CN102650640A - Method for detecting allergen ovomucoid or ovotransferrin in influenza vaccine quantificationally - Google Patents
Method for detecting allergen ovomucoid or ovotransferrin in influenza vaccine quantificationally Download PDFInfo
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- CN102650640A CN102650640A CN2012101216752A CN201210121675A CN102650640A CN 102650640 A CN102650640 A CN 102650640A CN 2012101216752 A CN2012101216752 A CN 2012101216752A CN 201210121675 A CN201210121675 A CN 201210121675A CN 102650640 A CN102650640 A CN 102650640A
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Abstract
The invention discloses a method for detecting allergen ovomucoid or ovotransferrin in influenza vaccine quantificationally. Based on the preparation of anti-ovomucoid or anti-ovotransferrin monoclonal antibody, after paired antibodies are screened, a double antibody sandwich ELIsA (enzyme linked immunosorbent assay) adsorption test is established to quantificationally detect allergen ovomucoid or ovotransferrin in influenza vaccine. The method aims to reduce the incidence rate of allergic reaction of inoculated people caused by ovomucoid or ovotransferrin remaining in influenza vaccine prepared in a chick embryo allantoic cavity in a purified manner, has the advantages of strong specificity, high sensitivity, good repeatability and simplicity and convenience in implementation, and is very suitable for quickly and quantificationally detecting ovomucoid and ovotransferrin in food or influenza vaccine in disease control.
Description
Technical field
The invention belongs to the immuno analytical method field, relate to a kind of detection method of albumen, particularly utilize the double-antibody sandwich EUSA to come the method for anaphylactogen ovomucoid or ovum transferrin new in the detection by quantitative influenza vaccines.
Background technology
Influenza is the disease of tool world pop potentiality, and be very popular and bring about great losses to the whole world in the grippal world last century for several times.Since the beginning of this century, the World Health Organization (WHO) continues the human case of issue H5N1 bird flu, and the novel HIN1 influenza (swine flu) of particularly issuing the H5N1 bird flu of epidemic situation all over the world successively and breaking out in recent years all might cause being very popular of the whole world.Influenza vaccines are that the prevention influenza takes place and popular most effectual way.The main mode of the influenza vaccines of countries in the world production at present is after utilizing the influenza virus epidemic strain in Embryo Gallus domesticus egg capsule chamber, to cultivate, through inactivation treatment, again with being prepared into vaccine behind the influenza antigen purifying.Therefore have a part of ovum gallinaceum residual protein in the vaccine, residual protein possibly can cause anaphylactic type (I type) allergic reaction of IgE mediation as anaphylactogen to the part individuality." 2010 editions the 3rd regulations of Chinese pharmacopoeia: adopt ELISA to detect, egg white protein content should not be higher than 500ng/mL in the influenza vaccines.
Contain multiple anaphylactogen in the egg white, except ovalbumin, also have ovomucoid and ovum transferrin etc.Wherein, egg white protein content is the highest in the egg white, accounts for 54% of egg white total protein.In addition, ovomucoid accounts for 11% of egg white total protein.The ovum transferrin accounts for 12% of egg white total protein.Significant be that some individual oral or injection can the generation severe anaphylactic reaction after containing food or the preparation of ovomucoid and ovum transferrin.By the prepared influenza vaccines of the influenza virus liquid of cultivating in the chick embryo allantoic liquid are residual a certain amount of chicken egg white arranged; At present ovalbumin in the influenza virus vaccine there is clear and definite regulation both at home and abroad, but does not have the relevant regulations of ovomucoid and ovum transferrin residual quantity.Up to the present, ovomucoid or the new anaphylactogen of ovum transferrin that exists in the influenza vaccines is not fully recognized that more there is not in the influenza vaccines regulation both at home and abroad to these two types of allergen content.
Summary of the invention
The objective of the invention is to; Anaphylactogen ovomucoid or ovum transferrin quantitative detecting method in a kind of new influenza vaccines are provided; This method not only can detection by quantitative; And high specificity, highly sensitive, good reproducibility, simple and easy to do, be very suitable for fast quantification and detect ovomucoid and ovum transferrin in the influenza vaccines.
