CN102175878A - ELISA (enzyme linked immunosorbent assay) kit of folic acid - Google Patents
ELISA (enzyme linked immunosorbent assay) kit of folic acid Download PDFInfo
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Abstract
An ELISA (enzyme linked immunosorbent assay) kit of folic acid belongs to the technical field of enzyme linked immunosorbent assay. The reagent components in the kit comprise: an enzyme label plate coated with a folic acid coating antigen (the antigen is prepared by coupling folic acid and ovalbumin); a folic acid polyclonal antibody; a goat anti-rabbit antibody marked by enzyme-labelled bi-antibody which is horseradish peroxidase; a folic acid standard solution; a concentrated phosphate buffer; a concentrated cleaning solution; a substrate colour developing solution A; a substrate colour developing solution B and a stop solution. The ELISA kit provided by the invention performs ELISA by using a polyclonal antibody, wherein IC50=15.5ng/ml. The lowest detecting limit in milk products is 11.9ng/ml; the coefficients of variation between batches and in batches are 11.8%; and the recovery is 89.1-110.3%. The ELISA kit provided by the invention has the characteristics of simple structure, fast operations, accurate detection result and high sensitivity; and the kit can be used for fast detecting folic acids contained in foods, feeds and vitamin products.
Description
Technical field
The present invention relates to a kind of enzyme-linked immunologic detecting kit that is used for detecting the folate content of food, feed and vitamin products, belong to the check and analysis technical field of food and feed ingredient.
Background technology
Folic acid (Folic acid) is a kind of water-soluble B family vitamin, claims Cobastab again
9Or vitamin(e) M, for body cell growth with breed necessary material.Folic acid works with the form of tetrahydrofolic acid in vivo, and tetrahydrofolic acid participates in the synthetic of purine nucleic acid and pyrimidine nucleotide in vivo and transforms.On manufacturing nucleic acid, play the part of important role.The necessary material of human body when utilizing sugar and amino acid.During folic acid deficiency, deoxythymidylic acid, the form of purine nucleotides and amino acid whose change are obstructed, and DNA is synthetic in the cell reduces, and the ripe obstacle that takes place of the division of cell causes megaloblastic anemia.In addition, research finds that also folic acid is even more important to the pregnant woman.As lacking folic acid in 3 months at conceived, can cause the fetal neural tube developmental defect, split animal brains, the incidence of anencephalus thereby increase.
Folic acid extensively is present in the various animals and plants food, but the most of content of the contained folic acid of wholefood is not high, exists with many glutamic acid folic acid form, and absorptivity is not high, and folic acid residence time in human body is not long, and people strengthen the high single glutamate form folic acid of absorptivity usually in food.The U.S. rose and forces to strengthen folic acid in some cereal foods on January 1st, 1998, and FDA stipulates that per 1 kilogram of cereal foods strengthens 1.4 mg folic acid.China strengthens folic acid in infant, pregnant woman, old people food.For fear of hypovitaminosis, nowadays, the manufacturer adds an amount of vitamin by the mode of mixing or spray in food, yet will make it be distributed in not a duck soup in the food equably.In addition, the vitamin content in the necessary mark shelf-life on the label of product.Consider the loss of part vitamin in food production stores, the suggestion of German chemical enterprise association, the vitamin amount that excess is added should not surpass label and indicate 50% of content.Therefore, manufacturer and superintendent office all are necessary the content of vitamin in the product is controlled.
Folic acid is a lot of as its analytical approach of B family vitamin, mainly contains chemical method, microbial method, thin layer chromatography scanning, fluorometry and vapor-phase chromatography etc.These methods are more numerous and diverse, time-consuming, poor reproducibility, detection limit are low, and vapor-phase chromatography easily damages vitamin.Use the report of high performance liquid chromatography (HPLC) mensuration B family vitamin more in recent years.Though the HPLC method has fast, accurately, the characteristics of favorable reproducibility, used instrument is expensive heavy, and needs a large amount of organic solvents, the sample pre-treatments complexity, time-consuming, effort is difficult to use in execute-in-place.Enzyme linked immunosorbent detection method (ELISA) has fast, and is accurately simple and easy, do not need advantages such as the professional operates, and the enzyme-linked immunologic detecting kit of therefore researching and developing a kind of folic acid has direct economic benefit and social benefit.
