CN107064504A - A kind of method of lysozyme in detection egg - Google Patents
A kind of method of lysozyme in detection egg Download PDFInfo
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Abstract
The invention discloses a kind of method for detecting lysozyme in egg, this method comprises the following steps：(1) elisa plate is coated with 2 μ g/mL monoclonal antibody 3E4,100 μ L/ holes, 4 DEG C of coatings are stayed overnight；(2) 0.5% PBST is washed 3 times, 5min/ times, is patted dry；(3) closing of 2.5% skimmed milk power solution, 200 μ L/ holes, 37 DEG C of coating 1h are added；(4) 0.5% PBST is washed 3 times, 5min/ times, is patted dry；(5) lysozyme standard product solution, the measuring samples and 1 being serially diluted are added:The polyclonal antibody of the horseradish peroxidase-labeled of 1600 times of dilutions, each 100 μ L/ holes, 37 DEG C of incubation 1h；(6) 0.5% PBST is washed 3 times, 5min/ times, is patted dry；(7) tmb substrate solution is added, 100 μ L/ holes, 37 DEG C of lucifuges react 15min；(8) 3M sulfuric acid terminating reactions, 50 μ L/ holes are added；(9) readings at ELIASA 450nm, records OD values；(10) standard curve is drawn, the content of lysozyme in egg white in measuring samples is calculated.The detection method of lysozyme of the invention, detection program is simple to operation, and detection time is not long, and high sensitivity, available for the antigen detection kit prepared quickly, effectively, inexpensive.
Description
Technical field
The present invention relates to bioassay quarantine field, more particularly to a kind of side for being used to detect lysozyme in egg
Method.
Background technology
Lysozyme is also known as N-acetylmuramide lycanohydrlase, muramidase, be one kind can hydrolyze glued in pathogenic bacteria it is many
The alkaline enzyme of sugar.It is most strong to the antibacterial ability of gram-positive bacteria, it can also suppress Gram-negative bacteria and fungi, also with antioxygen
Change ability.Lysozyme is not likely to produce the resistance to the action of a drug as the nospecific immunity factor in a kind of organism when pathogen is killed in suppression,
And with the excellent antibacterial characteristics such as nontoxic, harmless, noresidue, has a broad antifungal spectrum, activity stabilized, security be high.Lysozyme is not only
There is non-specific immune function in vivo, this function can also be passed to cub by mammal by milk, be improved
The premunition of cub, poultry passes to hatching egg by egg white, improves the resistance of embryonic development.Food industry (preservative),
The field such as medical (enzyme antimicrobial), feed (substitute antibiotics) and bioengineering (biology enzyme), lysozyme has been obtained extensively
With, and have the trend of substitute antibiotics.
Lysozyme content highest in egg, accounts for the 3.5% of albumen, is the best source for extracting lysozyme.Egg white
In lysozyme content it is higher, enzyme activity is stronger, is more conducive to the storage of egg, therefore determines the lysozyme pair in egg white
China's lysozyme production, poultry egg products are machined with important directive function, also provide an important parameter for Egg Quality evaluation.
The research to lysozyme starts from early 20th century, nasal mucus, saliva, the tears of nineteen twenty-two Britain's Fleming finder earliest
There is powerful Bacteriolytic activity, and this bacteriolyze factor is turned into lysozyme.Hereafter Various Tissues in humans and animals, juice with
And it has also been found that lysozyme content highest in the presence of lysozyme, wherein egg white in plant, microorganism, it has also become lysozyme
The primary raw material of production.But neither one more easily method is gone back in the quantitative determination of lysozyme from egg white.Traditional method is
The lysozyme in egg white is first extracted, then determines the protein content in extract solution.Due to the bacteriolyze in egg white can not possibly be extracted completely
Enzyme, the egg white lysozyme content that this method is calculated is more relatively low than actual numerical value.
