CN110058011A - A kind of luminous enzyme-linked immunologic detecting kit of tetracycline chemical - Google Patents
A kind of luminous enzyme-linked immunologic detecting kit of tetracycline chemical Download PDFInfo
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- CN110058011A CN110058011A CN201910436476.2A CN201910436476A CN110058011A CN 110058011 A CN110058011 A CN 110058011A CN 201910436476 A CN201910436476 A CN 201910436476A CN 110058011 A CN110058011 A CN 110058011A
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Abstract
The invention discloses a kind of luminous enzyme-linked immunologic detecting kits of tetracycline chemical.The kit includes reagent needed for tetracycline gene engineered antibody and other enzyme linked immunosorbent detections, is such as coated with the chemiluminescence ELISA Plate of tetracycline envelope antigen, tetracycline standard items, enzyme labelled antibody, chemical luminescence for liquid and cleaning solution.Kit of the invention has the advantages that high-throughput detection, high sensitivity, high specific, high precision, high stability, detection range is wide, analysis speed is fast, accurate；Standard curve maximum detection range is 15~120pg/mL, sensitivity 45pg/mL, detection limit 40pg/mL；And it is nontoxic, environmentally friendly, economical, mass production can be carried out by technique for gene engineering, to culture medium without particular/special requirement, density of fermenting is high, fermentation period is short, and manufacturing cost is low.The kit is highly suitable for the trace analysis of tetracycline residue and the screening of batch sample detects, and has important practical significance and popularization and application foreground.
Description
Technical field
The invention belongs to medicament residue detection technique fields.It shines enzyme-linked exempt from more particularly, to a kind of tetracycline chemical
Epidemic disease detection kit.
Background technique
Tetracycline medication (TCs) has effects that the sterilization antibiosis of broad-spectrum high efficacy, can add with most drugs or feed
Add agent mixed, has been widely used in Production of Livestock and Poultry.But it is excessively used and inevitably causes serious medicament residue, at
For one of the food safety hazards for seriously endangering human health.Countries in the world detection remaining to TCs thus is extremely paid close attention to.European Union
The total amount of tentatively tetracycline medication of the regulation in muscle and milk must not exceed 100 μ g/kg in 675/92nd command, in kidney
Tetracycline medication must not exceed 600 μ g/kg, 300 μ g/kg and 200 μ g/kg in dirty, liver and egg.Japanese Positive List System
The total amount that metric determines tetracycline compound in milk and egg must not exceed 100ng/mL and 400ng/mg respectively.
The reported remaining detection method of tetracycline medication has microbial method, thin-layered chromatography, efficient liquid phase measurement
Method, high performance liquid chromatography-tandem mass method, Capillary Electrophoresis, electrochemical analysis, enzyme-linked immunization, colloidal gold method etc., instrument inspection
Survey method requires height to the pre-treatment of sample to be tested, cumbersome, needs precision instrument and equipment.Enzyme-linked immunization (ELISA) though
Have the characteristics that simple, quick, sensitive, is also suitable for large-scale screening, but its sensitivity has certain restriction, Er Qiexian
The ELISA kit for detecting tetracycline is needed using with toxic or for carcinogen substrates such as corrosive sulfuric acid.
Therefore, establish it is a kind of it is economical, reliable, special, sensitive, quickly and effectively tetracycline medication detection method have it is very big
Practical significance and application prospect.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defects and deficiency of existing tetracycline medication detection technique, provide
One kind have high-throughput detection, high sensitivity, high specific, high precision, high stability, detection range is wide, analyze speed fastly,
Accurately, and tetracycline medication chemiluminescent enzyme-linked immunosorbent immunoassay technology the advantages that environmental protection and economy.
The object of the present invention is to provide a kind of luminous enzyme-linked immunologic detecting kits of tetracycline chemical.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of luminous enzyme-linked immunologic detecting kit of tetracycline chemical, contains tetracycline gene engineered antibody.
Preferably, the preparation method of the tetracycline gene engineered antibody: to be immunized and examine by tetracycline immunizing antigen
Surveying serum titer titre to reach the total serum IgE of the zooblast (spleen cell) of requirement is template, utilizes SEQ ID NO.1~4 institute
Show that primer RT-PCR amplifies the light chain of antibody, heavy chain variable region gene and connects, and is inserted into carrier
PCANTAB5E, in expression in escherichia coli.
Preferably, the amplification condition of light chain are as follows: 94 DEG C × 5min denaturation, be added 1 μ L 2.5U high-fidelity Pfu enzyme, carry out with
Lower circulation: 94 DEG C × 30s, 54 DEG C × 1min, 72 DEG C × 1min, totally 25 recycle, last 72 DEG C of extensions 10min.
Preferably, the amplification condition of heavy chain are as follows: 94 DEG C × 5min denaturation, be added 1 μ L 2.5U high-fidelity Pfu enzyme, carry out with
Lower circulation: 94 DEG C × 30s, 60 DEG C × 1min, 72 DEG C × 1min, totally 30 recycle, last 72 DEG C of extensions 10min.
Preferably, then after amplifying light chain and heavy chain fragment, kit according to claim 1, which is characterized in that with
The light chain and heavy chain fragment amplified template each other completes the pre-splicing of VH--VL segment by PCR, with the 1/ of pre-splicing product
50 dilutions are template, carry out the splicing of VH--VL full-length gene, and (contain Bgl I and Not I enzyme using primer Q-B/Q-F
Enzyme site) secondary PCR amplification is carried out to antibody, the full length antibody genetic fragment for being about 760bp is obtained, by antibody fragment Bgl
It after I and Not I double digestion, is connect with carrier pCANTAB5E, conversion Escherichia coli TG3 carries out IPTG inducing expression.Wherein, institute
State the condition of secondary PCR amplification are as follows: 94 DEG C × 5min initial denaturation adds 0.5 μ L high-fidelity Pfu enzyme, the following circulation of progress: 94 DEG C ×
45s, 50 DEG C × 1min, 72 DEG C × 1min.
