CN101893636A - Enzyme-linked immunosorbent assay method for egg allergen ovalbumin in foods - Google Patents
Enzyme-linked immunosorbent assay method for egg allergen ovalbumin in foods Download PDFInfo
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Abstract
The invention discloses an Enzyme-linked immunosorbent assay (ELISA) method for egg allergen ovalbumin in foods, which belongs to the technical field of immunoassay. In the invention, a polyclonal antibody is obtained by immunizing a healthy New Zealand white rabbit with high-purity ovalbumin, and an indirect ELISA method for ovalbumin in foods is established by using the antibody as a detecting agent and the high-purity ovalbumin as a standard product and a coating antigen. In the invention, a quick and high-efficiency detection means is provided for detecting the ovalbumin content of the foods is provided; due to the adoption of the polyclonal antibody, the cost is low and the stability and the repeatability are high; the sensitivity is 1ppm, the linear range is 1 to 128ppm; and the high specificity and affinity of an immunoreactions ensure that the ELISA has extremely high selectivity and sensitivity.
Description
Technical field
The present invention relates to the enzyme-linked immune detection method of egg allergen egg white protein content in a kind of detection by quantitative food, belonged to the immuno analytical method field.
Background technology
In recent years, food hypersenstivity has become the food-safety problem of a public character, and egg is one of modal eight larger food anaphylactogens.Anaphylactic disease serious harm people's is healthy, though excite the amount of anaphylactoid minimum anaphylactogen different along with crowd's difference, the anaphylactogen of trace just can make most patients produce allergic symptom.For fear of the anaphylactogen of contact trace, the detection of anaphylactogen becomes the task of top priority.Now, though there are many anaphylactogen detection methods on concrete the application, all to exist different separately problems for using for reference.Vivo experiment method can provide the most directly evidence, but because the consideration of aspects such as safety factor is only carried out in hospital under unavoidable situation, and the expense costliness, has a big risk; The experiment in vitro method has the advantage of convenience, safety in contrast to this, but exists the shortcoming of poor accuracy.The detection method of present external trace has immunization, PCR method, histamine release experimental method, anaphylactogen fingerprint fast detection method etc.Though the method that at present relevant anaphylactogen detects is a lot, all has different separately problems, the analytical instrument slow as detection speed, that cost is high, needs are specific etc., these have restricted the development of anaphylactogen detection method in the food.Therefore realize accurately, safety, economy, fast, high flux, high-sensitive vitro detection technical method have realistic meaning.
Summary of the invention
(1) technical matters that will solve
The objective of the invention is to set up a kind of have high sensitivity, high specific, pin-point accuracy, pinpoint accuracy, the simple enzyme-linked immune detection method of method of operating, be used for batch, the fast detecting of food ovalbumin.
(2) technical scheme
For achieving the above object, the present invention has set up the enzyme-linked immune detection method of egg allergen egg white protein content in a kind of food, comprises the optimization to detection method.
