CN102426222B - Magnetic lateral flow immunoassay for rapid detection of TTX and preparation of detection test strip - Google Patents
Magnetic lateral flow immunoassay for rapid detection of TTX and preparation of detection test strip Download PDFInfo
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Abstract
The invention relates to a magnetic lateral flow immunoassay (LFIA) for a rapid detection of Tetrodotoxin (TTX) and a preparation method of a detection test strip, belonging to the technical field of TTX detection. The magnetic LFIA for the rapid detection of TTX is characterized in that: a sample pad, a combination pad labeled by magnetic beads and modified by anti-TTX specific rat monoclonal antibody, a chromatography membrane pre-coated with a detection line T of TTX-BSA and a control line C of IgG, a water absorbent pad are successively pasted on a base plate staggeredly by the distance between adjacent part being 1 mm, then a transparent plastic sealing membrane is coated on the top layer to form a magnetic LFIA test strip for the rapid detection of TTX; immunomagnetic beads are captured by chromatography, the rapid qualitative detection of TTX is realized according to the formed 1-2 macroscopic color developing strips after sample chromatography; the prepared magnetic LFIA test strip through a magnetism analyzer is detected, and the quantitative determination of TTX can be realized according to the detection values of the formed magnetic signal of the detection line and control line after sample chromatography.
Description
Technical field
The invention belongs to field of food inspection, particularly relate to a kind of magnetic immuno-chromatographic method of quick detection TTX and the preparation of test strip, belong to detection technique class.
Background technology
Globe fish meat flavour is delicious, nutritious, the Asia resident such as Chinese, Japanese have edible custom, but the internal organ such as the ovary of globe fish, liver, enteron aisle are rich in tetraodotoxin (Tetrodotoxin, be called for short TTX), due to this toxin stable in properties, poisoning rear shortage effectively saves measure in addition, so often have the poisoning report because eating poisonous globe fish by mistake every year, the life security of serious threat consumer.
TTX is one of natural neurotoxin that toxicity is the strongest, in distributed in nature widely.Except being present in Tetraodontiformes fingerling China and foreign countries, in seawater, the fresh water animals such as some newt, toad, octopus, conch, starfish, and also once detected this toxin in the microbial body such as such as flatworm.TTX stable in physicochemical property, can be water-soluble, is insoluble to absolute ethyl alcohol and ordinary organic solvents.Stable to heat, daylight, enzyme, salt and acid, be soluble in spirit of vinegar.The chemical structural formula of TTX is as shown below:
This toxin toxicity ratio sodium cyanide is strong 1000 times, and toxin except directly acting on intestines and stomach and causing local excitation symptom, and is entered in blood by body absorption, and nerve endings and nerve center can be made rapidly to benumb.What be first injured is sensoparalysis, is secondly each random motor nerve ending paralysis, body physical motion can not be moved.Poison is measured when increasing and is then caused vagus nerve to be benumbed, and breathe and reduce, pulse is slow, body temperature and blood pressure drops time serious, vasomotor nerve maincenter or tabula flesh and respiratory nerve maincenter finally occurs and benumbs, cause breath stopped, dead rapidly.
The detection technique research of TTX starts from the sixties in 20th century, the Measurement for Biotechnique that it is representative that traditional detection technique mainly comprises with mouse detection (mouse bioassay, MBA); With the instrument detection technique that fluorescence spectrophotometry (fluorescence spectrophotometry), liquid chromatographic detection (high pressureliquid chromatography, HPLC) method are representative; And with the immuno analytical method etc. that enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) and immunochromatographic method (lateral flow immunoassay, LFIA) are representative.
(1) in the Measurement for Biotechnique of TTX, Mouse bioassay is internationally recognized detection method, and is decided to be official's detection method of TTX and widespread use in Japan.Mouse bioassay detects tetraodotoxin and has intuitively, fast, typical being easy to of symptom judge, be easy to advantages such as operating, expense is low, and do not need specific installation, its accuracy of detection can meet the features such as Safety of Aquatic Products detection requirement, is a kind of conventional method being widely used in detecting tetraodotoxin.After lumbar injection toxin, linear relationship is there is between the inverse of its death time and toxin dose, by the corrupt corpse toxin virulence of mouse, represents with mouse unit (mouse unit, MU) in the method based on the mouse of constant weight.The definition of current 1MU refers to the toxin amount of the male ddy mouse killing an about 20g in 30min, and namely 1MU is equivalent to 0.22 μ gTTX.But because it is wasted time and energy, poor repeatability and being vulnerable to coexist amino acid and inorganic ions impact and cause the shortcomings such as Lower result, extensively do not popularize at home.