The content of the invention
The present invention provides a kind of method for detecting lysozyme in egg, and this method comprises the following steps：
(1) with monoclonal antibody 3E4 (2 μ g/mL) the coating elisa plates diluted, 100 μ L/ holes, 4 DEG C of coatings are stayed overnight；
(2) 0.5% PBST is washed 3 times, 5min/ times, is patted dry；
(3) the skimmed milk power solution for adding 2.5% is closed, 200 μ L/ holes, 37 DEG C of coating 1h；
(4) 0.5% PBST is washed 3 times, 5min/ times, is patted dry；
(5) many of the lysozyme standard product solution be serially diluted or measuring samples and horseradish peroxidase-labeled are added
Clonal antibody (1:1600 times of dilutions), each 100 μ L/ holes, 37 DEG C of incubation 1h；
(6) 0.5% PBST is washed 3 times, 5min/ times, is patted dry；
(7) tmb substrate solution is added, 100 μ L/ holes, 37 DEG C of lucifuges react 15min；
(8) 3M sulfuric acid terminating reactions, 50 μ L/ holes are added；
(9) readings at ELIASA 450nm, records OD values；
(10) standard curve is drawn, the content of lysozyme in measuring samples is calculated.
Wherein described monoclonal antibody 3E4 is immunized BALB/c female mices by lysozyme and prepared；
Wherein described polyclonal antibody is obtained after new zealand white rabbit is immunized by lysozyme；
Specifically, monoclonal antibody 3E4 is prepared via a method which to obtain：
(a) subcutaneous inoculation scheme immune health BALB/c female mices are used：With 1mg/mL the μ L of lysozyme 100 and equivalent
Freund's complete adjuvant mixing, using ultrasonic emulsification, be defined by instilling indiffusion in water after emulsification, about 40min；Use alcohol swab
The position that mouse to be immunized carries out disinfection, subcutaneous multi-point injection；Head exempts from the latter 2 weeks μ L of lysozyme 100 and equivalent with 1mg/mL
Incomplete Freund's adjuvant it is well mixed after be subcutaneously injected mouse, two exempt from after 10 days, to immune mouse blood sampling, detect serum antibody
Level, exempts to determine the need for three；
(b) screening and cloning of cell fusion and positive hybridoma cell strain：
Fusion first 5 days with same dose, same procedure booster immunization in step (a) once.Tail vein blood, separates blood
Clear as positive control, mouse neck is dislocated lethal, 75% alcohol disinfecting body surface 5min, is taken out mouse spleen under aseptic condition, is used
Electrical pipette rifle draws DMEM culture mediums and rinses spleen repeatedly to prepare spleen cell suspension, is put into containing 10mL DMEM bases
In the glass dish of culture medium, spleen is caught broken with blunt-ended forceps, splenocyte suspension is made in piping and druming repeatedly.Removed with Omazene net filtration
Cell suspension after fats massive texture, filtering is gone to suck 15mL from pipe, 300g, 4 DEG C of centrifugation 10min.Exhaust supernatant, adds
The 0.17M NH of 5mL precoolings4Cell is resuspended in Cl, is put into 4 DEG C of refrigerators, stands 10min lysed erythrocytes, counts splenocyte.Press spleen
Cell and SP2/0 cells 8:1 ratio is sufficiently mixed, and cell fusion is carried out with 50%PEG1000 under 37 DEG C of water-baths.Will fusion
Cell afterwards is according to splenocyte 2 × 106/ ml, the requirement in 100 μ L/ holes, (SIGMA is public for the HT culture mediums of pre-temperature needed for gently adding
Department).After 24h, the HT culture mediums containing 2 times of amount A (Aminoperin) are added per hole, per the μ L of hole 100.After 4 days, half amount changes pre-temperature
HAT culture mediums (Aminopterin, referred to as HAT culture mediums are added in HT culture mediums).After 7 days, half amount changes the HT cultures of pre-temperature