Preferably, the kit also contains the chemiluminescence ELISA Plate for being coated with tetracycline envelope antigen, tetracycline mark
Quasi- product, enzyme labelled antibody, chemical luminescence for liquid and cleaning solution；
Preferably, the chemiluminescence ELISA Plate is the removable opaque white color luminescent screen in 96 holes；
Preferably, the tetracycline envelope antigen is the conjugate of tetracycline haptens and bovine serum albumin(BSA) BSA；
Preferably, the peridium concentration of the tetracycline envelope antigen is 0.5mg/L；
Preferably, 1.69g sodium carbonate and 2.95g sodium bicarbonate are preferably dissolved in 1L distilled water and obtaining by coating buffer, and four
The peridium concentration of ring element envelope antigen is 0.5mg/L.
Preferably, confining liquid preferably takes 0.1gBSA (bovine serum albumin(BSA)), and 5g glycine is dissolved in 100mLPBS
(0.01mol/L PH7.4) solution obtains.
Preferably, the preparation method of the tetracycline envelope antigen is the p-aminobenzoic acid and tetracycline after diazotising
Phenolic hydroxyl group contraposition occur azo, and then introduce carboxyl, carboxylic tetracycline haptens use DCC method and carrier protein
BSA coupling.
Specifically, the tetracycline antigen (envelope antigen, immunizing antigen) the preparation method is as follows:
(1) synthesis of tetracycline haptens:
A: p-aminobenzoic acid is dissolved in 1.5~2.5moL/L, 4 DEG C of HCl, and the nitrous of 0.4~0.5mg/mL is added
Acid sodium solution is placed in 0~8 DEG C of 8~15min of stirring, obtained reaction solution, referred to as A liquid；
B: weighing tetracycline raw medicine, is dissolved in 0.1~0.3moL/L NaOH, referred to as B liquid；
A liquid: and then being added drop-wise in B liquid by c the bottom of by, and mixture pH is maintained between 8~9, and in 10 DEG C of 1~3h of stirring,
Taking-up is cooled to room temperature, and after low acidified, filtered out liquid with concentrated hydrochloric acid and is adjusted to 3.0, and precipitating is then filtered to obtain, and precipitating uses cold distilled water
Washing, finally freeze-drying obtains p-aminobenzoic acid tetracycline；
(2) preparation of tetracycline envelope antigen:
P-aminobenzoic acid tetracycline, NHS and DCC are weighed, is dissolved in N-N- dimethylformamide, is stirred at room temperature
4~6h is centrifuged to obtain supernatant；BSA is dissolved in the phosphate buffer of 0.04~0.06moL/L, pH 8, is slowly stirred at 0~8 DEG C
Mix 20~30h；Reaction solution is transferred in the bag filter handled well after the reaction was completed to above-mentioned, uses phosphorus under 0~8 DEG C, stirring
Acid buffer dialysis, -20 DEG C save backup；
(3) preparation of tetracycline immunizing antigen:
P-aminobenzoic acid tetracycline, NHS and DCC are weighed, is dissolved in N-N- dimethylformamide, is stirred at room temperature
4~6h is centrifuged to obtain supernatant；KLH is dissolved in the phosphate buffer of 0.04~0.06moL/L, pH 8, is slowly stirred at 0~8 DEG C
Mix 20~30h；Reaction solution is transferred in the bag filter handled well after the reaction was completed to above-mentioned, uses phosphorus under 0~8 DEG C, stirring
Acid buffer dialysis, -20 DEG C save backup.
Wherein, the concentration of HCl is 2moL/L in step a, and the concentration of sodium nitrite solution is 0.48mg/mL.
P-aminobenzoic acid in step a: HCl: the ratio of sodium nitrite solution is 0.69g:16mL:1mL.
The concentration of NaOH is 0.2moL/L in step a.
Tetracycline in step a: the ratio of NaOH is 2.220g:60mL.
The concentration of phosphate buffer is 0.05moL/L in step (2), (3).
P-aminobenzoic acid tetracycline in step (2), (3): the ratio of NHS:DCC:N-N- dimethylformamide is
0.594g:0.115g:0.21g:5mL.
BSA or KLH in step (2), (3): the ratio of phosphate buffer is 0.8375g:10mL.
The concentration of phosphate buffer is 0.01moL/L in step (2), (3), and dialysis time 72h, preceding 8h every for 24 hours change liquid 1
It is secondary.
Furthermore it is preferred that being coated with the preparation of the chemiluminescence ELISA Plate of tetracycline envelope antigen: taking ELISA Plate, be coated with
Antigen is added in every hole after being diluted to 0.5mg/L with coating buffer, 37 DEG C overnight, liquid in hole of inclining, with washing lotion wash for several times,
It is patted dry on blotting paper；Then confining liquid is added in every hole, and 37 DEG C are incubated overnight, liquid in hole of inclining, and is placed in vacuum after 37 DEG C of drying
Seal 4 DEG C of preservations.
Preferably, the original content of tetracycline gene engineered antibody is 2mg/mL, is diluted when use with 0.01mol/L PBST
6000 times.
The formula of the 0.01mol/L PBST is NaH2PO4.12H2O 2.9g、NaCl 8.5g、KCl 0.2g、KH2PO4
0.2g, Tween-20 0.5mL, are settled to 1mL.
Preferably, the concentration of the tetracycline standard items is 1mg/mL, with 0.01mol/L PBST by standard when use
Product are diluted to a series of tetracycline standard solutions that concentration is 0,0.03,0.1,0.3,1,3 μ g/L.
