CN102827054B - Hapten of L-hydroxyproline, artificial antigen, monoclonal antibody, preparation method and application - Google Patents
Hapten of L-hydroxyproline, artificial antigen, monoclonal antibody, preparation method and application Download PDFInfo
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- CN102827054B CN102827054B CN201210315692.XA CN201210315692A CN102827054B CN 102827054 B CN102827054 B CN 102827054B CN 201210315692 A CN201210315692 A CN 201210315692A CN 102827054 B CN102827054 B CN 102827054B
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- oxyproline
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Abstract
The invention discloses hapten of L-hydroxyproline, artificial antigen, a monoclonal antibody, a preparation method and an application. The hapten of L-hydroxyproline contains a molecular structure of L-hydroxyproline, a connecter of hapten and carrier protein is taken as immunogen, thereby the antibody aimed at L-hydroxyproline can be generated. The hapten of L-hydroxyproline can prepare a specific antibody of L-hydroxyproline for immunological analysis and screening of L-hydroxyproline, so that the detection efficiency is enhanced and the detection cost is reduced.
Description
Technical field
The present invention relates to food safety field, be specifically related to a kind of haptens, artificial antigen and monoclonal antibody and preparation method thereof and application of L-oxyproline.
Background technology
Collagen protein is present in skin, bone, heel string, cartilage, blood vessel and the tooth of animal, accounts for 1/4th of Mammals total protein, and collagen hydrolysate be can be made into animal hydrolyzed protein.Hydrolyzed animal collagen proteins not only can affect mouthfeel and local flavor after mixing milk and milk products, and makes amino acid whose ratio unreasonable, and nutritive value reduces, and nondigestible, and absorption of human body utilization ratio is reduced.Some animal hydrolyzed protein is the protein that utilizes waste material, rubbish and hair etc. to be processed into, cheap, contain carcinogens, heavy metal, a large amount of miscellaneous bacteria, especially the heavy metal chromium containing in " tanning " lower horn scraps, if be absorbed by the body, accumulate, can cause that joint is loose, arthroncus, cause body weight for humans metal poisoning, very big to harm.For this reason, ministry of Health of China is listed second batch " the non-edible material from soybeans list of the illegal interpolation of possibility in food " in by " leather skin hydrolyzate ".
L-oxyproline is peculiar amino acid in animal collagen, and in collagen protein, content approximately 10.0～13.4%, in other albumen, do not exist.Utilize this characteristic just can carry out the monitoring of collagen protolysate, by detecting L-oxyproline, identify in breast and milk-product whether added hydrolyzed animal collagen proteins.
At present, the measuring method for L-oxyproline mainly contains colorimetry, high performance liquid chromatography, tablets by HPLC-MS, automatic analyzer for amino acids assay method etc.But above method is measured the L-oxyproline in milk and milk products, respectively has deficiency.Colorimetry specificity is strong, complex operation, precision are poor, and measuring result is subject to various factors.High performance liquid chromatography needs pre-column derivatization, complicated operation.The consumptive material of tablets by HPLC-MS and automatic analyzer for amino acids assay method is expensive, cost is high makes many laboratories hope and halt.
And immune analysis method has very high tolerance range, susceptibility and specificity, and not high to technical requirements, there is detection time short, be applicable to the advantages such as mensuration in enormous quantities.Up to the present, also do not have bibliographical information to adopt immunization to detect L-oxyproline.Preparation, for the specific antibody of L-oxyproline, can, for setting up immunodetection or the research and development rapid detection product for L-oxyproline, be carried out the quick Site Detection of protolysate in milk-product and be laid the foundation with extensive generaI investigation.
Summary of the invention
One of object of the present invention is to provide a kind of haptens of L-oxyproline.
Above-mentioned purpose of the present invention reaches by the following technical programs:
A haptens for L-oxyproline, its molecular structural formula as shown in Equation 1:
Formula 1.
Two of object of the present invention is to provide the haptenic preparation method of above-mentioned L-oxyproline.
A haptenic preparation method for L-oxyproline, comprises the steps,
(a) mol ratio of L-oxyproline and p-Hydroxybenzaldehyde be set to 1: 1～2, in ethanolic soln, heating reflux reaction is 1～4 hour,
(b) after reacting completely, cooling, suction filtration, washing obtains the L-oxyproline haptens of formula 1 structure:
Formula 1.
An optimal technical scheme, is characterized in that: in described step (a), the mol ratio of L-oxyproline used and p-Hydroxybenzaldehyde is 1: 1, and in ethanol, heating reflux reaction is 2 hours.
Three of object of the present invention is to provide a kind of artificial antigen of L-oxyproline.
Above-mentioned purpose of the present invention reaches by the following technical programs:
An artificial antigen for L-oxyproline, is combined into by aforesaid L-oxyproline haptens and carrier proteins, its molecular structural formula as shown in Equation 2:
Formula 2.