Base.Observation cell, marks the cell of fusion daily, when clone cell is grown to more than the area of bottom hole 1/4, using indirect
ELISA method carries out antibody titer detection to supernatant, screens positive hybridoma cell., cell growth state high to antibody titer
Good hole, clone purification is carried out using limiting dilution assay, is not less than 90% continuous 3 times until positive rate, then expand culture,
Freeze.The female BAl BIc that positive hybridoma cell intraperitoneal injection is treated through paraffin oil in advance/c mouse, 1 × 106Individual cell/
Only, note observing the ascites and survival condition of mouse, ascites is extracted in time.Mouse limitation of activity is such as found, to put to death and put immediately
Ascites.Impurity removing is removed in 5000r/min centrifugations, with saturated ammonium sulfate and Protein G Resin antibody purifications, through ultraviolet absorption method
MAb concentration is determined, passes through its purity of SDS-PAGE electrophoretic analysis.Successively the fusion rate of three cell fusions be respectively 17.7%,
19.4% and 78.2%, positive rate is respectively 0,0 and 1.89%, is screened by three time cloningizations, and finally obtaining one plant can be stable
The hybridoma cell strain of secretory antibody is named as 3E4, and its affinity constant is 4.1 × 108mol/L。
Wherein described polyclonal antibody is obtained after new zealand white rabbit is immunized by lysozyme；
Specifically described polyclonal antibody is prepared via a method which to obtain：
(a) lysozyme immune health new zealand white rabbit 3 is used, immunizing dose is 500 μ g/, and one exempts to be by lysozyme
Emulsified with isometric Freund's complete adjuvant, multi-point injection is in rabbit neck and dorsal sc, 2 Zhou Houjia after emulsification completely
Strong to be immunized once, booster immunization is lysozyme and isometric incomplete Freund's adjuvant, and immunizing dose and immunization method are with first
Secondary immune, third time booster immunization is the ear vein injection that not emulsified lysozyme is carried out to rabbit, and the μ g/ of injection volume 500 are only.
7 days collection vein hematometry serum titers after intravenous injection, when serum titer reaches 24And during the above, arteria carotis is taken a blood sample and 37
DEG C 2h is placed, 4 DEG C stand overnight rear 3000rpm centrifugation 20min, collect serum, that is, obtain polyclonal antibody.
(b) purifying of polyclonal antibody
Using the rabbit anteserum of caprylic acid-ammonium purified pool, comprise the following steps that：(1) 4 DEG C of rabbit anteserum, 12000rpm
15min is centrifuged, the removal of impurity is gone.(2) 1 part of serum and 2 parts of acetate buffers (0.06mol/L, pH 4.8) are taken to mix, room temperature is stirred
Mix down and the μ L/mL rabbit anteserums of caprylic acid 75 are added dropwise.(3) mixed at room temperature 30min.(4) 4 DEG C of standing more than 2h, make it fully sink
Form sediment.(5) 4 DEG C, 12000rpm centrifugation 30min abandon precipitation.(6) after supernatant is filtered through sand core funnel, in the 0.01M of 50 times of volumes
4 DEG C of dialysis 6h in PBS (pH 7.4).(7) isometric saturated ammonium sulfate is added in the supernatant after dialysis, makes into 50% saturation
Degree, stirring while adding, 4 DEG C of standing more than 1h.(8) 4 DEG C, 10000rpm centrifugation 30min abandon supernatant.(9) precipitation with containing in right amount
137mM NaCL, 2.6mM KCL, 0.2mM EDTA PBS (pH 7.4) dissolvings, 4 in the above-mentioned PBS of 50~100 times of volumes
DEG C dialyzed overnight.(10) take after a small amount of dialysis after sample suitably dilution, with UV spectrophotometer measuring protein content, SDS-
PAGE detects antibody concentration, and it is standby to dispense extremely -80 DEG C of preservation.