Preferably, the enzyme labelled antibody is sheep anti-mouse igg or goat anti-rabbit igg (the preferably sheep of horseradish peroxidase label
Anti- mouse IgG)；Its original content is 10mg/mL, and preferably 0.01mol/L PBST dilutes 6000 times when use.
Preferably, the chemical luminescence for liquid is made of A liquid and B liquid, and A liquid is to contain 20mg pairs in 100mL deionized water
Iodophenol, 8mg luminol, 1.21g Tris, pH8.4；B liquid is to contain volume fraction 0.40%H in 100mL deionized water2O2、
1.21g Tris, pH7.0；When use by A liquid and B liquid by volume 1:1 ratio mix.
Preferably, the cleaning solution is 20 times of concentrated cleaning solutions, and 20 times of concentrated cleaning solutions are containing volume fraction 0.5%
The phosphate buffer of the pH7.4 0.4mol/L of Tween20 is diluted to 1 times of cleaning solution with deionized water when use.
The above-mentioned application method for stating kit, includes the following steps:
S1. the pre-treatment of sample to be tested；
S2. kit detects: sequentially adding tetracycline standard solution or sample, tetracycline gene engineered antibody, competes
Enzyme labelled antibody is added after reaction, is eventually adding the quantitative inspection that chemical luminescence for liquid carries out tetracycline by chemical illumination immunity analysis instrument
It surveys；
S3. result treatment and analysis.
Wherein, when the sample to be tested described in the step S1 is milk sample, egg or tissue sample, processing method is as follows:
Milk sample: directly taking the milk of fresh milk or freeze thawing, diluted with PBST, the measurement of centrifuging and taking supernatant；(preferably
Ground directly takes the milk of fresh milk or freeze thawing, is diluted with 0.05M PBST with 1:10, takes supernatant to measure after centrifugation)；
Egg: egg liquid is diluted with PBS solution, the measurement of centrifuging and taking supernatant；Lead to x- containing 0.1% Qula in the PBS solution
100；(preferably, with egg liquid: PBS solution 1:10 dilution takes supernatant to measure after centrifugation；It is bent containing 0.1% in the PBS solution
Draw logical x-100)；
Tissue sample: taking chicken, the chicken of pig, liver or renal tissue sample respectively, adds Mcllvaine buffer, oscillation
Centrifugation, takes supernatant spare；Oasis HLB solid-phase extraction column successively uses anhydrous methanol, water prewashing；Spare supernatant is taken to cross column,
It is eluted, is extracted with water；It is eluted, is extracted with oxalic acid methanol solution, collect eluent；The eluent of collection is shaken up, it is molten with PBST
It is to be measured as sample solution after liquid dilution.
Preferably, tissue sample is taken, Mcllvaine buffer is added, oscillation centrifugation takes supernatant spare；Oasis HLB is solid
Phase extraction column successively uses anhydrous methanol, water prewashing；It takes spare supernatant to cross column, is eluted with water, extracted；With 20mmol/L methyl ethyl oxalate
Alcoholic solution 1ml elution, is extracted, and collects eluent；The eluent of collection is shaken up, is used after 10 times of PBST solution dilutions as sample
Solution is to be measured；The formula of the Mcllvaine buffer: 0.1M citrate buffer solution, 0.1M EDTA, pH3.8.
Preferably, the specific method is as follows by step S2:
S21. kit is placed in (25~27 DEG C) 30~40min of balance of room temperature；
S22. chemiluminescence ELISA Plate is taken out, the tetracycline standard solution of various concentration, sample are added into gauge orifice
Sample to be tested is added in hole, then tetracycline gene engineered antibody is added in every hole, covers the shaking of cover board film and mixes, is incubated for；
S23. the reaction solution in plate hole is removed, cleaning solution washing is added, ELISA Plate is patted dry；
S24. enzyme labelled antibody is added into each hole, pats mixing, be incubated for,
S25. chemiluminescence reaction liquid is added in every hole, pats mixing, covers cover board film, exempted from after 1~2min with chemiluminescence
The luminous value RLU in each hole of epidemic disease analysis-e/or determining.
The method of step S3 is calculated and is analyzed according to following formula:
Inhibiting rate (%)=B/B0 × 100 (%),
In formula: the luminous value of B-various concentration tetracycline standard solution hole (or sample well)；0 concentrations of tetracycline of B0-mark
Quasi- solution luminous value；Using inhibiting rate as ordinate, the logarithm of tetracycline concentration is that abscissa draws standard curve, so that it is determined that sample
The content of tetracycline in product.
Specifically, alternatively, the application method of the kit comprises the following steps:
(1) kit is placed in equilibrium at room temperature 30min or more, standard items, which are diluted to concentration, with 0.01mol/L PBST is
0, a series of tetracycline standard solutions of 0.03,0.1,0.3,1,3 μ g/L；
(2) chemiluminescence ELISA Plate is taken out, the tetracycline standard solution of 50 μ L various concentrations, sample is added in gauge orifice
50 μ L samples to be tested are added in hole, and then the tetracycline gene engineered antibody that 50 μ L have diluted is added in every hole, cover cover board film micro-
It measures after vibrating 10min on oscillator, is placed in 37 DEG C of incubation 50min；
(3) reaction solution in plate hole is absorbed, about 300 μ L of cleaning solution is added in each hole, stands 20 seconds or so, removes wherein liquid
Body is so washed 5 times altogether, for the last time pats dry plate；Class can also be washed with automatic washer 5 times, be upside down in micropore frame after washing
It pats on blotting paper to guarantee to completely remove the liquid in hole.