(1) the bag quilt of antigen
With the high-purity ovalbumin as envelope antigen, be cushioned the sodium carbonate buffer CBS dilution envelope antigen of liquid pH 9.6,0.05M with bag, envelope antigen concentration is that 0.125～256 μ g/mL is as coating buffer, in every hole of ELISA Plate, add 100 μ L coating buffers, 4 ℃ of overnight incubation are cushioned liquid with the bag that contains 0.1% gelatin and seal as confining liquid;
Confining liquid is the pH 9.6 that contains 0.1% gelatin, the CBS of 0.05M;
(2) competitive reaction
With the ovalbumin polyclonal antibody of high-purity ovalbumin immunity preparation with antibody diluent PBST by weight ratio dilution in 1: 4000 after, add in the ELISA Plate, every hole adds 50 μ L, add series concentration 0.125 μ g/mL simultaneously respectively with the standard items diluted, 0.25 μ g/mL, 0.5 μ g/mL, 1.0 μ g/mL, 2.0 μ g/mL, 4.0 μ g/mL, 8.0 μ g/mL, 16 μ g/mL, 32 μ g/mL, 64 μ g/mL, 128 μ g/mL, the high-purity ovalbumin standard items of 256 μ g/mL, every hole 50 μ L are hatched behind the 1h with PBST cleansing solution washing 4 times for 37 ℃;
Antibody diluent PBST be contain 1% gelatin, contain 0.5% Tween-20, the phosphate buffered solution of pH 7.4;
The prescription of PBST cleansing solution is to add sodium chloride 8g, potassium dihydrogen phosphate 0.2g, sodium hydrogen phosphate 2.9g, potassium chloride 0.2g, Tween-20 0.5mL in the 1000mL distilled water;
The prescription of standard items dilution is 5% sodium chloride for adding mass concentration in the PBS of the pH6.5 that contains 30% volumes methanol;
(3) add ELIAS secondary antibody
The goat-anti rabbit HRP-IgG of horseradish peroxidase-labeled is 1: 3000 with antibody diluent PBST dilution, and every hole adds 100 μ L, washs 4 times with the PBST cleansing solution behind 37 ℃ of incubation 1h;
(4) colour developing
Every hole adds 100 μ L colour developing liquid, and the 37 ℃ of reactions in dark place 15min takes out the sulfuric acid that every hole, back adds 100 μ L stop buffer 2mol/L, measures light absorption value OD with microplate reader
450
Colour developing liquid comprises A liquid and B liquid, and the A formula of liquid is to add 0.933g citric acid, 3.68g Na in every 100mL water
2HPO
412H
2O, 18 μ L 30%H
2O
2The B formula of liquid is dissolved in 100mL ethylene glycol for the 60mg tetramethyl benzidine; Press A: B=5 during use: 1 volume ratio is used;
Described high-purity ovalbumin is to purchase in the A7642 of SIGMA company high-purity ovalbumin.
The check and analysis principle of the inventive method is: each Kong Jun on the ELISA Plate is coated with the antigen of same amount, after adding the sample and ovalbumin polyclonal antibody that contains ovalbumin to be measured, ovalbumin in solid-phase coating antigen and the testing sample is vied each other and antibody response, because the solid phase antigen in each hole and the antibody content of adding are all consistent, so when ovalbumin concentration is high in the testing sample, the antibody that then is bonded on the solid phase antigen is few, the ELIAS secondary antibody that adds is few with the antibodies amount that is fixed, add substrate solution (liquid A liquid promptly develops the color) and colour developing liquid (being B liquid) with cleansing solution washing back, chromogenic reaction is shallow, the OD value that detects with microplate reader is low, shows the inhibiting rate height; Otherwise, when testing sample contains ovalbumin concentration when low, the OD value height of then being surveyed, inhibiting rate is low.According to the typical curve of being done, can extrapolate the concentration that testing sample contains ovalbumin.
(3) beneficial effect
Egg allergen ovalbumin detection method provided by the invention has adopted the ovalbumin polyclonal antibody, can accurately detect the concentration that contains ovalbumin in the food delicately, the pre-treatment process of sample is simple, consuming time few, can detect a large amount of samples simultaneously, the sample detection cost is low, and the inventive method good stability, "dead" pollution.The present invention has important practical significance to the online detection that solves egg white protein content in the batch samples.
Description of drawings
Fig. 1 ovalbumin ELISA typical curve.
Specific embodiments
Further specify the present invention by the following examples.
One, instrument:
TGL-40B table-type low-speed hydro-extractor, Anting Scientific Instrument Factory, Shanghai
The KFLOW water purification machine, Kai Folong company
The horizontal shaking table of ZD-9556, granary science and education equipment factory
Costar 96 holes 8 * 12 removable ELISA Plate, the lucky safe bio tech ltd in Shanghai
Multiska Mks microplate reader, Thermo Labsystems company
Can debug pipettor, Thermo Labsystems company
Turbine mixer, Shanghai Hu Xi instrumental analysis factory
Two, reagent:
The goat anti-rabbit igg of horseradish peroxidase-labeled (HRP-IgG), health become bio-engineering corporation's tetramethyl benzidine (TMB), Huamei Bio-Engrg Co.,
Other reagent are analytical reagent
Three, step
1. the preparation of antiserum (polyclonal antibody)
1) animal used as test: the healthy new zealand white rabbit that to select 22 monthly ages, body weight be 1.5-2kg is an animal used as test.
