CN105004866B

CN105004866B - Quickly detect test kit and the application thereof of vibrio parahaemolyticus TDH toxin in food

Info

Publication number: CN105004866B
Application number: CN201510441999.8A
Authority: CN
Inventors: 窦勇; 胡佩红; 顾鹏程; 董静; 姚妙爱; 吴存兵
Original assignee: Jiangsu Vocational College of Finance and Economics
Current assignee: Jiangsu Vocational College of Finance and Economics
Priority date: 2015-07-25
Filing date: 2015-07-25
Publication date: 2016-08-17
Anticipated expiration: 2035-07-25
Also published as: CN105004866A

Abstract

The present invention provides test kit and the application thereof of vibrio parahaemolyticus TDH toxin in quick detection food, and test kit includes being coated the capture ELISA Plate of antibody, TDH standard dilution, rabbit anti-vibrio parahaemolytious negative serum, rabbit anti-vibrio parahaemolyticus polyclonal antibody, the phosphate buffer of goat anti-rabbit igg HRP, the phosphate buffer containing polysorbas20, substrate solution, stop buffer: 2mol/L H₂SO₄.Present invention offer is with vibrio parahaemolyticus TDH toxin in detection food as object of study, anti-TDH polyclonal antibody is prepared by the TDH toxin protein immune balb/c mice prepared, prepare rabbit anti-vibrio parahaemolytious polyclonal antibody using pathogenic vibrio parahaemolytious thalline as immunogen immune new zealand white rabbit simultaneously, using anti-TDH polyclonal antibody as capture antibody, anti-somatic polyclonal antibody is detection antibody, set up double-antibody sandwich elisa detection method, and use vibrio parahaemolytious TDH toxin in this method detection food, develop vibrio parahaemolytious TDH toxin ELISA quick detection kit in food.

Description

Quickly detect test kit and the application thereof of vibrio parahaemolyticus TDH toxin in food

Technical field

The present invention relates to a kind of ELISA kit, quickly detect vibrio parahaemolyticus TDH poison in food particularly to one The double-antibody sandwich elisa detection kit of element, further relates to this test kit vibrio parahaemolyticus TDH poison in quickly detection food Application in element, belongs to technical field of food safety detection.

Background technology

The food-borne pathogenic that vibrio parahaemolytious (Vibrio parahaemolyticus is called for short V.p) is common in food is micro- Biology, is primarily present in aquatic products particularly marine product, and it is to cause China's Coastal Areas alimentary toxicosis and abdomen in summer every year The important pathogen rushed down.The hemotoxin that V.p produces is the main cause that this bacterium is caused a disease, including heat-resisting direct hemolysin (Thermostable Direct Hemolysin is called for short TDH), Thermostable direct hemolysin-related hemolysin (TDH-related Hemolysin, TRH) and thermo-labile hemolysin (Thermolabile Hemolysin is called for short TLH), epidemiological investigation Show that TDH is the main virulence factor of V.p.

In current domestic detection food, vibrio parahaemolytious is mainly carried out by GB 4789.7 method, the method remain by Carry out according to the process such as separation and Culture, biochemical identification, the most time-consumingly, and operation complexity, sensitivity is limited.Application round pcr detection Vibrio parahaemolyticus and TDH, TRH toxin thereof have been studied, but this method has the deficiency that cost is big；On the contrary, easy, quick, warp Ji, sensitive, special ELISA detection method are one of development trends of detecting safely and fast of modern food.

Showing according to the study, in environment, vibrio parahaemolytious contains virulence factor the most entirely, say, that not every secondary haemolysis Property vibrio all has pathogenic.The detection of vibrio parahaemolytious is studied more rare by ELISA method, and cannot distinguish between detected pair It is pathogenic whether hemolysis vibrion has.The relevant report of application elisa technique detection vibrio parahaemolytious TDH toxin is the rarest, Prepare vibrio parahaemolytious Thermostable direc t hemolysin TDH monoclonal antibody in particular with immunological technique, and set up ELISA method The research detecting pathogenic vibrio parahaemolyticus is the most extremely rare, and the ELISA of detection vibrio parahaemolytious TDH toxin is quick Detection kit not yet occurs.

Summary of the invention

Technical problem: invent a technical problem to be solved and be to overcome vibrio parahaemolyticus in existing detection food Detection time length, testing cost is high and is difficult to differentiate between whether having the deficiencies such as pathogenic, it is provided that secondary haemolysis in a kind of food Property vibrio virulence factor TDH toxin DAS-ELISA method quick detection kit, its detection accuracy is high, and the detection time is short, energy Enough realize ELISA detection method standardization detection.

Technical scheme: the test kit of vibrio parahaemolyticus TDH toxin in the quickly detection food that the present invention provides, including:

(1) ELISA Plate: be coated the ELISA Plate of capture antibody；Described capture antibody is mouse-anti TDH toxin polyclonal antibody blood Clearly；

(2) TDH standard dilution: vibrio parahaemolyticus TDH toxin；

(3) negative control: rabbit anti-vibrio parahaemolytious negative serum；

(4) detection antibody: rabbit anti-vibrio parahaemolyticus polyclonal antibody；

(5) ELIAS secondary antibody: goat anti-rabbit igg-HRP；

(6) blank liquid: phosphate buffer (the Phosphate Buffer Saline letter of 0.01mol/L, pH7.4 Claim PBS)；

(7) cleaning mixture: phosphate buffer (the Phosphate Buffer Saline of 0.01mol/L, pH7.4 Tween-20, is called for short PBST)；

(8) substrate solution: include A bottle and B bottle, A bottle is the o-phenylenediamine containing 4mg/100mL, pH5.0 phosphate-Fructus Citri Limoniae Acid buffer；B bottle is the H of mass percent concentration 30%₂O₂；

(9) stop buffer: 2mol/L H₂SO₄。

As preferably, the preparation method of described mouse-anti TDH toxin polyclonal antibody, comprise the following steps: pair is molten After courageous and upright vibrio TDH toxin is fully combined with the incomplete Freund's adjuvant of equivalent, the Freund's complete adjuvant of equivalent respectively, divide many Two groups of mices are injected in method abdominal cavity secondary, dosage escalation respectively；Fundamental immunity uses the 4 g TDH combining Freund's complete adjuvant, Carry out 1 booster immunization subsequently every 2 weeks, prepare mouse-anti TDH toxin polyclonal antibody.

Preferred as another kind, the preparation method of described rabbit anti-vibrio parahaemolyticus polyclonal antibody, including following Step: at 0.05% formaldehyde, the pathogenic vibrio parahaemolyticus producing TDH toxin is inactivated 2 h at 35 DEG C and prepare somatic antigen, point Repeatedly, dosage escalation enters ear vein injection；Immunity new zealand white rabbit, immunity concentration is 10⁸CFU/ mL；Exempt from the last time Epidemic disease terminates one week after, and arteria auricularis is taken a blood sample in a large number, obtains rabbit anti-vibrio parahaemolyticus polyclonal antibody.

Preferred as another kind, the dilution factor of rabbit anti-vibrio parahaemolyticus polyclonal antibody is 1:4000 ~ 1:8000； The dilution factor of described goat anti-rabbit igg-HRP is 1:1000 ~ 2000.

Preferred as another kind, the preparation method of described vibrio parahaemolyticus TDH toxin, comprise the following steps:

(1) preparation of TDH crude toxin: the vibrio parahaemolytious producing TDH toxin is inoculated in sodium chloride basic peptone water In solution in shaking table after amplification culture, centrifugal segregation thalline, supernatant adds (NH₄)₂SO₄, stand and saltout with fully precipitation TDH toxin；High speed centrifugation, resolution of precipitate, in PBS solution, is dialysed, is obtained TDH crude toxin；

(2) purification of TDH crude toxin:

(2.1) DEAE-cellulose ion-exchange column chromatography: the TDH crude toxin that obtains step (1) prepared is slowly dropped to In DEAE-cellulose ion exchange column, with the 1L PBST eluant solution of NaCl containing 0.2 mol/L, then 1L with containing 0.2 ~ The PBS solution linear gradient elution of 1.0mol/L NaCl, collects eluent；Add solid (NH₄)₂SO₄Saltout, precipitation PBS After dissolving, dialysed overnight；

(2.2) HAP column chromatography: the dialysis solution of step (2.1) is added drop-wise on HAP post, with 0.5L containing 0.1mol/L PBS liquid (pH7.0) eluting of NaCl, then with PBS liquid (pH7.0) eluting of the 0.1 ~ 0.3mol/L of 0.8L, collect eluent；Add Enter solid (NH₄)₂SO₄Saltout, precipitate after dissolving with PBS, dialysed overnight；

(2.3) Sephadex G-25 column chromatography: the dialysis solution of step (2.2) is crossed Sephadex G-25 post, uses The Tris-HCl buffer solution elution of 0.01mol/L pH 7.0, eluent PEG-20000 concentrates, and obtains vibrio parahaemolyticus TDH toxin.