(2) the instrumental analysis detection technique of TTX includes fluorescence, ultraviolet spectro-photometric analysis and stratographic analysis etc.Fluorescence spectrophotometry is the instrumental method of the quantitative detection TTX set up the earliest, and its principle is by detecting fluorescent chemicals C
9alkali measures the amount of TTX.Detect and be limited to 0.3mg/ml.TTX generates C in the basic conditions
9while alkali, also quantitative oxalic sodium, the latter has obvious absorption peak at 230nm place, detects be limited to 0.02 ~ 0.1mg/ml according to this ultraviolet spectrophotometry set up.Fluorescence method and ultraviolet spectrophotometry easy and simple to handle, but sensitivity is low, and selectivity is poor.
In chromatography, current domestic multiplex liquid phase chromatography (HPLC) detects TTX, the method sensitivity higher (0.2 ~ 9.6 μ g/ml).China has formulated corresponding GB mensuration---the liquid chromatography-fluorescence detection of tetraodotoxin " in the GB/T 23217-2008 aquatic products ".But the sample pre-treatments of the method is cumbersome, after needing to carry out post, then basic hydrolysis measures with fluorescence detector, and instrument is expensive, testing cost is relatively high.
In addition, Japanese scholars once adopted liquid phase-tandem mass spectrum (LC/MS/MS) analytic approach and liquid phase-flight time mass spectrum (LC/TOF-MS) analytic approach to detect TTX, and detectability reaches 10ng/m L and 0.02 μ g/g respectively.Gas chromatography-mass spectrum (GC/MS) combination analysis method was also once used to detect TTX, and detectability is consistent with LC/MS/MS, is 10ng/ml.Because red, orange, green, blue, yellow (ROGBY) testing process is simple, fast, be furnished with highly sensitive detecting device simultaneously, sensitiveer, be one of conventional detection method, but require higher to sample purity.Its shortcoming is that sample preparation is cumbersome, and instrument apparatus expensive, testing cost is high.
(3) advantages such as immune analysis method is quick, sensitive due to it, and the relatively simple and selectivity of operation is good, are developed rapidly in field of modern detection.Most immunoassay is based on TTX monoclonal antibody specific, also has based on antiserum exploitation.Most widely used is at present ELISA method, the method is the detection method based on solid-liquid antigen-antibody reaction system, with the GB mensuration of tetraodotoxin " in the fresh globe fish of GB/T 5009.206-2007 " for representative, current domestic existing commercially available TTX ELISA detection kit.The method not only ensure that specificity and the quantitative relationship of antigen, antibody response, and detection sensitivity high (detection is limited to 10ng/ml), but its operating process needs specialized equipment and professional to detect, and operation steps is comparatively loaded down with trivial details, generally needs detection time ~ and 3 hours.LFIA technology is the expanded application of elisa technique principle, generally use latex particle, electroselenium, collaurum, liposome and on turn phosphorus etc. as colored marker.When chromatography, the compound formed between label to determinand can catch by corresponding part and the detection line be gathered on nitrocellulose membrane and present label with color, therefore testing goal can be realized by the presence or absence of bar that tunica fibrosa develops the color, shade and reflection ray etc., owing to generally not needing special main equipment, thus there is advantage that is easy and simple to handle, rapid sensitive.Be a kind of method for quick being applicable to scene, in report disclosed at present, mostly using colloid gold particle as label, be therefore otherwise known as colloidal gold immunity chromatography.2010, the people such as Yu Zhou reported one " detection method based on tetraodotoxin in the globe fish tissue of colloidal gold immunochromatographimethod ", but there is not yet the patent report of tetraodotoxin immunochromatographydetecting detecting test strip both at home and abroad at present.
Magnetic immuno-chromatographic technology replaces traditional label to carry out immunochromatography with supperparamagnetic particles, the quantitative detection data to biological specimen are provided by the detection object be combined on superparamagnetic nano particle, recycle by magnetic particle marker antibody linear relationship between the immune complex of catching and magnetic signal can realize to biological specimen Quantitative detection object, magnetic immuno-chromatographic technology have highly sensitive, stability is strong, the range of linearity is wide, the advantage such as easy and simple to handle, quick.