Summary of the invention
The objective of the invention is provides a kind of enzyme-linked immunologic detecting kit of folic acid at the prior art above shortcomings.
The composition of reagent comprises in the enzyme-linked immunologic detecting kit of folic acid of the present invention:
(1) is coated with the ELISA Plate of folic acid envelope antigen;
(2) polyclonal antibody of folic acid;
(3) ELIAS secondary antibody is the antibody of the goat-anti rabbit of horseradish peroxidase-labeled;
(4) folic acid standard solution;
(5) concentrated phosphoric acid salt buffer;
(6) concentrated cleaning solution;
(7) substrate colour developing liquid A;
(8) substrate colour developing liquid B;
(9) stop buffer.
Wherein: described folic acid envelope antigen obtains folic acid haptens and ovalbumin coupling, and the concentration of folic acid envelope antigen is 1 μ g/ml.
The carrier protein of described conjugate is that molecular weight ranges is bovine serum albumin or the ovalbumin of 6.7KDa～6.8KDa.
The polyclonal antibody of described folic acid is that the conjugate by folic acid and carrier protein prepares as immunogen immune animal (new zealand white rabbit); The working concentration of described folic acid polyclonal antibody is 1:10000～1:20000, preferred 1:20000.
Described ELIAS secondary antibody adopts sodium periodate that horseradish peroxidase and goat anti-rabbit antibody are carried out coupling and obtains; The working concentration of described ELIAS secondary antibody is preferably 1:2000.
Described folic acid standard solution concentration is respectively 3ng/ml, 6 ng/ml, 12.5 ng/ml, 25 ng/ml, 50 ng/ml, 100 ng/ml.
Described concentrated cleaning solution is to contain the pH7.4 of volume fraction 0.05% Tween-20, the phosphate buffer of 0.1mol/L.
Described substrate colour developing liquid A is a TMB solution, and substrate colour developing liquid B is the urea peroxide solution of pH5.0～6.0, and colour developing liquid A mixes with colour developing liquid B equal-volume during use; Stop buffer is the sulfuric acid solution of 2mol/L.
Kit provided by the invention can be used for detecting the folate content in food, feed and the vitamin products.
Principle of the present invention is based on immunological response, the high degree of specificity of antigen, antibody response is combined with the efficient catalytic effect of enzyme to substrate, according to developing the color behind the zymolyte, judge test findings with change color, can do quantitative test through microplate reader.
Advantage of the present invention and beneficial effect:
Enzyme-linked immunologic detecting kit of the present invention has highly sensitive, easy characteristics fast and accurately, can detect folate content in food, feed and vitamin products and play a significant role.
Description of drawings
Fig. 1 is the inhibiting rate curve of folic acid antibody of the present invention.
Fig. 2 is the typical curve of the enzyme-linked immunologic detecting kit of folic acid of the present invention.
Embodiment
The preparation of the synthetic and antibody of embodiment 1, immunogene, envelope antigen
1, immunogenic synthetic
Adopt the EDC method to carry out coupling folic acid and bovine serum albumin (BSA) and obtain immunogene.Specifically may further comprise the steps:
A, take by weighing folic acid 22.7mg(51.5 μ mol respectively), water-soluble carbodiimide (EDC) 98.7mg(514.7 μ mol), N-hydroxy-succinamide (NHS) 29.6mg(257.4 μ mol) is dissolved among the 5mLDMF lucifuge, the reaction of room temperature magnetic agitation 24 hours successively;
B, with 100mg(1.5 μ mol) the BSA(molecular weight ranges is 6.7KDa～6.8 KDa) be dissolved in 10mL phosphate buffer (PBS) (0.01M, pH7.4) in;
C, " product of steps A " slowly joined in " product of step B " room temperature magnetic agitation reaction 3 hours; After reaction finishes, reactant liquor is transferred in the semi-permeable diaphragm, 0～4 ℃ with the PBS damping fluid (0.01M, pH7.4) dialysis is 3 days, during changed one time dislysate in per 4～6 hours; Used distill water dialysis subsequently 3 days, during changed one time dislysate in per 4～6 hours; Dialysis finishes, and uses the freeze drier freeze-drying, and obtain yellow cotton-shaped solid and be immunogene (conjugate of folic acid and bovine serum albumin) ,-20 ℃ of preservations, standby.