Wherein, in step (1), the mass percent concentration of the aqueous solution of described sodium chloride basic protein peptone is 3%；Expand Condition of culture is: 37 DEG C, 150r/min, 24h；Centrifugal rotational speed is 6000r/min；(NH₄)₂SO₄Addition is preferred 35.1g/ 100mL supernatant；Salting-out condition is preferably 4 DEG C, 10h；High speed centrifugation condition be 10000r/min, 4 DEG C；The concentration of PBS solution It is 7.0 for 0.010mol/L, pH.

Wherein, in step (2.1), solid (NH₄)₂SO₄The addition saltoutd is 40g/100mL；Dialysis temperature preferably 4 DEG C.

Preferred as another kind, described in be coated the preparation method of ELISA Plate of capture antibody, comprise the following steps: at new enzyme Every hole of target adds the mouse-anti TDH toxin polyclonal antibody 100 μ L of dilution, adds closing fluid-tight and close ELISA Plate after drying, 37 DEG C of constant temperature and humidities hatch 80min, discard unnecessary confining liquid, use cleaning mixture to fill each hole, jog 2 ~ 4min, pat dry, repeat 3 After secondary, button is dry, to obtain final product；The mouse-anti TDH toxin polyclonal antibody of described dilution be use 0.05mol/L, pH be the carbon of 9.6 The serum of hydrochlorate buffer solution dilution, dilution factor 1:2000 ~ 1:4000；Described confining liquid is the cattle of mass percent concentration 3 ~ 5% Serum albumin or 3 ~ 5% defatted milk powder.

Present invention also offers above-mentioned ELISA kit in quickly detection food in vibrio parahaemolyticus TDH toxin Application.

Described application, comprises the following steps:

(1) Sample pretreatment: weigh food samples with sterile working, adds the phosphate-buffered of 0.01mol/L, pH7.4 Liquid, homogenizing in homogenizing cup, make the equal liquid of sample；Equal for sample liquid thin,tough silk yarn is filtered, in 8000 ~ 10000r/min be centrifuged 8 ~ 10min, takes supernatant and is measuring samples extracting solution；Food samples is 25g:225mL with the amount ratio of phosphate buffer；

(2) in being coated the hole of ELISA Plate of capture antibody, it is separately added into: 100 μ L measuring samples extracting solution or 100 μ L TDH standard dilution (being used for replacing sample extracting solution when drawing standard curve), 100 μ L rabbit anti-vibrio parahaemolytious negative serums are made PBS for negative control, 100 μ L hatches 60 ~ 80min as blank, shrouding film shrouding, 37 DEG C of constant temperature and humidities；Every hole adds Entering the TDH standard dilution for drawing standard curve, concentration is respectively 2 μ g/mL, 4 μ g/mL, 6 μ g/mL, 8 μ g/mL, 10 μ g/ mL、12μg/mL；

(3) plate is washed: manual wash plate or wash trigger and wash plate；Manual plate washing method is: discarding liquid in hole, cleaning mixture fills respectively Hole, jog 3min, pat dry, after being repeated 3 times, button is dry；Washing trigger plate washing method is: after selecting washing 3 times and wash time 2min, Automatically washing plate, button is dry；

(4) adding 100 μ L rabbit anti-vibrio parahaemolyticus polyclonal antibodies in every hole, 37 DEG C of constant temperature and humidities hatch 60- 70min；

(5) plate is washed: manual wash plate or wash trigger and wash plate；Manual plate washing method is: discarding liquid in hole, cleaning mixture fills respectively Hole, jog 3min, pat dry, after being repeated 3 times, button is dry；Washing trigger plate washing method is: when using cleaning mixture to select washing 3 times and washing Between after 2min, automatically wash plate, button is dry；

(6) adding 100 μ L ELIAS secondary antibody goat anti-rabbit igg-HRP in every hole, 37 DEG C of constant temperature and humidities hatch 45 ~ 55min；

(7) plate is washed: manual wash plate or wash trigger and wash plate；Manual plate washing method is: discarding liquid in hole, cleaning mixture fills respectively Hole, jog 3min, pat dry, after being repeated 3 times, button is dry；Washing trigger plate washing method is: after selecting washing 3 times and wash time 2min, Automatically washing plate, button is dry；

(8) adding 100 μ L substrate solutions in every hole, 30 DEG C of constant temperature and humidity black outs hatch 15 ~ 25min；

(9) adding 50 μ L stop buffers in every hole, shake up, use microplate reader reading in 5min, reference wavelength is set to 630nm, Measure wavelength and be set to 492nm, return to zero with blank well, be then read out each hole OD₄₉₂Value；

(10) result judges:

A, qualitative analysis: sample OD₄₉₂The average OD of value/negative control₄₉₂Value >=2.1, it is judged that for the positive, be otherwise negative； Negative control OD₄₉₂When value is less than 0.05, calculate with 0.05；During higher than 0.05, with actual OD₄₉₂Value calculates；

B, quantitative analysis: with OD₄₉₂Value is vertical coordinate, with TDH standard dilution concentration (μ g/mL) as abscissa, draws mark Directrix curve；OD according to sample₄₉₂Value can find testing sample TDH concentration on standard curve, with this concentration be multiplied by 10 i.e. TDH content of toxins in sample, unit μ g/g.

Beneficial effect: the present invention provides with detection TDH toxin as object of study, is exempted from by the TDH toxin protein prepared Epidemic disease BALB/C mice prepares anti-TDH polyclonal antibody (TDH Polyclonal Antibody, be called for short TDH-PAb), simultaneously so that Characteristic of disease vibrio parahaemolytious thalline prepares rabbit anti-vibrio parahaemolytious polyclonal antibody as immunogen immune new zealand white rabbit (Vibrio parahaemolyticus Polyclonal Antibody is called for short V.p-PAb), make with anti-TDH polyclonal antibody For capture antibody, anti-somatic polyclonal antibody is detection antibody, sets up double-antibody sandwich elisa detection method (Double Antibody Sandwich ELISA, is called for short DAS-ELISA), and use vibrio parahaemolytious TDH toxin in this method detection food, Develop vibrio parahaemolytious TDH toxin ELISA quick detection kit in food.

Accompanying drawing explanation

Fig. 1 is that 100 DEG C of heat treatments affect result figure to TDH toxin hemolytic activity.

Fig. 2 is that vibrio parahaemolytious TDH toxin polyclonal antibody produces progress curve figure.

Fig. 3 is vibrio parahaemolytious TDH toxin multi-resistance cross reaction result of the test figure.

Fig. 4 is vibrio parahaemolytious TDH toxin sensitive result of the test figure.

Fig. 5 is capture antibody TDH-PAb and TDH antigen optimal reaction time result of the test figure.

Fig. 6 is ELISA Plate blocking test result figure.

Fig. 7 is the Selection experiment result figure of detection antibody V.p-PAb and TDH antigen optimal reaction time.

Fig. 8 is goat anti-rabbit igg-HRP and detection antibody V.p-PAb optimal reaction selection of time result of the test figure.

Fig. 9 is DAS-ELISA method specific test result figure.

Detailed description of the invention

Below in conjunction with embodiment, the technology of the present invention solution is further described, but the invention is not restricted to these and implement Example.