Summary of the invention
Object of the present invention: deficiency and the defect that can not detect TTX for existing detection technique fast, easily, providing a kind of take immunomagnetic beads as the magnetic immuno-chromatographic method of quick detection TTX and the preparation of marked by magnetic bead chromatograph test strip of label.Thus realize quick, easy and can quantitatively detect TTX object.
The magnetic immuno-chromatographic method of this quick detection TTX that the present invention proposes, is characterized in that:
The chromatographic film of sample pad, the pad of marked by magnetic bead modified through anti-TTX specific murine system monoclonal antibody, the detection line T of the pre-coated TTX-BSA of having detectable antigens and the control line C of goat anti-mouse IgG, adsorptive pads successively interlaced about 1mm are pasted onto on base plate, then cover transparent plastic diaphragm seal on upper strata, set up into a kind of magnetic immuno-chromatographic test paper strip that can detect TTX fast; Catch immunomagnetic beads by chromatography effect, according to forming macroscopic 1 ~ 2 colour developing band after sample chromatography, the fast qualitative realizing TTX detects; Or constructed magnetic immuno test strips detected by magnetic analytical meter, the detection line formed after foundation sample chromatography and the magnetic signal detected value of control line can realize the quantitative detection of TTX.
Described anti-TTX specific monoclonal antibody is the specific monoclonal antibody body adopting hybridoma technology preparation screening gained, TTX is had to specific reaction.
The preparation method of described chromatograph test strip is as follows:
A. the preparation of detectable antigens TTX-BSA conjugate:
TTX is mixed by 1: 5 weight ratio (W/W) with bovine serum albumin(BSA) (BSA), add 4% formalin as coupling agent, revolving reaction 3d at 30 DEG C, after having reacted, with business-like PD-10 desalting column after being taken out by potpourri, desalination medium is the 10mM PBS of pH 7.4; According to appended product operation instructions, desalination is carried out to TTX-BSA conjugate during desalting processing.
B. the preparation and purification of antibody:
Using TTX-KLH as immunogene, immune balb/c mice, hybridoma technology routinely and limiting dilution method are prepared and screening monoclonal antibody cell line, then by the specific antibody cell line Multiplying culture of anti-TTX, be expelled in BALB/C mice and prepare ascites, prepare gained ascites after 50% (w/v) sulphur ammonium is concentrated, adopt business-like Protein-G affinity column to carry out the purifying of antibody; When specifically carrying out affinitive layer purification process, carry out according to appended product operation instructions.
C. the preparation of immunomagnetic beads:
Select diameter to be the magnetic bead of 50-200nm, use the mode of carbon dimethylamine (EDC) and succinimide (NHS) covalent cross-linking to be marked on magnetic bead by anti-TTX monoclonal antibody and form immunomagnetic beads;
D. the process of pad:
The immunomagnetic beads suspending liquid specking prepared is marked pad to form immunomagnetic beads on pad; Also hydrojet or quantitative liquid-jet device manually can be adopted during specking to carry out.
E. the bag quilt of chromatographic film:
With automatic spray film instrument, with the speed of 0.8 μ L/cm, by the TTX-BSA of selected concentration, goat anti-mouse IgG, specking is at the T line of chromatographic film and C line place respectively, and spacing distance during specking between line and line is 0.5-1.0cm; Wrap by after chromatographic film in the drying box of 37 DEG C, dry 4-6 hour, be placed in exsiccator and save backup;
F. the assembling of test strips and preparation:
Sample pad, the pad combining anti-TTX immunomagnetic beads, coated film, adsorptive pads successively interlaced about 1mm are pasted onto on base plate, then cover transparent plastic diaphragm seal on upper strata; Width cutting can obtain magnetic immuno-chromatographic test paper strip as requested.
