CN109971727A - It is a kind of secrete chloramphenicol resistance monoclonal antibody hybridoma cell strain and its application - Google Patents
It is a kind of secrete chloramphenicol resistance monoclonal antibody hybridoma cell strain and its application Download PDFInfo
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- CN109971727A CN109971727A CN201910206505.6A CN201910206505A CN109971727A CN 109971727 A CN109971727 A CN 109971727A CN 201910206505 A CN201910206505 A CN 201910206505A CN 109971727 A CN109971727 A CN 109971727A
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- chloramphenicol
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
Abstract
The invention discloses a kind of hybridoma cell strain for secreting chloramphenicol resistance monoclonal antibody and its applications.The hybridoma cell strain is named as hybridoma cell strain G-1-2, and deposit number is CGMCC No.16688.Mouse is immunized using the chloromycetin artificial antigen of designed, designed in the present invention, hybridoma cell strain is prepared using immune mouse boosting cell, the chloramphenicol resistance monoclonal antibody of hybridoma cell strain secretion, potency is high, high specificity can be used for carrying out fast and accurately immune detection and immunoassay to chloramphenicol.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of hybridoma for secreting chloramphenicol resistance monoclonal antibody
Strain and its application, the detection particularly suitable for chloramphenicol residue in aquatic products, milk, honey, animal tissue's equal samples.
Background technique
Chloramphenicol ((Chloramphenicol, CAP) is a kind of broad-spectrum antibiotic of Cheap highly effective, to Gram-positive and
Negative bacteria has good inhibiting effect, therefore is once widely used in farming and animal husbandry.But animal derived food with
Food chain is taken in for a long time by human body, can cause a variety of diseases.Less serious case destroys the equilibrium state of normal flora in human body, and flora loses
It adjusts, so that human body is generated drug-fast bacteria pearl, bring adverse effect using antibiotic treatment to illness from now on；The people of antibiotic allergic constitution
It will appear allergic reaction, jeopardize health.It may interfere with the synthesis of bone marrow cell protein when serious, and juvenile cell DNA inhibited to close
At causing granulocyte to reduce, cause the malignant diseases such as alpastic anemia, haemolysis, purple paralysis.
In view of the toxic side effect of chloramphenicol, international food educational circles is classified as banning drugs, and European Union, U.S. etc. advise in regulation
Determining residual chloromycetin limit standard is " zero tolerance " (Zerotolerance), i.e., must not detect, according to European Union " 2002/657/
EC " standard provides that it is 0.3 μ g/kg that the maximum of chloramphenicol, which requires detection limit, in animal derived food.Soon U.S. FDA is also done
Corresponding regulation out.The Ministry of Agriculture, China defines CAP and must not detect in the edible tissues of all food animals, and by its from
It is deleted in " Chinese veterinary pharmacopoeia ", is classified as banning drugs.
To adapt to higher examination criteria, detection technique level must be correspondingly improved, last decade chloramphenicol detection technique
Always one of the popular project of international expert scholar research.Currently, the detection method of chloramphenicol mainly has liquid chromatography
(LC), high performance liquid chromatography (HPLC), mass spectrography (MS) etc..These are all the traditional instrumental methods of comparison, and current
Confirmation method.The application of laboratories mass detection sample is usually immunization method, therefore obtains one plant with specificity
The chloramphenicol resistance monoclonal antibody hybridoma cell strain of affinity is very important.The present invention prepares a kind of secretion chloramphenicol
The hybridoma cell strain of monoclonal antibody, and it is applied to immunoassay product, the residual of chloramphenicol drug in measurement aquatic products, milk
Amount has many advantages, such as that low detection limit, high specificity, easy to operate, detection speed is fast, testing cost is low, is very easy to promote.
Summary of the invention
It is an object of that present invention to provide one plant to have the monoclonal antibody hybridoma cell of specific affinity to chloramphenicol
The preparation method of strain.
The present invention provides one plant of chloramphenicol monoclonal antibody hybridoma cell strain, and the antibody prepared by the cell strain is mould to chlorine
Element has preferable affinity and detection sensitivity, can be used to establish the immunological detection method of chloramphenicol total amount, including enzyme
Linked immunoassay reagent kit, colloidal gold colloidal gold detection test paper strip, immune affinity column etc..
