CN108459166A - Method that is a kind of while detecting quinolone antibiotics residual quantity - Google Patents
Method that is a kind of while detecting quinolone antibiotics residual quantity Download PDFInfo
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Abstract
The invention discloses methods that is a kind of while detecting quinolone antibiotics residual quantity, using direct competition method reaction principle, the monoclonal antibody of a variety of quinolone drugs is fixed on microwell plate bottom with microarray formats, it is prepared into microwell plate Biochip arrays, and it is reacted successively with the mixing artificial antigen of quinolones nano material label, quinolone drugs standard items, finally developed the color with human lymph node, and fast scan imaging is carried out to microwell plate colour developing result using visual chip scanner, image is handled using chip analysis software.The method of the present invention has greatly shortened detection time, improves sensitivity, and experimental implementation is simple；9 kinds of antibiotic of quinolones can be detected respectively simultaneously, improve detection efficiency, it is of great significance to the multinomial medicament residue analysis method research of quinolones, there is good application and popularization value, certain reference is provided for the residue detection of Fluorine in Foods quinolones.
Description
Technical field
The invention belongs to medicament residue detection techniques, and quinolone antibiotics residual quantity is detected simultaneously more particularly to a kind of
Method.
Background technology
Quinolone drugs (Fluoroquinolones, FQs) is a kind of with the fluoro- 7- piperazines -4- promises ketone ring structures of 6-
The antibacterials of (mother nucleus structure).Quinolone drugs is a kind of broad spectrum antibiotic that developed recently gets up, and in recent years
The new varieties with application are competitively developed in countries in the world, are up to the present launched on the market there are many drug.In order to control
With treatment Animal diseases, the case where either simultaneously or alternately being used there are many quinolone drugs during cultivation and treatment animal,
Which results in the how remaining generations of drug.Quinolones medicament relict in animal food is exceeded potential to human body presence
It threatens, this kind of residue of veterinary drug problem is increasingly paid close attention to by people.
The exceeded food of quinolones medicament relict, which is eaten for a long time, in people may cause the digestive discomfort of human body, easily cause abdomen
Bitterly, diarrhea and vomiting；It is also possible to causing adverse reaction such as twitch, the dizziness of cental system that can possibly even cause epilepsy.
Food and Drug Adminstration of the US emphasizes that quinolone antibiotics can cause nerve permanent damage.Quinolone drugs may
It influences the cartilage development of children and causes skin phototoxicity reaction such as nettle rash, erythema and flush with itch, or even deposit
In teratogenesis, carcinogenic, mutagenic effect.Quinolone drugs systemic damage is mainly shown as that allergic reaction and anaphylaxis are stopped
Gram, other include fever, shiver with cold, hidrosis, weak, oedema etc..Wherein, anaphylactic shock is mainly anaphylactic type, more
Number is cured after treatment or rescue, also has small number of patients dead.Quinolone drugs is mainly shown as kidney to urinary system damage
Functional lesion, including frequent micturition, oliguresis, crystalluria, cloudy urine, albuminuria etc..
Currently, multiple countries and mechanism define the use standard and its maximum residue limit of quinolone drugs.Day
Originally it is 10~100 μ g/kg to define the maximum allowable residual quantity of quinolone drugs, and different cultivars quinolones in China's regulation
The maximum residue limit of drug is between 30~500 μ g/kg.Fat, muscle, liver, the kidney food of the animals such as chicken, ox, sheep, rabbit, pig
10~1900 μ g/kg of the quinolones veterinary drug such as middle Norfloxacin, pefloxacin maximum residue limit.