The preparation method of immunomagnetic beads is as follows:
1) 2mg carboxyl magnetic bead is drawn in centrifuge tube, with 500 μ L 0.01M containing 0.5% (v/v) Tween-20, pH is that the MES solution of 5.0 is as activation buffer solution cleaning magnetic bead, being placed in by centrifuge tube on Magneto separate frame makes magnetic bead be separated with activated solution, repeated washing several times, last resuspended magnetic bead;
2) freshly prepared EDC and NHS is added activated carboxyl 30min in suspension containing magnetic beads, reaction terminates first to use MEST buffer solution magnetic bead afterwards, then uses 0.005M borate tween solution (being called for short BST) as coupling buffer washing magnetic bead 2 times;
3) add the anti-TTX monoclonal antibody of 150 μ g, impeller reacts 3 hours;
4) 30min is reacted under adding 1% (w/v) BSA solution room temperature;
5) use BST solution washing immunomagnetic beads 4 times, magnetic bead is resuspended, and 4 DEG C save backup.
The present invention compared with prior art tool has the following advantages:
Superparamagnetic nanomaterial, immunochromatography technique and magnetism detector are combined, be the immuno-chromatographic test paper strip of label with immunomagnetic beads by structure, both can meet qualitative detection demand, and can Quantitative detection be realized again, have highly sensitive, consuming time short, facilitate the features such as easy-to-use.
Accompanying drawing explanation
Fig. 1 is the structural representation of the magnetic immuno-chromatographic test paper strip taking immunomagnetic beads as label.
Magnetic immuno-chromatographic test paper strip shown in Fig. 1 is made up of the adsorptive pads 1 of sequential, chromatographic film 2 (nitrocellulose filter), pad 3, sample pad 4 and PVC base plate 7.Chromatographic film 2 is coated with two lines, wherein 5 is detection line T, and specking has TTX-BSA; 6 is control line C, and specking has goat anti-mouse IgG.
The result that TTX detection is carried out for the constructed magnetic immuno-chromatographic test paper strip of application in Fig. 2 ~ 5 sets schematic diagram.
Wherein:
Fig. 2 adopts described magnetic immuno-chromatographic test paper strip to the testing result of TTX negative sample: namely detection line T and control line C all has colour developing band.
Fig. 3 adopts described magnetic immuno-chromatographic test paper strip to the strong positive testing result of TTX: namely detection line T place is without colour developing band, and there is colour developing band at control line C place.
Fig. 4 adopts described magnetic immuno-chromatographic test paper strip to detect invalid result to TTX: namely there is colour developing band at detection line T place, and control line C place is without colour developing band.
Fig. 5 adopts described magnetic immuno-chromatographic test paper strip to detect invalid result to TTX: namely detection line T and control line C is all without colour developing band.
Fig. 6 adopts described magnetic immuno-chromatographic test paper strip to the quantitative detection curve of mark-on TTX in globe fish musculature.
Fig. 7 adopts described magnetic immuno-chromatographic test paper strip to the quantitative detection curve of mark-on TTX in globefish livers tissue.
Fig. 8 adopts described magnetic immuno-chromatographic test paper strip to detect the qualitative detection result of globe fish sample to TTX.
Wherein:
1. number be the contrast of negative muscle;
2. number be the contrast of negative liver;
3. number be wild Puffer fish globefish muscle samples;
4. number be the yellow globefish muscle samples of wild chrysanthemum;
5. number be wild yellowfin globefish muscle samples;
6. number be the yellow globefish liver specimens of wild chrysanthemum;
7. number be wild chrysanthemum yellow globefish ovary sample.
For further illustrating magnetic immuno-chromatographic test paper strip of quick detection TTX of the present invention and preparation method thereof, be described especially exemplified by following embodiment, this embodiment is to explain instead of limiting the present invention by any way.
Embodiment
The magnetic immuno-chromatographic test paper strip of quick detection TTX of the present invention as shown in Figure 1, staggered 1mm this test strips pastes chromatographic film 2, immunomagnetic beads mark pad 3, sample pad 4, adsorptive pads 1 successively on base plate 7, and in the test strips that upper strata covering transparent plastic diaphragm seal assembles, pre-coated in chromatographic film have TTX-BSA detection line T and controlling C.