Technical solution of the present invention: one plant of chloramphenicol monoclonal antibody hybridoma cell strain is named as G-1-2, has protected
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.16688.
Chloramphenicol monoclonal antibody, it is miscellaneous by the chloramphenicol monoclonal antibody that the deposit number is CGMCC No.16688
Hand over tumor cell strain G-1-2 is secreted to generate.
The application of the chloramphenicol monoclonal antibody, the detection for chloromycetin content in food safety.
The preparation step of cell strain G-1-2 provided by the invention are as follows:
(1) preparation and identification of artificial antigen: it is anti-to obtain chloramphenicol half for chloramphenicol Base and glutaric acid mono ethyl ester acyl chloride reaction
Original, haptens and protein are coupled, and prepare artificial antigen, i.e. immunogene, coating antigen；
(2) preparation of monoclonal antibody: by the way that mouse is immunized, monoclonal antibody is obtained；
(3) preparation of cell strain G-1-2: by cell fusion and cloning, chloramphenicol monoclonal antibody hybridoma is obtained
Cell strain G-1-2.
(4) application of cell strain G-1-2: it is applied to enzyme linked immunological kit.
(5) application of cell strain G-1-2: it is applied to colloidal gold immuno-chromatography test paper strip.
(6) application of cell strain G-1-2: it is applied to chemiluminescence immune detection reagent kit, immuno magnetic cell separation enrichment examination
Agent box, magnetic granule chemiluminescence kit, time-resolved fluoroimmunoassay chromatograph test strip, fluorescent micro-ball immune chromatography test paper strip,
Other immune detection products such as immune affinity column.
Detailed description of the invention
Fig. 1: chloramphenicol hapten synthesis route map
Specific embodiment
Below with reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this
Invention, and be not intended to limit the scope of the invention.
The preparation of 1 chloramphenicol reagent constituents of embodiment
1, the preparation of chloramphenicol haptens
Extracting chloromycetin alkali 1.0g adds acetone 30ml to dissolve, adds glutaric acid mono ethyl ester acyl chlorides 1.31g, 3h is stirred at room temperature, and stops
Reaction, revolving remove acetone, add water 50ml, ethyl acetate 50ml*3, and extraction three times, merges organic phase, is evaporated, upper silicagel column,
Ethyl acetate/petroleum ether (v/v, 1/1) elution separation, obtains midbody acid amide chloramphenicol Base 1.5g, yield 90.3%；
Amide chloramphenicol Base is taken, the sodium hydrate aqueous solution of 0.5mol/L is added to dissolve, 4h is stirred at room temperature, stops reaction, adds
Ethyl acetate extraction removes organic phase, and water phase adjusts pH value to 6, adds 60ml ethyl acetate to extract, be evaporated, and obtains grease, and two
Chloromethanes/petroleum ether (v/v, 1/3) 80ml, recrystallization, obtains glutaric acid chloramphenicol Base 1.2g, as haptens product, yield
86.96%.
2, the preparation of antigen
Immunogene preparation --- chloramphenicol haptens and bovine serum albumin(BSA) (BSA) coupling obtain immunogene.
Glutaric acid chloramphenicol Base haptens 16mg is taken, adds DMSO 3ml to dissolve, adds 50 μ l of triethylamine, chlorination iso-butyl formate
33 μ l, are stirred at room temperature 5h, obtain haptens activating solution A liquid；Bovine serum albumin(BSA) (BSA) 50mg is taken, 3ml 0.05mol/LPB is added
Buffer solution obtains B liquid, and A drop is added in B liquid, and for 24 hours, 0.02mol/L PBS dialyses three days for stirring, obtains chloramphenicol-
BSA immunogene, -20 degree save, spare.
Coating antigen preparation --- chloramphenicol haptens and ovalbumin (OVA) coupling obtain immunogene.
Glutaric acid chloramphenicol Base haptens 12mg is taken, adds DMSO 3ml to dissolve, adds 40 μ l of triethylamine, chlorination iso-butyl formate
19 μ l, are stirred at room temperature 5h, obtain haptens activating solution A liquid；Oralbumin (OVA) 50mg is taken, adds 3ml 0.05mol/LPB slow
Fliud flushing dissolution, obtains B liquid, A drop is added in B liquid, and for 24 hours, 0.02mol/L PBS dialyses three days for stirring, obtains chloramphenicol-
OVA coating antigen, -20 degree save, spare.