In order to realize above-mentioned task, the present invention takes following technical solution:
Anaphylactogen ovomucoid or ovum transferrin quantitative detecting method in a kind of influenza vaccines; It is characterized in that; This method is on the basis of anti-ovomucoid of preparation or anti-ovum transferrin monoclonal antibody; Through the screening of pairing antibody and the double-antibody sandwich EUSA of foundation, come ovomucoid or ovum transferrin in the detection by quantitative influenza vaccines.
Described quantitative detecting method comprises: the MONOCLONAL ANTIBODIES SPECIFIC FOR of anti-ovomucoid or anti-ovum transferrin and the screening of pairing antibody and the foundation of double-antibody sandwich EUSA.
The screening technique of described anti-ovomucoid or anti-ovum transferrin MONOCLONAL ANTIBODIES SPECIFIC FOR and pairing antibody is as immunogene with ovomucoid or ovum transferrin; Adopt conventional hybridization knurl technology to obtain the monoclonal antibody of anti-ovomucoid or anti-ovum transferrin; And the conventional mark horseradish peroxidase of difference; Respectively with unlabelled monoclonal antibody as coated antibody; The monoclonal antibody of mark as enzyme labelled antibody, is adopted Array Method to match in twos and selects the pairing antibody that susceptibility is the highest and specificity is best.
The method of described double-antibody sandwich EUSA is; In 96 orifice plates; Order adds coated antibody, sample to be measured and standard items, enzyme labelled antibody and substrate; Measure absorbance value in wavelength 410nm, an above-mentioned best antagonist of selecting is used separately as coated antibody and enzyme labelled antibody detects ovomucoid or ovum transferrin.Encapsulate 96 orifice plates with the 5mg/L coated antibody, seal 96 orifice plates with the damping fluid that contains bovine serum albumin(BSA) after 24 hours, the washing back adds the ovomucoid or the ovum transferrin standard items of sample to be measured (influenza infection allantoic fluid stoste, the influenza vaccines of dilution certain multiple produce intermediate product and influenza vaccines finished product) and doubling dilution; Hatch and wash the back and add enzyme labelled antibody; Hatch and wash the back once more with 2, the colour developing of 2 '-azine group-two (3-ethyl benzo thiophene pyrrolin-sulfonic acid) substrate is measured absorbance value in wavelength 410nm; Absorbance value with standard items is an ordinate; Respective concentration is a horizontal ordinate, makes typical curve, according to the absorbance value of sample to be measured; Through curve match or conversion, draw the concentration of ovomucoid in the sample to be measured or ovum transferrin.
Anaphylactogen ovomucoid in the influenza vaccines of the present invention or ovum transferrin quantitative detecting method; It is the new method of on the monoclonal antibody basis, setting up; Utilization detection by quantitative ovomucoid and the EUSA of ovum transferrin double-antibody sandwich; Can detect ovomucoid or ovum transferrin by fast quantification, the content that detects ovomucoid in the influenza virus stock solution first at home and abroad is between 0.9～258ng/mL, and the concentration of ovum transferrin is between 2～450ng/mL.Be to improve and control influenza vaccines quality, reduce and prevent that the postvaccinal allergic reaction of influenza virus vaccine from providing important evidence.
Monoclonal antibody is the antibody of the single epi-position of identification, so high specificity of the present invention; Again because screen, so of the present invention highly sensitive through the pairing of antibody; The present invention is based upon on the basis of EUSA, if take the scheme of preparatory coated antibody, testing process only needs about 3 hours; Because the monoclonal antibody quality is easy to control, error is very little between the absorbance value during detection between each multiple hole, so the present invention is simple and easy to do in addition, the characteristics of good reproducibility.
Embodiment
According to technical scheme of the present invention, on the basis of anti-ovomucoid of preparation or anti-ovum transferrin monoclonal antibody, pass through the screening of pairing antibody and set up the double-antibody sandwich EUSA, come detection by quantitative ovomucoid or ovum transferrin.
Anti-ovomucoid or anti-ovum transferrin MONOCLONAL ANTIBODIES SPECIFIC FOR are: as immunogene, adopt conventional hybridization knurl technology to obtain the monoclonal antibody of anti-ovomucoid or anti-ovum transferrin ovomucoid or ovum transferrin.