An optimal technical scheme, is characterized in that: described carrier proteins is ovalbumin or bovine serum albumin.
Four of object of the present invention is to provide the preparation method of the artificial antigen of above-mentioned L-oxyproline.
The preparation method of L-oxyproline artificial antigen, comprises the steps:
(a) haptens of the L-oxyproline of formula 1 structure is dissolved in distilled water, obtain solution A, by N, N-carbonyl dimidazoles is dissolved in anhydrous propanone, obtain solution B, L-oxyproline haptens and the N of formula 1 structure used, the mol ratio of N-carbonyl dimidazoles is 1:3～8, solution A and solution B mix and blend are reacted 4～6 hours, obtain intermediate product C;
(b) described carrier proteins is dissolved in phosphate buffer soln, obtains solution D, solution C is added drop-wise in solution D, stirring reaction 11～14 hours, obtains the artificial antigen of formula 2 structures.
Five of object of the present invention is to provide a kind of monoclonal antibody of L-oxyproline.
Above-mentioned purpose of the present invention reaches by the following technical programs:
A monoclonal antibody for L-oxyproline, be can with the immunoglobulin (Ig) of the artificial antigen generation specific immune response of aforesaid L-oxyproline.
Six of object of the present invention is to provide the application of above-mentioned L-oxyproline monoclonal antibody.
Above-mentioned purpose of the present invention reaches by the following technical programs:
Described monoclonal antibody is for the immunodetection of L-oxyproline.
The molecular structure of L-oxyproline is fairly simple, is directly connected with carrier proteins as immunogen and goes immune animal can not produce specific antibody.Therefore, the structure of L-oxyproline must be carried out complicatedly, increase molecular weight, expand molecular structure.The molecular structure that the above-mentioned haptens based on L-oxyproline contains L-oxyproline, is connected preparation immunogen with carrier proteins with this haptens, can produce the antibody for L-oxyproline.Therefore, L-oxyproline compound provided by the invention can be prepared the specific antibody of L-oxyproline, for immunoassay and the screening of L-oxyproline, improves detection efficiency, reduces testing cost.
Below by specific embodiment, the present invention will be further described, but and do not mean that limiting the scope of the invention.
Embodiment
Experimental technique described in following embodiment, if no special instructions, is ordinary method; Described reagent and biomaterial, if no special instructions, all can obtain from commercial channel.
Embodiment 1
Experiment 1: the haptenic synthetic example 1 of formula 1 structure
1. in 25mL round-bottomed flask, add 131mg (0.001mol) L-oxyproline and 122mg (0.001mol) p-Hydroxybenzaldehyde;
2. add 4mL ethanol solution and several acetic acid, back flow reaction 2 hours, drains, and obtains product 175.9mg, productive rate 70%.
Formula 1.
245 ℃ of this product fusing points; Infrared spectra (IR) characterization data: (KBr) V
max3284,3300-2450,3137,2950,2732,1639,1600,1480,1400,1321,1068,962cm
-1.With the infrared data comparison of L-oxyproline, had more phenyl ring and-CO-N key.
Experiment 2: the haptenic synthetic example 2 of formula 1 structure
1. in 25mL round-bottomed flask, add 131mg (0.001mol) L-oxyproline and 244mg (0.002mol) p-Hydroxybenzaldehyde;
2. add 7mL ethanol solution and several acetic acid, back flow reaction 4 hours, drains, and obtains product 190.9mg, productive rate 76%.
Product structure and characterization data are the same.
Experiment 3: the haptenic synthetic example 3 of formula 1 structure
1. in 25mL round-bottomed flask, add 131mg (0.001mol) L-oxyproline and 183mg (0.0015mol) p-Hydroxybenzaldehyde;
2. add 5mL ethanol solution and several acetic acid, back flow reaction 1 hour, drains, and obtains product 180.9mg, productive rate 72%.
Product structure and characterization data are the same.
Experiment 4: the synthetic example 1 of the artificial antigen of formula 2 structures
1. the compound dissolution of formula 1 structure of the above-mentioned preparation of 12.6mg (0.05mmol) is in distilled water, and magnetic agitation is dissolved it completely, obtains solution A, and by 40.5mg (0.25mmol) N, N-carbonyl dimidazoles is dissolved in 3mL anhydrous propanone, obtains solution B; A solution and B solution are mixed to magnetic agitation reaction 5 hours, obtain solution C;
2. 65mg (0.001mmol) bovine serum albumin (BSA) is dissolved in the phosphate buffer solution (PBS) of the 0.01moL/L of 5mL pH7.2, obtains solution D, above-mentioned solution C is dropwise joined in solution D, magnetic agitation reaction 12 hours;
3. above-mentioned reaction solution is packed in dialysis tubing, 4 ℃ with normal saline solution dialysis 72 hours, during change dialyzate 6 times; Outwell dialyzate, the solution in dialysis tubing is carried out centrifugal, rotating speed is 4000rpm, 5 minutes time, get supernatant liquor, and be the artificial antigen of formula 2 structures, the artificial antigen of formula 2 structures is sub-packed in ampere bottle to-20 ℃ of preservations.
