CN104076154A - Enzyme linked immunosorbent assay kit detecting folic acid and application thereof - Google Patents
Enzyme linked immunosorbent assay kit detecting folic acid and application thereof Download PDFInfo
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Abstract
The invention provides an enzyme linked immunosorbent assay kit detecting folic acid. The enzyme linked immunosorbent assay kit comprises an elisa plate coated with a coating antigen, a folic acid antibody working solution, an enzyme marker, a folic acid standard substance solution, a substrate developing solution, a stop solution, a scrubbing solution and a compound solution. The coating antigen is a folic acid coupling antigen, and the enzyme marker is an enzyme marking antibody. The invention further discloses a method applying the enzyme linked immunosorbent assay kit detecting the folic acid. The method comprises the steps that sample pretreatment is carried out at first, then the kit is used for detection, and finally the detection result is analyzed. The enzyme linked immunosorbent assay kit can be used for detecting the content of the folic acid in finished milk, milk powder and cereals such as rice, millet, corns, soybeans and flour. The kit is easy and convenient to operate, low in cost, high in sensitivity, capable of monitoring the folic acid on site, and suitable for screening a large number of samples.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, be specifically related to a kind of enzyme linked immunological kit for detection of folic acid, it is particularly suitable for the detection of finished milk, milk powder and cereal (rice, millet, corn, soya bean, flour etc.) Folic Acid.
Background technology
The compound that folic acid (Folic Acid) is comprised of petrin pyridine, p-aminobenzoic acid and glutamic acid etc. is a kind of water-soluble B family vitamin.Folic acid has important trophism to human body, lacks folic acid and can cause macrocytic anemia and leukopenia, also can cause weak, irritability, have no appetite and psychotic symptoms.In addition, folic acid is even more important to pregnant woman, as lacked folic acid at conceived in 3 months, can cause fetal neural tube developmental defect, thereby increase the incidence of splitting animal brains, anencephalus.The effect of folic acid is now familiar with by people just gradually, many countries have added consciously folic acid in people's diet, in June, 2009, the 21 Ministry of Public Health announced to start 6 public health programs, augment folic acid prevention neural tube defects project, this has just proposed higher requirement for the detection of folic acid.
Folic acid detection method comprises at present: GC-MS (GC-MS), Liquid Chromatography-Tandem Mass Spectrometry method (LC-MS-MS), liquid phase chromatography, colourimetry, thin layer chromatography, microbial method and isotope radioimmunology etc., instrumental method is sensitive, accurate, high specificity, degree of separation are good, can measure multi-medicament simultaneously, but need the complex pretreatment of expensive instrument, sample, loaded down with trivial details cost time-consuming, that detect higher, can not execute-in-place, and need professional to operate, so limited its application.By comparison, enzyme-linked immunoassay method has the advantages such as screening detection that specificity is good, highly sensitive, easy and simple to handle, testing cost is low, be suitable for batch sample, and equipment needed thereby is few, simple to operate easy to learn, with low cost, can meet better China's food enterprise, government function supervision department etc. and carry out testing.
Summary of the invention
The object of the present invention is to provide a kind of simple in structure, easy to use, low price, the portable enzyme linked immunological kit detecting for folic acid, and a kind of efficient, accurate, easy, qualitative and quantitative analysis method of being suitable for batch samples screening is provided.
Kit of the present invention, it comprises: the ELISA Plate, folic acid specific antibody, enzyme labeling thing, folic acid standard solution, substrate nitrite ion, stop buffer, cleansing solution, the redissolution liquid that are coated with coating antigen, described coating antigen is folacin coupled antigen, and described enzyme labeling thing is enzyme labeling antiantibody.
Described folacin coupled antigen is to be obtained by folic acid and carrier protein couplet, and described carrier protein can be mouse haemocyanin, thyroprotein, bovine serum albumin(BSA), rabbit anteserum albumen, human albumin, ovalbumin, hemocyanin or fibrinogen.
Described folic acid specific antibody is to using folacin coupled antigen to prepare as immunogene, and described folic acid specific antibody can be folic acid monoclonal antibody or folic acid polyclonal antibody, wherein preferred folic acid monoclonal antibody.
Described antiantibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody, wherein preferred sheep anti mouse antiantibody.