2, envelope antigen is synthetic
Adopt mixed anhydride method to carry out coupling folic acid and ovalbumin (OVA) and obtain envelope antigen.Concrete steps are as follows:
A, taking by weighing 49mg(111.1 μ mol) folic acid is dissolved in vibration dissolving in the 5mL dry DMF (anhydrous N-N dimethyl formamide), add 29 μ L (122.2 μ mol) tri-n-butylamine, ice bath reaction 30 minutes, dropwise add 20.2 μ L (155.5 μ mol) isobutyl chlorocarbonate then, room temperature reaction 1 hour.
B, take by weighing 100mg OVA (ovalbumin) (2.22 μ mol) and be dissolved among the PBS.
C, " product of steps A " dropwise joined in " product of step B ", the stirring at room reaction is spent the night.After reaction finishes, successively use the PBS damping fluid (0.01M, pH7.4) and distill water dialysis 6 days, during change dislysate; Dialysis finishes, and uses the freeze drier freeze-drying, and obtain yellow cotton-shaped solid and be envelope antigen (conjugate of folic acid and ovalbumin) ,-20 ℃ of preservations, standby.
3, folic acid Polyclonal Antibody Preparation
Adopting male new zealand white rabbit as immune animal, is immunogene with the conjugate of folic acid and bovine serum albumin.First immunisation is mixed into water in oil emulsion with Freund's complete adjuvant and immunogene by 1:1, and every rabbit injection 0.5mg immunogene is carried out the subcutaneous multi-point injection in back (5～10 point); Booster immunization for the first time after 20 days mixes with the equal-volume immunogene with incomplete Freund, and it is immune to carry out the second time, and dosage reduces by half, and method is exempted from head; Later on once, do not add adjuvant for the 5th time and only carry out the last immunity, inject and carry out heart after seven days and take a blood sample with physiological saline every two all booster immunizations.Blood is 4 ℃ of standing over night, and next day, 10000rpm got antiserum in centrifugal 15 minutes, was the folic acid polyclonal antibody.
The foundation of embodiment 2, ELISA detection method
1, preferred (the square formation method) of antibody and envelope antigen concentration
Vertically with the series concentration coated elisa plate of envelope antigen by 8.0 μ g/mL, 4.0 μ g/mL, 2.0 μ g/mL, 1.0 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 100 μ L/ holes, placed 2 hours, and washed plate three times for 37 ℃ with cleansing solution 300 μ L/ holes; With the sealing of lock solution 250 μ L/ holes, 0 ~ 4 ℃ of placement is spent the night, and washes plate three times again; The antibody (1:2500 to 1:80000) that laterally adds the 100 a series of dilutions in μ L/ hole was placed 30 minutes, and was washed plate three times for 37 ℃; The ELIAS secondary antibody that adds 100 μ L/ hole 1:2000 is the goat anti-rabbit antibody of horseradish peroxidase mark, places 30 minutes, and washes plate three times for 37 ℃; Add the substrate colour developing liquid in 100 μ L/ holes, measure absorbance.With the concentration of envelope antigen the envelope antigen concentration of obvious graded and antibody dilution being arranged with absorbance is that optium concentration is carried out specific assay.