TTX-BSA detectable antigens adopted in a particular embodiment and anti-TTX specific monoclonal antibody are all non-commercially produced products.What the magnetic immuno-chromatographic test paper strip described in utilization adopted when carrying out TTX detection is competition law Cleaning Principle, and when dividing the period of the day from 11 p.m. to 1 a.m containing TTX in sample to be tested, the immunomagnetic beads of TTX molecule first on pad is combined.Along with the carrying out of chromatography effect, when through detection line T, being combined with the TTX-BSA detectable antigens at T line place in conjunction with the immunomagnetic beads of TTX molecule thus resting on T line place, the immunomagnetic beads of detected antigen capture then continues to move ahead; When through control line C, C line place two anti-to combine with immunomagnetic beads thus the same magnetic beads held that makes at this place.Whole chromatography reaction completed in 30 minutes, and General reactions, after 15 minutes, detection line T and control line C occurs the apparent colour developing band of naked eyes.Test strips is put into the card reading slot of magnetic analytical meter, corresponding magnetic signal detected value can be obtained at T line place.The T place magnetic signal value of positive is generally weaker than negative sample, and difference is larger just represents that positive reaction is stronger.
Embodiment 1: magnetic immuno-chromatographic method detects the TTX in Muscle vector
(1) preparation of detectable antigens (TTX-BSA conjugate): TTX is mixed by 1: 5 (w/w) with bovine serum albumin(BSA) (BSA), adds 4% formalin as coupling agent, revolving reaction 3d at 30 DEG C.After having reacted, with business-like PD-10 desalting column after being taken out by potpourri, carry out desalination according to appended product operation instructions to TTX-BSA conjugate, desalination medium is 10mMPBS (pH 7.4).
(2) preparation and purification of antibody: using TTX-KLH as immunogene, immune balb/c mice, hybridoma technology routinely and limiting dilution method are prepared and screening monoclonal antibody cell line.Then by the specific antibody cell line Multiplying culture of anti-TTX, be expelled in BALB/C mice and prepare ascites.Prepare gained ascites after 50% (w/v) sulphur ammonium is concentrated, adopt business-like Protein-G affinity column, carry out the purifying of antibody according to appended product operation instructions.
(3) preparation of immunomagnetic beads: select diameter to be the magnetic bead of 200nm, draw 2mg carboxyl magnetic bead in centrifuge tube, with 500 μ L0.01M MEST as activation buffer solution cleaning magnetic bead, being placed in by centrifuge tube on Magneto separate frame makes magnetic bead be separated with activated solution, and repeated washing is rear resuspended magnetic bead several times.EDC and NHS is added activated carboxyl 30min in suspension containing magnetic beads subsequently, reaction terminates first to use MEST buffer solution magnetic bead afterwards, then washs magnetic bead 2 times with 0.005M BST as coupling buffer.Then add the anti-TTX monoclonal antibody of 150 μ g, impeller reacts 3 hours.Then capping 30min under 1% (w/v) BSA solution room temperature is added.Reaction terminate rear BST solution washing close after immunomagnetic beads 4 times, discard cleansing solution, magnetic bead is resuspended, and 4 DEG C save backup.
(4) by the immunomagnetic beads prepared manually specking be prepared on pad immunomagnetic beads mark pad.
(5) the bag quilt of chromatographic film: use 10mM PBS, the bag of pH7.4 is buffered liquid and TTX-BSA detectable antigens, goat anti-mouse IgG is diluted to 0.4mg/ml, 1mg/ml respectively.Then with automatically spraying film instrument, with the even specking of speed of 0.8 μ L/cm in chromatographic film, carrying out the bag quilt of chromatographic film, forming TTX-BSA detection line T, and goat anti-mouse IgG control line C; Wrap by time line and line between spacing distance be 0.5-1.0cm; Wrap by after chromatographic film (abbreviation coated film) in the drying box of 37 DEG C, dry 4-6 hour, be placed in exsiccator and save backup.
(6) preparation of test strips: sample pad, immunomagnetic beads mark pad, coated film, adsorptive pads successively interlaced about 1mm are pasted onto on base plate, then cover transparent plastic diaphragm seal on upper strata; The test strips cutting into 0.5cm wide is for subsequent use.
(7) detection of sample: with through confirming that nontoxic globe fish Muscle vector gradient dilution TTX standard items are as sample.The sample pad of test strips adds 100 μ L samples, observations after chromatography 20min.Negative findings is that obvious band all appears in T line and C line; Positive findings is that T line color is more shallow or do not occur band, and obvious band appears in C line.Also the magnetic immuno-chromatographic test paper strip of analyte sample fluid can being had to load special test strips draw-in groove by dripping, reading magnetic signal in insertion magnetic signal reader MAR instrument and carrying out quantitative test, drawing standard curve.