The domestic and international residual research method of fluoroquinolones mainly has microbiological method, chromatography, immunoassay
Method.Wherein chromatography includes that high performance liquid chromatography (HPLC), hygroplasm combination analysis method (LC/MC), high performance liquid chromatography-are glimmering
Light method (HP) LC/FL, high performance thin layer chromatography (HPTLC) etc., immunoassay detection method includes enzyme linked immunosorbent assay
(ELISA), colloidal gold strip method, protein chip etc..The advantages of microbial method is that easy to operate, at low cost, detection is quick,
But the detection limitation of the method is limited the quantity higher than the lowest detection of various countries' defined；Sensitivity is not high, poor specificity.Although chromatography is examined
It is accurate to survey result, but that there are technical operations is complicated, detection time is long, pre-treatment is cumbersome, equipment is expensive and costly etc. lacks
Point, so it is unsuitable for large-scale field quick detection.Enzyme-linked Immunosorbent Assay (ELISA) measures in immunization and colloidal gold tries
Paper slip technology is widely used in various fields, still as a kind of convenience, low cost, screening means quickly, high-throughput
ELISA can only detect one pack system residual, and colloidal gold strip technology is susceptible to high false positive rate or false negative rate.Albumen
Chip method has high specificity, reacts very sensitive, the quick detection for a large amount of samples, and there is detection multicomponent to remain, is special
The advantages that anisotropic high, easy to operate, high-throughput, detection time is very short, at low cost, it is widely used in research at home and abroad
The fields such as Food Monitoring, clinical monitoring, virus monitor, it is believed that we are with scientific and technological fast development, and immunoassay will
Most possible substitution chromatographic apparatus is parsed into as the most common method of such drug.
Invention content
Goal of the invention：In view of the above-mentioned problems existing in the prior art, visible protein core is utilized the present invention provides a kind of
Piece analytical technology is to detection method while a variety of carbostyril antibiotic relict contents in sample to be tested.
Technical solution：Method that is of the present invention a kind of while detecting quinolone antibiotics residual quantity, including it is following
Step：
(1) visible protein chip is prepared：Quinolones monoclonal antibody is subjected to printing operation on chip carrier, it is permanent
Temperature, which is incubated, fixes, and sealer is added and continues constant-temperature incubation, is washed out and pats dry；
(2) specific binding reaction：It takes step (1) to prepare gained visible protein chip, quinolone is added into orifice plate
The hybrid standard product or detected sample of class various concentration gradient, each standard items or sample repeat at least two holes；It adds
The quinolones hybrid antigen of nano silver label, constant temperature oscillation is incubated after sticking epiphragma plate, is washed and is patted dry after the completion of being incubated；
(3) chromogenic reaction：Nanometer human lymph node developing solution is added in orifice plate after being reacted to step (2), constant temperature oscillation is incubated,
Then it is patted dry with milli-Q water；
(4) processing analysis：Using visible biological chip scanner, to step (3), treated, and protein chip is swept
It retouches, and result treatment analysis is carried out using chip analysis software.
In step (1), the constant-temperature incubation each means 4-37 DEG C of incubation 0.5-16h of holding；In step (2) and step (3),
The constant temperature oscillation incubation refers to incubation 5-60min at 4-37 DEG C.
In the method for the present invention, the quinolone antibiotics are selected from Ofloxacin, Lomefloxacin, pefloxacin, Yi Nuosha
It is one or more in star, Norfloxacin, Enrofloxacin, Ciprofloxacin, acidum nalidixicum and flumequine.
In step (1), the chip carrier uses microwell plate, slide, film or macromolecule chip base, prepares quinolones Dan Ke
Grand antibody array, 1-10 point is repeated per sample quinolones monoclonal antibody, and point sample amount is 50nL/ points；It is described in step (1)
Sealer refers to bovine serum albumin(BSA) (BSA).
In step (1), the Ofloxacin, Lomefloxacin, pefloxacin, Enoxacin, Norfloxacin, Ciprofloxacin,
The sessile antibody a concentration of 1 of acidum nalidixicum, flumequine, Enrofloxacin:1-1:100 dilutions, point sample amount are 10-100nL/ points.
In step (2), the amount of the hybrid standard product or detected sample that are added in each hole is 10-100 μ L, concentration
Ranging from 0.1-40ng/mL.
In step (2), the amount of the quinolones hybrid antigen for the nano silver label being added in each hole is 10-100 μ
L, concentration range 1:2-1:128 dilutions.
In step (2), the quinolones hybrid antigen of nano silver label be Ofloxacin, Lomefloxacin, acidum nalidixicum,
Flumequine is respectively 1:4-1:128 dilutions.
In step (3), the human lymph node developing solution is developing solution A and developing solution B volume ratios 1:1 mixing, it is now with the current.Its
In, developing solution A, developing solution B can directly be bought from auspicious middle science and technology, and name of product visualizes developing solution.The human lymph node developing solution
Dosage is the holes 10-100 μ L/.