The marker enzyme of described enzyme labeling thing is that horseradish peroxidase or bacterium are extracted alkaline phosphatase, wherein preferred horseradish peroxidase; The antiantibody of enzyme labeling adopts glutaraldehyde method or sodium periodate method that marker enzyme and antiantibody are carried out to coupling and obtains.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises folic acid standard solution, substrate nitrite ion, stop buffer, cleansing solution, redissolution liquid.
6 bottles of described folic acid standard solutions, concentration is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L, 81 μ g/L.
When marker enzyme is horseradish peroxidase, described substrate nitrite ion is comprised of substrate solution A liquid and substrate solution B liquid, A liquid is hydrogen peroxide or urea peroxide, and B liquid is o-phenylenediamine or tetramethyl benzidine, the sulfuric acid that described stop buffer is 1 ~ 2mol/L or hydrochloride buffer; When marker enzyme is bacterium extraction alkaline phosphatase, described substrate nitrite ion is to nitro phosphate buffer, and described stop buffer is 1 ~ 2mol/L sodium hydroxide solution.
It is 7.4 that described cleansing solution is preferably pH value, the phosphate buffer that contains 0.5% ~ 1.0% Tween-20, biological preservative, 0.1 ~ 0.3mol/L, and described number percent is percent weight in volume.
The pH value of described redissolution liquid is 7.0, contains sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride.
Wherein in ELISA Plate preparation process, coated damping fluid used is that pH value is 9.6, the carbonate buffer solution of 0.05mol/L, confining liquid is that pH value is 7.1 ~ 7.5, the phosphate buffer that contains 1% ~ 3% casein, 0.1 ~ 0.3mol/L, and described number percent is percent weight in volume.
In the present invention, the preparation process of ELISA Plate is: with coated damping fluid, coating antigen is diluted to 20 μ g/ml, every hole adds 100 μ l, 37 ℃ of lucifuges are hatched 2h or 4 ℃ and are spent the night, and liquid in the hole of inclining, with cleansing solution washing 2 times, each 30s, pat dry, then in every hole, add 150 ~ 200 μ l confining liquids, 37 ℃ of lucifuges are hatched 1 ~ 2h, the liquid in hole that inclines pats dry, and preserves after dry with the vacuum seal of aluminium film.
Detection principle of the present invention is:
Pre-coated folacin coupled antigen on capillary strip, add after sample solution or standard solution, add again folic acid specific antibody solution, the anti-folic acid specific antibody of folacin coupled antigenic competition being coated with in folic acid in sample and ELISA Plate, add enzyme labeling antiantibody to carry out amplification, with nitrite ion colour developing, the content of sample absorbance and folic acid is negative correlation, relatively can obtain the content of sample Folic Acid with typical curve; Simultaneously according to the depth of color in ELISA Plate, with the comparison of the standard solution color of the series concentration concentration range of judgement sample Folic Acid roughly.
The present invention also provides a kind of method that above-mentioned enzyme linked immunological kit detects folic acid of applying, and it comprises step:
(1) sample pre-treatments;
(2) with kit, detect;
(3) analyzing and testing result.
The enzyme linked immunological kit that the present invention detects folic acid mainly adopts competitive ELISA method qualitative or quantitatively detect the content of sample Folic Acid; Pre-treatment requirement to sample is low, and sample pretreatment process is simple, simultaneously fast detecting batch samples; Main agents provides with the form of working fluid, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, degree of accuracy is high, accuracy high.Enzyme linked immunological kit of the present invention, simple in structure, easy to use, low price, carrying convenience, detection method be efficient, accurate, easy, be suitable for the qualitative, quantitative of batch samples screening.
Accompanying drawing explanation
Fig. 1: kit canonical plotting
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only for the present invention is described, and be not used for limiting the scope of the invention.
The preparation of embodiment 1 reagent constituents
1, the preparation of antigen
Immunogene preparation---folic acid and bovine serum albumin(BSA) (BSA) coupling obtains immunogene.
Get 8mg folic acid, be dissolved in 1ml dimethyl formamide (DMF); Get after 15mg carbodiimides (EDC) fully dissolves with 0.2ml water and add in folic acid solution, under room temperature, stir 24h, can obtain reactant liquor A; Take BSA 30mg, make it to be fully dissolved in 3ml 0.1mol/L CB(pH 9.6) in, reactant liquor A is dropwise slowly added drop-wise in protein solution, and stir 24h under room temperature, with 4 ℃ of dialysis 3d of 0.01mol/L PBS, change dislysate every day 3 times, to remove unreacted small-molecule substance; Packing, saves backup in-20 ℃.