3, the preparation of chloramphenicol monoclonal antibody
Animal immune: the immunogene that above-mentioned steps are obtained is injected into Balb/c Mice Body, and immunizing dose is 150 μ g/
Only, it is made to generate antiserum.
Cell fusion and cloning: after mice serum measurement result is higher, taking its splenocyte, compares by 8:1 (quantitative proportion)
Example is merged with SP2/0 myeloma cell, measures cell supernatant using indirect competitive ELISA, screens positive hole.Using limited dilute
Interpretation of the law carries out cloning to positive hole, until obtaining the hybridoma cell strain of secretion chloramphenicol monoclonal antibody.
Biological material specimens preservation: it is micro- to be preserved in China by one plant of chloramphenicol monoclonal antibody hybridoma cell strain G-1-2
Biological inoculum preservation administration committee common micro-organisms center, abbreviation CGMCC, address are as follows: BeiChen West Road, Chaoyang District, BeiJing City 1
Institute 3, Institute of Microorganism, Academia Sinica, deposit number are CGMCC No.16688.The deposit date is October 29 in 2018
Day.
Cell cryopreservation and recovery: monoclonal hybridoma strain is made 1 × 10 with frozen stock solution6The cell suspension of a/ml,
It is saved for a long time in liquid nitrogen.Cryopreservation tube is taken out when recovery, 37 DEG C of water-bath middling speeds is immediately placed in and melts, and after centrifugation removal frozen stock solution, is moved
Enter to cultivate culture in glassware.
The production and purifying of monoclonal antibody: Balb/c mouse peritoneal is injected into sterilizing paraffin oil 0.5ml/ only, abdomen after 7 days
Chamber injects stable monoclonal hybridoma strain 5 × 105A/only, ascites is acquired after 7 days.With octanoic acid-saturated ammonium sulfate method into
The purifying of row ascites, -20 DEG C of preservations.
4, the preparation of sheep anti mouse antiantibody
It is that immune animal obtains sheep anti mouse antiantibody using source of mouse antibody as immunogen immune pathogen-free domestic sheep with sheep.
5, the preparation of enzyme label antiantibody
Sheep anti mouse antiantibody is coupled with horseradish peroxidase (HRP) using the Over-voltage protection after improvement.It passes
It is 4:1 that the Over-voltage protection of system, which requires the molar concentration rate of enzyme and antibody in reaction system, since horseradish peroxidase is strong
Many sites in conjunction with antibody are generated under oxidation, the horseradish peroxidase molecule activated in this way acts as each point of connection
The bridge of son, reduces the enzymatic activity of enzyme marker, makes to be mixed with many condensates in the conjugate of preparation.It is asked to solve this
Topic, we are improved traditional method, it may be assumed that
(1) closed process of amino is eliminated, because the amino that can generate the connection of itself amino is actually seldom；
(2) molar concentration ratio of horseradish peroxidase and antibody is reduced to 2:1, and the method after improvement is than traditional side
Method is easy, reduces to the loss of enzymatic activity.
6, the preparation of ELISA Plate
Coating antigen is diluted to 20 μ g/ml with coating buffer, 100 μ l are added in every hole, and 37 DEG C are protected from light incubation 2h, hole of inclining
Middle liquid is washed 2 times with cleaning solution, and each 30s is patted dry, and 200 μ l confining liquids is then added in every hole, 37 DEG C are protected from light incubation
2h, liquid pats dry in hole of inclining, and is saved after dry with aluminium film vacuum sealing.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of chloramphenicol
The enzyme linked immunological kit for setting up detection chloramphenicol, makes that it includes following components:
(1) it is coated with the ELISA Plate of chloramphenicol coupled antigen；
(2) 6 bottles of chloramphenicol standard solution, concentration be respectively 0 μ g/L, 0.025 μ g/L, 0.075 μ g/L, 0.3 μ g/L,
1.2μg/L,4.8μg/L；
(3) chloramphenicol monoclonal antibody working solution；
(4) the sheep anti mouse antiantibody of horseradish peroxidase-labeled is used；
(5) substrate developing solution is made of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine；
(6) terminate liquid is 2mol/L sulfuric acid；
(7) it is 7.4 that cleaning solution, which is pH value, anti-containing 0.5%~1.0% Tween-20,0.01 ‰~0.03 ‰ sodium azide
Rotten agent, 0.1~0.3mol/L phosphate buffer, the percentage be percent weight in volume；
(8) redissolve liquid be pH value be 7.0, the phosphate buffer of 0.02mol/L, the percentage be weight volume basis
Than.