Embodiment 1

The TDH Toxic extraction of vibrio parahaemolyticus and purification, comprise the following steps:

(1) product TDH toxin vibrio parahaemolytious is inoculated in 3% sodium chloride basic peptone water at 37 DEG C, carries out 150r/ After min shaking table amplification culture 24h, through 6000r/min centrifugal segregation thalline, add in supernatant by the amount of 35.1g/100mL (NH₄)₂SO₄, stand at 4 DEG C after fully dissolving and saltout overnight, with fully precipitation TDH toxin, in 10000r/min, at 4 DEG C from Heart 10min, abandons supernatant, and resolution of precipitate is in the PBS solution of a small amount of 0.01mol/L pH=7.0, and dialyses with this PBS solution At night, obtain TDH crude toxin；

(2) DEAE-cellulose ion-exchange column chromatography: by Raw toxin liquid, is used in DEAE-cellulose DE-25 and carries out post Chromatography, method is as follows: is slowly dropped in chromatographic column by the TDH crude toxin balanced overnight in above-mentioned PBS liquid 4 DEG C, uses simultaneously About 1L NaCl Han 0.2mol/L above-mentioned PBST eluting pillar, then carry out linear ladder with the 1L above-mentioned PBS containing 0.2 ~ 1.0/L NaCl Degree eluting, collects eluent, adds solid (NH by the amount of 40g/100mL₄)₂SO₄Saltouing, precipitation is dissolved with above-mentioned PBS again Rear 4 DEG C of dialysed overnight；

(3) HAP column chromatography: by the dialysis solution HAP(2.2 × 35cm of step (2)) carry out column chromatography, with about 500mL's PBS liquid (pH7.0) eluting of 0.1mol/L, then with PBS liquid (pH7.0) eluting of the 0.1 ~ 0.3mol/L of 800mL, collect eluting Liquid, dialysed overnight method is ibid.

(4) Sephadex G-25 column chromatography: the product through HAP column chromatography is crossed Sephadex G-25 post and (uses 0.01mol/L Tris-HCl pH of buffer 7.0 balances), with same buffer solution eluting pillar, eluent PEG-20000 Concentrate a small size, be the TDH toxin of this research purification, after its lyophilization, obtain the TDH poison of vibrio parahaemolyticus Element.

Prepared by the polyclonal antibody of pathogenic vibrio parahaemolyticus and TDH toxin thereof, comprise the following steps:

(1) prepared by mouse-anti vibrio parahaemolyticus TDH toxin polyclonal antibody:

Choose 6-8 week old Healthy female BALB/c mouse 4 (numbering BALB/c-1 ~ 4), after breeding observing 1 week is without difference, Take a blood sample from eyeball, do tube agglutination test with TDH toxin, be used for preparing negative serum ,-20 DEG C of preservations without agglutination person.

Take a certain amount of toxin of TDH after purification fully to combine respectively at incomplete Freund's adjuvant and the Freund's complete adjuvant of equivalent After, several times, the method abdominal cavity of dosage escalation inject mice respectively, fundamental immunity uses 4 g combining Freund's complete adjuvant TDH, carried out 1 booster immunization (immunizing dose is shown in Table 1) subsequently every 2 weeks, carried out 1 rathole ball blood sampling 0.5ml every 2 weeks, Measuring immune serum titer, immunity terminates one week after the last time, and eyeball is taken a blood sample in a large number, and blood moves to 4 DEG C of refrigerator overnight, takes Supernatant, is centrifuged 10min in 3000r/min, prepares mouse-anti TDH toxin polyclonal antibody, puts-20 DEG C of Refrigerator stores.

Table 1 TDH toxin immunity BALB/c mouse process

(2) prepared by rabbit anti-vibrio parahaemolyticus polyclonal antibody:

Choose the body weight healthy Male New Zealand White Rabbit at 2 ~ 2.5 kg, after breeding observing 1 week is without difference, ear vein Blood sampling, does tube agglutination test with vibrio parahaemolyticus, is used for preparing negative serum ,-20 DEG C of preservations without agglutination person.To produce The pathogenic vibrio parahaemolyticus of TDH toxin, through 0.05% formaldehyde, 35 DEG C vibrio parahaemolytious is inactivated 2 h and prepares somatic antigen, Immunity concentration is 10⁸CFU/ mL(injection dosage see attached list 2), several times, dosage escalation enter ear vein injection, the last time Immunity terminates one week after, and arteria auricularis is taken a blood sample in a large number, and room temperature underlying inclined-plane 2h moves to 4 DEG C of refrigerator overnight, and supernatant is that the anti-pair of rabbit is molten Courageous and upright vibrio polyclonal antibody, is centrifuged 5min if any erythrocyte with 4000 r/min rotating speed and removes, be sub-packed in colourless In polypropylene cryogenic ,-20 DEG C of Refrigerator stores.

Table 2 vibrio parahaemolyticus immunity new zealand white rabbit process

Immune time	1st time	2nd time	3rd time	The 4th	The 5th	6th time
							Immunizing dose (mL)	1.0	1.5	2.0	2.5	3.0	3.5
Interval time (d)	0	7	7	7	7	7

Embodiment 2

The application of DAS-ELISA quick detection kit, method is as follows:

(1) Sample pretreatment, weighs big (little) the Channa argus sample of 25g with sterile working, adds 225mL 0.01mol/L, The phosphate buffer of pH7.4, homogenizing in homogenizing cup, make the equal liquid of sample (1:10).Equal for sample liquid thin,tough silk yarn is filtered, in 8000r/min is centrifuged 10min, takes supernatant and carries out DAS-ELISA detection.

(2) taking-up has been coated the ELISA Plate capturing antibody, and every hole adds 100 μ L measuring samples extracting solution and TDH standards Diluent (draws standard curve use, concentration is respectively 2 μ g/mL, 4 μ g/mL, 6 μ g/mL, 8 μ g/mL, 10 μ g/mL, 12 μ g/mL), It is simultaneously introduced 100 μ L rabbit anti-vibrio parahaemolytious negative serums as negative control, adds the PBST of 100 μ L as blank, Shrouding film shrouding, 37 DEG C of constant temperature and humidities hatch 60min.

(3) washing plate by hand: discarding liquid in hole, cleaning mixture fills each hole, jog 3min, pats dry, after being repeated 3 times, button is dry. Washing trigger and wash plate: after selecting washing 3 times and wash time 2min, automatically wash plate, button is dry.

(4) every hole adds 100 μ L rabbit anti-vibrio parahaemolyticus immune serums, and 37 DEG C of constant temperature and humidities hatch 70min.

(5) washing plate according to step (3), button is dry.

(6) every hole adds enzyme conjugates goat anti-rabbit igg-HRP 100 μ L, and 37 DEG C of constant temperature and humidities hatch 45min.

(7) washing plate according to step (3), button is dry.

(8) every hole adds 100 μ L substrate solutions, and 30 DEG C of constant temperature and humidity black outs hatch 15min.

(9) every hole adds 50 μ L stop buffers, uses microplate reader reading in shaking up 2min, 5min, and reference wavelength is set to 630nm, measures wavelength and is set to 492nm, return to zero with blank well, be then read out each hole OD₄₉₂Value.

(10) result judges:

A, qualitative analysis: sample OD₄₉₂The average OD of value/negative control₄₉₂Value >=2.1, it is judged that for the positive, be otherwise negative. Negative control OD₄₉₂When value is less than 0.05, calculate with 0.05；During higher than 0.05, with actual OD₄₉₂Value calculates.

B, quantitative analysis: with OD₄₉₂Value is vertical coordinate, with TDH standard concentration (μ g/mL) as abscissa, draws standard bent Line.OD value according to sample can find testing sample TDH concentration on standard curve, with this concentration be multiplied by 10 i.e. in sample TDH content of toxins, unit μ g/g.

Embodiment 3

The application of DAS-ELISA quick detection kit, method is as follows:

(1) Sample pretreatment, weighs the Species of Crustacea samples such as 25g prawn, crab with sterile working, adds 225mL The phosphate buffer of 0.01mol/L, pH7.4, homogenizing in homogenizing cup, make the equal liquid of sample (1:10).Equal for sample liquid is used Thin,tough silk yarn filters, and is centrifuged 8min in 10000r/min, takes supernatant and carry out DAS-ELISA detection.

(2) taking-up has been coated the ELISA Plate capturing antibody, and every hole adds 100 μ L measuring samples extracting solution and TDH standards Diluent (draws standard curve use, concentration is respectively 2 μ g/mL, 4 μ g/mL, 6 μ g/mL, 8 μ g/mL, 10 μ g/mL, 12 μ g/mL), It is simultaneously introduced 100 μ L rabbit anti-vibrio parahaemolytious negative serums as negative control, adds the PBST of 100 μ L as blank, Shrouding film shrouding, 37 DEG C of constant temperature and humidities hatch 80min.

(4) every hole adds 100 μ L rabbit anti-vibrio parahaemolyticus immune serums, and 37 DEG C of constant temperature and humidities hatch 65min.