In step (1) and step (2), the washing refers to being washed 2-4 times using cleaning solution, each 10-15s；Step (3)
In, the washing refers to using milli-Q water 2-4 times, each 10-15s.
In step (2), the detected sample can be milk, milk powder, cheese, feed, urine, animal tissue, serum,
Honey, bee milk, egg and aquatic products etc..
For synthesis, the present invention uses direct competition method reaction principle, by the monoclonal antibody of a variety of quinolone drugs
Be fixed on microwell plate bottom with microarray formats, be prepared into microwell plate Biochip arrays, and successively with quinolones nanometer material
Mixing artificial antigen, the reaction of quinolone drugs standard items for expecting label, are finally developed the color with human lymph node, and use visual chip
Scanner carries out fast scan imaging to microwell plate colour developing result, and image is handled using chip analysis software.
Advantageous effect：Be compared to the prior art, the method for the present invention using sessile antibody, labelled antigen method, greatly
Ground has shortened detection time, improves sensitivity, and experimental implementation is simple；9 kinds of antibiotic of quinolones can be examined respectively simultaneously
It surveys, improves detection efficiency, be of great significance to the multinomial medicament residue analysis method research of quinolones, have and answer well
With promotional value, certain reference is provided for the residue detection of Fluorine in Foods quinolones.9 kinds of quinolines are detected by the method for the invention
The recovery of standard addition of promise ketone drug is respectively between 70%~130%, and relative standard deviation is within 15%, Neng Gouyong
The quick primary dcreening operation while residual of 9 kinds of quinolone drugs.
Description of the drawings
Fig. 1 is the application testing principle schematic diagram；
Fig. 2 is sample point sample sequential schematic；
Fig. 3 is various concentration standard items testing result scanning figure；
Fig. 4 is quinolone drugs standard curve；
Fig. 5 is quinolone drugs mark-on testing result scanning figure in milk.
Specific implementation mode
The application is explained in detail with reference to specific embodiment.
Reagent and equipment source needed for embodiment：
Quinolones monoclonal antibody (Ofloxacin, Lomefloxacin, pefloxacin, Enoxacin, Norfloxacin, En Nuo
Sha Xing, Ciprofloxacin, acidum nalidixicum, flumequine), nano silver label quinolones artificial antigen (Ofloxacin, Lomefloxacin,
Pefloxacin, Enoxacin, Norfloxacin, Enrofloxacin, Ciprofloxacin, acidum nalidixicum, flumequine), quinolones standard items (oxygen
Flucloxacillin, Lomefloxacin, pefloxacin, Enoxacin, Norfloxacin, Enrofloxacin, Ciprofloxacin, acidum nalidixicum, flumequine),
Nanometer human lymph node developing solution etc. is provided by the Nanjing bio tech ltd Xiang Zhong.NaCl, KCl, NA2HPO4·12H2O、
KH2PO4, polysorbas20, ethylenediamine tetra-acetic acid (EDTA) be to be bought by Nanjing Chemistry Reagent Co., Ltd..Bovine serum albumin(BSA) (BSA)
It is to be bought from Sigma companies.
18.25MΩ.cm-1Ultra-pure water generated by Milli-Q water systems.10mM PBS (phosphate buffer) pH 7.2
As analysis buffer.Under conditions of pH is 7.2, the preparation of milk sample and dilute is carried out with the 10mM PBS of the EDTA containing 1mM
It releases.Washing buffer is the PBS (PBST) containing 0.05% polysorbas20.Sealer is the 10mM PBS containing 1%BSA.Milk sample
Dilution is EDTA containing 1mM in PBST.All buffer solutions are filtered using preceding by 0.22 μm of bore filter device.
Biochip point sample instrument, Q-array2000 visible biological chips analyzer, microwell plate constant temperature oscillation instrument, multitube
Vortex DL instrument (the Nanjing bio tech ltd Xiang Zhong)；(Suzhou hundred obtains the limited public affairs of Laboratory Instruments to single track adjustable pipette
Department), KQ218 types ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), microwell plate (the auspicious middle limited public affairs of biotechnology in Nanjing
Department).