(4) the sheep anti-mouse igg enzyme mark antiantibody for the horseradish peroxidase label that 100 μ L have diluted is added into each hole, gently
It claps after mixing, is placed in 37 DEG C of incubation 30min；
(5) 100 μ LA liquid and B liquid mixed chemical luminescence for liquid in equal volume is added in every hole, pats mixing, covers cover board film,
1~2min, after the luminous value RLU in each hole is measured with chemical illumination immunity analysis instrument, save data；
(6) testing result is calculated and is analyzed: inhibiting rate (%)=B/B0 × 100 (%), in formula: B-various concentration Fourth Ring
The luminous value of plain standard solution hole (or sample well)；0 concentrations of tetracycline's standard solution luminous value of B0-；It is vertical sit with inhibiting rate
Mark, the logarithm of tetracycline concentration are that abscissa draws standard curve, so that it is determined that in sample tetracycline content.
The invention has the following advantages:
The present invention provides a kind of luminous enzyme-linked immunologic detecting kits of tetracycline chemical, and luminescent substance is directly tagged to
It is true by the chemiluminescence intensity for measuring marker after specific immune response occurs with antibody or antigen on antigen or antibody
Determine the content of measured antibody or antigen.With high-throughput detection, high sensitivity, detection range is wide, analysis speed is fast, inexpensive economy
The advantages that.The tetracycline chemical that the present invention the constructs enzyme-linked immunoassay technology that shines has the advantage that
(1) environmental protection and economy: compared with the ELISA kit of used detection tetracycline, not needing to reuse has corrosion
Property sulfuric acid and most of toxic or for carcinogen substrate, it is more environmentally-friendly.Genetic engineering antibody can be in various expression
It is expressed in system, and mass production can be carried out by technique for gene engineering, to culture medium without particular/special requirement, density of fermenting is high, sends out
The ferment period is short, can reduce manufacturing cost.
(2) highly sensitive, high specific: compared with the ELISA kit of existing detection tetracycline, kit is overcome
The drawbacks of in detection vulnerable to endogenous enzyme interference, the detection of absorbance also influence vulnerable to a variety of external factors, the present invention
Using high specific, the antibody of high-affinity, the chemiluminescent enzyme-linked immunosorbent sensitivity of immune detection for detecting tetracycline is higher, can reach
0.045ng/mL。
(3) high precision, high stability: carrying out the coating of ELISA Plate using envelope antigen, is coated with relative to antibody, more has
Conducive to preferable coating effect and longer holding time is reached, to improve the precision and stability of kit detection.
(4) quickly, accurately: pre-treating method is simple and quick, meets the fast and accurately testing requirements of kit.
It is highly suitable for the trace analysis and batch detection of tetracycline residue based on above this kit of advantage, has important
Realistic meaning and popularization and application foreground.
Detailed description of the invention
Fig. 1 is tetracycline canonical plotting.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, agents useful for same and material of the present invention are commercially available.
Reagent used in following embodiment is as follows:
Coating buffer: 1.69g sodium carbonate and 2.95g sodium bicarbonate are dissolved in 1L distilled water and obtained.
20 times of concentrated cleaning solutions: the phosphate buffer of the pH7.4 0.4mol/L of volume fraction 0.5%Tween20 makes
Used time dilutes 1 times with deionized water.
Confining liquid: 0.1gBSA (bovine serum albumin(BSA)), 5g glycine is taken to be dissolved in 100mL PBS solution (0.01mol/L
Obtaining pH7.4)
Tetracycline standard solution: it is spare that tetracycline standard items are diluted to 1mg/mL with chromatographic grade acetonitrile；It uses again
0.01mol/L PBST by standard items be diluted to concentration be 0,0.03,0.1,0.3,1,3 μ g/L tetracycline standard solution, 4
DEG C save.
Chemical luminescence for liquid: being made of A liquid and B liquid, and A liquid is preferably by 20mg to iodophenol, 8mg luminol, 1.21g
Tris is dissolved in 100mL deionized water, with hydrochloric acid tune pH to 8.4；B liquid is preferably by volume fraction 0.40%H2O2、1.21g Tris
It is dissolved in 100mL deionized water, is obtained with hydrochloric acid tune pH to 7.0；When use by A liquid and B liquid by volume 1:1 ratio mix.
The preparation of embodiment 1 tetracycline gene engineered antibody, envelope antigen, immunizing antigen
(1) preparation of envelope antigen, immunizing antigen
The synthesis of tetracycline haptens:
A: 0.69g p-aminobenzoic acid is dissolved in 16mL, in the HCl that 4 DEG C of 2moL/L, the Asia of 1mL0.48mg/mL is added
Nitric acid takes solution, is placed in 4 DEG C of stirring 10min, obtained reaction solution, referred to as A liquid
B: weighing 2.220g tetracycline raw medicine, is dissolved in 60mL 0.2moL/L NaOH, referred to as B liquid.
C: and then be added drop-wise to A liquid in B liquid the bottom of by, mixture pH is maintained between 8~9, and in 10 DEG C of stirring 2h, is taken out
It is cooled to room temperature, after low acidified, filter out liquid with concentrated hydrochloric acid and be adjusted to 3.0, then filter to precipitate, precipitating is washed with cold distilled water
It washs, finally freeze-drying obtains p-aminobenzoic acid tetracycline.
The preparation of tetracycline envelope antigen:
0.594g p-aminobenzoic acid tetracycline, 0.115gNHS and 0.21g DCC are weighed, the N-N- diformazan of 5mL is dissolved in
In base formamide, 5h is stirred at room temperature, is centrifuged to obtain supernatant.The BSA of 0.8375g is dissolved in 10mL 0.05moL/L (pH 8)
Phosphate buffer in, be slowly stirred for 24 hours at 4 DEG C.
Reaction solution is transferred in the bag filter handled well after the reaction was completed to above-mentioned, in 4 DEG C of refrigerators, stirs lower use
0.01moL/L phosphate buffer is dialysed 72h, and preceding 8h every for 24 hours changes liquid 1 time, dispenses the cross-linking products after dialysis after the completion of dialysis,
It is saved backup in -20 DEG C.