2) antigen configuration: the immunogene ovalbumin is diluted with physiological saline, be made into the solution of 2mg/mL.
3) emulsification: with complete with equivalent or the incomplete freund adjuvant of above-mentioned solution with mixing paddling process with its emulsification, until an emulsion is splashed in the water, is not scattered float on the water surface till.
4) immunization method: initial immunity reagent that emulsification is good is in rabbit back multi-point injection, and 1mL/ only.Immunity behind the initial immunity is called booster immunization, booster immunization freund 's incomplete adjuvant emulsification, booster immunization is intramuscular injection, per two all booster immunizations once, dosage is identical with initial immunity.
5) blood sampling: from ear edge vein exploitating blood, adopt indirect non-competing euzymelinked immunosorbent assay (ELISA) to measure antiserum titre behind 4 booster immunizations.Wait to tire reach requirement after, adopt the ear vein bloodletting acquisition antiserum that combines with the heart bloodletting, be collected in 50mL and sterilize in the plastic centrifuge tube.
6) purifying antibody and preservation: antiserum XmL is diluted to 2XmL with the physiological saline of equivalent, under agitation dropwise adds the saturated ammonium sulfate with dilution back antiserum equivalent (2X) then, place for 4 ℃ and it was fully precipitated in 3 hours.Centrifugal (3000r/min) 20min abandons supernatant and is precipitated to XmL with physiological saline solution, drips saturated ammonium sulfate X/2mL more gradually.Placing for 4 ℃ fully precipitated it in 3 hours, repeat the above-mentioned second step process 1 time, the centrifugal back of last gained sediment is dissolved to XmL with 0.02mol/L PBS (pH 7.4), the fully dialysis of 0.02mol/L PBS (pH 7.4) in the bag filter of packing into, change liquid during this time 3 times, having dialysed concentrates, and packing is put into-20 ℃ of refrigerators and preserved standby.
2, ELISA course of reaction:
The antibody titer determination step:
1) coating antigen is cushioned liquid with bag and makes the serial dilution bag by 96 hole ELISA Plate, 100 μ L/ holes are in 4 ℃ of refrigerator overnight.Take out ELISA Plate and be back to room temperature next day, and 200 μ L PBST solution are injected in every hole, and the 3min that vibrates on the shaking table firmly gets rid of cleansing solution, pats dry on thieving paper, continues washing 2 times.Following washing methods is identical.
2) after the abundant washing, with sealing damping fluid sealase target, 200 μ L/ holes, taking-up is dried stand-by behind the incubation 2h in 37 ℃ of incubation casees.
3) positive serum dilution back correspondence is joined preceding 7 ranks of ELISA Plate, eighth row adds negative serum, 50 μ L/ holes, 37 ℃ hatch behind the 1h with the PBST cleansing solution wash 4 times, pat dry.
4) every hole adds 100 μ L, the goat anti-rabbit igg of HRP mark of dilution in 1: 3000,37 ℃ hatch behind the 1h with the PBST cleansing solution wash 4 times, pat dry.
5) every hole adds 100 μ L colour developing liquid (TMB and substrate solution volume ratio are 1: 5), and the 37 ℃ of reactions in dark place 15min takes out every hole, back and adds 100 μ L stop buffers (sulfuric acid of 2mol/L), measures light absorption value OD with microplate reader
450
The antibody specificity determination step:
A, bag quilt: use the coating antigen bag of setting concentration by enzyme-linked reaction plate, 100 μ L/ holes, 4 ℃ are spent the night.
B, washing: use PBST washing reaction plate 4 times, each 3min, 200 μ L/ holes dry reaction plate then.
C, sealing: contain the PBS of 0.1% gelatin, 200 μ L/ holes, 37 ℃ of sealing 2h.
D, washing: same b.