Testing principle：9 kinds of quinolone antibody are fixed in 96 orifice plate bottoms, sample and labeled antigen is added, label
Antigen and simple competitive are combined with fixed antibody specificity, after washing away the labelled antigen and sample not being combined, are added aobvious
Color liquid develops the color, developing solution and labeled antigen binding catalyzed coloration.As sample concentration increases, labeled antigen and
Sessile antibody combines fewer, and signal value is lower.So sample concentration and detected signal value are negatively correlated.Testing principle schematic diagram
See Fig. 1.
Embodiment 1
Method that is a kind of while detecting 9 kinds of quinolone antibiotics residual quantities：
(1) preparation of visible protein chip：6 × 5 matrixes are prepared on 96 microwell plates with biochip point sample instrument, respectively
9 kinds of quinolones (Ofloxacin, Lomefloxacin, pefloxacin, Enoxacin, Norfloxacin, Enrofloxacin, rings are put in order
Third husky star, acidum nalidixicum, flumequine) monoclonal antibody, point sample amount is 50nL/ points, repeats 3 points respectively per sample；After point sample,
It is put in 37 DEG C of insulating boxs and is incubated 2h, antibody is made to be fixed on board bottom；200 μ L chips sealers (BSA) are added per hole, are put in 37 DEG C
It is incubated 2h in insulating box, is then washed 3 times with washing buffer, each 10s is patted dry, and it is for use to be placed in 4 DEG C of refrigerators；
(2) being separately added into 25 μ L per hole in the orifice plate that step (1) prepares, (Ofloxacin, Lomefloxacin, training fluorine are husky
Star, Enoxacin, Norfloxacin, Enrofloxacin, Ciprofloxacin, acidum nalidixicum, flumequine) various concentration gradient hybrid standard
Product or sample, each standard items or sample repeat 2 holes；The quinolones mixing that 25 μ L nano silvers label is added per hole again is anti-
Original sticks cover board film, is put in 37 DEG C of constant temperature orifice plate shakers and is incubated 30min, is then washed 3 times, each 15s with cleaning solution,
It pats dry；
(3) the nanometer human lymph node developing solution of 50 μ L is added in step (2) treated chip is per hole, is put into 37 DEG C of constant temperature
Orifice plate shaker is incubated 12min, and then with ultrapure washing 3 times, each 15s is patted dry；
(4) utilizing visible biological chip scanner, treated, and protein chip is scanned to step (3), and utilizes core
Piece analysis software carries out result treatment analysis.
Antibody antigen condition optimizing.
The condition optimizing refers to according to optimum signal value and being mixed under different antibody fixed concentrations or antigen concentration
Standardization product concentration and detected signal value are negatively correlated, and thus screening obtains optimal conditions.
Concrete operations：In step (1), Ofloxacin, Lomefloxacin, pefloxacin, Enoxacin antibody difference 1:5、1:
10、1:20 dilutions, Norfloxacin, Ciprofloxacin, acidum nalidixicum, flumequine antibody difference 1:1、1:2、1:4 dilutions, Enrofloxacin are anti-
Body difference 1:10、1:20、1:30 dilutions.In step (2), the quinolones antigen difference 1 of nano silver label:16、1:32、1:
64、1:128 dilutions.
Optimum results：The result shows that the best sessile antibody of Ofloxacin, Lomefloxacin, pefloxacin, Enoxacin is dense
Degree is 1:10, Norfloxacin, Ciprofloxacin, acidum nalidixicum, flumequine best sessile antibody a concentration of 1:2, Enrofloxacin is fixed
Antibody concentration is 1:30.The quinolones antigen final choice Ofloxacin 1 that nano silver marks after optimization:32 dilutions, Lip river
U.S. sand star 1:32 dilutions, acidum nalidixicum 1:128 dilutions, flumequine 1:32 dilution hybrid antigens.The results are shown in Table 1.
Table 1：The corresponding combination antigen of quinolones antibody and corresponding signal
Embodiment 2
The specificity experiments of quinolones
The specificity of quinolones antibody judges usually using competition inhibition curve.Use the antigen of various concentration and dry
It disturbs object and calculates their own combination ratio (B/B0), competition inhibition curve is drawn out, and calculate their own IC50.Under
Formula (1) calculates cross reacting rate.
CR=[IC50(analyte)/IC50(chaff interferent)] × 100% (1)
IC50It is that analyte or chaff interferent inducement signal inhibit concentration needed for 50%.