(5) washing plate according to step (3), button is dry.

(6) every hole adds enzyme conjugates goat anti-rabbit igg-HRP 100 μ L, and 37 DEG C of constant temperature and humidities hatch 50min.

(7) washing plate according to step (3), button is dry.

(8) every hole adds 100 μ L substrate solutions, and 30 DEG C of constant temperature and humidity black outs hatch 20min.

(10) result judges:

B, quantitative analysis: with OD₄₉₂Value is vertical coordinate, with TDH standard concentration (μ g/mL) as abscissa, draws standard bent Line.OD according to sample₄₉₂Value can find testing sample TDH concentration on standard curve, with this concentration be multiplied by 10 i.e. sample Middle TDH content of toxins, unit μ g/g.

Embodiment 4

The application of DAS-ELISA quick detection kit, method is as follows:

(1) Sample pretreatment, weighs the marine product samples such as 25g marine seabass, Trichiurus haumela with sterile working, adds 225mL The phosphate buffer of 0.01mol/L, pH7.4, homogenizing in homogenizing cup, make the equal liquid of sample (1:10).Equal for sample liquid is used Thin,tough silk yarn filters, and is centrifuged 9min in 9000r/min, takes supernatant and carry out DAS-ELISA detection.

(2) taking-up has been coated the ELISA Plate capturing antibody, and every hole adds 100 μ L measuring samples extracting solution and TDH standards Diluent (draws standard curve use, concentration is respectively 2 μ g/mL, 4 μ g/mL, 6 μ g/mL, 8 μ g/mL, 10 μ g/mL, 12 μ g/mL), It is simultaneously introduced 100 μ L rabbit anti-vibrio parahaemolytious negative serums as negative control, adds the PBST of 100 μ L as blank, Shrouding film shrouding, 37 DEG C of constant temperature and humidities hatch 70min.

(4) every hole adds 100 μ L rabbit anti-vibrio parahaemolyticus immune serums, and 37 DEG C of constant temperature and humidities hatch 60min.

(5) washing plate according to step (3), button is dry.

(6) every hole adds enzyme conjugates goat anti-rabbit igg-HRP 100 μ L, and 37 DEG C of constant temperature and humidities hatch 55min.

(7) washing plate according to step (3), button is dry.

(8) every hole adds 100 μ L substrate solutions, and 30 DEG C of constant temperature and humidity black outs hatch 25min.

(10) result judges:

B, quantitative analysis: with OD₄₉₂Value value is vertical coordinate, with TDH standard concentration (μ g/mL) as abscissa, draws standard Curve.OD according to sample₄₉₂Value can find testing sample TDH concentration on standard curve, with this concentration be multiplied by 10 i.e. sample TDH content of toxins in product, unit μ g/g.

Embodiment 5

1 materials and methods

1.1 material

1.1.1 main equipment

Freund's complete adjuvant, incomplete Freund's adjuvant, hydroxyapatite (HAP) (Sigma company)；Sheep anti-mouse igg-HRP, Raw emerging biotechnology (Nanjing) company limited of bovine serum albumin, Sephadex G-25()；Ammonium sulfate, o-phenylenediamine OPD, PBST (Huaian traditional Chinese medicines chemical reagent company limited, AR)；DEAE cellulose DE-52, bag filter (the Nanjing limited public affairs of Sen Beijia biotechnology Department)；Full-automatic microplate reader StatFax3200(Awareness company)；Micropipette rifle Nichpet EX(Japan NICHIRYO Company)；Ultra-violet and visible spectrophotometer UV2300 (Shanghai Techcomp Instrument Ltd.)；Detachable ELISA Plate (Shanghai It is Yi Liu Science and Technology Ltd. product).

1.1.2 for examination animal and bacterial strain

6-8 week old Healthy female BALB/c mouse (Medical Center of Fudan University's Experimental Animal Center)；Produce TDH toxin pair haemolysis Vibrio ATCC33846, vibrio parahaemolytious 1.2164(Shanghai Ocean University food safety laboratory provide)；Staphylococcus aureus Bacterium, Salmonella, escherichia coli, Listeria, non-pathogenic vibrio parahaemolytious (numbering V.p1 ~ 4, Jiangsu finance and economics occupation skill Microbiological Lab of art institute preservation)

1.2 method

1.2.1 the former preparation of TDH toxin immunity

Product TDH toxin vibrio parahaemolytious ATCC33846 is inoculated in 3% sodium chloride basic peptone water and carries out at 37 DEG C After 150r/min shaking table amplification culture 24h, through 6000r/min centrifugal segregation thalline, by the amount of 35.1g/100mL in supernatant Add (NH₄)₂SO₄, stand at 4 DEG C after fully dissolving and saltout overnight, with fully precipitation TDH toxin, in 10000r/min, 4 DEG C Under be centrifuged 10min, abandon supernatant, resolution of precipitate is in the PBS solution of a small amount of 0.01mol/L pH=7.0 and saturating with this PBS solution Analysis overnight, obtains TDH crude toxin.DEAE-cellulose ion-exchange column chromatography: by this Raw toxin liquid, be used in DEAE-fiber Element DE-25 carries out column chromatography, and method is as follows: the TDH crude toxin balanced overnight in above-mentioned PBS liquid 4 DEG C is slowly dropped to layer In analysis post, simultaneously with about 1L NaCl Han 0.2mol/L above-mentioned PBST eluting pillar, then contain the above-mentioned of 0.2 ~ 1.0/L NaCl with 1L PBS carries out linear gradient elution, collects eluent, adds solid (NH by the amount of 40g/100mL₄)₂SO₄Saltouing, precipitation is again Rear 4 DEG C of dialysed overnight are dissolved with above-mentioned PBS.HAP column chromatography: by toxin filtrate HAP(2.2 × 35cm) carry out column chromatography, use PBS liquid (pH7.0) eluting of the 0.1mol/L of about 500mL, then wash with the PBS liquid (pH7.0) of the 0.1 ~ 0.3mol/L of 800mL De-, collect eluent, dialysed overnight method is ibid.Sephadex G-25 column chromatography: by the product mistake through HAP column chromatography Sephadex G-25 post (balances by 0.01mol/L Tris-HCl pH of buffer 7.0), with same buffer solution eluting post Son, eluent PEG-20000 concentrates a small size, is the TDH toxin of this research purification, after its lyophilization, To TDH toxin after purification.After the incomplete Freund's adjuvant of this TDH toxin and equivalent and Freund's complete adjuvant are fully combined, Prepare TDH immunogen.

1.2.2 TDH toxin hemolytic measures

Reference literature (Yang Jingya etc. the development of vibrio parahaemolytious TDH monoclonal antibody and Property Identification) method carries out.

1.2.3 TDH toxin thermostability and oxicity analysis

Owing to TDH has bigger toxicity, during immune mouse, used in amounts strictly to control in order to avoid causing mice to die like a rat, TDH multi-resistance cannot be prepared.This research have chosen the Healthy female BALB/c mouse 36 of 6-8 week old, and body weight differs super up and down Cross 3g, be divided into 12 groups, prepare according to dosage (see Table 1) this research of lumbar injection of 14,12,10,6,2 g/ respectively TDH toxin, records the time-to-live of each group of mice, determines its toxicity dose to mice, provides good raising in process of the test Support environment.

Separately take the TDH toxin of this test preparation, after dissolving with the PBS solution of 0.01mol/L pH=7.0, be uniformly divided into 6 Equal portions, are respectively placed in the thermostat water bath of 50,60,70,80,90,100 DEG C and heat, every 3min, and separately sampled mensuration Its hemolytic activity, the heat resistance of research TDH.

1.2.4 prepared by TDH toxin polyclonal antibody

Choose 6-8 week old Healthy female BALB/c mouse 4 (numbering BALB/c-1 ~ 4), after breeding observing 1 week is without difference, Take a blood sample from eyeball, do tube agglutination test with TDH toxin, be used for preparing negative serum ,-20 DEG C of preservations without agglutination person.Take The a certain amount of toxin of TDH after purification after the incomplete Freund's adjuvant of equivalent and Freund's complete adjuvant fully combine, several times, Two groups of mices are injected in the method abdominal cavity of dosage escalation respectively, and fundamental immunity uses and combines 4 g(or 12 of Freund's complete adjuvant g) TDH, carried out 1 booster immunization (immunizing dose is shown in Table 3) subsequently every 2 weeks, carried out 1 rathole ball blood sampling 0.5ml every 2 weeks, Measuring immune serum titer, immunity terminates one week after the last time, and eyeball is taken a blood sample in a large number, and blood moves to 4 DEG C of refrigerator overnight, takes Supernatant, is centrifuged 10min in 3000r/min, prepares mouse-anti TDH toxin polyclonal antibody, puts-20 DEG C of Refrigerator stores.