Coating antigen preparation---folic acid and ovalbumin (OVA) coupling obtains immunogene.
Get 10mg folic acid, be dissolved in 1ml DMF; Get glutaraldehyde water solution 0.1ml and add in folic acid solution, under room temperature, stir 24h, can obtain reactant liquor A; Take OVA 30mg, make it to be fully dissolved in 3ml 0.1mol/L CB(pH 9.6) in, reactant liquor A is dropwise slowly added drop-wise in protein solution, and stir 24h under room temperature, sodium borohydride aqueous solution 0.2ml reduction reaction 4h with 5mol/L, with 4 ℃ of dialysis 3d of 0.01mol/L PBS, change dislysate every day 3 times, to remove unreacted small-molecule substance; Packing, saves backup in-20 ℃.
2, the preparation of folic acid monoclonal antibody
Animal immune: the immunogene that above-mentioned steps is obtained is injected in Balb/c Mice Body, immunizing dose is 150 μ g/, makes it produce antiserum.
Fusion of Cells and cloning: after mice serum measurement result is higher, get its splenocyte, in 8:1(quantitative proportion) ratio and SP2/0 myeloma cell's fusion, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay to carry out cloning to positive hole, until obtain secreting the hybridoma cell strain of folic acid monoclonal antibody.
Cell cryopreservation and recovery: monoclonal hybridoma strain is made to 1 * 10 with cryopreserving liquid
6the cell suspension of individual/ml is preserved for a long time in liquid nitrogen.During recovery, take out cryopreservation tube, put into immediately 37 ℃ of water-bath middling speeds and melt, after centrifugal removal cryopreserving liquid, move in culture flask and cultivate.
The production of monoclonal antibody and purifying: Balb/c mouse peritoneal is only injected to sterilizing paraffin oil 0.5ml/, and within 7 days, pneumoretroperitoneum is injected stable monoclonal hybridoma strain 5 * 10
5individual/only, after 7 days, to gather ascites.By sad-saturated ammonium sulfate method, carry out ascites and purify ,-20 ℃ of preservations.
3, the preparation of sheep anti mouse antiantibody
Take sheep as immune animal, take mouse source antibody as immunogen immune pathogen-free domestic sheep, obtain sheep anti mouse antiantibody.
4, the preparation of enzyme labeling antiantibody
Adopt the sodium periodate method after improvement to carry out coupling sheep anti mouse antiantibody and horseradish peroxidase (HRP).In traditional sodium periodate method requirement reaction system, the molar concentration rate of enzyme and antibody is 4:1, because horseradish peroxidase produces many sites of being combined with antibody under strong oxidation, the horseradish peroxidase molecule of activation has served as the bridge that connects each molecule like this, reduce the enzymatic activity of enzyme labeling thing, in the conjugate that makes to prepare, be mixed with many condensates.In order to address this problem, we improve traditional method, that is:
(1) saved amino closed process, because can produce self amino amino reality connecting seldom;
(2) the volumetric molar concentration ratio that reduces horseradish peroxidase and antibody is to 2:1, and the method after improvement is easier than traditional method, and the loss of enzymatic activity is reduced.
5, the preparation of ELISA Plate
With coated damping fluid, coating antigen is diluted to 20 μ g/ml, every hole adds 100 μ l, 37 ℃ of lucifuges are hatched 2h, and liquid in the hole of inclining, with cleansing solution washing 2 times, each 30s, pat dry, then in every hole, add 200 μ l confining liquids, 37 ℃ of lucifuges are hatched 2h, the liquid in hole that inclines pats dry, and preserves after dry with the vacuum seal of aluminium film.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of folic acid
Set up the enzyme linked immunological kit that detects folic acid, make it comprise following component:
(1) ELISA Plate of coated folic acid coupled antigen;
(2) folic acid standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L, 81 μ g/L;
(3) folic acid monoclonal antibody working fluid;
(4) use the sheep anti mouse antiantibody of horseradish peroxidase-labeled;
(5) substrate nitrite ion is comprised of A liquid and B liquid, and A liquid is hydrogen peroxide, and B liquid is tetramethyl benzidine;
(6) stop buffer is 2mol/L sulfuric acid;
(7) the pH value of cleansing solution is 7.4, the phosphate buffer that contains 0.5% ~ 1.0% Tween-20, biological preservative, 0.1 ~ 0.3mol/L, and described number percent is percent weight in volume;
(8) the pH value of redissolution liquid is 7.0, contains sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride.