Experimental result is as shown in table 2, show quinolones antibody and 9 kinds of quinolone medicine cross reacting rates 1%~
Between 5%.Illustrate that the specificity of quinolone antibody is good, mutually without intersection, each quinolone antibiotics can be detected respectively.
2 quinolone drugs cross reacting rate of table
Embodiment 3
The foundation of quinolones standard curve
It explores quinolones standard curve and uses indirect competitive.Each antibody concentration 3 parallel microarray points of point, signal
Value takes its average value, and in a certain range, with the increase of competition test product concentration, fixed antibody combines labeled artificial
The content of antigen can be fewer and fewer, thus the signal value detected will be lower, be more than this range after, detected signal value not with
The increase of competition test product concentration and change.With Ofloxacin standard items, Lomefloxacin standard items, pefloxacin standard items, according to
Promise sand star standard items, Norfloxacin standard items, Enrofloxacin standard items, Ciprofloxacin standard items, acidum nalidixicum standard items, flumequine
Standard items (as shown in table 3) mixed standard solution is at war with Inhibition test, and Fig. 2, Fig. 3 show while measuring 9 kinds of quinolones
Scanning figure after albuminoid chip Competitive assays.
3 quinolone drugs standard concentration of table
Using mapping software to inhibiting rate (B/B0) with the logarithm (logC) of concentration make competition inhibition curve, as shown in figure 4,
The standard curve range of Ofloxacin drug is 2.5~40ng/ml, the standard curve range of Lomefloxacin drug is 2.5~
The standard curve range of 40ng/ml, pefloxacin drug are 0.1~9ng/ml, and the standard curve range of Enoxacin drug is
The standard curve range of 0.1~3.2ng/ml, Norfloxacin drug are 0.2~3.2ng/ml, and the standard of enrofloxacin is bent
The standard curve range of line ranging from 0.1~16ng/ml, oxolinic acid drug are 2~32ng/ml, and the standard of acidum nalidixicum drug is bent
The standard curve range of line ranging from 1.0~16ng/ml, flumequine drug are 1.25~20ng/ml.
Embodiment 4
Under conditions of above-mentioned optimization is good, the standard curve range of linearity of 9 kinds of substances, detection limit, as a result such as 4 institute of table are measured
Show.
The regression curve equation of 4 quinolone drugs of table, related coefficient, the range of linearity
Embodiment 5
The recovery of standard addition of milk sample
Under the experiment condition of above-mentioned optimization, accurate measuring 1mL blank milk samples add Ofloxacin, Lome respectively
The standard items of Sha Xing, pefloxacin, Enoxacin, Norfloxacin, Enrofloxacin, Ciprofloxacin, acidum nalidixicum, flumequine, mark-on
Concentration is as shown in table 5, milk sample sample diluting liquid 1:It is detected after 1 dilution, each sample does multiple holes, testing result and sample-adding
Sequence is as shown in Fig. 5 and table 6.Each sample repeats to survey 5 times, according to testing result as it can be seen that the rate of recovery of quinolones exists respectively
Between 70%~130%, CV% can meet the requirement of quinolones residue limits within 15%.