(c) horseradish peroxidase (HRP) mark polyclonal antibody
A kind of horseradish peroxidase kit (HRP, Galaxybio company) of activation of polyclonal antibody after purification
It is marked, labeling method is carried out according to kit specification, and concrete operation method is as follows：(1) 100 μ L antibody-solutions are taken
(1mg/mL) is transferred to the activation horseradish peroxidases of REAGENT I, slightly centrifuges, horseradish peroxidase is sunk to ttom of pipe, horseradish enzyme
Dry powder HRP on tube wall will rinse dissolving completely.Total reaction system control is in 100 μ L/mg activation HRP or so, system increase
Mark rate is influenceed, according to by mg activation HRP correspondence antibody 100-500 μ g, HRP：X molecule mol ratios are maintained at 30:15~1 is left
It is right.(2) with REAGENT II adjust pH value to 9.5 or so, 4 DEG C overnight.(3) (the REAGENT of about 3 times of volumes of REAGENT III are added
II) adjustment of REAGENT III, it is ensured that pH is 7.0 or so for enzyme combination, can be added, terminating reaction is mixed.(4) glycerol adding is extremely
50%, -20 DEG C save backup.
Present invention also offers a kind of kit for detecting lysozyme, the kit includes：
Foregoing monoclonal antibody 3E4 and the polyclonal antibody of horseradish peroxidase-labeled；And coating buffer, closing
Liquid, washing lotion, the lysozyme standard product solution being serially diluted, the how anti-dilution of enzyme mark, substrate solution and terminate liquid；
Also including the use of specification in the kit.
Wherein monoclonal antibody 3E4 is coated antibody；The polyclonal antibody of horseradish peroxidase-labeled is detection antibody；
Coating buffer is the phosphate buffer PBS of pH=7.2 ± 0.1；Confining liquid is 2.5% (weight content) skimmed milk power solution；Washing lotion
For 0.5%PBST；The serial dilutions of lysozyme standard product solution are respectively 0,1.5,3,6,12 and 24 μ g/ml；Enzyme mark is more
Anti- dilution is PBST (0.01M PBS, containing tween-20 0.5%), and substrate solution is TMB solution, and terminate liquid is 3M sulfuric acid.
The present invention is anti-as detection using the polyclonal antibody that HRP is marked using the high monoclonal antibody of specificity as coated antibody
Body, experimental procedures are simple, and the time is short, and sensitivity is high, and minimum detection limit can reach 0.3125ng/mL, can prepare fast
Speed, bacteriolyze enzyme detection kit effectively, inexpensive, are easily realized, with good promotional value.
Brief description of the drawings
Obtained standard curve is drawn in Fig. 1 embodiments 3
Embodiment
BALB/c mouse (Shanghai medical college of Fudan University) is immunized in the lysozyme of embodiment 1, prepares monoclonal antibody
(1) immunization method of body is as follows：
Mixed with the 1mg/mL μ L of lysozyme (Bioengineering Research Institute is built up in Nanjing) 100 and the Freund's complete adjuvant of equivalent
Close, ultrasonic emulsification 40min.Abdominal cavity and the μ g/100 μ L of dorsal sc multi-point injection 100 lysozyme, carry out secondary exempt from after two weeks
Epidemic disease, 100 μ g/100 μ L lysozymes and equivalent incomplete Freund's adjuvant mixing and emulsifying, the every minor tick of booster immunization two weeks, after three exempt from
Tail vein detects mice serum potency within 10 days.
(2) screening and cloning of cell fusion and positive hybridoma cell strain：
It is strong to exempt from latter 5 days, mouse tail vein blood sampling, separate serum and give over to positive control, aseptic collection mouse spleen uses liquid relief
Rifle draws DMEM culture mediums (Hyclone companies) and rinses spleen repeatedly to prepare splenocyte suspension, NH4It is red in Cl processing splenocytes
Cell, 8 are pressed by splenocyte:1 ratio is sufficiently mixed with SP2/0 cells, by the cell after fusion according to splenocyte 2 × 106/
Ml, the requirement in 100 μ L/ holes, the HT culture mediums of pre-temperature needed for gently adding.After 24h, added per hole containing 2 times of amount A
The HT culture mediums of (Aminoperin, SIGMA company), per the μ L of hole 100.After 4 days, half amount changes the HAT culture mediums (SIGMA of pre-temperature
Company).After 7 days, half amount changes the HT culture mediums of pre-temperature.Observation cell, marks the cell of fusion, treats that clone cell is grown to daily
When more than the area of bottom hole 1/4, antibody titer detection is carried out to supernatant using indirect elisa method, positive hybridoma cell is screened.