The detection of embodiment 3 finished milks, milk powder and cereal (rice, millet, corn, soya bean, flour etc.) sample Folic Acid
1, sample pre-treatments
Finished milk Sample pretreatment method: measure 100 μ l milk samples to 2ml polystyrene centrifuge tube; Add respectively 50 μ l 2% sodium chloride and 850 μ l redissolution working fluid; With vortex instrument 3000r/min whirling motion 30s, its sample is fully mixed; Get 50 μ l for analyzing.
Milk powder, cereal (rice, millet, corn, soya bean, flour etc.) Sample pretreatment method: take sample after 0.5g ± 0.05g homogeneous to 10ml polystyrene centrifuge tube, add 5ml 2% sodium chloride; With vortex instrument 3000r/min whirling motion 2min, its sample is fully mixed; Standing 5min; Pipette 100 μ l supernatants to 2ml polystyrene centrifuge tube, add 900 μ l redissolution working fluids, with vortex instrument 3000r/min whirling motion 30s, sample is fully mixed; Get 50 μ l for analyzing.
2, with kit, detect
To being coated with, in the ELISA Plate micropore of folacin coupled antigen, add folic acid standard solution or through the sample solution 50 μ l/ holes of pre-treatment, the sheep anti mouse antiantibody 50 μ l/ holes that add again horseradish peroxidase-labeled, finally add folic acid monoclonal antibody 50 μ l/ holes, vibration mixes gently, with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate; Pour out liquid in hole, every hole adds 250 μ l cleansing solutions fully to wash 4 ~ 5 times, and every minor tick 10s, pats dry with thieving paper; Every hole adds substrate nitrite ion A liquid hydrogen peroxide 50 μ l, substrate nitrite ion B liquid tetramethyl benzidine (TMB) 50 μ l, vibration mixes gently, with the rearmounted 25 ℃ of lucifuges colour developing of cover plate membrane cover plate 15min, every hole adds stop buffer 2mol/L sulfuric acid 50 μ l, vibration mixes gently, by microplate reader wavelength set, at 450nm place, measures every hole absorbance (OD value).
3, Analysis of test results
The mean light absorbency value (diplopore) of standard items or sample is divided by the mean light absorbency value of first standard items (0 standard), then is multiplied by 100%, obtains the percentage absorbance of standard items or sample.The logarithm of folic acid standard items concentration (μ g/L) of take is horizontal ordinate, and the standard items percentage absorptance of take is ordinate, drawing standard curve map, as shown in Figure 1.By in the percentage absorptance substitution typical curve of sample, from typical curve, read the corresponding concentration of sample, be multiplied by the actual concentrations that its corresponding extension rate is sample Folic Acid.
Definite test of embodiment 4 folic acid enzyme linked immunological kit technical parameters
1, kit sensitivity and detectability
According to conventional method, measure kit sensitivity, kit typical curve minimum point is 1 μ g/L, and the scope of typical curve is 1 ~ 81 μ g/L, IC
50(50% inhibition concentration) domain of walker is 2 ~ 4 μ g/L; 20 parts of blank samples are detected, from typical curve, find the concentration corresponding to each percentage absorbance, mean value with 20 parts of concentration of specimens adds that 3 times of standard deviations represent detectability, result obtains the method the detection of finished milk sample is limited to 10 μ g/L, and the detectability of milk powder sample and cereal sample is to 100 μ g/L.
2, sample preci-sion and accuracy test
Using the recovery as accuracy estimating index, and the testing result relative standard deviation (RSD%) of a certain concentration sample of replication is as precision evaluation index.Computing formula is: the recovery (%)=practical measurement value/theoretical value * 100%, the interpolation concentration that wherein theoretical value is sample; Relative standard deviation RSD%=SD/X * 100%, wherein SD is standard deviation, the mean value that X is determination data.