The preparation of tetracycline immunizing antigen:
0.594g p-aminobenzoic acid tetracycline, 0.115gNHS and 0.21g DCC are weighed, the N-N- diformazan of 5mL is dissolved in
In base formamide, 5h is stirred at room temperature, is centrifuged to obtain supernatant.The KLH of 0.8375g is dissolved in 10mL 0.05moL/L (pH 8)
Phosphate buffer in, be slowly stirred for 24 hours at 4 DEG C.
Reaction solution is transferred in the bag filter handled well after the reaction was completed to above-mentioned, in 4 DEG C of refrigerators, stirs lower use
0.01moL/L phosphate buffer is dialysed 72h, and preceding 8h every for 24 hours changes liquid 1 time, dispenses the cross-linking products after dialysis after the completion of dialysis,
It is saved backup in -20 DEG C.
(2) preparation of tetracycline gene engineered antibody
Design degenerate primer:
Heavy chain primer
VH-B (SEQ ID NO.1): 5 '-AATACGGCCCAACCGGCCTGAGTCGG-3 '
VH-F (SEQ ID NO.2): 5 '-TGAGGAGATTTGCGGCCGCGTCCCTTGG-3 '
Light chain primer
VL-B (SEQ ID NO.3): 5 '-GACATACGGCCCAACCGGCCGTCACCA-3 '
VL-F (SEQ ID NO.4): 5 '-CATTTTAGCGGCCGCAGCTTGGTGCC-3 '
Overall length primer
Q-B (SEQ ID NO.5):
5‘-GTTGCCATTATGGCTACGGCCCAACCGGCCTGAGTCGG-3’
Q-F (SEQ ID NO.6):
5‘-GAGTCGCGGCCGCATTTAGCGGCCGCAGCTTGGTGCC-3’
Primer R1 (SEQ ID NO.7): 5 '-CCATGATTACGCCAAGCTTTGGAGGC-3 '
Primer R2 (SEQ ID NO.8): 5 '-CGATCTAATGTTTTGTCGTCTTACC-3 '
Wherein primer VH (Back) restriction enzyme site containing SfiI, VH (For) I restriction enzyme site containing Not；VL (For) enzyme of I containing Not
Enzyme site, VL (Back) restriction enzyme site containing SfiI, overall length primer (Back) restriction enzyme site of I containing Bgl, overall length primer (For) contain
Not I restriction enzyme site, R1, R2 are vector-specific primers, and the PCR for Insert Fragment is identified.
Mouse is immunized with the tetracycline immunizing antigen of synthesis, detection serum titer titre reaches requirement；It takes small
Mouse spleen is extracted total serum IgE, has been gone out a full set of light and heavy chain of immune mouse through RT-PCR Successful amplification using the degenerate primer of design
Variable region gene, the amplification condition of light chain are as follows: 1 μ L high-fidelity Pfu enzyme (2.5U) is added in 94 DEG C × 5min denaturation, carries out following
Circulation: 94 DEG C × 30s, 54 DEG C × 1min, 72 DEG C × 1min, totally 25 recycle, last 72 DEG C of extensions 10min.The amplification of heavy chain
Condition are as follows: 1 μ L high-fidelity Pfu enzyme (2.5U) is added, the following circulation of progress in 94 DEG C × 5min denaturation: 94 DEG C × 30s, 60 DEG C ×
1min, 72 DEG C × 1min, totally 30 recycle, last 72 DEG C of extensions 10min.VH genetic fragment size is about 345bp, VL gene piece
Duan great little is about 330bp.Take each 150ng of the VH-VL of purifying, by PCR complete VH-VL segment it is pre-splicing (pre-splicing program:
94℃2min；94 DEG C of 1min, 60 DEG C of 1min, 72 DEG C of 1min, 30 circulations；72℃10min).It is dilute with the 1/50 of pre-splicing product
Releasing liquid is template, carries out the splicing of VH-VL full-length gene, and use two upstream and downstream containing Bgl I and Not I restriction enzyme site
Primer (i.e. primer pair Q-B/Q-F, sequence is as shown in SEQ ID NO.5-6) carries out secondary PCR amplification to antibody, and condition is 94 DEG C
× 5min initial denaturation adds 0.5 μ L high-fidelity Pfu enzyme, the following circulation of progress: 94 DEG C × 45s, 50 DEG C × 1min, and 72 DEG C × 1min,
The full length antibody genetic fragment for being about 760bp is obtained, after antibody fragment Bgl I and Not I double digestion, with carrier
PCANTAB5E connection, converts Escherichia coli TG3, and IPTG induces tetracycline resistance antibody to carry out solubility expression.Using osmotic shock
Method is extracted the soluble tetracycline resistance antibody in somatic cells pericentral siphon chamber, and to expressing in culture supernatant and pericentral siphon chamber extract
Antibody carry out SDS-PAGE, Western-Blotting and ELISA identification, and it is purified using affinity chromatography.
The foundation of 2 chemiluminescence enzyme immunization method of embodiment
(1) envelope antigen and antibody concentration is preferred
1) envelope antigen is pressed into 2.5mg/L, 1.25mg/L, 0.833mg mg/L, 0.625mg/L, 0.5mg/L coating buffer
(0.05mol/L pH5.0 carbonate buffer solution) dilutes and is longitudinally coated with opaque white color luminescent screen, and 100 holes μ L/, 37 DEG C are for 24 hours,
It is washed 2 times with washing lotion, is patted dry on blotting paper.
2) configured 150 hole μ L/ of confining liquid is added to be closed, 37 DEG C overnight, and baking oven drying is put into after drying.
3) 50 holes the μ L/ diluted tetracycline standard items serial solution of 0.01mol/L PBST is added.
4) be added 50 holes μ L/ with the diluted tetracycline gene engineered antibody of 0.01mol/L PBST (1:4000,1:5000,
1:6000,1:7000,1:8000), 37 DEG C of 60min board-washing 5 times, are patted dry on blotting paper.