The detection of chloramphenicol in 3 aquatic products of embodiment, milk sample
1, sample pre-treatments
(1) aquatic products
With homogenizer homogeneous aquatic products sample；Aquatic products (fish, shrimp etc.) sample after weighing 3.0 ± 0.05g homogeneous is poly- to 50ml
In styrene centrifuge tube, 6ml ethyl acetate is added, vibrates 5min, (20-25 DEG C) centrifugation 5min of 3000g room temperature with oscillator；It moves
Take 4ml upper organic phase (sample for being approximately equivalent to 2g) into 10ml clean dried glass tube, under 50-60 DEG C of water-bath nitrogen stream
Drying；1ml n-hexane is added, with vortex instrument whirling motion 1min, adds 1ml and redissolves working solution, with vortex instrument whirling motion 15s, 3000g
(20-25 DEG C) centrifugation 5min of room temperature；Upper organic phase is removed, takes 50 μ l of lower layer's water phase for analyzing.
(2) milk
2ml fresh milk sample is measured into 50ml polystyrene centrifuge tube, is separately added into 50 μ l protein precipitants and 8ml
Ethyl acetate is mixed, (20-25 DEG C) centrifugation 5min of 3000g room temperature with vortex instrument whirling motion 30s；Pipette 4ml upper organic phase extremely
10ml clean dried glass tube, dries up under 50-60 DEG C of water-bath nitrogen stream；0.5ml n-hexane is added, with vortex instrument whirling motion
1min adds 0.5ml and redissolves working solution, with vortex instrument whirling motion 30s, mixes；By all liq be transferred to 2ml polystyrene from
In heart pipe, (20-25 DEG C) centrifugation 5min of 3000g room temperature；Upper organic phase is removed, takes 50 μ l of lower layer's water phase for analyzing.
2, it is detected with kit
50 μ l of standard items/sample is added into corresponding micropore.Amount combines enzyme conjugates concentrate with enzyme as needed
Object dilution is diluted according to the volume ratio of 1:10.50 hole μ l/ of enzyme conjugates working solution is added, gently oscillation mixes, with lid
30min is reacted in plate membrane cover plate 25 DEG C of light protected environments of postposition.Cover board film carefully is opened, liquid in hole is dried, washers are added
Make 250 hole μ l/ of liquid, sufficiently washing 5-6 times, every minor tick 20s, sprinkles cleaning solution in board falling hole, patted dry with blotting paper.Substrate is added
50 hole μ l/ of liquid A liquid adds 50 hole μ l/ of substrate solution B liquid, and gently oscillation mixes, with cover board membrane cover plate 25 DEG C of light protected environments of postposition
Middle reaction 20min.50 hole μ l/ of terminate liquid is added, gently oscillation mixes, and sets microplate reader at 450nm, measures every hole OD value.
3, Analysis of test results
The percentage absorptance of standard items or sample be equal to the average value (diplopore) of the absorbance value of standard items or sample divided by
The average value of the absorbance value of first standard items (0 standard) obtains the percentage extinction of standard items or sample multiplied by 100%
Angle value.Using standard items percentage absorptance as ordinate, using the logarithm of chloramphenicol standard concentration (μ g/L) as abscissa, mark is drawn
Directrix curve figure.The percentage absorptance of sample is substituted into standard curve, concentration corresponding to sample is read from standard curve, is multiplied
It is the actual concentrations of chloramphenicol in sample with its corresponding extension rate.
4 chloramphenicol technical parameter of embodiment determines test
1, kit sensitivity and detection limit
Conventionally assay kit sensitivity, the range of standard curve are 0.025~4.8 μ g/L, IC50(50%
Inhibition concentration) floating range be 0.11~0.27 μ g/L；20 parts of samples are detected, finds and corresponds to respectively from standard curve
The concentration of percentage absorbance value indicates detection limit plus 3 times of standard deviations with the average value of 20 parts of concentration of specimens, the results show that should
Method is respectively 0.025 μ g/kg, 0.0125 μ g/kg to the detection limit of aquatic products, milk.