2, the mensuration of antibody sensitivity
According to the optimization experiment of above-mentioned antagonist and envelope antigen concentration, selection and definite antibody concentration are 1:20000, and envelope antigen concentration is the mensuration that 1 μ g/mL carries out the sensitivity of antibody:
A, bag quilt: with the carbonate buffer solution of 0.05M pH9.6 the envelope antigen of folic acid is made into the solution of 1 μ g/mL, adds 100 μ L in the reacting hole of each ELISA Plate, 37 ℃ of placements 2 hours, discard solution in the hole, wash 3 times, 300 μ L/ holes with lavation buffer solution, each 3 clocks pat dry.(this step is called for short washing, down together).
B, sealing: with the above-mentioned ELISA Plate of having wrapped quilt of lock solution sealing, 250 μ L/ holes, 0 ~ 4 ℃ of placement is spent the night, then washing.
C, application of sample: add dilution folic acid antibody (1:20000) 50 μ L/ holes with without the folic acid standard solution 50 μ L/ holes of concentration in the above-mentioned reacting hole that has sealed, placed 30 minutes for 37 ℃, then washing.
D, add ELIAS secondary antibody: in each reacting hole, add goat anti-rabbit antibody (1:2000) the 100 μ L/ holes of the horseradish peroxidase-labeled of fresh dilution, 30 minutes, washing.
E, colour developing: in each reacting hole, add the colour developing liquid 100 μ L/ holes (colour developing liquid A50 μ L+ colour developing liquid B50 μ L) of provisional configuration, placed 15 minutes for 37 ℃.
F, termination: in each reacting hole, add stop buffer 100 μ L/ holes, detect with microplate reader immediately.
G, computation process: testing result is calculated with inhibiting rate, and % inhibiting rate=%B/ B0, B are to add the light absorption value of medicine as the rival, and B0 is the light absorption value that does not add the rival.Drug concentrations is the sensitivity of this antibody when calculating 50% inhibiting rate.
The assembling of the enzyme-linked immunologic detecting kit of embodiment 3, detection folic acid
1, detects the composition of the enzyme-linked immunologic detecting kit of folic acid
A, be coated with the solid phase carrier (ELISA Plate) of envelope antigen (conjugate of folic acid and ovalbumin);
B, folic acid antibody working fluid (volume by volume concentration is 1:20000);
6 bottles of C, folic acid standard solution, concentration is respectively: 3ng/ml, 6 ng/ml, 12.5 ng/ml, 25 ng/ml, 50 ng/ml, 100 ng/ml;
The goat anti-rabbit igg antibody working fluid of D, horseradish peroxidase-labeled (working concentration is 1:2000);
E, concentrated phosphoric acid salt buffer are every liter and contain NaCl 80g, KH
2PO
42.0g, Na
2HPO
412H
2O
229.0g, the aqueous solution of KCl 2.0g.
F, concentrated cleaning solution: be that above-mentioned concentrated phosphoric acid salt buffer adding volume by volume concentration is 0.05% polysorbas20 (Tween20);
G, substrate colour developing liquid: colour developing liquid A is a TMB solution; Colour developing liquid B is the urea peroxide solution of pH5.0～6.0;
H, stop buffer: concentration is the sulfuric acid solution of 2mol/L.
2, the preparation of ELISA Plate
With carbonate buffer solution (0.05M pH9.6) is diluted to envelope antigen 1 μ g/mL, and every hole of ELISA Plate adds 100 μ L, hatches 2 h for 37 ℃, the coating buffer that inclines, every hole adds 300 μ L cleansing solutions washings 3 times, pats dry; Every then hole adds confining liquid 250 μ L, and 4 ℃ are spent the night, liquid in the hole of inclining, and cleansing solution washing 3 times pats dry, with 4 ℃ of preservations of masking foil vacuum seal.
The application of the enzyme-linked immunologic detecting kit of embodiment 4, detection folic acid
1, the preparation of reagent
A, sample diluting liquid: the concentrated phosphoric acid salt buffer that provides in the kit is used with behind 10 times of the distilled water dilutings.