And positive height is selected, the good hole of cell growth state is cloned using limiting dilution assay, is then expanded culture, is frozen, until
Untill all clone cell holes are all for the positive.Successively the fusion rate of three cell fusions is respectively 17.7%, 19.4% and
78.2%, positive rate is respectively 0,0 and 1.89%, is screened by three time cloningizations, and finally obtaining one plant can stably excreting antibody
Hybridoma cell strain be named as 3E4, its affinity constant be 4.1 × 108mol/L。
(3) identification of monoclonal antibody hypotype and ascites are largely prepared：
From Sigma companies mouse monoclonal antibody MAb hypotypes identification kit (Mouse monoclonal
Antibody Isotype Reagent), monoclonal antibody hypotype is identified using ELISA method according to operating instruction.Specific method is as follows：
(1) the μ g/mL coated elisa plates of lysozyme 2 are used, 4 DEG C overnight；
(2) antigen is discarded, PBST is washed 3 times, 5min/ times, dried；
(3) ascites or cells and supernatant suitably diluted, 37 DEG C, 1h are added；
(4) PBST is washed 3 times, 5min/ times；
(5) 2.5% skimmed milk power solution is added as confining liquid, 200 μ L/ holes, 37 DEG C of effect 1h, washing methods is ibid；
(6) Monoclonal Antibody Cell nutrient solution supernatant is diluted with 2.5% skimmed milk power solution, 100 μ L/ holes are added, 37 DEG C
1h is acted on, washing methods is ibid；
(7) by specification adds 1:Goat Anti-Mouse IgG1, IgG2a, IgG2b, IgG3, IgM of 1000 dilutions
And IgA antibody, 100 μ L/ holes, it is incubated at room temperature 30min, washing；
(8) the HRP mark rabbit-anti sheep IgG secondary antibodies of dilution, 100 μ L/ holes, 37 DEG C of effect 1h, washing are added；
(9) the μ L of tmb substrate solution 100, room temperature lucifuge effect 15min are added per hole；
(10) 3M H are added2SO4, 50 μ L/ holes, terminating reaction；
(11) ELIASA is preheated in advance, readings result is shown in such as table 1 below, judges that 3E4 antibody belongs to IgG2b hypotypes.
The MAb hypotype qualification results of table 1
The concrete operations that mouse ascites are largely prepared：Take 8~10 week old female BAl BIcs/c mouse of stalwartness, every mouse abdomen
Chamber injects the 500 autoclaved paraffin oils of μ L, 5 days hybridomas 1~2 × 10 of the pneumoretroperitoneum injection in exponential phase6
Individual/only, injection cell is after 5~7 days, and selection abdominal cavity substantially expands, and syringe needle is inserted mouse abdomen by handicapped mouse
Intracavitary, the intraperitoneal weak yellow liquid of drainage is into centrifuge tube, and 5000rpm centrifugation 10min take faint yellow supernatant, packing, mark
- 80 DEG C save backup afterwards.About it is spaced 2 days, after ascites is built up again in Mice Body, same method obtains ascites.
(4) octanoic acid-ammonium sulfate precipitation purifying ascites：
Concrete operations are as follows：
(1) 4 DEG C of ascites, 12000rpm centrifugation 15min, go the removal of impurity.
(2) take 1 part of ascites to be mixed with 2 parts of acetate buffers (0.06mol/L, pH 4.8), be stirred at room temperature down and add dropwise
Enter the μ L/mL ascites of caprylic acid 33.
(3) mixed at room temperature 30min.
(4) 4 DEG C of standing more than 2h, make it fully precipitate.
(5) 4 DEG C, 12000rpm centrifugation 30min abandon precipitation.
(6) after supernatant is filtered through sand core funnel, the 0.1M PBS (pH 7.4) of 1/10 volume are added, pH is adjusted with 2M NaOH
It is worth 7.4.
(7) 45% saturation degree is made into the ammonium sulfate that 0.277g/mL is added in 30min under ice bath.