2, sample preci-sion and accuracy is tested
Using the rate of recovery as accuracy estimating index, the testing result relative standard deviation of a certain concentration samples of replication
(RSD%) it is used as precision evaluation index.Calculation formula are as follows: the rate of recovery (%)=actual measured value/theoretical value × 100%,
Middle theoretical value is the addition concentration of sample；Relative standard deviation RSD%=SD/X × 100%, wherein SD is standard deviation, and X is
The average value of determination data.
Recycling measurement is added to flesh of fish sample respectively by the chloramphenicol of 0.05 μ g/kg, 0.1 two concentration of μ g/kg, is pressed
0.025 μ g/kg, 0.05 two concentration of μ g/kg chloramphenicol recycling measurement is added to milk sample respectively, each sample does
4 parallel, is measured with three batches of different reagents, the average recovery rate and precision result for calculating sample see the table below.
1 precision of table and accuracy test
Recycling measurement is added to flesh of fish sample respectively by the chloramphenicol of 0.05 μ g/kg, 0.1 two concentration of μ g/kg, is put down
The equal rate of recovery is between 76.2%~85.7%；By 0.025 μ g/kg, 0.05 two concentration of μ g/kg chloramphenicol respectively to milk
Sample is added recycling measurement, and average recovery rate is between 83.6%~94.2%；Relative standard deviation is small in batch, between criticizing
In 10%.
3, stabilization of kit is tested
Kit preservation condition is 2~8 DEG C, by measurement in 12 months, the maximum absorbance value (zero standard) of kit,
50% inhibition concentration, chloramphenicol addition actual measured value are within normal range (NR).Consider to have in transport and use process
Improper preservation condition occurs, and kit is placed 7 days under 37 DEG C of preservation conditions, carries out accelerated aging tests, the results showed that
The kit indices comply fully with requirement.In view of kit freezing happens, it is cold that kit is put into -20 DEG C of refrigerators
Freeze 7 days, measurement result also indicates that kit indices are completely normal.It can show that kit can be at 2~8 DEG C from result above
At least save 12 months or more.
The preparation of 5 colloidal gold immuno-chromatography test paper strip of embodiment
The preparation method of the test strips mainly comprises the steps that
1) preparation is coated with chloramphenicol monoclonal antibody-colloid gold label object conjugate release pad；
2) preparation has the detection line for being coated with chloramphenicol hapten-carrier protein conjugate and is coated with sheep anti mouse and resists
The reaction film of the nature controlling line of body；
And 2) 3) 1) conjugate release pad, reaction film and the sample absorption pad, water absorption pad and PVC bottom plate that prepare are assembled
At test strips.
Substep narration in detail below:
1, chloramphenicol monoclonal antibody-colloid gold label object preparation
(1) preparation of colloidal gold
1% gold chloride is diluted to 0.01% (mass fraction) with double distilled deionized water, 100ml is taken to be placed in conical flask,
It is heated to boiling with thermostatic electromagnetic blender, in continuous high temperature, is persistently added with stirring 1% trisodium citrate of 2.5ml, continue
Solution is at the uniform velocity heated with stirring in stopping when bright red, is restored to original volume, 4 DEG C of guarantors with deionized water after being cooled to room temperature
It deposits.The colloidal gold appearance prepared is pure, it is bright, without precipitating and floating material.
(2) chloramphenicol monoclonal antibody-colloid gold label object preparation
Under magnetic stirring, molten by every milliliter of colloidal gold with the pH value of 0.2mol/L solution of potassium carbonate tune colloidal gold to 7.0
Chloramphenicol monoclonal antibody is added into colloidal gold solution for the standard that 20~50 μ g are added in liquid, continues to stir and evenly mix 30min, add
Enter 10%BSA, make its final concentration of 1% (volume fraction) in colloidal gold solution, stands 10min.12000r/min,4℃
It is centrifuged 40min, abandons supernatant, it is the redissolution of initial colloid gold volume 1/10 with volume that precipitating is washed twice with redissolution buffer
Buffer will precipitating be resuspended, set 4 DEG C it is spare.