Table 3 TDH toxin immunity BALB/c mouse process

Group	Immune time	1st time	2nd time	3rd time	The 4th	The 5th
							1	BALB/c-1 ~ 2 mouse immune dosage (g/ is only)	4	6	8	10	12
2	BALB/c-3 ~ 4 mouse immune dosage (g/ is only)	12	10	8	6	4
								Interval time (d)	0	14	14	14	14

1.2.5 indirect elisa method measures TDH toxin polyclonal antibody titer

During by 2.1 times of negative serum light absorption values of immune serum light absorption value, the inverse of the maximum dilution multiple of immune serum It is defined as mouse-anti TDH toxin polyclonal antibody titer.Preliminary experiment chessboard method is used to determine that the suitableeest concentration that is coated of TDH is 12 g/mL Coated elisa plate, 37 DEG C of constant temperature and humidities hatch 2h, discard and unnecessary are coated liquid, wash plate 4 times with PBST, pat dry, the calf serum of 5% Blocking antigen, 37 DEG C of constant temperature and humidities of shrouding hatch 2 h.Discard unnecessary calf serum, wash plate 4 times with PBST, pat dry.TDH is exempted from Epidemic disease serum carries out doubling dilution, and every hole adds immune serum and the Mus negative serum of 100 l, replaces serum to arrange with PBS simultaneously Blank, 37 DEG C of constant temperature and humidities of shrouding are hatched 2h, are discarded unnecessary serum, wash plate with PBST and pat dry for 4 times, and every hole adds 100 l 1:1000(description recommend consumption) sheep anti-mouse igg-HRP, hatch 1h, PBST for 37 DEG C and wash plate 4 times, pat dry, every hole add The OPD of 100 l, 30 DEG C of constant temperature lucifuge reaction 20min, every hole adds the sulphuric acid of 50 l 2mol/L and terminates enzyme reaction, in microplate reader Measure OD₄₉₂Value, and calculate TDH toxin polyclonal antibody titer.

1.2.6 specific test

By pathogenic staphylococcus aureus, Salmonella, escherichia coli, Listeria and non-pathogenic pair haemolysis Vibrio V.p1 ~ 4 bacterial strain is as strains tested, and after in antibacterial enrichment liquid, 24h cultivated by 37 DEG C of shaking tables, culture fluid is in 10000r/min Centrifugal 10min, takes each for trying bacterium supernatant and the TDH solution 100 l coated elisa plate of 12 g/mL, every hole addition 1 respectively: The TDH immune serum of 64000, replaces serum to arrange blank with PBST simultaneously, and subsequent operation method is indirect with 1.2.5 ELISA method, measures each hole OD₄₉₂.Positive judgement standards is P/N >=2.1, wherein。

1.2.7 sensitivity tests

TDH toxin is diluted to 10 g/mL, 9 g/mL, 8 g/mL, 7 g/ with the PBST solution of 0.01mol/L pH=7.0 ML, 6 g/mL, 4 g/mL, 2 g/mL, 1 g/mL, take 100 l coated elisa plates respectively, carries out indirect ELISA test, follow-up behaviour Make the same 1.2.5 of step, according to the criterion of P/N in 1.2.6, as positive judgment basis, determine the quick of TDH polyclonal antibody Perception.

2 results and analysis

2.1 TDH toxin thermostability and toxicity

Table 4 is it can be seen that the abdominal cavity direct injection TDH toxin without Freund adjuvant, and along with injection dosage increases, survival is flat All time substantially reduces, every injected in mice dosage be 2 below g without the phenomena of mortality, start dead at 2 more than g, but every note Penetrating dosage when being increased to 16 g by 4 g, mice mean survival time is reduced to 100.8min by 350.3min；And it is left to combine Freund Agent obtains TDH toxin, white mice is obtained lethal dose and significantly improves, and injection combines the cause to mice of the TDH toxin of Freund's complete adjuvant Extremely measure at 8 more than g, and combine the incomplete Freund's adjuvant lethal dose to mice at 12 more than g, it can be seen that left dose of Freund is right The toxicity of TDH produces a certain degree of impact, and tracing it to its cause is likely to be due to TDH toxin and is combined into " Water-In-Oil " breast with left dose of Freund Shape liquid slow down toxin and enters amount and the time of blood, thus reduces its toxicity, reduces the mechanism of TDH toxicity about left dose of Freund Need further experimental study.According to by table 2 result of the test, for avoiding the phenomena of mortality of white mice in immunization schedule, preferably Preparation TDH polyclonal antibody, can primarily determine that the combination incomplete Freund's adjuvant TDH dosage control of immune mouse is at 12 g/ Only, in conjunction with Freund's complete adjuvant TDH dosage control 8 g/ only within.

The table 4 TDH toxin toxicity data to BALB/c mouse

Table 5 result shows, 60 DEG C of process groups, part haemolysis occurs when 12min, and hemolytic activity starts to reduce, and reaches During to 15min, hemolytic activity completely loses.And in addition to 60 DEG C of process groups, other process groups, within 18min, all cannot eliminate TDH hemolytic activity.Illustrate TDH as a kind of proteotoxin, heat resistance is very strong, and this toxin protein may occur at 60 DEG C The change of some group, causes hemolytic activity to lose, and its mechanism awaits studying further.This toxin is boiled at 100 DEG C Certain time, in Fig. 1, pipe No. 1 ~ 6 represents boiling time 6 ~ 21min successively, measures a hemolytic activity every 3min, and Fig. 1 shows There is haemolysis in 1 ~ No. 4 pipe, and No. 6 pipes start without haemolysis, illustrates that TDH loses hemolytic activity, namely when 18min Say that TDH hemolytic activity at 100 DEG C can maintain 15 ~ 18min.

Table 5 different temperatures, the heat treatment time impact on TDH hemolytic activity

Note: " ++ " represents complete hemolysis, "+" represent part haemolysis, " one " indicates without haemolysis.

2.2 TDH toxin multi-resistance produce process

Fig. 2, it can be seen that the titer of 1, No. 2 mice generation antibody, was imitated apparently higher than 3, No. 4 mouse antibodies from the 42nd day Valency, illustrates to use the 1st group of mouse immunization protocol can obtain high-titer antibody, namely little according to the method immunity of dosage escalation Mus, more can stimulate lymphocytic emiocytosis polyclonal antibody.Fig. 2 result shows, many at the TDH creating high price on the 56th day of immunity Clonal antibody, antibody titer is up to 6.4 × 10⁶。

2.3 TDH multi-resistance specificitys

Fig. 3 result shows, two strains pathogenic vibrio parahaemolyticus P/N value is noticeably greater than 2.1, presents strong positive, rather than causes The vibrio parahaemolytious V.p1-4 of characteristic of disease, pathogenic staphylococcus aureus, Salmonella, shigella, Listeria P/N value is significantly lower than 2.1, presents feminine gender, illustrates that this polyclonal antibody has the strongest specificity, with above-mentioned bacterial strains without any friendship Fork reaction.

2.4 TDH multi-resistance sensitivity

From fig. 4, it can be seen that along with TDH is coated the continuous reduction of concentration, P/N value reduces rapidly, when being coated lowering of concentration During to 6 g/mL, P/N is 1.75 < 2.1, i.e. the minimum concentration that TDH can be detected of this TDH multi-resistance is 6 g/mL.