According to 2,4 of bibliographical information, the coupling ratio that 6-trinitro-benzene-sulfonic acid method is measured haptens and carrier proteins is 14: 1, proves the coupling of haptens and carrier proteins.2,4,6-trinitro-benzene-sulfonic acid can react with the free amine group in albumen, and the concentration of products therefrom is directly proportional to the absorbancy at ultraviolet 335nm place.Control artificial antigen and equate with the carrier proteins concentration of not coupling, with the reaction of 2,4,6-trinitro-benzene-sulfonic acid, the absorbancy of product is different, illustrates that a part of free amine group in carrier proteins has occurred to react with haptens respectively.With the difference of absorbancy, calculate the coupling ratio of haptens and carrier proteins.
Experiment 5: the synthetic example 2 of the artificial antigen of formula 2 structures
1. the compound dissolution of formula 1 structure of the above-mentioned preparation of 12.6mg (0.05mmol) is in distilled water, and magnetic agitation makes it
Dissolve completely, obtain solution A, by 24.3mg (0.15mmol) N, N-carbonyl dimidazoles is dissolved in 3mL anhydrous propanone,
Obtain solution B; A solution and B solution are mixed to magnetic agitation reaction 4 hours, obtain solution C;
2. 81mg (0.00125mmol) bovine serum albumin (BSA) is dissolved in the phosphate buffer solution (PBS solution) of the 0.01moL/L of 5mLpH7.2, obtain solution D, above-mentioned solution C is dropwise joined in solution D to magnetic agitation reaction 11 hours;
3. above-mentioned reaction solution is packed in dialysis tubing, 20 ℃ with normal saline solution dialysis 48 hours, during change dialyzate 6 times; Outwell dialyzate, the solution in dialysis tubing is carried out centrifugal, rotating speed is 4000rpm, 5 minutes time, get supernatant liquor, and be the artificial antigen of formula 2 structures, the artificial antigen of formula 2 structures is sub-packed in ampere bottle to-20 ℃ of preservations.
Experiment 6: the synthetic example 3 of the artificial antigen of formula 2 structures
1. the compound dissolution of formula 1 structure of the above-mentioned preparation of 12.6mg (0.05mmol) is in distilled water, and magnetic agitation makes it
Dissolve completely, obtain solution A, by 64.8mg (0.4mmol) N, N-carbonyl dimidazoles is dissolved in 3mL anhydrous propanone,
Obtain solution B; A solution and B solution are mixed to magnetic agitation reaction 6 hours, obtain solution C;
2. by 23mg (0.0005mmol) ovalbumin molten (OA) solution in the phosphate buffer solution (PBS solution) of the 0.01moL/L of 5mL pH7.2, obtain solution D, above-mentioned solution C is dropwise joined in solution D to magnetic agitation reaction 14 hours;
3. above-mentioned reaction solution is packed in dialysis tubing, 20 ℃ with normal saline solution dialysis 84 hours, during change dialyzate 6 times; Outwell dialyzate, the solution in dialysis tubing is carried out centrifugal, rotating speed is 4000rpm, 5 minutes time, get supernatant liquor, and be the artificial antigen of formula 2 structures, the artificial antigen of formula 2 structures is sub-packed in ampere bottle to-20 ℃ of preservations.
Embodiment 2: the preparation of antibody and evaluation
1) immune animal is prepared antiserum(antisera)
Using the artificial antigen of above-mentioned formula 2 structures as immunogen immune Balb/c mouse respectively, with the NaCl solution of 0.15mol/L, immunogen is diluted to the concentration of 1mg/mL, add equivalent Fu Shi Freund's complete adjuvant and make water in oil emulsifying agent, in the back of Babl/c mouse intracutaneous multi-point injection, carry out head and exempt from; 2 weeks, interval, gets after same amount antigen adds the emulsification of equivalent Fu Shi Freund's incomplete adjuvant and carries out the subcutaneous multi-point injection in back, is booster immunization; Booster immunization is 5 times altogether, and last booster immunization does not add adjuvant, abdominal injection.
Last immunity is extractd eyeball by mouse in latter 3 days and is got blood, and every about 2mL of the desirable blood of mouse places 5-6 hour by the blood sample of collection in 4 ℃ of refrigerators, and then 5000rpm is centrifugal 10 minutes, separation of serum.
