of " fluorescent material is sprayed on backboard " can be as follows:
(1) RB 200 is taken, the Tris-HCl buffer solution for being 0.05-0.2M (such as 0.2M) with concentration obtains
Concentration is the RB 200 solution of 0.05-0.2% (such as 0.01%) (m/v).
(2) after completing step (1), the RB 200 solution is taken, is added ProClin 300 (as preservative),
Obtain working solution；In working solution, the concentration of ProClin 300 is 0.01-0.03% (such as 0.02%) (v/v).
(3) by the uniform spraying of working solution that step (2) obtains in the backboard in PVC board, obtaining spraying fluorescent material.To keep away
Exempt from fluorescent material to fall off in storage and use process, after the uniform spraying of working solution is in PVC board, can also cover light transmission above
Good plastic packaging film.
In above-mentioned colloid gold test paper, the reaction film can be nitrocellulose filter.
In above-mentioned colloid gold test paper, the material of the quenching antibody bonding pad can be glass fibre element film.
In above-mentioned colloid gold test paper, the Quality Control line position, the concentration for being coated with the antibody of the melamine antibody can be
0.1-0.3mg/mL (such as 0.2mg/mL).In an embodiment of the present invention, the PBS buffer solution of pH7.4,0.1-1mM are specifically used
Sheep anti mouse secondary antibody is diluted, the Quality Control that concentration is 0.2mg/mL is obtained and draws film liquid.Drawing film amount can be 0.5-0.6 μ L/cm.
In above-mentioned colloid gold test paper, the detection line position, the concentration for being coated with the antigen of the melamine antibody can be
0.1-0.2mg/mL (such as 0.15mg/mL).In an embodiment of the present invention, specifically dilute with the PBS buffer solution of pH7.4,150mM
Melamine-bovine serum albumin(BSA) conjugate is released, the detection that concentration is 0.15mg/mL is obtained and draws film liquid.Drawing film amount can be 0.5-
0.6μL/cm。
In above-mentioned colloid gold test paper, the spacing of nature controlling line and detection line can be 2-4mm (such as 2-3mm, 3-4mm, 2mm, 3mm
Or 4mm).
The present invention, which also protects, a kind of to be detected melamine in solution to be measured using any of the above-described colloid gold test paper and contains
The method of amount, it may include following steps:
(1) sample pad of any of the above-described colloid gold test paper is immersed into solution to be measured, stands 10min or more, then
Colloid gold test paper is inserted into fluorescence immunoassay quantitative analysis instrument, obtains F1/F2 ratio；
Fluorescence intensity (F1)/detection line fluorescence intensity of the blank area of F1/F2 ratio=between nature controlling line and detection line
(F2)；
(2) sample pad of any of the above-described colloid gold test paper is immersed into melamine standard solution, stand 10min with
On, colloid gold test paper is then inserted into fluorescence immunoassay quantitative analysis instrument, obtains corresponding F1/F2 ratio；
(3) standard is drawn according to the concentration of melamine in the melamine standard solution and corresponding F1/F2 ratio
Curve, the F1/F2 ratio for then obtaining the step (1) substitute into the standard curve, obtain melamine in solution to be measured
Content.
In the above method, the solution to be measured can be milk sample.
It is demonstrated experimentally that can quickly detect containing for melamine in milk sample using colloid gold test paper provided by the present invention
Amount, easy to operate, time-consuming short, accuracy rate is high, and high sensitivity, and minimum detection limit is up to 5 μ g/L.Colloid provided by the present invention
Golden test paper has important application value in quickly detection content of melamine.
Detailed description of the invention
Fig. 1 is the structural schematic diagram for detecting the colloid gold test paper of melamine.
Fig. 2 is the standard curve of melamine.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
Test material as used in the following examples is unless otherwise specified to buy from routine biochemistry reagent shop
It arrives.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
Fluorescence intensity (F1)/detection line fluorescence intensity of the blank area of F1/F2 ratio=between nature controlling line and detection line
(F2)
Embodiment 1, detect melamine colloid gold test paper preparation
One, the preparation of antigen
1, the preparation of compound (i.e. melamine hapten) shown in formula (I)
Preparing compound (i.e. melamine hapten) shown in formula (I), specific step is as follows:
(1) chloro- 4,6 diamino triazine of 100mg 2- is substantially soluble in 10mL ethyl alcohol, obtains solution 1.
(2) 130mg NapABA is substantially soluble in the sodium bicarbonate aqueous solution that 10mL concentration is 0.1mol/L,
Obtain solution 2.