5) 100 holes the μ L/ sheep anti-mouse igg that diluted horseradish peroxidase marks is added, 37 DEG C of 30min are washed with washing lotion
It plate 5 times, is patted dry on blotting paper.
6) chemical luminescence for liquid now matched in 100 holes μ L/ is added, measures luminous value with chemical illumination immunity analysis instrument.To shine
Value has the envelope antigen concentration of obvious change of gradient with envelope antigen concentration and antibody dilution is that optium concentration carries out specificity
Measurement.Obtaining envelope antigen optium concentration is 0.5mg/L, and antibody (concentration 2mg/mL) extension rate is 1:6000.
(2) measurement of antibody sensitivity
It is 0.5mg/L with envelope antigen concentration, tetracycline gene engineered antibody (concentration 2mg/mL) extension rate is 1:
6000, carry out the measurement of antibody sensitivity.
1) 96 hole opaque white color luminescent screens are taken, envelope antigen are diluted to 0.5mg/L with coating buffer, 100 μ are added in every hole
L, 37 DEG C overnight, is patted dry on blotting paper with washing lotion flushing 2 times.
2) 150 hole μ L/ of confining liquid is added to be closed, 37 DEG C overnight, and baking oven drying is put into after drying.
3) first plus various concentration 50 hole μ L/ of tetracycline titer, then plus extension rate be 1:6000 tetracycline gene
50 hole μ L/ of engineered antibody, 37 DEG C of 60min board-washing 5 times, are patted dry on blotting paper.
4) sheep anti-mouse igg that the horseradish peroxidase that 100 μ L/ hole extension rates of addition are 1:6000 marks, 37 DEG C
30min board-washing 5 times, is patted dry on blotting paper.
5) chemistry now matched is added to give out light reaction solution, 100 holes μ L/ measure luminous value.
Testing result is calculated with inhibiting rate, inhibiting rate (%)=B/B0× 100 (%), B are that various concentration standard solution is competing
The luminous value striven, B0It is the luminous value that standard items are not added.The concentration of standard items is tetracycline gene when calculating 50% inhibiting rate
The sensitivity of engineered antibody is 0.045ng/mL.
The building of the luminous enzyme-linked immunologic detecting kit of 3 tetracycline chemical of embodiment
(1) composition of kit
1) be coated with the chemiluminescence ELISA Plate of tetracycline envelope antigen: ELISA Plate shines for the removable opaque white color in 96 holes
Plate has been coated with tetracycline envelope antigen and confining liquid；Tetracycline envelope antigen is tetracycline haptens and bovine serum albumin(BSA)
Conjugate, peridium concentration 0.5mg/L.
The preparation of ELISA Plate: taking the removable opaque white color luminescent screen in 96 holes, and envelope antigen is diluted to 0.5mg/ with coating buffer
100 μ L are added in every hole by L, and 37 DEG C overnight, and liquid in hole of inclining is washed 2 times with washing lotion, patted dry on blotting paper.Then every hole
150 μ L of confining liquid is added, 37 DEG C are incubated overnight, liquid in hole of inclining, and are placed in 37 DEG C of baking ovens close with aluminium foil bag vacuum after drying
Seal 4 DEG C of preservations.
2) tetracycline serial standards solution (0,0.03,0.1,0.3,1,3 μ g/L of concentration).
3) tetracycline gene engineered antibody 2mg/mL, working concentration 1:6000.
4) the sheep anti-mouse igg 10mg/mL, working concentration 1:6000 of horseradish peroxidase label.
5) it chemical luminescence for liquid: is made of A liquid and B liquid.
6) 20 times of concentrated cleaning solutions.
(2) reagent dispenses: by aseptic subpackaged, 1mL/ bottles of tetracycline standard items (10 μ g/mL) after each reagent measurement qualification,
7mL/ bottles of diluted tetracycline gene engineered antibody, 7mL/ bottles of sheep anti-mouse igg of diluted horseradish peroxidase label, A liquid
7mL/ bottles, 7mL/ bottles of B liquid, 20 times 50mL/ bottles of concentrated cleaning solution.It labels after packing, indicates lot number and validity period, 4 DEG C of preservations.
(3) assembling of kit: respectively by the chemiluminescence ELISA Plate 1 for being coated with tetracycline envelope antigen of step (1)
Sheep anti-mouse igg, the A liquid, B liquid, 20 times that block and tetracycline standard items, tetracycline gene engineered antibody, horseradish peroxidase mark
Each 1 bottle and 1 part of operation instructions of concentrated cleaning solution is set designated position in kit, and kit encapsulates after the assay was approved, 4 DEG C of guarantors
It deposits.
The application method of the luminous enzyme-linked immunologic detecting kit of 4 tetracycline chemical of embodiment
(1) sample pre-treatments
1) milk: sample to be tested is shaken up, and weighs 1ml sample and 9ml 0.05M PBST solution is added, vibrate 2min,
4000r/min is centrifuged 5min, and supernatant can be used to detect
2) egg: weighing the egg liquid of 1g mixing, and the PBS solution 9mL for leading to x-100 containing 0.1% Qula is added, vibrates 2min,
4000r/min is centrifuged 5min, and supernatant is taken to measure.
3) tissue sample: taking chicken, the chicken of pig, liver respectively, and each tissue sample of kidney respectively weighs 2g, adds Mcl l vai
Ne buffer (0.1M citrate buffer solution, 0.1M EDTA, pH3.8) 8mL, vibrates 5min, and 4000r/min is centrifuged 5min, takes
Clear liquid is spare.Oasis HLB solid-phase extraction column successively uses anhydrous methanol 3mL, water 2mL prewashing.It takes reserve liquid 5mL to cross column, uses water
2mL elution, is extracted.It is eluted, is extracted with 20mmol/L oxalic acid methanol solution 1ml, collect eluent.The eluent of collection is shaken
It is even, use 10 times of PBST solution dilution after it is to be measured as sample solution.