Redissolve buffer: casein containing protein 0.02%~0.1% (mass fraction), 0.05%~0.2% (quality of Tween-80
Score), the 0.02mol/L phosphate buffer of pH7.2.
2, the preparation of conjugate release pad
Conjugate release pad is soaked in (concentration of the bovine serum albumin(BSA) in buffer is containing bovine serum albumin(BSA)
0.5%), in the phosphate buffer of pH 7.2,0.5mol/L, 1h is uniformly soaked, 37 DEG C of baking 3h are spare.With Isoflow point film
Instrument by the chloramphenicol prepared monoclonal antibody-colloid gold label object even application in conjugate release pad, every 1cm conjugate
After release pad sprays 0.01ml chloramphenicol monoclonal antibody-colloid gold label object, (humidity < 20%) is placed in 37 DEG C of environment
It is taken out after 60min, is placed in dry environment (humidity < 20%) and saves backup.
3, the preparation of reaction film
Detection line will be constituted in chloramphenicol haptens-ovalbumin conjugate coating to reaction film, by sheep anti mouse antiantibody
It is coated on reaction film and constitutes nature controlling line.
Coating process: being diluted to 10mg/ml for chloramphenicol haptens-ovalbumin conjugate with phosphate buffer, uses
Isoflow point film instrument is coated in the detection line (T line) on nitrocellulose filter, and package amount is 0.8 μ l/cm；With
Sheep anti mouse antiantibody is diluted to 200 μ g/ml by the phosphate buffer of 0.01mol/L, pH7.4, with Isoflow point film instrument by its
The nature controlling line (C line) being coated on nitrocellulose filter, package amount are 1.0 μ l/cm.The reaction film being coated with is placed in 37 DEG C of items
Dry 2h, spare under part.
4, the preparation of sample absorption pad
Sample absorption pad is placed in slow containing 0.5% bovine serum albumin(BSA) (volume fraction), pH7.2,0.1mol/L phosphate
2h is impregnated in fliud flushing, 37 DEG C of baking 2h are spare.
5, the assembling of test strips
Sample absorption pad, conjugate release pad, reaction film, water absorption pad are successively pasted on PVC bottom plate in order；In conjunction with
Object release pad has 1/3 region to be absorbed by the sample pad covering from starting point, and the end of conjugate release pad and the beginning of reaction film connect
It connects, the end of reaction film is connected with the beginning of water absorption pad, and the beginning of sample absorption pad is aligned with the beginning of PVC bottom plate, water absorption pad
End be aligned with the end of PVC bottom plate；There are detection line and nature controlling line, detection line (T line) and nature controlling line (C on the reaction film
Line) it is the strip tape perpendicular with the length of the test strips；Detection line is located at the side close to the end of conjugate release pad；
Nature controlling line is located remotely from the side of the end of conjugate release pad；Test strips are cut into the small item of 3mm wide with machine, mounted in special
Plastics fabrication in, under the conditions of 4~30 DEG C can be reserved for 12 months.
The detection of residual chloromycetin in 6 sample of embodiment
1, it is detected with test strips
Measuring samples solution, which to be drawn, with suction pipe 3 drops is vertically added dropwise in well, liquid starts timing when flowing, reaction 5~
10min determines result.
2, analysis detection result
Negative (-): the colour developing of T line is better than the colour developing of C line or develops the color no significant difference with C line, indicates mould without containing chlorine in sample
Element or its concentration are lower than detection limit.
Positive (+): the colour developing of T line is markedly less than the colour developing of C line or T line does not develop the color, indicate in sample chloramphenicol concentration be equal to or
It is limited higher than detection.
It is invalid: not occur C line, show the deterioration failure of incorrect operating process or test strips.It in the case, should be again
It is secondary to read over specification, and retested with new test strips.
7 sample detection example of embodiment
1, detection limit test
Take blank honey, milk, pork, the flesh of fish, shrimp sample, add respectively chloramphenicol to final concentration of 0.05,0.1,
0.2ng/ml takes test strips to be detected, and each sample is repeated three times.
When detecting honey, milk, pork, the flesh of fish, shrimp sample with test strips, judgement detection limit is shown according to test strips,
Show that this test strips is limited to 0.1ng/ml to the detection of chloramphenicol.