(3) after completing step (1) and (2), it is slowly added to solution 1 in Xiang Suoshu solution 2, mixes, then 70 DEG C of stirrings are anti-
It should for 24 hours.
(4) reaction system of step (3) is taken into, the aqueous hydrochloric acid solution adjusting pH value that concentration is 0.1mol/L, which is added, is
6.0, precipitating is collected in filtering.
(5) precipitating for taking step (4) to collect, is added a small amount of water washing, is then dried in vacuo, obtained powder is trimerization
Cyanamide haptens.
2, the preparation of compound shown in formula (II) (i.e. melamine-bovine serum albumin(BSA) conjugate)
Preparing compound (i.e. melamine-bovine serum albumin(BSA) conjugate) shown in formula (II), specific step is as follows:
(1) 40mg melamine hapten is taken, 5mL n,N-Dimethylformamide (DMF) first is added, is sufficiently dissolved；Then
38 μ L tri-n-butylamines are added under condition of ice bath, are stirred to react 10min；22 μ L isobutyl chlorocarbonates are eventually adding, are reacted at room temperature
1h, the melamine hapten solution activated.
(2) it takes 100mg bovine serum albumin(BSA) (BSA), the aqueous sodium carbonate that 10mL concentration is 0.1mol/L is added, sufficiently
Dissolution；Then the melamine hapten solution that a dropping step (1) obtains dropwise, is stirred overnight at room temperature；Finally with pH7.4's
0.01MPBS buffer obtains dialysis product as dialyzate, 4 DEG C of dialysis 72h (period changes dialyzate 6 times).
(3) the dialysis product for taking step (2) to obtain aseptically passes through 0.2 μm of filter membrane, obtains filtrate.
The filtrate is melamine-bovine serum albumin(BSA) conjugate.
Melamine-bovine serum albumin(BSA) conjugate is as melamine immunizing antigen.
3, the preparation of compound shown in formula (III) (i.e. melamine-oralbumin conjugate)
Preparing compound (i.e. melamine-oralbumin conjugate) shown in formula (III), specific step is as follows:
(1) 40mg melamine hapten is taken, 5mL n,N-Dimethylformamide (DMF) first is added, is sufficiently dissolved；Then
38 μ L tri-n-butylamines are added under condition of ice bath, are stirred to react 10min；22 μ L isobutyl chlorocarbonates are eventually adding, are reacted at room temperature
1h, the melamine hapten solution activated.
(2) it takes 100mg oralbumin (OVA), the aqueous sodium carbonate that 10mL concentration is 0.1mol/L is added, it is sufficiently molten
Solution；Then the melamine hapten solution that a dropping step (1) obtains dropwise, is stirred overnight at room temperature；Finally with pH7.4's
0.01MPBS buffer obtains dialysis product as dialyzate, 4 DEG C of dialysis 72h (period changes dialyzate 6 times).
(3) the dialysis product for taking step (2) to obtain aseptically passes through 0.2 μm of filter membrane, obtains filtrate.
The filtrate is melamine-oralbumin conjugate.
Melamine-oralbumin conjugate is as melamine envelope antigen.
Two, the preparation of melamine monoclonal antibody
Balb/c mouse is that the healthy Balb/c female mice of 8 week old (ties up the limited public affairs of tonneau China experimental animal technology in Beijing
The product of department).
Melamine-bovine serum albumin(BSA) conjugate is diluted using the 0.1MPBS buffer of pH7.4, obtains solution first.
1, fundamental immunity
(1) 1mL solution first and 1mL Freund's complete adjuvant (product of sigma company, the U.S.) are mixed, 4 DEG C of stirrings to abundant cream
Change (mixture instill water in not will disperse be considered as it is fully emulsified), the comlete antigen emulsified.
(2) comlete antigen of emulsification, the subcutaneous multi-point injection Balb/c mouse of the nape of the neck are taken, injection dosage is 50-100 μ g/
Only.
2, booster immunization
(1) 1mL solution first and 1mL Freund's incomplete adjuvant (product of sigma company, the U.S.) are mixed, 4 DEG C are stirred to abundant
Emulsification (mixture instill water in not will disperse be considered as it is fully emulsified), the incomplete antigen emulsified.
(2) incomplete antigen for taking emulsification, it is immune to completing the Balb/c mouse progress of step 1 after two weeks 4 times, every time
Immunization interval two weeks.Immune step is as follows every time: taking the incomplete antigen of emulsification, the subcutaneous multi-point injection Balb/c of the nape of the neck is small
Mouse.Injection dosage is 50-100 μ g/ (in terms of BSA).