Table 5：Quinolone drugs spiked levels table
Quinolones recovery of standard addition in 6 milk of table
Detection limit is calculated according to following formula (2)：
Concentration+the 3*SD (2) of detection limit=blank group
Embodiment 6
Method that is a kind of while detecting 9 kinds of quinolone antibiotics residual quantities：
(1) preparation of visible protein chip：6 × 9 matrixes are prepared on slide with biochip point sample instrument, are pressed respectively suitable
(Ofloxacin, Lomefloxacin, pefloxacin, Enoxacin, Norfloxacin, Enrofloxacin, ring third are husky for 9 kinds of quinolones of sequence point
Star, acidum nalidixicum, flumequine) monoclonal antibody, concentration is respectively 1:20 dilutions, point sample amount are 20nL/ points, repeat 6 respectively per sample
A point；After point sample, 4 DEG C of incubation 12h are put in, antibody is made to be fixed on board bottom；20 μ L chips sealers (BSA) are added per hole,
It is put in 25 DEG C of insulating boxs and is incubated 4h, then washed 3 times with washing buffer, each 15s is patted dry, and it is for use to be placed in 4 DEG C of refrigerators；
(2) being separately added into 10 μ L per hole in the orifice plate that step (1) prepares, (Ofloxacin, Lomefloxacin, training fluorine are husky
Star, Enoxacin, Norfloxacin, Enrofloxacin, Ciprofloxacin, acidum nalidixicum, flumequine) various concentration gradient hybrid standard
Product or sample, each standard items or sample repeat 2 holes；The quinolones mixing that 10 μ L nano silvers label is added per hole again is anti-
The quinolones hybrid antigen of original, nano silver label is Ofloxacin, Lomefloxacin, acidum nalidixicum, flumequine, hybrid antigen concentration
Respectively 1:32 dilutions, stick cover board film, are put in 25 DEG C of constant temperature orifice plate shakers and are incubated 60min, then wash 3 with cleaning solution
Secondary, each 15s is patted dry；
(3) it is added the nanometer human lymph node developing solution of 20 μ L in step (2) treated chip is per hole, developing solution A and aobvious
Color liquid B volume ratios 1:1 mixing, it is now with the current, it is put into 25 DEG C of constant temperature orifice plate shakers and is incubated 20min, then with ultrapure washing 3
Secondary, each 15s is patted dry；
(4) utilizing visible biological chip scanner, treated, and protein chip is scanned to step (3), and utilizes core
Piece analysis software carries out result treatment analysis.
Embodiment 7
Method that is a kind of while detecting 9 kinds of quinolone antibiotics residual quantities：
(1) preparation of visible protein chip：6 × 3 matrixes are prepared on film with biochip point sample instrument, respectively in order
9 kinds of quinolones of point (Ofloxacin, Lomefloxacin, pefloxacin, Enoxacin, Norfloxacin, Enrofloxacin, Ciprofloxacin,
Acidum nalidixicum, flumequine) monoclonal antibody, concentration is respectively 1:20 dilutions, point sample amount are 50nL/ points, and 2 points are distinguished per sample；Point
After sample, 25 DEG C of incubation 4h are put in, antibody is made to be fixed on board bottom；100 μ L chips sealers (BSA) are added per hole, are put in 25
It is incubated 4h in DEG C insulating box, is then washed 3 times with washing buffer, each 15s is patted dry, and it is for use to be placed in 4 DEG C of refrigerators；
(2) being separately added into 50 μ L per hole in the orifice plate that step (1) prepares, (Ofloxacin, Lomefloxacin, training fluorine are husky
Star, Enoxacin, Norfloxacin, Enrofloxacin, Ciprofloxacin, acidum nalidixicum, flumequine) various concentration gradient hybrid standard
Product or sample, each standard items or sample repeat 2 holes；The quinolones mixing that 50 μ L nano silvers label is added per hole again is anti-
The quinolones hybrid antigen of original, nano silver label is Ofloxacin, Lomefloxacin, acidum nalidixicum, flumequine, hybrid antigen concentration
Respectively 1:32 dilutions, stick cover board film, are put in 25 DEG C of constant temperature orifice plate shakers and are incubated 60min, then wash 3 with cleaning solution
Secondary, each 15s is patted dry；
(3) it is added the nanometer human lymph node developing solution of 100 μ L in step (2) treated chip is per hole, developing solution A and aobvious
Color liquid B volume ratios 1:1 mixing, it is now with the current, it is put into 25 DEG C of constant temperature orifice plate shakers and is incubated 20min, then with ultrapure washing 3
Secondary, each 15s is patted dry；
(4) utilizing visible biological chip scanner, treated, and protein chip is scanned to step (3), and utilizes core
Piece analysis software carries out result treatment analysis.
Claims (10)
1. method that is a kind of while detecting quinolone antibiotics residual quantity, which is characterized in that include the following steps：
(1) visible protein chip is prepared：Quinolones monoclonal antibody is subjected to printing operation on chip carrier, constant temperature is incubated
Fixation is educated, sealer is added and continues constant-temperature incubation, is washed out and pats dry；
(2) specific binding reaction：It takes step (1) to prepare gained visible protein chip, quinolones is added not into orifice plate
With the hybrid standard product or detected sample of concentration gradient, each standard items or sample repeat at least two holes；Add nanometer
The quinolones hybrid antigen of silver label note, constant temperature oscillation is incubated after sticking epiphragma plate, is washed and is patted dry after the completion of being incubated；
(3) chromogenic reaction：Nanometer human lymph node developing solution is added in orifice plate after being reacted to step (2), constant temperature oscillation is incubated, then
It is patted dry with milli-Q water；
(4) processing analysis：Using visible biological chip scanner, to step (3), treated, and protein chip is scanned, and
Result treatment analysis is carried out using chip analysis software.