B, wash solution: the concentrated cleaning solution that provides in the kit is used with behind 10 times of the distilled water dilutings.
C, substrate colour developing liquid: colour developing liquid A mixes the back with colour developing liquid B equal-volume to be used.
2, sample pre-treatments
A, milk: with milk sample at 4 ℃, 10000 rev/mins centrifugal 15 minutes, remove fat deposit; The milk of degreasing is diluted to 1:10 with phosphate buffer, obtains testing sample.
B, milk powder: take by weighing 10g milk powder and add the 90ml dissolved in distilled water, then at 4 ℃, 10000 rev/mins centrifugal 15 minutes, remove fat deposit; The milk of degreasing is diluted to 1:10 with phosphate buffer, obtains testing sample.
C, vitamin drinks: with sample at 4 ℃, 10000 rev/mins centrifugal 15 minutes, get supernatant, be diluted to 1:100 with phosphate buffer, obtain testing sample.
D, multivitamin or capsule: take by weighing 1g sample 100ml dissolved in distilled water, then at 4 ℃, 10000 rev/mins centrifugal 15 minutes, get supernatant, be diluted to 1:10 with phosphate buffer, obtain testing sample.
3, detect step
A, application of sample: add folic acid series concentration standard solution or sample solution 50 μ L in the ELISA Plate micropore, add folic acid antibody-solutions 50 μ L then, room temperature (25 ℃) constant temperature is hatched 1 h;
B, washing: the middle liquid that portals that inclines, every hole adds wash solution 300 μ L, washs 3 times, pats dry;
C, enzyme-added mark goat anti-rabbit antibody solution: every hole adds enzyme mark goat anti-rabbit antibody solution 100 μ L, and room temperature constant temperature is hatched 1 h;
D, washing: the middle liquid that portals that inclines, every hole adds wash solution 300 μ L, washs 3 times, pats dry;
E, add colour developing liquid: every hole adds colour developing liquid 100 μ L, and room temperature (25 ℃) constant temperature was hatched 15 minutes;
F, termination: every hole adds stop buffer 100 μ L;
F, detection: the absorbance of measuring the 450nm place with microplate reader.
4, the result judges
The drafting of typical curve: horizontal ordinate is the logarithm value of folic acid concentration, and ordinate is the ratio of the light absorption value of 0ng/ml for the light absorption value and the titer of each standard items and sample, i.e. inhibiting rate, and the concentration of each sample can be read from typical curve.
% inhibiting rate=% standard items light absorption value (or sample)/0 standard items light absorption value.
Embodiment 5, kit precision and accuracy test
Get the folic acid standard specimen of 20,50,100 ppb, add in the milk sample, detect the recovery of folic acid.The interassay coefficient of variation of each concentration all calculates with 5 repeating datas of different 5 days, and the variation within batch coefficient calculates with 5 repeating datas on the same day.
Carry out the quantitative Analysis of the recovery according to the linear equation of the typical curve of formulating.The results are shown in following table.
From the said determination result, the coefficient of variation is lower than 11.8%, and the recovery is between 89.1～110.3%.Show that this kit has good repeatability and accuracy.
Claims (9)
1. the enzyme-linked immunologic detecting kit of a folic acid is characterized in that the composition of reagent in this kit comprises:
(1) is coated with the ELISA Plate of folic acid envelope antigen;
(2) polyclonal antibody of folic acid;
(3) ELIAS secondary antibody is the antibody of the goat-anti rabbit of horseradish peroxidase-labeled;
(4) folic acid standard solution;
(5) concentrated phosphoric acid salt buffer;
(6) concentrated cleaning solution;
(7) substrate colour developing liquid A;
(8) substrate colour developing liquid B;
(9) stop buffer.
2. kit according to claim 1 is characterized in that described folic acid envelope antigen obtains folic acid haptens and ovalbumin coupling; The concentration of described folic acid envelope antigen is 1 μ g/ml.