E, competition: with PBST the ovalbumin mother liquor is diluted to 0.125,0.25,0.5,1,2,4,8,16,32,64,128,256 μ g/mL series concentration, other establishes a PBST blank, 50 μ L/ holes.Every then hole adds the antiserum of 4000 times of 50 μ L dilutions, in 37 ℃ of incubation 1h.
F, washing: same b.
G, add ELIAS secondary antibody (goat-anti rabbit HRP-IgG, 1: 3000), 100 μ L/ holes, 37C reacts 1h.
H, washing: same b.
I, colour developing: add substrate TMB 100 μ L/ holes, colour developing 15min.
J, termination: add stop buffer 100 μ L/ holes.
K, mensuration: detect OD with microplate reader
450
The mensuration of the recovery:
The ovalbumin mother liquor accurately is diluted to 50 μ g/mL, 20 μ g/mL, 5 μ g/mL, 1.25 μ g/mL, and each concentration is done 6 times and is measured mean value, does repeated experiments 3 times, with the OD that measures
450Mean value is ordinate, and corresponding ovalbumin standard items concentration is horizontal ordinate, calculates the measured value of egg white protein content according to the linear regression equation of gained.
The calculating of the recovery: add the sample OD value calculating corresponding inhibition ratio of concentration according to difference, find separately concentration according to corresponding inhibition ratio from typical curve again.Detectable concentration is the recovery of corresponding concentration with the ratio of actual concentration.
Test findings is as follows:
1, typical curve: the range of linearity of the Detection of antigen that this experiment obtained is to be 1～128 μ g/mL, as shown in drawings.
2, the concentration of the pairing standard items of gained 50% maximum light absorption value, i.e. IC
50Be 63 μ g/mL.
3, sample recovery rate:
| Theoretical value (μ g/mL) detectable concentration mean value (μ g/mL) recovery (%) |
| 50 46.67 93.3420 20.03 100.155 4.61 92.21.25 1.17 93.6 |
Claims (2)
1. the enzyme-linked immune detection method of egg allergen egg white protein content in the food is characterized in that
(1) the bag quilt of antigen
With the high-purity ovalbumin as envelope antigen, be cushioned the sodium carbonate buffer CBS dilution envelope antigen of liquid pH 9.6,0.05M with bag, envelope antigen concentration is that 0.125～256 μ g/mL is as coating buffer, in every hole of ELISA Plate, add 100 μ L coating buffers, 4 ℃ of overnight incubation are cushioned liquid with the bag that contains 0.1% gelatin and seal as confining liquid;
Confining liquid is the pH 9.6 that contains 0.1% gelatin, the CBS of 0.05M;
(2) competitive reaction
With the ovalbumin polyclonal antibody of high-purity ovalbumin immunity preparation with antibody diluent PBST by weight ratio dilution in 1: 4000 after, add in the ELISA Plate, every hole adds 50 μ L, add series concentration 0.125 μ g/mL simultaneously respectively with the standard items diluted, 0.25 μ g/mL, 0.5 μ g/mL, 1.0 μ g/mL, 2.0 μ g/mL, 4.0 μ g/mL, 8.0 μ g/mL, 16 μ g/mL, 32 μ g/mL, 64 μ g/mL, 128 μ g/mL, the high-purity ovalbumin standard items of 256 μ g/mL, every hole 50 μ L are hatched behind the 1h with PBST cleansing solution washing 4 times for 37 ℃;
Antibody diluent PBST be contain 1% gelatin, contain 0.5% Tween-20, the phosphate buffered solution of pH 7.4;
The prescription of PBST cleansing solution is to add sodium chloride 8g, potassium dihydrogen phosphate 0.2g, sodium hydrogen phosphate 2.9g, potassium chloride 0.2g, Tween-20 0.5mL in the 1000mL distilled water;
The prescription of standard items dilution is 5% sodium chloride for adding mass concentration in the PBS of the pH 6.5 that contains 30% volumes methanol;
(3) add ELIAS secondary antibody
The goat-anti rabbit HRP-IgG of horseradish peroxidase-labeled is 1: 3000 with antibody diluent PBST dilution, and every hole adds 100 μ L, washs 4 times with the PBST cleansing solution behind 37 ℃ of incubation 1h;
(4) colour developing
Every hole adds 100 μ L colour developing liquid, and the 37 ℃ of reactions in dark place 15min takes out the sulfuric acid that every hole, back adds 100 μ L stop buffer 2mol/L, measures light absorption value OD with microplate reader
450
Colour developing liquid comprises A liquid and B liquid, and the A formula of liquid is to add 0.933g citric acid, 3.68g Na in every 100mL water
2HPO
412H
2O, 18 μ L30%H
2O
2The B formula of liquid is dissolved in 100mL ethylene glycol for the 60mg tetramethyl benzidine; Press A: B=5 during use: 1 volume ratio is used.