(8) 4 DEG C of standing more than 1h.
(9) 4 DEG C, 10000rpm centrifugation 30min abandon supernatant.
(10) precipitation appropriate NaCL containing 137mM, 2.6mM KCL, 0.2mM EDTA PBS (pH 7.4) dissolves, in 50
4 DEG C of dialyzed overnights in the above-mentioned PBS of~100 times of volumes.
(11) take after a small amount of dialysis after sample suitably dilution, with UV spectrophotometer measuring protein content, SDS-PAGE
Antibody concentration is detected, and it is standby to dispense extremely -80 DEG C of preservation.
(5) indirect ELISA detects titer of ascites after purification：
Concrete operations are as follows：
(1) with antalzyme protein (Bioengineering Research Institute is built up in Nanjing) coated elisa plate, 4 DEG C overnight；
(2) antigen is discarded, PBST is washed 3 times, 5min/ times, dried；
(3) doubling dilution since 5,000 times of ascites after purification, each concentration has 2 repeating holes, and 37 DEG C are incubated 1h, together
When set negative control hole, negative control is the ascites that injection of bone marrow oncocyte is produced；
(4) PBST is washed 3 times, 5min/ times, is dried；
(5) the sheep anti mouse secondary antibody (1 of 100 μ L horseradish peroxidase-labeleds is added per hole:10000 times of dilutions), 37 DEG C incubate
Educate 1h；
(6) washing methods is ibid；
(7) the μ L of tmb substrate solution 100 are added per hole, color development at room temperature 15min, ELIASA reads OD450nm values record result.
(6) monoclonal antibody molecule amount and ascties protein assay：
By ascites before purification and after purification after being denatured SDS-PAGE electrophoresis, with Marker marker proteins in gel
Relative mobility be abscissa, by ordinate of the logarithm of labelled protein molecular weight drafting standard curve.According to antibody light chain
With migration situation of the heavy chain in gel, it is 154,368Da to calculate monoclonal antibody 3E4 molecular weight.Protein concentration is determined using BCA methods,
Standard curve is drawn, according to absorbance, standard curve is searched, the ascties protein content for obtaining 3E4 is 11.78mg/mL.
(7) monoclonal antibody affinity is determined：
Concrete operations are as follows：
By antigen lysozyme according to 8 μ g/ml, 4 μ g/ml, 2 μ g/ml, 1 μ g/ml coated elisa plates, by monoclonal antibody from certain dense
Degree starts doubling dilution, and remaining step is consistent with indirect elisa method.Logarithm value using antibody concentration (mol/L) is right as abscissa
The absorbance answered is ordinate, and 4 sigmoid curves can be made in a coordinate system.The top of sigmoid curve is found out, is set as
ODmax.Find out 4 curves each corresponding antibody concentrations of 50%ODmax respectively in curve.By 4 concentration one group, root two-by-two
The affinity constant of monoclonal antibody is calculated according to following equation.
Ka=(n-1)/2 (n [Ab '] t- [Ab] t)
Wherein n is the multiple of two coating concentration in every group, and [Ab '] t and [Ab] t are respectively two 50% in every group
The corresponding antibody concentrations of ODmax (mol/L)
The affinity constant that can try to achieve 3E4 monoclonal antibodies according to the formula is 4.1 × 108Mol/L, belongs to high-affinity antibody,
It is adapted to ELISA detections.
The preparation of the lysozyme polyclonal antibody of embodiment 2
(1) it is immunized and obtains polyclonal antibody
With lysozyme immune health new zealand white rabbit (Shanghai medical college of Fudan University) 3, immunizing dose is 500 μ g/
Only, one exempts to be to be emulsified lysozyme with isometric Freund's complete adjuvant, and multi-point injection is in rabbit neck after emulsification completely
And dorsal sc, once, booster immunization is lysozyme and isometric incomplete Freund's adjuvant, immunizing agent to booster immunization after 2 weeks
Amount and immunization method with being immunized for the first time, and third time booster immunization is the ear vein note that not emulsified lysozyme is carried out to rabbit
Penetrate, the μ g/ of injection volume 500 are only.7 days collection vein hematometry serum titers after intravenous injection, when serum titer reaches 24And more than
When, arteria carotis blood sampling simultaneously places 2h at 37 DEG C, and 4 DEG C stand overnight rear 3000rpm centrifugations 20min, collect serum, that is, obtain many grams
Grand antibody.