The screening technique of pairing double antibody is: with the conventional mark horseradish peroxidase of monoclonal antibody difference that obtains; Respectively with unlabelled monoclonal antibody as coated antibody; With the monoclonal antibody of mark as enzyme labelled antibody; Adopt Array Method to match the pairing antibody of selecting best conditions in twos, set up the system of double-antibody sandwich EUSA.The selection method of the pairing antibody of best conditions is: select susceptibility the highest; The absorbance value of ovomucoid or ovum transferrin that promptly detects same concentrations is the highest; And the pairing antibody of specificity high (absorbance value that promptly detects irrelevant albumen is minimum, also is that non-special background is minimum).
The method of double-antibody sandwich EUSA is in 96 orifice plates, to add the 5mg/L coated antibody; Seal 96 orifice plates with the damping fluid that contains bovine serum albumin(BSA) after 24 hours, the influenza virus stock solution sample that extracts in the diluted chick embryo allantoic liquid of washing back adding and the ovomucoid or the ovum transferrin standard items of doubling dilution are hatched and are washed the back and add enzyme labelled antibody; Hatch and wash the back once more with 2, the colour developing of 2 '-azine group-two (3-ethyl benzo thiophene pyrrolin-sulfonic acid) substrate is measured absorbance value in wavelength 410nm; Absorbance value with ovomucoid or ovum transferrin standard items is an ordinate; Respective concentration is a horizontal ordinate, makes typical curve, according to the absorbance value of sample to be measured; Through curve match or conversion, draw the concentration of ovomucoid in the influenza vaccines or ovum transferrin.
Below be the embodiment that the inventor provides:
Embodiment 1: ovomucoid in the detection by quantitative influenza virus stock solution:
Ovomucoid as immunogene, is adopted conventional hybridization knurl technology, through Fusion of Cells, screening and cloning, obtain the monoclonal antibody of the anti-ovomucoid of 8 strains altogether, respectively called after FMU-OM1～FMU-OM8.
After the pairing of antibody screening, with the FMU-OM5 monoclonal antibody as coated antibody, and with behind the FMU-OM6 labeling of monoclonal antibody horseradish peroxidase as enzyme labelled antibody.Na with pH9.6,0.05mol/L
2CO
3-NaHCO
3Damping fluid dilution coated antibody FMU-OM5 to 5mg/L adds 96 orifice plates, and preserving moisture in 100 μ l/ holes, placed 24 hours for 4 ℃.Wash 96 orifice plates 3 times with lavation buffer solution, add the damping fluid that contains 1% bovine serum albumin(BSA), room temperature sealing 30 minutes.Wash plate 3 times, add the influenza virus stock solution sample that in the dilution chick embryo allantoic liquid, extracts to be measured and the ovomucoid standard items of doubling dilution, hatched 1 hour for 37 ℃ in 100 μ l/ holes.Wash plate 3 times, add the enzyme labelled antibody FMU-OM6 of dilution in 1: 500 respectively, hatched 1 hour for 37 ℃ in 100 μ l/ holes.Wash plate 3 times, with 2, the colour developing of 2 '-azine group-two (3-ethyl benzo thiophene pyrrolin-sulfonic acid) substrate, placed 5 minutes for 37 ℃ in 100 μ l/ holes, measures absorbance value in wavelength 410nm.Absorbance value with standard items is an ordinate, and respective concentration is a horizontal ordinate, makes typical curve, and its sensitivity reaches 0.5ng/mL.According to the absorbance value of sample to be measured, through the curve match, the content that draws ovomucoid in 7 batches of different influenza virus stock solutions is between 0.9～258ng/mL.Standard items are the Sigma product, are the ovomucoid that is used for immune mouse of concentration known.
Embodiment 2: ovum transferrin in the detection by quantitative influenza virus stock solution:
The ovum transferrin as immunogene, is adopted conventional hybridization knurl technology, through Fusion of Cells, screening and cloning, obtain the monoclonal antibody of the anti-ovum transferrin of 18 strains respectively, respectively called after FMU-OT1～FMU-OT18.