(2) detection method of kit
1) kit is taken out, (25~27 DEG C) balance 30min or more of room temperature are placed in, takes out chemiluminescence ELISA Plate, is used
Tetracycline standard items are diluted to 0,0.03,0.1,0.3,1,3 μ g/L of concentration by 0.01mol/L PBST.
2) the tetracycline standard solution of 50 μ L various concentrations is added in gauge orifice, 50 μ L samples to be tested are added in sample well,
The tetracycline gene engineered antibody that 50 μ L have been diluted with 0.01mol/L PBST is added in every hole later, covers cover board film in micro vibration
It swings after vibrating 10min on device, is placed in 37 DEG C of incubation 50min.
3) reaction solution in plate hole is got rid of, 300 μ L of washing lotion is added in each hole, stands 20 seconds, gets rid of the total liquid of Qi, so wash altogether
Plate 5 times, plate is patted dry for the last time；It can also use automatic washer board-washing 5 times.Micropore frame is upside down on blotting paper after washing and is clapped
(every wheel board-washing is patted 3 times) is beaten to guarantee to completely remove the liquid in hole.
4) sheep anti-mouse igg for the horseradish peroxidase label that 100 μ L have diluted is added in ELISA Plate, after patting mixing, sets
In 37 DEG C of incubation 30min.
5) 100 μ L substrate buffer solutions and the mixed chemiluminescence reaction liquid of substrate solution same volume is added in every hole, pats mixed
It is even, cover board film is covered, measures the luminous value RLU in every hole after 1~2min with chemical illumination immunity analysis instrument, saves data.
(2) testing result is calculated and is analyzed
Inhibiting rate (%)=B/B0× 100 (%)
In formula: the luminous value of B-various concentration tetracycline standard solution hole (or sample well)；B0- 0 concentrations of tetracycline mark
Quasi- solution luminous value；
Using inhibiting rate as ordinate, the logarithm of tetracycline concentration is that abscissa draws standard curve, so that it is determined that in sample
The content of tetracycline.
5 kit of embodiment and accuracy are tested
(1) repetitive test of tetracycline standard solution
In the ELISA Plate prepared from 3 batches according to the method in embodiment 3,20 micropores are respectively extracted out, according to 4 pilot scale of embodiment
The luminous value of detection method the measurement 0.1 μ g/L and 0.3 μ g/L tetracycline standard solution of agent box, are repeated 20 times, calculate variation lines
Number CV%, the results are shown in Table 1.
1 tetracycline table honor solution repetitive test of table
(2) sample repeatability and accuracy test
Accuracy refers to the Compound Degree of measured value and true value, and in enzyme-linked immunosorbent assay, accuracy is often with rate of recovery table
Show, precision is often indicated with the coefficient of variation.In milk, egg, tissue sample, addition tetracycline to final concentration of 0.1,
0.3,1 μ g/L, each concentration each 10 parallel, measures 3 batches.Calculate average value, TIANZHU XINGNAO Capsul and batch interior and batch variation system
Number.It the results are shown in Table 2.
The rate of recovery (%)=(detected level-blank amount)/additive amount × 100%；
CV (%)=(SD/X) × 100%；
2 sample repeatability of table and accuracy test result
The result shows that the TIANZHU XINGNAO Capsul of milk, egg, tissue sample in range, in variation within batch coefficient range, is criticized
Between the coefficient of variation in range, meet country for kit indices standard.
The test of 6 storage life of embodiment
(1) kit of embodiment 3 is placed in 2~8 DEG C, picks and places the examination for setting 0,2,4,6,8,9,10 and 12 months respectively
Agent box respectively joins the luminous value of tetracycline standard items (0.1 μ g/L), 50% inhibition concentration, TIANZHU XINGNAO Capsul, variation within batch coefficient
Number is measured.
(2) kit is placed 12 days under conditions of 37 DEG C of preservations, is shone daily to 0.1 μ g/L of tetracycline standard items
Value, 50% inhibition concentration, TIANZHU XINGNAO Capsul, each parameter of variation within batch coefficient are measured.
(3) kit is saved 12 days in -20 DEG C of refrigerators, daily to the luminous value of tetracycline standard items (0.1 μ g/L),
50% inhibition concentration, TIANZHU XINGNAO Capsul, each parameter of variation within batch coefficient are measured.
It can be seen that from result, by three kinds of condition food preservation tests, the luminous value decline of tetracycline standard items (0.1 μ g/L) is small
In 5%, 50% inhibiting rate between 0.03~0.06 μ g/L the day sword rate of recovery between 78%~105%；Interassay coefficient of variation
Less than 10%；Indices conform to quality requirements, and therefore, kit can save 12 months at 2~8 DEG C.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>Guangzhou Yuan Rui Biotechnology Co., Ltd
<120>the luminous enzyme-linked immunologic detecting kit of a kind of tetracycline chemical
<130>
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 26
<212> DNA
<213>primer VH-B
<400> 1
aatacggccc aaccggcctg agtcgg 26
<210> 2
<211> 28
<212> DNA
<213>primer VH-F
<400> 2
tgaggagatt tgcggccgcg tcccttgg 28
<210> 3
<211> 27
<212> DNA
<213>primer VL-B
<400> 3
gacatacggc ccaaccggcc gtcacca 27
<210> 4
<211> 26
<212> DNA
<213>primer VL-F
<400> 4
cattttagcg gccgcagctt ggtgcc 26
<210> 5
<211> 38
<212> DNA
<213>primer Q-B
<400> 5
gttgccatta tggctacggc ccaaccggcc tgagtcgg 38
<210> 6
<211> 37
<212> DNA
<213>primer Q-F
<400> 6
gagtcgcggc cgcatttagc ggccgcagct tggtgcc 37
<210> 7
<211> 26
<212> DNA
<213>primer R1
<400> 7
ccatgattac gccaagcttt ggaggc 26
<210> 8
<211> 25
<212> DNA
<213>primer R2
<400> 8
cgatctaatg ttttgtcgtc ttacc 25
Claims (10)
1. a kind of tetracycline gene engineered antibody, which is characterized in that the preparation method of the tetracycline gene engineered antibody: with warp
The total serum IgE for crossing the immune zooblast of tetracycline immunizing antigen is template, utilizes primer RT-PCR shown in NO.1~4 SEQ ID
Light-chain variable region gene, the heavy chain variable region gene of antibody are amplified, and is inserted into carrier pCANTAB5E after connecting,
Tetracycline gene engineered antibody is obtained in expression in escherichia coli.