3 conclusions

It for examination bacterium, is first increased by this research using pathogenic vibrio parahaemolytious ATCC 33846 as TDH Toxic extraction After bacterium cultivates certain time so that it is fully after secretion TDH toxin, use ammonium sulfate salting-out process to extract Raw toxin then this is the most malicious Element uses DEAE-cellulose chromatography, HAP column chromatography and Sephadex G-25 column chromatography successively, is further purified TDH toxin, Obtain TDH toxin after purification after freeze-dried, analyze the toxicity of this albumen, thermostability and hemolytic.By this toxin protein After being combined with Freund's complete adjuvant and incomplete Freund's adjuvant respectively prepare TDH immunogen, several times, various dose immunity BALB/c mouse, blood sampling prepares mouse-anti TDH toxin polyclonal antibody, use indirect elisa method measure this antibody titer, Sensitivity and specificity.Result shows: when utilizing this TDH toxin immunity mice, in conjunction with incomplete Freund's adjuvant TDH dosage control At 12 g/ only, in conjunction with Freund's complete adjuvant TDH dosage control 8 g/ only within.This TDH toxin hemolytic activity can be 100 Maintain 15 ~ 18min at DEG C, use incremental dose method immune mouse can obtain high-titer TDH polyclonal antibody, immunity the 56th day Rising, antibody titer significantly improves, and up to 6.4 × 10⁶, this antibody has the strongest specificity and sensitivity, to TDH toxin Detection sensitivity is 6 g/ml.

Embodiment 6

1 materials and methods

1.1 material

1.1.1 main equipment

1.1.2 for examination animal and bacterial strain

6-8 week old Healthy female BALB/c mouse, healthy Male New Zealand White Rabbit (Medical Center of Fudan University's laboratory animal Center)；Produce TDH toxin vibrio parahaemolytious ATCC33846, the food safety experiment of vibrio parahaemolytious 1.2164(Shanghai Ocean University Room provides)；Staphylococcus aureus, Salmonella, escherichia coli, Listeria, non-pathogenic vibrio parahaemolytious (numbering V.p1 ~ 4, the preservation of Microbiological Lab of Jiangyin Vocational and Technical College of Finance & Economics)；The marine products such as Carnis Pseudosciaenae, Trichiurus haumela, prawn, Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) (are purchased In Yong Hui supermarket, Huai'an).

1.2 method

1.2.1 the preparation of TDH-PAb Yu V.p-PAb

Choose 6-8 week old Healthy female BALB/c mouse, after breeding observing 1 week is without difference, take a blood sample from eyeball, with TDH poison Element does tube agglutination test, is used for preparing negative serum ,-20 DEG C of preservations without agglutination person.Take a certain amount of TDH toxin after purification After the incomplete Freund's adjuvant of equivalent and Freund's complete adjuvant fully combine, several times, the method abdominal cavity of dosage escalation Injecting two groups of mices respectively, fundamental immunity uses the 4 g TDH combining Freund's complete adjuvant, carried out 1 time subsequently every 2 weeks and strengthens Immunity (immunizing dose is shown in Table 6), carried out 1 rathole ball blood sampling 0.5ml, measures immune serum titer, the last time every 2 weeks Immunity terminates one week after, and eyeball is taken a blood sample in a large number, and blood moves to 4 DEG C of refrigerator overnight, takes supernatant, is centrifuged in 3000r/min 10min, prepares mouse-anti TDH toxin polyclonal antibody, puts-20 DEG C of Refrigerator stores.The preparation of V.p-PAb can use existing often Rule method.

Table 6 TDH toxin immunity BALB/c mouse process

Immune time	1st time	2nd time	3rd time	The 4th	The 5th
						Immunizing dose (g/ is only)	4	6	8	10	12
Interval time (d)	0	14	14	14	14

1.2.2 DAS-ELISA method testing process

Be coated TDH-PAb → wash, pat dry → close → wash, pat dry → add testing sample (or TDH Venom antigens) → Washing, pat dry → add V.p-PAb → wash, pat dry → add ELIAS secondary antibody → wash, pat dry → add substrate → wash → add termination Liquid → mensuration OD₄₉₂Value.

1.2.3 the determination of the suitableeest working condition of DAS-ELISA method

1.2.3.1 Checkerboard titration method selects that TDH-PAb is the suitableeest is coated concentration and the suitableeest working concentration of TDH antigen

Treat coated TDH-PAb immune serum by prepare, by 1:1000 doubling dilution to 1:32000, be coated to enzyme On target, after 50 DEG C dry, the calf serum blocking antigen of 5%, 37 DEG C of constant temperature and humidities hatch 3h, by 3 g/ml, 6 g/ml, 9 G/ml, 12 g/ml, 15 g/ml, 18 g/ml concentration add the TDH Venom antigens with PBS dilution of 100 l, simultaneously with PBST It is respectively provided with blank and negative control, shrouding 37 DEG C with negative serum, after constant temperature and humidity hatches 2 h, washs, pat dry, often Hole adds the V.p-PAb immune serum of 1:2000, and 37 DEG C of constant temperature and humidities hatch 2h, wash, pat dry, and every hole adds the 1 of 100 l: 1000(description recommend consumption) goat anti-rabbit igg-HRP, hatch 1h for 37 DEG C, wash, pat dry, every hole adds the OPD of 100 l Substrate, 30 DEG C of constant temperature lucifuge reaction 20min, every hole adds the sulphuric acid of 50 l 2mol/L and terminates enzyme reaction, measures in microplate reader OD₄₉₂Value, by formulaCalculate P/N value, with OD₄₉₂Close to 1.0, P/N value is bigger Antigen, antibody concentration are coated concentration and TDH toxin working concentration as optimal TDH-PAb is the suitableeest.

1.2.3.2 capture antibody TDH-PAb and TDH antigen optimal reaction selection of time

To capture the suitableeest working concentration that antibody TDH-PAb and TDH antigen diluent determines to 1.2.3.1, the response time sets Being set to 10min, 20min, 30min, 40min, 50min, 60min, 70min, 80min, other reaction conditions and operation are same 1.2.3.1, select OD₄₉₂It is worth the indeclinable shortest time as capture antibody TDH-PAb and TDH antigen optimal reaction time.

1.2.3.3 ELISA Plate selects off-period

The suitableeest TDH-PAb that determines of 1.2.3.1,1.2.3.2 is used to be coated concentration, TDH antigen concentration and antigen-antibody In the suitable response time, arrange and be respectively 20min, 40min, 60min, 80min, 100min, 120min, 150min off-period, OD is measured after 180min, other reaction conditions and operation same 1.2.3.1, DAS-ELISA test₄₉₂Value, selects OD₄₉₂Value does not changes Shortest time be optimal off-period.

1.2.3.4 the selection of the suitableeest working concentration of antibody V.p-PAb is detected

The suitableeest TDH-PAb using 1.2.3.1 ~ 1.2.3.3 to determine is coated concentration, TDH antigen concentration and off-period, will V.p-PAb is by 1:1000 doubling dilution to 1:32000, and it is same that every hole adds the 100 above-mentioned diluents of l, other reaction conditions and operation 1.2.3.1, OD is measured after DAS-ELISA test₄₉₂Value, with OD₄₉₂Close to 1.0, the V.p-PAb dilution factor that P/N value is bigger is made For its best effort concentration.

1.2.3.5 detection antibody V.p-PAb and the selection of TDH antigen optimal reaction time

Using the best operating condition that 1.2.3.1 ~ 1.2.3.4 determines, the response time is set to 20min, 30min, Other reaction conditions of 40min, 50min, 60min, 70min, 80min, 90min and the same 1.2.3.1 of operation, select OD₄₉₂It is worth constant The shortest time changed is as the optimal reaction time of detection antibody V.p-PAb with TDH antigen.

1.2.3.6 goat anti-rabbit igg-HRP and detection antibody V.p-PAb optimal reaction selection of time

The best operating condition using 1.2.3.1 ~ 1.2.3.5 to determine, is respectively provided with goat anti-rabbit igg-HRP and V.p- The PAb response time is 30min, 40min, 45min, 50min, 60min, 65min, 70min, the dilution of goat anti-rabbit igg-HRP Degree measures after using commercial product recommending optium concentration 1:1000, other reaction conditions and operation same 1.2.3.1, DAS-ELISA test OD₄₉₂Value, selecting the OD value indeclinable shortest time is that goat anti-rabbit igg-HRP selects optimum response with the V.p-PAb response time Time.

1.2.4 DAS-ELISA method sensitivity test

TDH antigen concentration is arranged 3 g/ml, 4 g/ml, 5 g/ml, 6 g/ml, 7 g/ml, 8 g/ml, 10 g/ml, 12 G/ml, uses the suitableeest working condition determined by 1.2.4 to carry out DAS-ELISA test, judges mark using P/N >=2.1 as positive Standard, determines this law lowest detectable limit to TDH toxin.