2. method that is according to claim 1 while detecting quinolone antibiotics residual quantity, which is characterized in that the quinoline
It is husky that promise ketone antibiotic is selected from Ofloxacin, Lomefloxacin, pefloxacin, Enoxacin, Norfloxacin, Enrofloxacin, ring third
It is one or more in star, acidum nalidixicum and flumequine.
3. method that is according to claim 1 while detecting quinolone antibiotics residual quantity, which is characterized in that step
(1) in, the chip carrier uses microwell plate, slide, film or macromolecule chip base, prepares quinolones monoclonal antibody square
Battle array repeats 1-10 point per sample quinolones monoclonal antibody.
4. method that is according to claim 1 while detecting quinolone antibiotics residual quantity, which is characterized in that step
(1) in, the Ofloxacin, Lomefloxacin, pefloxacin, Enoxacin, Norfloxacin, Ciprofloxacin, acidum nalidixicum, fluorine first
The sessile antibody a concentration of 1 of quinoline, Enrofloxacin:1-1:100 dilutions, point sample amount are 10-100nL/ points.
5. method that is according to claim 1 while detecting quinolone antibiotics residual quantity, which is characterized in that step
(2) in, the amount of the hybrid standard product or detected sample that are added in each hole is 10-100 μ L, concentration range 0.1-
40ng/mL。
6. method that is according to claim 1 while detecting quinolone antibiotics residual quantity, which is characterized in that step
(2) in, the amount of the quinolones hybrid antigen for the nano silver label being added in each hole is 10-100 μ L, and concentration range is
1:2-1:128 dilutions.
7. method that is according to claim 1 while detecting quinolone antibiotics residual quantity, which is characterized in that step
(2) in, the quinolones hybrid antigen of the nano silver label is Ofloxacin, Lomefloxacin, acidum nalidixicum, flumequine are respectively
1:4-1:128 diluted hybrid antigens.
8. method that is according to claim 1 while detecting quinolone antibiotics residual quantity, which is characterized in that step
(3) in, the human lymph node developing solution is developing solution A and developing solution B volume ratios 1:1 mixing, now with the current, dosage is 10-100 μ
The holes L/.
9. method that is according to claim 1 while detecting quinolone antibiotics residual quantity, which is characterized in that step
(1) and in step (2), the washing refers to being washed 2-4 times using cleaning solution, each 10-15s；In step (3), the washing
Refer to using milli-Q water 2-4 times, each 10-15s.
10. method that is according to claim 1 while detecting quinolone antibiotics residual quantity, which is characterized in that step
(2) in, the detected sample is milk, milk powder, cheese, feed, urine, animal tissue, serum, honey, bee milk, egg
Or aquatic products.
Priority Applications (1)
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| CN103257233A (en) * | 2013-05-31 | 2013-08-21 | 南京祥中生物科技有限公司 | Biochip and method capable of simultaneously visually detecting multiple antibiotics, illegal addition agents and biotoxins |
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| CN106520704A (en) * | 2016-11-28 | 2017-03-22 | 江南大学 | Anti-quinolone antibiotic class specific monoclonal antibody hybridoma cell strain YH6 and application thereof |
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| CN103257233A (en) * | 2013-05-31 | 2013-08-21 | 南京祥中生物科技有限公司 | Biochip and method capable of simultaneously visually detecting multiple antibiotics, illegal addition agents and biotoxins |
| CN103439514A (en) * | 2013-08-05 | 2013-12-11 | 徐州工程学院 | Pesticide and veterinary drug multi-residue detection method based on microarray detection chip |
| CN106520704A (en) * | 2016-11-28 | 2017-03-22 | 江南大学 | Anti-quinolone antibiotic class specific monoclonal antibody hybridoma cell strain YH6 and application thereof |
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