By 100 μ g/L(μ g/kg), 300 μ g/L(μ g/kg), 500 μ g/L(μ g/kg) three concentration folic acid add to reclaim to finished milk, milk powder and grain sample respectively and measure, each sample do 4 parallel, with three batches of different reagent, measure, average recovery rate and the precision of calculation sample the results are shown in following table.
Table 1 finished milk precision and accuracy test
Table 2 milk powder precision and accuracy test
Table 3 grain precision degree and accuracy test
With 100,300,500 μ g/L(μ g/kg) folic acid of three concentration adds finished milk, milk powder and grain sample respectively, and average recovery rate is between 81.1% ~ 115.6%; Batch in, batch between relative standard deviation be all less than 10%.
3, kit stability test
Kit preservation condition is 2 ~ 8 ℃, and through the mensuration of 12 months, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, folic acid added practical measurement value all within normal range.Consideration, in transportation and use procedure, has improper preservation condition and occurs, kit is placed 7 days under 37 ℃ of preservation conditions, carries out accelerated deterioration experiment, and result shows that this kit indices meets the requirements completely.Consider that the freezing situation of kit occurs, kit is put into-20 ℃ of refrigerator freezings 7 days, measurement result also shows that kit indices is completely normal.From above result, can show that kit can at least preserve more than 12 months at 2 ~ 8 ℃.
Claims (9)
1. an enzyme linked immunological kit that detects folic acid, it is characterized in that comprising: the ELISA Plate, folic acid specific antibody, enzyme labeling thing, folic acid standard solution, substrate nitrite ion, stop buffer, cleansing solution, the redissolution liquid that are coated with coating antigen, described coating antigen is folacin coupled antigen, and described enzyme labeling thing is enzyme labeling antiantibody.
2. kit as claimed in claim 1, it is characterized in that described folacin coupled antigen is to be obtained by folic acid and carrier protein couplet, described carrier protein can be mouse haemocyanin, thyroprotein, bovine serum albumin(BSA), rabbit anteserum albumen, human albumin, ovalbumin, hemocyanin or fibrinogen.
3. kit as claimed in claim 1, is characterized in that described folic acid specific antibody is to using folacin coupled antigen to prepare as immunogene, and described folic acid specific antibody is folic acid monoclonal antibody or folic acid polyclonal antibody.
4. kit as claimed in claim 1, is characterized in that described antiantibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody.
5. kit as claimed in claim 1, the marker enzyme that it is characterized in that described enzyme labeling thing is that horseradish peroxidase or bacterium are extracted alkaline phosphatase, when marker enzyme is horseradish peroxidase, substrate nitrite ion A liquid is hydrogen peroxide or urea peroxide, substrate nitrite ion B liquid is o-phenylenediamine or tetramethyl benzidine, the sulfuric acid that stop buffer is 1 ~ 2mol/L or hydrochloride buffer; When marker enzyme is bacterium extraction alkaline phosphatase, substrate nitrite ion is to nitro phosphate buffer, and stop buffer is 1 ~ 2mol/L NaOH.
6. kit as claimed in claim 1, is characterized in that described cleansing solution is that pH value is 7.4, the phosphate buffer that contains 0.5% ~ 1.0% Tween-20,0.01 ‰ ~ 0.03 ‰ sodium azide antiseptic, 0.1 ~ 0.3mol/L.
7. kit as claimed in claim 1, is characterized in that described redissolution liquid is that pH value is 7.0, contains sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride.
8. kit as claimed in claim 1, is characterized in that the concentration of described folic acid standard solution is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L, 81 μ g/L.
9. a method that detects sample Folic Acid, comprises step:
(1) sample pre-treatments;
(2) with the kit described in claim 1 ~ 7 any one, detect;
(3) analyzing and testing result.
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| CN109061172A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | It is a kind of detect butralin enzyme linked immunological kit and its application |
| CN111458525A (en) * | 2020-01-16 | 2020-07-28 | 卢氏实验室公司 | Test strip and kit for detecting human body physiological substance concentration and preparation method |
| CN111234024A (en) * | 2020-01-17 | 2020-06-05 | 上海领潮生物新材料有限公司 | Mouse-derived anti-human FA monoclonal antibody and preparation method thereof |
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