1.2.5 DAS-ELISA method specific test

By pathogenic staphylococcus aureus, Salmonella, escherichia coli, Listeria and non-pathogenic pair haemolysis Vibrio V.p1 ~ 5 bacterial strain is as strains tested, and after in antibacterial enrichment liquid, 24h cultivated by 37 DEG C of shaking tables, culture fluid is in 10000r/min Centrifugal 10min, takes each TDH solution for examination bacterium supernatant and 10 g/mL respectively and is determined as detection sample, employing 1.2.4 The suitableeest working condition carry out DAS-ELISA test, Positive judgement standards is P/N >=2.1, it is determined that the detection of DAS-ELISA method Accuracy.

1.2.6 vibrio parahaemolytious TDH in DAS-ELISA method detection food

Weigh 25 g food samples with sterile working, add the 0.01mol/L of 225 mL, the phosphate buffer of pH7.4, It is placed in homogenizing in sterilizing homogenizing cup, makes the equal liquid of sample (1:10).The equal liquid of sample is centrifuged 10min in 10000r/min, takes supernatant Night carries out DAS-ELISA detection.Test according to food safety mandatory national standard method GB 4789.7-2013 simultaneously, compare The accuracy of DAS-ELISA detection.

2 results and analysis

Antibody TDH-PAb is the suitableeest is coated concentration and the suitableeest working concentration of TDH antigen and the determination in response time in 2.1 captures

As can be seen from Table 7, OD can be met₄₉₂Close to 1.0, what P/N value had a certain rule row has two groups of antigens-anti- Body, be respectively TDH concentration be 12 g/ml and two groups of 15 g/ml.When TDH concentration is 12 g/ml, TDH-PAb dilution factor exists OD under 1:4000 and 1:8000₄₉₂All about 1, but the P/N value of 1:8000 is the 19.72 P/N values 15.36 more than 1:4000, When TDH concentration is 12 g/ml, reacting optimal TDH-PAb dilution factor is 1:8000；When TDH concentration is 15 g/ml Time, TDH-PAb dilution factor OD under 1:4000 and 1:8000₄₉₂All about 1, but the P/N value of 1:4000 is 17.12 to be more than The P/N value 11.23 of 1:8000, when TDH concentration is 12 g/ml, reacting optimal TDH-PAb dilution factor is 1: 8000；This research is chosen bigger for the P/N in two groups one group and is coated concentration and the suitableeest work of TDH antigen as TDH-PAb is the suitableeest The determination of concentration, namely capture antibody TDH-PAb the suitableeest be coated concentration be 1:8000, the TDH the suitableeest working concentration of antigen be 12 g/ml。

Table 7 Checkerboard titration method selects that TDH-PAb is the suitableeest is coated concentration and the suitableeest working concentration of TDH antigen

Note: in table, the first row data are OD₄₉₂Value, the second row data are P/N value.

Fig. 5 shows, the response time is at 10 ~ 60min, prolongation OD over time₄₉₂Value, in obvious ascendant trend, illustrates this During capture antibody TDH-PAb just react with TDH antigen.And after 60min, OD₄₉₂All about 1.2, and substantially Remain unchanged, illustrate that now capture antibody TDH-PAb and TDH antigen has reacted complete, therefore we determined that capture antibody TDH- PAb Yu TDH antigen optimum reacting time is 60min.

The determination of 2.2 ELISA Plate off-periods

Fig. 6 shows, off-period is at 20-80min, prolongation OD over time₄₉₂Value declines notable, and this stage is described, little Ox blood serum is just at the hole that sealase target is unnecessary.And after 80min, OD₄₉₂All about 1.0, and substantially remain unchanged, Illustrate that now ELISA Plate hole has been closed completely, therefore we determined that the 80min optimal off-period of ELISA Plate.

The determination of the 2.3 detection the suitableeest working concentrations of antibody V.p-PAb

As can be seen from Table 8, when the dilution factor of V.p-PAb is 1:4000 and 1:8000, corresponding OD₄₉₂Value is all left 1.0 The right side, and P/N corresponding to dilution factor 1:4000 is the 18.11 P/N values 11.45 far above 1:8000, admittedly have selected 1:4000 conduct The suitableeest working concentration of detection antibody V.p-PAb.

Table 8 detects the selection of the suitableeest working concentration of antibody V.p-PAb

2.4 detection antibody V.p-PAb and the selection of TDH antigen optimal reaction time

Fig. 7 shows, the response time is at 20 ~ 40min, prolongation OD over time₄₉₂It is worth in rising trend inconspicuous, may Detection antibody is in the identification TDH antigen stage.And between 20 ~ 40min, OD₄₉₂Notable rising, illustrates that antigen-antibody is just fast Speed reaction.And after 70min, OD₄₉₂All about 1.2, and substantially remain unchanged, illustrate that now antigen antibody reaction is complete, Therefore we determined that detection antibody V.p-PAb is 70min with TDH antigen optimum reacting time.

2.5 goat anti-rabbit igg-HRP determined with the detection antibody V.p-PAb optimal reaction time

Fig. 8 shows, the response time is at 30-55min, prolongation OD over time₄₉₂Value, in obvious ascendant trend, illustrates this During ELIAS secondary antibody just react with V.p-PAb.And after 50min, OD₄₉₂All about 1.2, and basic maintenance is not Become, illustrate that now ELIAS secondary antibody is complete with detection antibody response, therefore we determined that goat anti-rabbit igg-HRP and detection antibody The optimum reacting time of V.p-PAb is 55min.

2.6 DAS-ELISA method detection sensitivities

As can be seen from Table 9, from TDH toxin concentration be 6 g/ml start raise after, testing result all presents the positive, explanation The lowest detection limit of this law is 6 g/ml, and the bottom line being converted into the detection of reagent food samples should be 60 g/g.

Table 9 DAS-ELISA method detection sensitivity

2.7 DAS-ELISA method specificity

Fig. 9 result shows, two strains pathogenic vibrio parahaemolyticus P/N value is noticeably greater than 2.1, presents strong positive, rather than causes The vibrio parahaemolytious V.p1-4 of characteristic of disease, pathogenic staphylococcus aureus, Salmonella, shigella, Listeria P/N value is significantly lower than 2.1, presents feminine gender, illustrates that DAS-ELISA method has the strongest specificity, and accuracy in detection is high.

Pathogenic vibrio parahaemolytious in 2.8 DAS-ELISA method detection food

Table 10 result shows, uses pathogenic vibrio parahaemolytious and GB 4789.7-in DAS-ELISA method detection food 2013 testing result coincidence rates are essentially 100%, and in Carnis Pseudosciaenae sample detection, national standard method method has a sample not detect, former Because of be probably some death in aquatic products or cannot not cultivation conditions bacterium, National Standard Method cannot detect, and the inspection of DAS-ELISA method Surveying is not thalline to liking TDH toxin, and therefore testing result is by not affected by cultivation conditions bacterium.All in all, DAS- ELISA detection method is accurately, reliably.

Table 10 DAS-ELISA method detection food result

3 conclusions

This research is with TDH⁺Immunogen prepared by vibrio parahaemolytious thalline and TDH toxin thereof, respectively immunity new zealand white rabbit And BALB/c mouse, it is prepared for rabbit anti-vibrio parahaemolyticus polyclonal antibody and mouse-anti TDH toxin polyclonal antibody, with Mus Anti-TDH toxin polyclonal antibody is capture antibody, and rabbit anti-vibrio parahaemolytious polyclonal antibody is detection antibody, sets up DAS- ELISA detection method, the detection parameter of this method is as follows: the concentration that is coated of capture antibody mouse-anti TDH polyclonal antibody is 1:8000, The best effort concentration of TDH Venom antigens is 12 g/mL, the suitableeest work of detection antibody rabbit anti-vibrio parahaemolytious polyclonal antibody Concentration is 1:4000, and ELIAS secondary antibody best effort concentration is 1:1000, and detection total time is 5h, and this method has the strongest special Property, it is 60 g/g to the detection lower bound of TDH toxin in food.