(3) incomplete antigen of emulsification is taken, the Balb/c mouse of step (2) after two weeks is completed in intraperitoneal injection.Injection dosage
Only (in terms of BSA) for 50-100 μ g/.
3, the acquisition of hybridoma and clone
(1) Balb/c mouse is extractd eyeball bloodletting till death, then takes the spleen of Balb/c mouse by the 4th day for completing step 2
Cell.
(2) after completing step (1), by the splenocyte of Balb/c mouse and SP2/0 myeloma cell, (ratio is (4-8): 1)
Cell fusion is carried out, several fused cells are obtained.
(3) indirect competitive ELISA (melamine-oralbumin conjugate is as envelope antigen) determination step is used
(2) the fused cell supernatant obtained screens positive hole.Cloning is carried out to positive hole using limiting dilution assay, until obtaining
The hybridoma cell strain of stably excreting monoclonal antibody.It is frozen after the hybridoma is expanded culture.
4, the preparation and purification of melamine monoclonal antibody
Melamine monoclonal antibody is prepared using the method for induction animal body endocrine monoclonal antibody.Specific steps
Are as follows:
(1) to every Balb/c mouse peritoneal injection sterilizing paraffin oil 0.5mL.
(2) step (1) is completed after 7-14 days, and the hybridoma that the intraperitoneal injection step 3 of Xiang Suoshu Balb/c mouse obtains is thin
Born of the same parents.The injection dosage of hybridoma is 5 × 105-106A/only.
(3) step (1) is completed after 7-10 days, collects ascites.
(4) ascites for taking step (3) to collect, carries out ascites with octanoic acid-saturated ammonium sulfate method and purifies, obtain melamine list
Clonal antibody.
Through detecting, 1 institute of sequence of the amino acid sequence of the variable region of the heavy chain of melamine monoclonal antibody such as sequence table
Show, the amino acid sequence of the variable region of the light chain of melamine monoclonal antibody is as shown in the sequence 2 of sequence table.
Three, the preparation of the colloid gold test paper of melamine is detected
1, the preparation of antibody bonding pad is quenched
(1) the 0.5-5% citric acid three sodium solution of 2-4mL is added in 1% aqueous solution of chloraurate of the 100mL of boiling,
Obtain colloidal gold solution；Colloid gold particle diameter is about 20-50nm in colloidal gold solution.
(2) 500 μ g melamine monoclonal antibodies are taken, 5mL is diluted to the PBS buffer solution of pH7.4,150mM, obtains list
Clonal antibody dilution.
(3) 10-25g colloidal gold solution is added in the monoclonal antibody dilution for taking step (2) to obtain, and mixes, 4 DEG C of standings
Overnight, gold labeling antibody suspension is obtained.
(4) tiling of glass fibre element film is soaked in gold labeling antibody suspension, takes out 37 DEG C of bakings overnight, being cut into width is
The strip of 18mm, a length of 300mm, as quenching antibody bonding pad.
2, it is coated with the preparation of the nitrocellulose filter of nature controlling line and detection line
(1) melamine-bovine serum albumin(BSA) conjugate is diluted with the PBS buffer solution of pH7.4,150mM, obtaining concentration is
Film liquid is drawn in the detection of 0.15mg/mL.
(2) PBS buffer solution of pH7.4,0.1-1mM dilute sheep anti mouse secondary antibody (U.S. Jackson ImmunoResearch
The product of company), it obtains the Quality Control that concentration is 0.2mg/mL and draws film liquid.
(3) Quality Control stroke film liquid and detection is taken to draw a film liquid, (draw a film amount is 0.5- to uniform stroke of difference on nitrocellulose filter
0.6 μ L/cm), the nature controlling line (C) being separated from each other and detection line (T), room temperature naturally dry are formed, coating nature controlling line and inspection are obtained
The nitrocellulose filter of survey line.It is coated on nature controlling line and the nitrocellulose filter of detection line, the spacing of nature controlling line and detection line is
2-4mm。
3, the preparation of fluorescent back plate
(1) RB 200 is taken, the Tris-HCl buffer solution for being 0.1M with concentration, obtaining concentration is 0.01%
(m/v) RB 200 solution.
(2) after completing step (1), the RB 200 solution is taken, is added ProClin 300 (as preservative),
Obtain working solution；In working solution, the concentration of ProClin 300 is 0.02% (v/v).