(2) caprylic acid-ammonium purified polyclonal antibodies：
Concrete operations are as follows：
(1) 4 DEG C of rabbit anteserum, 12000rpm centrifugation 15min, go the removal of impurity.
(2) take 1 part of serum and 2 parts of acetate buffers (0.06mol/L, pH 4.8) to mix, be stirred at room temperature down and add dropwise
Enter the μ L/mL rabbit anteserums of caprylic acid 75.
(3) mixed at room temperature 30min.
(4) 4 DEG C of standing more than 2h, make it fully precipitate.
(5) 4 DEG C, 12000rpm centrifugation 30min abandon precipitation.
(6) after supernatant is filtered through sand core funnel, 4 DEG C of dialysis 6h in the 0.01M PBS (pH 7.4) of 50 times of volumes.
(7) isometric saturated ammonium sulfate is added in the supernatant after dialysis, stirring while adding, 4 DEG C of standing more than 1h.
(8) 4 DEG C, 10000rpm centrifugation 30min abandon supernatant.
(9) precipitation appropriate NaCL containing 137mM, 2.6mM KCL, 0.2mM EDTA PBS (pH 7.4) dissolves, in 50
4 DEG C of dialyzed overnights in the above-mentioned PBS of~100 times of volumes.
(10) take after a small amount of dialysis after sample suitably dilution, with UV spectrophotometer measuring protein content, SDS-PAGE
Antibody concentration is detected, and it is standby to dispense extremely -80 DEG C of preservation.
(3) horseradish peroxidase-labeled polyclonal antibody
The horseradish peroxidase kit (Galaxybio companies) of polyclonal antibody activation after purification enters rower
Note, labeling method is carried out according to kit specification, and concrete operation method is as follows：(1) 100 μ L antibody-solutions (1mg/mL) are taken to turn
Enter the activation horseradish peroxidases of REAGENT I, slightly centrifuge, sink to horseradish peroxidase dry on ttom of pipe, horseradish enzyme tube wall
Powder HRP will rinse dissolving completely.Total reaction system control is in 100 μ L/mg activation HRP or so, and system increase influences mark rate,
According to by mg activation HRP correspondence antibody 100-500 μ g, HRP：X molecule mol ratios are maintained at 30:15~1 or so.(2) with
REAGENT II adjust pH value to 9.5 or so, 4 DEG C overnight.(3) REAGENT III (REAGENT II of about 3 times of volumes) is added, it is ensured that
PH is 7.0 or so for enzyme combination, can add the adjustment of REAGENT III, mixes terminating reaction.(4) glycerol adding to 50%, -20 DEG C guarantor
Deposit standby.
The operation of the lysozyme double crush syndrome detection method of embodiment 3
(1) with the μ g/ml coated elisa plates of monoclonal antibody 3E4 2 of the anti-lysozyme prepared in embodiment 1,100 μ L/ holes, 4
DEG C coating is stayed overnight；
(2) 0.5% PBST is washed 3 times, each 5min；
(3) 2.5% skimmed milk power (Shanghai Sheng Gong bioengineering Co., Ltd) solution of Fresh is as sealer,
200 μ L/ holes, 37 DEG C of closing 1h；
(4) 0.5% PBST is washed 3 times, each 5min；
(5) lysozyme standard product are diluted to 0,1.5,3,6,12,24 μ g/ml concentration (correspondence respectively with physiological saline
A1-A6 holes) and carry out with physiological saline measuring samples-egg white of 1000 times of dilutions (totally 86, correspond to remaining 86 respectively
Hole, Shanghai poultry breeding Co., Ltd), many anti-(being prepared in embodiment 2) (1 with horseradish peroxidase-labeled:
1600), each 100 μ L/ holes, 37 DEG C of incubation 1h；
(6) 0.5% PBST is washed 3 times, each 5min；
(7) tmb substrate solution is added, 100 μ L/ holes, 37 DEG C of incubation 15min add 3M sulfuric acid terminating reactions, 50 μ immediately
L/ holes, ELIASA determines OD450nm values.