Claims

1.ELISA test kit is the application in vibrio parahaemolyticus TDH toxin in quickly detection food, it is characterised in that: include Following steps:

(1) Sample pretreatment: weigh food samples with sterile working, adds the phosphate buffer of 0.01mol/L, pH7.4, in Homogenizing in homogenizing cup, makes the equal liquid of sample；Equal for sample liquid thin,tough silk yarn is filtered, is centrifuged 8-10min in 8000-10000r/min, Take supernatant and be measuring samples extracting solution；Food samples is 25g:225mL with the amount ratio of phosphate buffer；

(2) in being coated the hole of ELISA Plate of capture antibody, it is separately added into: 100 μ L measuring samples extracting solution, 100 μ L TDH marks Quasi-diluent, 100 μ L rabbit anti-vibrio parahaemolytious negative serums as negative control, 100 μ L PBST as blank, shrouding Film shrouding, 37 DEG C of constant temperature and humidities hatch 60-80min；TDH standard dilution is in order to draw standard curve, and the TDH that every hole adds marks Quasi-diluent concentration is respectively 2 μ g/mL, 4 μ g/mL, 6 μ g/mL, 8 μ g/mL, 10 μ g/mL, 12 μ g/mL；

(3) plate is washed: manual wash plate or wash trigger and wash plate；Manual plate washing method is: discarding liquid in hole, cleaning mixture fills each hole, Jog 3min, pats dry, and after being repeated 3 times, button is dry；Washing trigger plate washing method is: after selecting washing 3 times and wash time 2min, automatically Washing plate, button is dry；

(5) plate is washed: manual wash plate or wash trigger and wash plate；Manual plate washing method is: discarding liquid in hole, cleaning mixture fills each hole, Jog 3min, pats dry, and after being repeated 3 times, button is dry；Washing trigger plate washing method is: use cleaning mixture to select washing 3 times and wash time After 2min, automatically washing plate, button is dry；

(6) adding 100 μ L ELIAS secondary antibody goat anti-rabbit igg-HRP in every hole, 37 DEG C of constant temperature and humidities hatch 45-55min；

(7) plate is washed: manual wash plate or wash trigger and wash plate；Manual plate washing method is: discarding liquid in hole, cleaning mixture fills each hole, Jog 3min, pats dry, and after being repeated 3 times, button is dry；Washing trigger plate washing method is: after selecting washing 3 times and wash time 2min, automatically Washing plate, button is dry；

(8) adding 100 μ L substrate solutions in every hole, 30 DEG C of constant temperature and humidity black outs hatch 15-25min；

(9) adding 50 μ L stop buffers in every hole, shake up, use microplate reader reading in 5min, reference wavelength is set to 630nm, measures Wavelength is set to 492nm, returns to zero with blank well, is then read out each hole OD₄₉₂Value；

(10) result judges:

A, qualitative analysis: sample OD₄₉₂The average OD of value/negative control₄₉₂Value >=2.1, it is judged that for the positive, be otherwise negative；Negative When comparison OD value is less than 0.05, calculate with 0.05；During higher than 0.05, calculate with actual OD value；

B, quantitative analysis: with OD₄₉₂Value is vertical coordinate, with TDH standard dilution concentration as abscissa, unit μ g/mL, draws mark Directrix curve；OD value according to sample can find testing sample TDH concentration on standard curve, with this concentration be multiplied by 10 i.e. sample TDH content of toxins in product, unit μ g/g；

The ELISA test kit of vibrio parahaemolyticus TDH toxin in described quick detection food, including

(1) ELISA Plate: be coated the ELISA Plate of capture antibody；Described capture antibody is mouse-anti TDH toxin polyclonal antibody；

(2) TDH standard dilution: vibrio parahaemolyticus TDH toxin；

(3) negative control: rabbit anti-vibrio parahaemolytious negative serum；

(5) ELIAS secondary antibody: goat anti-rabbit igg-HRP；

(6) phosphate buffer of blank liquid: 0.01mol L, pH7.4；

(7) cleaning mixture: containing the phosphate buffer of 0.01mol/L, pH7.4 of 0.05% tween 20；

(8) substrate solution: include A bottle and B bottle, A bottle be the o-phenylenediamine containing 4mg/100mL, pH5.0 phosphate-citric acid delay Rush liquid；B bottle is the H of mass percent concentration 30%₂O₂；

(9) stop buffer: 2mol/L H₂SO₄；

Wherein, the preparation method of described mouse-anti TDH toxin polyclonal antibody, comprise the following steps: by vibrio parahaemolyticus After TDH toxin is fully combined with the incomplete Freund's adjuvant of equivalent, the Freund's complete adjuvant of equivalent respectively, several times, dosage passs Two groups of mices are injected in the method abdominal cavity increased respectively；Fundamental immunity uses the 4 g TDH combining Freund's complete adjuvant, subsequently every 2 1 booster immunization of Zhou Jinhang, prepares mouse-anti TDH toxin polyclonal antibody；

Wherein, the preparation method of described rabbit anti-vibrio parahaemolyticus polyclonal antibody, comprise the following steps: in 0.05% first Aldehyde, the pathogenic vibrio parahaemolyticus producing TDH toxin being inactivated 2 h at 35 DEG C and prepare somatic antigen, several times, dosage escalation enters Ear vein is injected；Immunity, immunity concentration is 10⁸CFU/ mL；Immunity terminates one week after the last time, and arteria auricularis is taken a blood sample in a large number, Obtain rabbit anti-vibrio parahaemolyticus polyclonal antibody；

Wherein, the dilution factor of described rabbit anti-vibrio parahaemolyticus polyclonal antibody is 1:4000 ~ 1:8000；Described goat resists The dilution factor of rabbit igg-HRP is 1:1000；

Wherein, the preparation method of described vibrio parahaemolyticus TDH toxin, comprise the following steps:

(1) preparation of TDH crude toxin: the vibrio parahaemolytious producing TDH toxin is inoculated in sodium chloride basic peptone water solution In in shaking table after amplification culture, centrifugal segregation thalline, supernatant adds (NH₄)₂SO₄, stand and saltout fully to precipitate TDH Toxin；High speed centrifugation, resolution of precipitate, in PBS solution, is dialysed, is obtained TDH crude toxin；Described sodium chloride basic protein peptone The mass percent concentration of aqueous solution is 3%；Amplification culture condition is: 37 DEG C, 150r/min, 24h；Centrifugal rotational speed is 6000r/ min；(NH₄)₂SO₄Addition is 35.1g/100mL supernatant；Salting-out condition is 4 DEG C, 10h；High speed centrifugation condition is 10000r/ min、4℃；The concentration of PBS solution be 0.010mol/L, pH be 7.0；

(2) purification of TDH crude toxin:

(2.1) DEAE-cellulose ion-exchange column chromatography: the TDH crude toxin that obtains step (1) prepared is slowly dropped to In DEAE-cellulose ion exchange column, contain the PBST eluant solution of the NaCl of 0.2 mol/L with 1L, then 1L is with containing 0.2-1.0 The PBS solution linear gradient elution of mol/L NaCl, collects eluent；Add solid (NH₄)₂SO₄Saltouing, precipitation PBS is molten Xie Hou, dialysed overnight；Solid (NH₄)₂SO₄The addition saltoutd is 40g/100mL；Dialysis temperature is 4 DEG C；

(2.2) HAP column chromatography: the dialysis solution of step (2.1) is added drop-wise on HAP post, with 0.5 L containing 0.1mol/L NaCl PBS liquid pH7.0 eluting, then with the PBS liquid pH7.0 eluting of the 0.1-0.3mol/L of 0.8L, collect eluent；Add solid (NH₄)₂SO₄Saltout, precipitate after dissolving with PBS, dialysed overnight；

(2.3) Sephadex G-25 column chromatography: the dialysis solution of step (2.2) is crossed Sephadex G-25 post, uses 0.01mol/L The Tris-HCl buffer solution elution of pH7.0, eluent PEG-20000 concentrates, and obtains vibrio parahaemolyticus TDH toxin；

Wherein, described in be coated the preparation method of ELISA Plate of capture antibody, comprise the following steps: add in every hole of new ELISA Plate The mouse-anti TDH toxin polyclonal antibody 100 μ L of dilution, adds closing fluid-tight and closes ELISA Plate after 40 DEG C of drying, 37 DEG C of constant temperature perseverances Wet hatching 80min, discard unnecessary confining liquid, use cleaning mixture to fill each hole, jog 2-4min, pat dry, after being repeated 3 times, button is dry, Obtain；The mouse-anti TDH toxin polyclonal antibody of described dilution is that the carbonate buffer using 0.05mol/L, pH to be 9.6 is molten The serum of liquid dilution, dilution factor 1:2000 ~ 1:4000；Described confining liquid is the bovine serum albumin of mass percent concentration 3-5% Or 3-5% defatted milk powder.