(3) the uniform spraying of working solution for obtaining step (2) in PVC board, (keep away by the top covering good plastic packaging film of light transmission
Exempt from fluorescent material to fall off in storage and use process).
4, the assembling of the colloid gold test paper of melamine is detected
The colloid gold test paper of melamine is detected by water absorption pad (i.e. water-absorbent material, such as filter paper), coating nature controlling line and detection
Nitrocellulose filter, quenching antibody bonding pad, sample pad (material is glass fibre cotton) and the fluorescent back plate composition of line.
The assembling steps for detecting the colloid gold test paper of melamine are as follows: by water absorption pad sticking double faced adhesive tape in coating Quality Control
One end (overlap length 0.2cm) of line and the nitrocellulose filter of detection line, then in the nitre of coating nature controlling line and detection line
With one end (overlap length 0.2cm) of sticking double faced adhesive tape quenching antibody bonding pad on the other end of acid cellulose film, will quench
With sticking double faced adhesive tape sample pad (overlap length 0.2cm) on the other end of antibody bonding pad, finally by them with two-sided gluing
(coating nature controlling line and the nitrocellulose filter of detection line are located at the center of fluorescent back plate) is attached in fluorescent back plate, and being cut into width is
The colloid gold test paper of the detection melamine of 4-5mm (specially 4.72mm).It is put after the colloid gold test paper is sealed with plastic packaging bag
Drying box saves backup.
Overlay region in colloid gold test paper ensure that the continuity of sample liquid flowing, occur that reaction can sufficiently, effectively
Ground reduces experimental error.The fluorescence area of fluorescent back plate will discharge fluorescence signal under the action of corresponding exciting light, in colloid
In golden immunochromatography reaction, combine the colloid gold particle of aggregation that can quench the fluorescence signal of the position at detection line and nature controlling line,
The quenching degree of fluorescence signal and the colored intensity of detection line and nature controlling line are positively correlated, to realize the quantitative inspection of melamine
The purpose of survey.
The structural schematic diagram for detecting the colloid gold test paper of melamine is shown in Fig. 1.As needed, it can also be set in sample pad
Set well.
The method that embodiment 2, colloid gold test paper detect the content of melamine in solution to be measured
1, melamine standard curve is drawn
(1) melamine standard items (product of Town in Shanghai spectrum experiment Science and Technology Co., Ltd.) is taken, is dissolved with water and dilute
It releases, obtains melamine standard dilutions；The concentration of melamine standard dilutions is 0 μ g/L, 5 μ g/L, 10 μ g/L, 25
μ g/L, 50 μ g/L or 100 μ g/L.
(2) dilution of 120 μ L melamine standard items is added in sample pad for the colloid gold test paper for taking detection melamine
Colloid gold test paper after standing 10min, is inserted into fluorescence immunoassay quantitative analysis instrument, obtains corresponding F1/F2 ratio by liquid.
(3) using the concentration of melamine standard dilutions as abscissa, corresponding F1/F2 ratio is ordinate, is drawn
Melamine standard curve.
The standard curve of melamine is shown in Fig. 2.
Melamine calibration curve equation are as follows: y=-0.04+ (3.12+0.04)/(1+ (x/7.85)1.24)。
2, the content of melamine in solution to be measured is detected
(1) take detection melamine colloid gold test paper, be added in sample pad 120 μ L solution to be measured (such as liquid milk sample,
Milk power solution), after standing 10min, colloid gold test paper is inserted into fluorescence immunoassay quantitative analysis instrument, obtains the F1/F2 of solution to be measured
Ratio.
(2) the F1/F2 ratio of solution to be measured is substituted into melamine calibration curve equation, obtains the trimerization in solution to be measured
Cyanamide content.
Solution to be measured can be milk sample or milk power solution.
Embodiment 3, detect melamine colloid gold test paper minimum detection limit
1, melamine standard curve is drawn
With step 1 in embodiment 2.
2, the content of melamine in 20 fresh milk samples is detected respectively.
The step of detecting content of melamine in each fresh milk sample is as follows: taking the colloidal gold examination of detection melamine
120 μ L fresh milk samples are added in sample pad for paper, and after standing 10min, colloid gold test paper is inserted into fluorescence immunoassay quantitative analysis
Instrument obtains the F1/F2 ratio of the fresh milk sample；The F1/F2 ratio of the fresh milk sample is substituted into melamine standard curve
Equation obtains the content of melamine in the fresh milk sample.