(8) draw standard curve and see Fig. 1, and according to the content of lysozyme in standard curve calculating section measuring samples, meter
Result is calculated to see such as table 2 below.
The concentration of lysozyme in each sample of table 2
The assembling of the lysozyme double crush syndrome detection kit of embodiment 4
Kit includes：The monoclonal antibody 3E4 of coating, the polyclonal antibody of horseradish peroxidase-labeled, coating
Liquid, sealer, washing lotion, the lysozyme standard product solution being serially diluted, the how anti-dilution of enzyme mark, substrate solution and terminate liquid；With
And specification is a；
Wherein coated antibody is the monoclonal antibody 3E4 prepared in embodiment 1, and detection antibody is system in embodiment 2
The polyclonal antibody of standby obtained horseradish peroxidase-labeled；Sealer is 2.5% skimmed milk power solution；Washing lotion is 0.5%
PBST；The serial dilutions of lysozyme standard product solution are respectively 0,1.5,3,6,12 and 24 μ g/ml；The how anti-dilution of enzyme mark
For PBST (0.01M PBS, containing tween-20 0.5%)；Substrate solution is TMB solution；Terminate liquid is 3M sulfuric acid.
Claims (2)
1. a kind of method for detecting lysozyme in egg, it is characterised in that this method comprises the following steps：
(1) elisa plate is coated with 2 μ g/mL monoclonal antibody 3E4,100 μ L/ holes, 4 DEG C of coatings are stayed overnight；
(2) 0.5% PBST is washed 3 times, 5min/ times, is patted dry；
(3) closing of 2.5% skimmed milk power solution, 200 μ L/ holes, 37 DEG C of coating 1h are added；
(4) 0.5% PBST is washed 3 times, 5min/ times, is patted dry；
(5) lysozyme standard product solution, the measuring samples and 1 being serially diluted are added:The horseradish peroxidase of 1600 times of dilutions
The polyclonal antibody of mark, each 100 μ L/ holes, 37 DEG C of incubation 1h；
(6) 0.5% PBST is washed 3 times, 5min/ times, is patted dry；
(7) tmb substrate solution is added, 100 μ L/ holes, 37 DEG C of lucifuges react 15min；
(8) 3M sulfuric acid terminating reactions, 50 μ L/ holes are added；
(9) readings at ELIASA 450nm, records OD values；
(10) standard curve is drawn, the content of lysozyme in egg white in measuring samples is calculated；
Wherein described monoclonal antibody 3E4 is immunized BALB/c female mices by lysozyme and prepared；
Wherein described polyclonal antibody is obtained after new zealand white rabbit is immunized by lysozyme.
2. a kind of kit for detecting lysozyme in egg, it is characterised in that the kit is included：
Monoclonal antibody 3E4, the polyclonal antibody of horseradish peroxidase-labeled, coating buffer, confining liquid, washing lotion, it is serially diluted
Lysozyme standard product solution, the how anti-dilution of enzyme mark, substrate solution and terminate liquid；
Wherein described monoclonal antibody 3E4 is immunized BALB/c female mices by lysozyme and prepared；Anti-TNF-α
Body is to be immunized after new zealand white rabbit to obtain by lysozyme；Coating buffer is the phosphate buffer PBS of pH=7.2 ± 0.1；Confining liquid
For 2.5% skimmed milk power solution；Washing lotion is 0.5%PBST；The serial dilutions of lysozyme standard product solution are respectively 0,
1.5th, 3,6,12 and 24 μ g/ml；The how anti-dilution of enzyme mark is PBST (0.01M PBS, containing tween-20 0.5%), and substrate is molten
Liquid is TMB solution, and terminate liquid is 3M sulfuric acid.
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