2. enzyme-linked immune detection method according to claim 1 is characterized in that described high-purity ovalbumin is to purchase in the A7642 of SIGMA company high-purity ovalbumin.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102221616A (en) * | 2011-04-15 | 2011-10-19 | 华南农业大学 | Indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic kit of mycoplasma gallisepticum |
| CN102353771A (en) * | 2011-06-23 | 2012-02-15 | 湖南中医药大学 | Comprehensive detection method for allergen in traditional Chinese medicine (TCM) injection |
| CN102680696A (en) * | 2012-06-08 | 2012-09-19 | 江南大学 | Double antibody sandwich enzyme-linked immuno sorbent assay (ELISA) method for detecting peanut allergic component Arah1 |
| CN102967712A (en) * | 2012-11-28 | 2013-03-13 | 昆明理工大学 | ELISA (Enzyme Linked Immunosorbent Assay) kit for qualitative and quantitative detection of walnut protein and detection method of kit |
| CN104569124A (en) * | 2014-05-29 | 2015-04-29 | 天津出入境检验检疫局动植物与食品检测中心 | Egg mass spectrometric detection signature sequence group and detection kit |
| CN105301258A (en) * | 2015-09-23 | 2016-02-03 | 集美大学 | Detection method of egg white content in heated foods |
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| CN101334409A (en) * | 2008-07-22 | 2008-12-31 | 江南大学 | Methyl paraoxon enzyme-linked immunosorbent assay method |
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| CN101334409A (en) * | 2008-07-22 | 2008-12-31 | 江南大学 | Methyl paraoxon enzyme-linked immunosorbent assay method |
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| JENNIFER M. ROLLAND ET AL.: "Specific and sensitive enzyme-linked immunosorbent assays for analysis of residual allergenic food proteins in commercial bottled wine fined with egg white, milk, and nongrape-derived tannins", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102221616A (en) * | 2011-04-15 | 2011-10-19 | 华南农业大学 | Indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic kit of mycoplasma gallisepticum |
| CN102221616B (en) * | 2011-04-15 | 2014-01-15 | 华南农业大学 | Indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic kit of mycoplasma gallisepticum |
| CN102353771A (en) * | 2011-06-23 | 2012-02-15 | 湖南中医药大学 | Comprehensive detection method for allergen in traditional Chinese medicine (TCM) injection |
| CN102353771B (en) * | 2011-06-23 | 2013-12-11 | 湖南中医药大学 | Comprehensive detection method for allergen in traditional Chinese medicine (TCM) injection |
| CN102680696A (en) * | 2012-06-08 | 2012-09-19 | 江南大学 | Double antibody sandwich enzyme-linked immuno sorbent assay (ELISA) method for detecting peanut allergic component Arah1 |
| CN102967712A (en) * | 2012-11-28 | 2013-03-13 | 昆明理工大学 | ELISA (Enzyme Linked Immunosorbent Assay) kit for qualitative and quantitative detection of walnut protein and detection method of kit |
| CN104569124A (en) * | 2014-05-29 | 2015-04-29 | 天津出入境检验检疫局动植物与食品检测中心 | Egg mass spectrometric detection signature sequence group and detection kit |
| CN105301258A (en) * | 2015-09-23 | 2016-02-03 | 集美大学 | Detection method of egg white content in heated foods |
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