Testing result is shown in Table 1.
Table 1
| Fresh milk sample number into spectrum | Content of melamine (μ g/L) | Fresh milk sample number into spectrum | Content of melamine (μ g/L) |
| 1 | 1.10 | 11 | 1.56 |
| 2 | 2.30 | 12 | 1.71 |
| 3 | 2.92 | 13 | 2.96 |
| 4 | 1.57 | 14 | 1.35 |
| 5 | 3.25 | 15 | 1.18 |
| 6 | 4.34 | 16 | 1.34 |
| 7 | 1.87 | 17 | 1.28 |
| 8 | 1.40 | 18 | 1.09 |
| 9 | 2.03 | 19 | 2.49 |
| 10 | 2.82 | 20 | 2.55 |
3, after completing step 2, the average and standard deviation of the content of melamine in 20 fresh milk samples is calculated.
The result shows that the average value of the content of melamine in 20 fresh milk samples is 2.06 μ g/L, standard deviation is
0.88μg/L。
4, after completing step 3, the minimum detection limit of colloid gold test paper is further obtained.
Minimum detection limit=average value+3 × standard deviation.
The result shows that the minimum detection of colloid gold test paper is limited to 4.70 μ g/L.For the stability for ensureing product, it is believed that inspection
The colloid gold test paper of melamine is surveyed in fresh milk detection limit about 5 μ g/L.
Embodiment 4, the accuracy for the colloid gold test paper for detecting melamine are tested
One, accuracy experiment one
1, melamine standard curve is drawn
With step 21 in embodiment 2.
2, the content of melamine in solution to be measured is detected
Solution to be measured is the fresh milk containing 5 μ g/L melamines or the fresh milk containing 10 μ g/L melamines.
Experiment, which is repeated 3 times, to be averaged (between criticizing).In repeating every time, each solution to be measured need to do 5 and be averaged in parallel
Value (in criticizing).It is duplicate every time that steps are as follows:
(1) 120 μ L solution to be measured are added in sample pad for the colloid gold test paper for taking detection melamine, stand 10min
Afterwards, colloid gold test paper is inserted into fluorescence immunoassay quantitative analysis instrument, obtains the F1/F2 ratio of solution to be measured.
(2) the F1/F2 ratio of solution to be measured is substituted into melamine calibration curve equation, obtains the trimerization in solution to be measured
Cyanamide content (mean+SD).
(3) rate of recovery (%) and coefficient of variation CV (%) are counted.
The rate of recovery=(measurement average value/addition concentration) × 100%.
Coefficient of variation CV=(measured value standard deviation/measurement average value) × 100%.
Experimental result is shown in Table 2.The result shows that the rate of recovery, between 88.70~109.20%, coefficient of variation CV is below
10%.
Table 2
Two, accuracy experiment two
Solution to be measured is 50 parts of blind samples of fresh milk.The blind sample number consecutively of 50 parts of fresh milks is 1-50.
1, the content of melamine in 50 parts of blind samples of fresh milk is detected using the colloid gold test paper of detection melamine
(1) melamine standard curve is drawn
With step 21 in embodiment 2.
(2) content of melamine in 50 parts of blind samples of fresh milk is detected
(1) colloid gold test paper for taking detection melamine, the 120 blind samples of μ L fresh milk are separately added into in sample pad, are stood
After 10min, colloid gold test paper is inserted into fluorescence immunoassay quantitative analysis instrument, obtains the F1/F2 ratio of solution to be measured.
(2) the F1/F2 ratio of solution to be measured is substituted into melamine calibration curve equation, obtains the trimerization in solution to be measured
Cyanamide content.
The testing result of colloid gold test paper is shown in Table the 2nd column in 3.
2, the liquid chromatogram-according to GBT 22388-2008 " raw milk and melamine in dairy products detection method "
Mass spectrum/mass spectrography is operated, and is detected the melamine in 50 parts of blind samples of fresh milk using gas chromatography-mass spectrum/mass spectrography and is contained
Amount.
Gas chromatography-mass spectrum/mass spectrography testing result is shown in Table the 3rd column in 3.
The result shows that the colloid gold test paper detection content of melamine using detection melamine provided by the invention is gentle
Phase chromatography-mass spectroscopy/mass spectrography content of melamine result is almost the same.Colloid gold test paper provided by the invention detects melamine
Amine content accuracy with higher.
Table 3
Note: melamine is not detected in " ND " expression.