Embodiment 2: magnetic immuno-chromatographic method detects the TTX in globe fish viscera tissue
Except in sample detection step, detecting sample is with through confirming nontoxic liver extract gradient dilution TTX standard items.Other step is with example 1.
Embodiment 3: magnetic immuno-chromatographic method detects the TTX in wild globe fish tissue
(1) sample preparation of globe fish tissue
For globe fish musculature.Get 5g globe fish musculature, shred, add the acetic acid of 25ml 0.1%, boil and stir 10min.Be cooled to the centrifugal 10min of 4000rpm after room temperature, supernatant is transferred to new 50ml centrifuge tube.Precipitation adds 20ml 0.1% acetic acid again, then boils 3min.After being cooled to room temperature, all liquid and the sample boiled all are added in centrifuge tube, the centrifugal 10min of 4000rpm.Get supernatant, amount volume, puts in separating funnel, adds equal-volume ether, degreasing 2 times.Water layer is put into 50ml volumetric flask.Regulate pH value to 6.5 ~ 7.4 in extract with 1M NaOH, then add PBS to 50ml, extract 4 DEG C saves backup.
(2) sample detection
The sample pad forming immunochromatographydetecting detecting test strip adds 100 μ L samples, observations after chromatography 20min.Negative findings is that obvious band all appears in T line and C line; Positive findings is that T line color is more shallow or do not occur band, and obvious band appears in C line.Also the magnetic immuno-chromatographic test paper strip of analyte sample fluid can be had to load special test strips draw-in groove by dripping, read magnetic signal in insertion magnetic signal reader MAR instrument and quantitatively detect, combined standard curve, calculates the concentration of TTX in testing sample.
Constructed magnetic immuno-chromatographic test paper strip magnetic analytical meter (Magnetic Assay Reader is called for short MAR instrument) detects, and the detection line formed after foundation sample chromatography and the magnetic signal detected value of control line, can realize the quantitative detection of TTX.
(3) ELISA kit method detects
Adopt commercially available ELISA kit (CDC), according to product description processing sample, prepare graticule, calculate the content of TTX according to graticule.
The testing result of two kinds of methods is as shown in the table:
Note: N.D: lower than the lowest detectable limit of this detection method.
Claims (5)
1. one kind is detected the magnetic immuno-chromatographic test paper strip of TTX fast, it is characterized in that: the chromatographic film of sample pad, the pad of marked by magnetic bead modified through anti-TTX specific murine system monoclonal antibody, the detection line T of the pre-coated TTX-BSA of having detectable antigens and the control line C of goat anti-mouse IgG, adsorptive pads successively interlaced about 1mm are pasted onto on base plate, then cover transparent plastic diaphragm seal on upper strata, namely width cutting is detected the magnetic immuno-chromatographic test paper strip of TTX fast as requested; Wherein, the magnetic bead that anti-TTX specific murine system monoclonal antibody is modified is referred to as immunomagnetic beads; (1) preparation method of immunomagnetic beads selects diameter to be the magnetic bead of 50-200nm, uses the mode of EDC and NHS covalent cross-linking by anti-TTX specific murine system labeling of monoclonal antibody on magnetic bead; (2) method for coating of chromatographic film is: with automatically spraying film instrument, with the speed of 0.8 μ L/cm, by TTX-BSA, goat anti-mouse IgG solution, respectively specking is at the detection line T of chromatographic film and control line C place, and spacing distance during specking between line and line is 0.5-1.0cm; Wrap by after chromatographic film in the drying box of 37 DEG C, dry 4-6 hour, be placed in exsiccator and save backup; Catch immunomagnetic beads by chromatography effect during detection, after sample chromatography, form macroscopic 1 ~ 2 colour developing band;
Wherein, TTX-BSA detectable antigens preparation method is: mixed by 1: 5 (w/w) with bovine serum albumin(BSA) (BSA) by TTX, add 4% formalin as coupling agent, revolving reaction 3d at 30 DEG C, after having reacted, with business-like PD-10 desalting column after being taken out by potpourri, desalination medium is the 10mM PBS of pH7.4; Described TTX refers to tetraodotoxin.