3. kit according to claim 1, the polyclonal antibody that it is characterized in that described folic acid are to be that the conjugate that the bovine serum albumin coupling of 6.7KDa one 6.8KDa is made prepares as the immunogen immune new zealand white rabbit by folic acid and molecular weight ranges; The working concentration of wherein said folic acid polyclonal antibody is 1:10000～1:20000.
4. kit according to claim 1 is characterized in that described ELIAS secondary antibody adopts sodium periodate that horseradish peroxidase and goat anti-rabbit antibody are carried out coupling and obtains; The working concentration of described ELIAS secondary antibody is 1:2000.
5. kit according to claim 1 is characterized in that the concentration of described folic acid standard solution is followed successively by 3ng/ml, 6 ng/ml, 12.5 ng/ml, 25 ng/ml, 50 ng/ml, 100 ng/ml.
6. kit according to claim 1 is characterized in that described concentrated phosphoric acid salt buffer is every liter and contains NaCl 80g, KH
2PO
42.0g, Na
2HPO
412H
2O
229.0g, the aqueous solution of KCl 2.0g.
7. kit according to claim 1 is characterized in that described concentrated cleaning solution is to contain the pH7.4 of volume fraction 0.05% Tween-20, the phosphate buffer of 0.1mol/L.
8. kit according to claim 1 is characterized in that described substrate colour developing liquid A is a TMB solution; Described substrate colour developing liquid B is the urea peroxide solution of pH5.0～6.0, and colour developing liquid A mixes the back use with colour developing liquid B equal-volume during use; Described stop buffer is the sulfuric acid solution of 2mol/L.
9. the application of the described enzyme linked immunological kit of claim 1 folate content in detecting food, feed and vitamin products.
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| CN201110004107XA CN102175878A (en) | 2011-01-11 | 2011-01-11 | ELISA (enzyme linked immunosorbent assay) kit of folic acid |
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| CN104076154A (en) * | 2013-03-28 | 2014-10-01 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay kit detecting folic acid and application thereof |
| CN111024962A (en) * | 2019-12-31 | 2020-04-17 | 上海复星长征医学科学有限公司 | Dissociating agent for detecting folic acid content from serum and detection method |
| CN111735785A (en) * | 2020-07-02 | 2020-10-02 | 无锡紫杉药业有限公司 | Detection method for tetrahydrofolic acid production |
| CN112683885A (en) * | 2020-12-29 | 2021-04-20 | 深圳泰乐德医疗有限公司 | 5-methyltetrahydrofolate chemiluminescence detection kit and preparation method thereof |
| CN116120430A (en) * | 2023-02-27 | 2023-05-16 | 浙江准策生物技术有限公司 | Folic acid complete antigen and antibody, and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN102998469A (en) * | 2012-11-20 | 2013-03-27 | 博奥赛斯（天津）生物科技有限公司 | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for folic acid, and preparation method of kit |
| CN104076154A (en) * | 2013-03-28 | 2014-10-01 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay kit detecting folic acid and application thereof |
| CN111024962A (en) * | 2019-12-31 | 2020-04-17 | 上海复星长征医学科学有限公司 | Dissociating agent for detecting folic acid content from serum and detection method |
| CN111024962B (en) * | 2019-12-31 | 2023-09-19 | 复星诊断科技(上海)有限公司 | Dissociating agent for detecting folic acid content in serum and detection method |
| CN111735785A (en) * | 2020-07-02 | 2020-10-02 | 无锡紫杉药业有限公司 | Detection method for tetrahydrofolic acid production |
| CN112683885A (en) * | 2020-12-29 | 2021-04-20 | 深圳泰乐德医疗有限公司 | 5-methyltetrahydrofolate chemiluminescence detection kit and preparation method thereof |
| CN116120430A (en) * | 2023-02-27 | 2023-05-16 | 浙江准策生物技术有限公司 | Folic acid complete antigen and antibody, and preparation method and application thereof |
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Application publication date: 20110907 |