2) purifying of antibody
Adopting sad-ammonium sulfate salting-out process to above-mentioned steps 1) antiserum(antisera) that obtains carries out purifying.Sadly under the condition of slant acidity, the protein except IgG in serum all can be precipitated, in supernatant, only have IgG.Three repetitions are established in experiment, and result shows, adopts the rate of recovery of aforesaid method IgG to reach more than 90%.
3) antiserum(antisera) lowest detectable limit (LOD value) and half amount of suppression (IC
50) detection
By square formation volumetry, determine above-mentioned steps 2) antibody of acquisition and the working concentration of artificial antigen prepared by above-described embodiment 1.
With the L-oxyproline standard solution of different concns, do experimental solutions, concentration is 0.025,0.05,0.1,0.2,0.4,0.8,1.6,3.2,6.4,12.8 (concentration unit of standard solution is μ g/mL).Adopt 8 groups of parallel tests (n=8).
Enzyme linked immuno sorbent assay (ELISA): with the artificial antigen coated elisa plate of formula 2 structures of above-mentioned working concentration, experimental solutions and antibody-solutions are added in enzyme plate aperture simultaneously, zero standard hole is set simultaneously (to be replaced the experimental solutions of interpolation with high purity water, other is consistent) and blank hole (change the antibody-solutions of interpolation into high purity water, other is consistent), 37 ℃ of incubations 0.5 hour, pour out liquid in hole, with washings (10x PBST) washing 2-5 time, enzyme plate is upside down on thieving paper and is patted; Add ELIAS secondary antibody solution in enzyme plate aperture, 37 ℃ of incubations 0.5 hour, repeat to wash 3-5 time with washings, blot; Add substrate chromophoric solution in enzyme plate aperture, under room temperature, react 10-15 minute, by microplate reader, at wavelength 450nm place, measure absorbance (OD).Take absorbancy as ordinate zou, and the logarithm (Log) of standard substance experimental solutions concentration of take is X-coordinate, draws semilog canonical plotting.
According to typical curve, draw 10% amount of suppression (LOD) and half amount of suppression (IC
50).
Inhibiting rate is calculated as follows;
In formula: the light absorption value (zero standard hole) of ODmax when not adding standard substance experimental solutions, ODx is the light absorption value of standard substance concentration while being x, ODmin is the light absorption value in blank hole.
Result shows, the half amount of suppression (IC of above-mentioned antibody to L-oxyproline
50) be 0.2 μ g/mL, lowest detectable limit (LOD) is 0.05 μ g/mL, and within the scope of 0.1-12.8 μ g/mL, the logarithmic value of inhibiting rate and L-oxyproline concentration is significant linear relationship, and relation conefficient is r=0.9984.
4) specific detection of antibody
The ability that sero-fast specificity just refers to its homospecificity antigen combination and comparison with this antigen-analogues ability.Conventional cross reacting rate is as the major criterion of evaluating.Cross reaction is less, and the specificity of antibody is better.
Several analogues of L-oxyproline (proline(Pro), D-oxyproline) being carried out to serial dilution, then with above-mentioned steps 2) antibody that obtains reacts, production standard curve.Half amount of suppression (IC while finding out respectively this several analogues generation 50% inhibition on curve
50), the cross reacting rate of calculating antibody and L-oxyproline analogue.
Cross reacting rate (%)=(the half amount of suppression of half amount of suppression/other analogue of L-oxyproline) * 100%.
Three repetitions are established in experiment, get the mean value of three repetitions as experimental result.Result shows, above-mentioned steps 2) antibody that obtains only identifies L-oxyproline, and specificity is fine, all there is no cross reactivity with proline(Pro) and D-oxyproline.Therefore, can be used as detection reagent L-oxyproline is carried out to immunoassay.
Claims (4)
1. a haptens for L-oxyproline, its molecular structural formula as shown in Equation 1:
2. an artificial antigen for L-oxyproline, its molecular structural formula as shown in Equation 2:
3. the artificial antigen of L-oxyproline as claimed in claim 2, is characterized in that, described carrier proteins is ovalbumin or bovine serum albumin.
4. the preparation method of the artificial antigen of L-oxyproline as described in claim 2 or 3, comprises the steps:
(a) haptens of the L-oxyproline of formula 1 structure is dissolved in distilled water, obtain solution A, by N, N-carbonyl dimidazoles is dissolved in anhydrous propanone, obtain solution B, haptens and the N of the L-oxyproline of formula 1 structure used, the mol ratio of N-carbonyl dimidazoles is 1: 3～8, solution A and solution B mix and blend are reacted 4～6 hours, obtain solution C;
(b) described carrier proteins is dissolved in phosphate buffer soln, obtains solution D, solution C is added drop-wise in solution D, stirring reaction 11～14 hours, obtains the artificial antigen of formula 2 structures,
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