<110>Beijing, Beijing Wdwkbio Biotechnology Co., Ltd feed supervises institute
<120>a kind of method for detecting content of melamine and its dedicated colloid gold test paper
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 107
<212> PRT
<213>artificial sequence
<220>
<223>
<400> 1
Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
1 5 10 15
Ser Ser Ala Ser Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu
20 25 30
Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro
35 40 45
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
50 55 60
Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met
65 70 75 80
Tyr Tyr Cys Ala Arg His Phe Tyr Tyr Tyr Ser Tyr Ala Met Asp Tyr
85 90 95
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
100 105
<210> 2
<211> 117
<212> PRT
<213>artificial sequence
<220>
<223>
<400> 2
Met Ala Trp Ile Ser Leu Ile Leu Ser Leu Leu Ala Leu Ser Ser Gly
1 5 10 15
Gly Ala Gly Ala Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr
20 25 30
Ser Pro Gly Glu Thr Val Gly Glu Thr Cys Arg Ser Ser Thr Gly Ala
35 40 45
Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His
50 55 60
Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val
65 70 75 80
Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr
85 90 95
Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu
100 105 110
Trp Tyr Ser Asn His
115
Claims (10)
1. the colloid gold test paper for detecting content of melamine, it is characterised in that: be coated with trimerization on quenching antibody bonding pad
The colloidal gold of cyanamide antibody label, the Quality Control line position on reaction film are coated with the antibody of the melamine antibody, detection line position
It sets the antigen for being coated with the melamine antibody, sprays fluorescent material on backboard or under reaction film；
The melamine antibody be prepared using the conjugate of formula (I) compound represented and carrier protein as immunogene it is anti-
Body；
2. colloid gold test paper as described in claim 1, it is characterised in that: the melamine antibody is a1) or a2):
A1) conjugate of formula (I) compound represented and carrier protein is obtained as after immunogen immune animal through somatic hybridization
The melamine monoclonal antibody obtained；
A2) conjugate of formula (I) compound represented and carrier protein is as immunogene obtained to animal booster immunization three
Poly cyanamid polyclonal antibody.
3. colloid gold test paper as described in claim 1, it is characterised in that: the antibody of the melamine antibody is sheep anti mouse
IgG antibody.
4. colloid gold test paper as described in claim 1, it is characterised in that: the antigen of the melamine antibody is claim
The conjugate of formula (I) compound represented and carrier protein in 1.
5. the colloid gold test paper as described in Claims 1-4 is any, it is characterised in that: the carrier protein is bovine serum albumin
White or oralbumin.
6. colloid gold test paper as described in claim 1, it is characterised in that: the fluorescent material is RB 200.
7. colloid gold test paper as described in claim 1, it is characterised in that: the reaction film is nitrocellulose filter.
8. colloid gold test paper as described in claim 1, it is characterised in that: the material of the quenching antibody bonding pad is glass fibers
Tie up plain film.
9. a kind of side for detecting content of melamine in solution to be measured using any colloid gold test paper of claim 1 to 8
Method includes the following steps:
(1) sample pad of any colloid gold test paper of claim 1 to 8 is immersed into solution to be measured, stands 10min or more,
Then colloid gold test paper is inserted into fluorescence immunoassay quantitative analysis instrument, obtains F1/F2 ratio；
Fluorescence intensity (F1)/detection line fluorescence intensity (F2) of the blank area of F1/F2 ratio=between nature controlling line and detection line；
(2) sample pad of any colloid gold test paper of claim 1 to 8 is immersed into melamine standard solution, stood
Then colloid gold test paper is inserted into fluorescence immunoassay quantitative analysis instrument, obtains corresponding F1/F2 ratio by 10min or more；
(3) standard curve is drawn according to the concentration of melamine in the melamine standard solution and corresponding F1/F2 ratio,
Then the F1/F2 ratio step (1) obtained substitutes into the standard curve, obtains content of melamine in solution to be measured.
10. method as claimed in claim 9, it is characterised in that: the solution to be measured is milk sample.
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| CN111537715A (en) * | 2020-07-09 | 2020-08-14 | 北京维德维康生物技术有限公司 | Cryptosporidium parvum antibody detection test strip and application thereof |
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| CN101477124A (en) * | 2008-12-17 | 2009-07-08 | 北京望尔康泰生物技术有限公司 | Method for detecting melamine and its special test paper |
| CN102401829A (en) * | 2010-09-17 | 2012-04-04 | 李久彤 | Immune response analysis method based on principle of fluorescent quenching |
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