After the pairing of antibody screening, with the FMU-OT12 monoclonal antibody as coated antibody, and with behind the FMU-OT6 labeling of monoclonal antibody horseradish peroxidase as enzyme labelled antibody.Na with pH9.6,0.05mol/L
2CO
3-NaHCO
3Damping fluid dilution coated antibody FMU-OT12 to 5mg/L adds 96 orifice plates, and preserving moisture in 100 μ l/ holes, placed 24 hours for 4 ℃.Wash 96 orifice plates 3 times with lavation buffer solution, add the damping fluid that contains 1% bovine serum albumin(BSA), room temperature sealing 30 minutes.Wash plate 3 times, add the influenza virus stock solution sample that in the dilution chick embryo allantoic liquid, extracts to be measured and the ovum transferrin standard items of doubling dilution, hatched 1 hour for 37 ℃ in 100 μ l/ holes.Wash plate 3 times, add the enzyme labelled antibody FMU-OT6 of dilution in 1: 500, hatched 1 hour for 37 ℃ in 100 μ l/ holes.Wash plate 3 times, with 2, the colour developing of 2 '-azine group-two (3-ethyl benzo thiophene pyrrolin-sulfonic acid) substrate, placed 5 minutes for 37 ℃ in 100 μ l/ holes, measures absorbance value in wavelength 410nm.Absorbance value with standard items is an ordinate, and respective concentration is a horizontal ordinate, makes typical curve.According to the absorbance value of sample to be measured, convert through curve match and molecular weight, the concentration that draws ovum transferrin in 7 batches of different influenza virus stock solutions is between 2ng/mL～450ng/mL.
Claims (2)
1. anaphylactogen ovomucoid or the ovum transferrin quantitative detecting method in the influenza vaccines; It is characterized in that; On the basis of anti-ovomucoid of preparation or anti-ovum transferrin monoclonal antibody; Pass through the screening of pairing antibody and set up the double-antibody sandwich EUSA, come detection by quantitative ovomucoid or ovum transferrin.
2. the method for claim 1 is characterized in that:
The preparation method of described anti-ovomucoid or anti-ovum transferrin is: as immunogene, adopt conventional hybridization knurl technology to obtain the monoclonal antibody of anti-ovomucoid or anti-ovum transferrin ovomucoid or ovum transferrin;
The screening technique of described pairing double antibody is: anti-ovomucoid that will obtain or anti-ovum transferrin monoclonal antibody be conventional mark horseradish peroxidase respectively; Respectively with unlabelled monoclonal antibody as coated antibody; As enzyme labelled antibody, adopt Array Method to match in twos the monoclonal antibody of mark, select and select susceptibility the highest; The absorbance value of ovomucoid or ovum transferrin that promptly detects same concentrations is the highest, and the high pairing antibody of specificity; Set up the system of double-antibody sandwich EUSA;
The method of described double-antibody sandwich EUSA is in 96 orifice plates, to add the 5mg/L coated antibody; Seal 96 orifice plates with the damping fluid that contains bovine serum albumin(BSA) after 24 hours, the influenza virus stock solution sample that extracts in the diluted chick embryo allantoic liquid of washing back adding and the ovomucoid or the ovum transferrin standard items of doubling dilution are hatched and are washed the back and add enzyme labelled antibody; Hatch and wash the back once more with 2, the colour developing of 2 '-azine group-two (3-ethyl benzo thiophene pyrrolin-sulfonic acid) substrate is measured absorbance value in wavelength 410nm; Absorbance value with ovomucoid or ovum transferrin standard items is an ordinate; Respective concentration is a horizontal ordinate, makes typical curve, according to the absorbance value of sample to be measured; Through curve match or conversion, draw the concentration of ovomucoid in the influenza vaccines or ovum transferrin.
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Cited By (3)
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| CN105181954A (en) * | 2015-10-12 | 2015-12-23 | 天津科技大学 | Preparation method of rabbit anti ovotransferrin allergen polyclone antibody and immunoassay method thereof |
| CN109239337A (en) * | 2018-09-27 | 2019-01-18 | 天津欣普赛尔生物医药科技有限公司 | The test paper and preparation method of bovine serum albumin(BSA) in a kind of detection biological products |
| CN112710837A (en) * | 2020-11-03 | 2021-04-27 | 浙江大学 | Method for quantifying nsLTP allergen in artemisia pollen |
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Cited By (3)
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| CN105181954A (en) * | 2015-10-12 | 2015-12-23 | 天津科技大学 | Preparation method of rabbit anti ovotransferrin allergen polyclone antibody and immunoassay method thereof |
| CN109239337A (en) * | 2018-09-27 | 2019-01-18 | 天津欣普赛尔生物医药科技有限公司 | The test paper and preparation method of bovine serum albumin(BSA) in a kind of detection biological products |
| CN112710837A (en) * | 2020-11-03 | 2021-04-27 | 浙江大学 | Method for quantifying nsLTP allergen in artemisia pollen |
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Application publication date: 20120829 |