2. one kind is detected the magnetic immuno-chromatographic method of TTX fast, it is characterized in that: the chromatographic film of sample pad, the pad of marked by magnetic bead modified through anti-TTX specific murine system monoclonal antibody, the detection line T of the pre-coated TTX-BSA of having detectable antigens and the control line C of goat anti-mouse IgG, adsorptive pads successively interlaced about 1mm are pasted onto on base plate, then cover transparent plastic diaphragm seal on upper strata, namely width cutting is detected the magnetic immuno-chromatographic test paper strip of TTX fast as requested; Wherein, the magnetic bead that anti-TTX specific murine system monoclonal antibody is modified is referred to as immunomagnetic beads; (1) preparation method of immunomagnetic beads selects diameter to be the magnetic bead of 50-200nm, uses the mode of EDC and NHS covalent cross-linking by anti-TTX specific murine system labeling of monoclonal antibody on magnetic bead; (2) method for coating of chromatographic film is: with automatically spraying film instrument, with the speed of 0.8 μ L/cm, by TTX-BSA, goat anti-mouse IgG solution, respectively specking is at the detection line T of chromatographic film and control line C place, and spacing distance during specking between line and line is 0.5-1.0cm; Wrap by after chromatographic film in the drying box of 37 DEG C, dry 4-6 hour, be placed in exsiccator and save backup; (3) testing process catches immunomagnetic beads by chromatography effect, and according to forming macroscopic 1 ~ 2 colour developing band after sample chromatography, the fast qualitative realizing TTX detects; Constructed magnetic immuno test strips detected by magnetic analytical meter, the magnetic signal detected value according to the detection line T formed after sample chromatography and control line C realizes the quantitative detection of TTX;
Wherein, TTX-BSA detectable antigens preparation method is: mixed by 1: 5 (w/w) with bovine serum albumin(BSA) (BSA) by TTX, add 4% formalin as coupling agent, revolving reaction 3d at 30 DEG C, after having reacted, with business-like PD-10 desalting column after being taken out by potpourri, desalination medium is the 10mM PBS of pH7.4, and described TTX refers to tetraodotoxin.
3. magnetic immuno-chromatographic method as claimed in claim 2, is characterized in that: described anti-TTX specific murine system monoclonal antibody is the monoclonal antibody adopting hybridoma technology preparation screening gained, TTX is had to specific reaction.
4. magnetic immuno-chromatographic method as claimed in claim 2, is characterized in that:
A. the preparation and purification of monoclonal antibody: using TTX-KLH as immunogene, immune balb/c mice, hybridoma technology routinely and limiting dilution method are prepared and screening cell strain of monoclonal antibody, then by the specific antibody monoclonal cell strain Multiplying culture of anti-TTX, be expelled in BALB/C mice and prepare ascites, prepare gained ascites after 50% (w/v) sulphur ammonium is concentrated, adopt business-like Protein-G affinity column to carry out the purifying of antibody;
B. the disposal route of pad is: the immunomagnetic beads suspending liquid specking prepared is marked pad to form immunomagnetic beads on pad.
5. magnetic immuno-chromatographic method as claimed in claim 2, it is characterized in that, the preparation method of immunomagnetic beads is as follows:
1) 2mg carboxyl magnetic bead is drawn in centrifuge tube, with 500 μ L, 0.01M is containing 0.5% (v/v) Tween-20, pH is that the MES solution of 5.0 is as activation buffer solution cleaning magnetic bead, being placed in by centrifuge tube on Magneto separate frame makes magnetic bead be separated with activation buffer, repeated washing several times, last resuspended magnetic bead;
2) freshly prepared EDC and NHS is added activated carboxyl 30min in suspension containing magnetic beads, reaction terminates rear first with above-mentioned MES solution washing magnetic bead, then washs magnetic bead 2 times with 0.005M borate tween solution as coupling buffer;
3) add 150 μ g anti-TTX specific murine system monoclonal antibody, impeller reacts 3 hours;
4) 30min is reacted under adding 1% (w/v) BSA solution room temperature;
5) wash immunomagnetic beads 4 times with borate tween solution, magnetic bead is resuspended, and 4 DEG C save backup.
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| CN112730857A (en) * | 2020-12-14 | 2021-04-30 | 上海海洋大学 | Magnetic immunochromatographic test strip and method for rapidly detecting MS-222 |
| CN113219114A (en) * | 2021-05-04 | 2021-08-06 | 中洋生物科技(上海)股份有限公司 | Rapid and efficient purification method of tetrodotoxin |
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