2. tetracycline gene engineered antibody according to claim 1, which is characterized in that the amplification condition of light chain are as follows: 94 DEG C ×
1 μ L 2.5U high-fidelity Pfu enzyme is added, the following circulation of progress in 5min denaturation: 94 DEG C × 30s, 54 DEG C × 1min, 72 DEG C ×
1min, totally 25 recycle, last 72 DEG C of extensions 10min.
3. tetracycline gene engineered antibody according to claim 1, which is characterized in that the amplification condition of heavy chain are as follows: 94 DEG C ×
1 μ L 2.5U high-fidelity Pfu enzyme is added, the following circulation of progress in 5min denaturation: 94 DEG C × 30s, 60 DEG C × 1min, 72 DEG C ×
1min, totally 30 recycle, last 72 DEG C of extensions 10min.
4. tetracycline gene engineered antibody according to claim 1, which is characterized in that with the light chain segments and heavy chain amplified
Segment template each other completes the pre-splicing of VH--VL segment by PCR, is template progress VH-- using the dilution of pre-splicing product
The splicing of VL full-length gene, and secondary PCR amplification is carried out to antibody using primer Q-B/Q-F, full length antibody genetic fragment is obtained,
It after segment Bgl I and Not I double digestion, is connect with carrier pCANTAB5E, conversion Escherichia coli TG3 carries out IPTG induction
Expression.
The enzyme-linked immunologic detecting kit 5. a kind of tetracycline chemical shines, which is characterized in that contain any institute of Claims 1 to 4
State tetracycline gene engineered antibody.
6. kit according to claim 5, which is characterized in that also containing the chemiluminescence for being coated with tetracycline envelope antigen
ELISA Plate, tetracycline standard items, enzyme labelled antibody, chemical luminescence for liquid and cleaning solution.
7. kit according to claim 6, which is characterized in that the tetracycline envelope antigen be diazotising after to ammonia
Azo occurs for the contraposition of the phenolic hydroxyl group of yl benzoic acid and tetracycline, and then introduces carboxyl, carboxylic tetracycline haptens with
The conjugate of bovine serum albumin(BSA) BSA；The peridium concentration of tetracycline envelope antigen is 0.5mg/L.
8. kit according to claim 6, which is characterized in that the enzyme labelled antibody is the sheep of horseradish peroxidase label
Anti- mouse IgG or goat anti-rabbit igg.
9. kit according to claim 6, which is characterized in that the chemical luminescence for liquid is made of A liquid and B liquid, and A liquid is
Containing 20mg to iodophenol, 8mg luminol, 1.21g Tris, pH8.4 in 100mL deionized water；B liquid is 100mL deionized water
In contain volume fraction 0.40%H2O2, 1.21g Tris, pH7.0；The ratio of A liquid and B liquid 1:1 by volume are mixed when use
It closes；The cleaning solution is 20 times of concentrated cleaning solutions, and 20 times of concentrated cleaning solutions are containing volume fraction 0.5%Tween20
The phosphate buffer of pH7.4 0.4mol/L.
10. kit according to claim 5, which is characterized in that when use such as to the pretreatment processing method of sample to be tested
Under:
Sample to be tested is milk sample: being diluted with PBST, the measurement of centrifuging and taking supernatant；
Sample to be tested is egg: egg liquid is diluted with PBS solution, the measurement of centrifuging and taking supernatant；It is bent containing 0.1% in the PBS solution
Draw logical x-100；
Sample to be tested is tissue sample: taking chicken, the chicken of pig, liver or renal tissue sample respectively, Mcllvaine is added to buffer
Liquid, oscillation centrifugation, takes supernatant spare；Oasis HLB solid-phase extraction column successively uses anhydrous methanol, water prewashing；Take spare supernatant
Liquid crosses column, is eluted with water, extracts；It is eluted, is extracted with oxalic acid methanol solution, collect eluent；The eluent of collection is shaken up, is used
It is to be measured as sample solution after the dilution of PBST solution.
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| US5849576A (en) * | 1994-05-19 | 1998-12-15 | Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. | Tetracycline promoter for the stringently regulated production of recombinant proteins in prokaryotic cells |
| US20100035847A1 (en) * | 2008-08-07 | 2010-02-11 | Hrvoje Petkovic | Genes for biosynthesis of tetracycline compounds and uses thereof |
| CN102928413A (en) * | 2011-08-09 | 2013-02-13 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescence kit for detecting tetracyclines, and applications thereof |
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Effective date of registration: 20210909 Address after: 510535 Room 301, building C5, No. 11, Kaiyuan Avenue, Huangpu District, Guangzhou, Guangdong Applicant after: Guangzhou Yian Biotechnology Co.,Ltd. Address before: 510670 room b304, No. 17, Xiangshan Road, Huangpu District, Guangzhou City, Guangdong Province Applicant before: Guangzhou Yuanrui Biotechnology Co.,Ltd. |
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Application publication date: 20190726 |