2, false positive rate, false negative rate test
Known chloromycetin content is taken to be greater than the honey of detection limit, milk, pork, the flesh of fish, shrimp positive sample each 20 respectively
Part and content are less than each 20 parts of negative sample of detection limit, are detected with three batches of test strips, calculate its yin and yang attribute rate.As a result see
Following table.
2 false positive rate of table, false negative rate test result
The result shows that: positive honey, milk, pork, the flesh of fish, shrimp sample are detected with the test strips of 3 batch productions respectively
As a result this when, is all positive, it is known that positive sample coincidence rate is 100%, false negative rate 0；Detect respectively 20 parts of negative honey,
When milk, pork, the flesh of fish, shrimp sample, as a result it is all negative, it is known that negative match-rate 100%, false positive rate 0.Explanation
The test strips of detection chloramphenicol of the invention can carry out residual chloromycetin in honey, milk, pork, the flesh of fish, shrimp quick
Detection.
3, specific test
The medicines such as aminoglycoside, Tetracyclines, fluoroquinolones, sulfamido with chloramphenicol test strips detection 500ng/ml
Object.The results show that test strips nature controlling line and detection line develop the color, it is negative.Illustrate this test strips to the amino sugar of 500ng/ml
The drugs no cross reaction such as glycoside, Tetracyclines, fluoroquinolones, sulfamido.
Claims (8)
1. a kind of hybridoma cell strain for secreting chloramphenicol resistance monoclonal antibody, which is characterized in that be named as G-1-2, preservation is compiled
Number be CGMCC No.16688.
2. preparing hybridoma cell strain as described in claim 1, need first to prepare chloramphenicol haptens, the preparation side of haptens
Method is as follows:
Extracting chloromycetin alkali 1.0g adds acetone 30ml to dissolve, adds glutaric acid mono ethyl ester acyl chlorides 1.31g, and 3h is stirred at room temperature, and stops anti-
It answers, revolving removes acetone, adds water 50ml, ethyl acetate 50ml*3, and extraction three times, merges organic phase, is evaporated, upper silicagel column, second
Acetoacetic ester/petroleum ether (v/v, 1/1) elution separation, obtains midbody acid amide chloramphenicol Base 1.5g, yield 90.3%；
Amide chloramphenicol Base is taken, the sodium hydrate aqueous solution of 0.5mol/L is added to dissolve, 4h is stirred at room temperature, stops reaction, adds acetic acid
Ethyl ester extraction, removes organic phase, and water phase adjusts pH value to 6, adds 60ml ethyl acetate to extract, be evaporated, obtain grease, dichloromethane
Alkane/petroleum ether (v/v, 1/3) 80ml, recrystallization, obtains glutaric acid chloramphenicol Base 1.2g, as haptens product, yield
86.96%.
3. application of the hybridoma cell strain as described in claim 1 in the enzyme linked immunological kit of preparation detection chloramphenicol.
4. hybridoma cell strain as described in claim 1 is in the colloidal gold immuno-chromatography test paper strip of preparation detection chloramphenicol
Using.
5. hybridoma cell strain as described in claim 1 preparation detection chloramphenicol chemiluminescence immune detection reagent kit,
Immuno magnetic cell separation enrichment kit, magnetic granule chemiluminescence kit, time-resolved fluoroimmunoassay chromatograph test strip, fluorescence are micro-
Application in other immune detection products such as ball immuno-chromatographic test paper strip, immune affinity column.
6. a kind of chloramphenicol resistance monoclonal antibody, which is characterized in that by hybridoma cell strain described in claim 1 or its passage
Cell strain secretion generates.
7. application of the chloramphenicol resistance monoclonal antibody as claimed in claim 6 in detection chloramphenicol.
8. a kind of detection kit of chloramphenicol, which is characterized in that anti-containing chloramphenicol resistance monoclonal as claimed in claim 6
Body.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021113736A1 (en) * | 2019-12-05 | 2021-06-10 | Massachusetts Institute Of Technology | Single-domain antibody to chloramphenicol |
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| CN101942414A (en) * | 2010-01-21 | 2011-01-12 | 泰州康正生物技术有限公司 | Hybridoma cell line and chloramphenicol-resistant monoclonal antibody produced by same |
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| WO2021113736A1 (en) * | 2019-12-05 | 2021-06-10 | Massachusetts Institute Of Technology | Single-domain antibody to chloramphenicol |
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