CN100357736C - Fast detection device for salmonella - Google Patents
Fast detection device for salmonella Download PDFInfo
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- CN100357736C CN100357736C CNB031337988A CN03133798A CN100357736C CN 100357736 C CN100357736 C CN 100357736C CN B031337988 A CNB031337988 A CN B031337988A CN 03133798 A CN03133798 A CN 03133798A CN 100357736 C CN100357736 C CN 100357736C
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- salmonella
- antigen
- monoclonal antibody
- detecting device
- quality control
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Abstract
The present invention relates to a rapid salmonella detecting device which comprises a carrier with a carried biological agen. The rapid salmonella detecting device is characterized in that the biological agent on the carrier comprises specific anti-salmonella IgG, colloidal gold labeled anti-salmonella H-antigen monoclonal antibody IgG, colloidal gold labeled quality control antigens and corresponding antibodies of the quality control antigens. In this way, the rapid salmonella detecting device is formed. The present invention has the advantages that due to the formation of the rapid salmonella detecting device, the detecting time is shorted greatly, and after enrichment culture, the detecting process only needs about ten minutes; operating procedures are simple, and the use is simple.
Description
Technical field
The present invention relates to a kind of device that is used for detecting food, feed salmonella, particularly salmonella device for fast detecting.
Background technology
Salmonella is a modal pathogenic microorganisms in food poisoning and the animal feed, infects in meat, blood, internal organ, egg and the manufactured goods of the animal of this pathogenic microorganisms to contain a large amount of salmonellas.Detection for salmonella mainly is to carry out in the laboratory at present, promptly finally obtains testing result by preceding increasing steps such as bacterium, selective enrichment, selectivity plate isolation, biological chemistry screening and serological Identification.Its whole process need 5-7 days, time and effort consuming.
Summary of the invention
The object of the present invention is to provide a kind of salmonella device for fast detecting that the salmonella in food and the animal feed is carried out fast detecting that is applicable to.
The object of the present invention is achieved like this: the salmonella device for fast detecting, comprise carrier, it is characterized in that preparation has biopreparate on the carrier, the composition of biopreparate is specific anti-salmonella IgG, colloid gold label anti-salmonella H antigen monoclonal antibody IgG, the Quality Control antigen of colloid gold label and the antibody of corresponding Quality Control antigen, the content of each composition is at least 55 microlitres, working concentration is 1: 55---1: 200u, form thus can the fast detecting salmonella pick-up unit.
The antibody of former and corresponding Quality Control antigen.
Above-mentioned biopreparate can obtain by following method:
The preparation of biopreparate: comprise the preparation of monoclonal antibody, (how anti-) and the anti-mouse serum of rabbit (how anti-) of salmonella H antigen, and monoclonal antibody and many anti-purification (extraction Immunoglobulin IgG) and Preparation of Colloidal Gold and mark.
1. the extraction of salmonella H antigen and purifying: the salmonella inoculation was cultivated 16-22 hour to nutrient agar panel, carry out enlarged culture results bacterium again.Bacterium is mixed the back transfer pH to 2.0 with physiological saline with 1M hydrochloric acid, stirring certain hour (20-40 minute) back is centrifugal, abandons precipitation.It is centrifugal once more to get supernatant, and then supernatant being transferred to pH with NaOH is 7.0-7.5, adds saturated ammonium sulfate and spends the night.Centrifugal once more, abandon supernatant, the collecting precipitation thing.Dialyse after sediment is dissolved in distilled water, the freeze-drying bag is deposited.
2. anti-salmonella H antigen monoclonal antibody cell line and monoclonal antibody are to set up like this and preparation:
(1) foundation of secretion anti-salmonella H antigen monoclonal antibody cell line; A, animal immune: get salmonella H antigen, dilute with physiological saline, mixing with the complete freund adjuvant of equivalent, through peritoneal immunity Balb/C mouse, mix booster immunization with measuring salmonella H antigen together with the complete freund adjuvant of equivalent again after 2 weeks, booster immunization is once again after 2 weeks, can consider to do cell about 2 weeks behind the booster immunization for the second time and merge, merge preceding 3 days, directly use the interior supplementary immunization of salmonella H antigen spleen once; B, Fusion of Cells: Fusion of Cells recovery the last week SP2/0 myeloma cell, be cultured to increased logarithmic phase, merge and to get feeder cells the previous day and spread in the 96 porocyte culture plates and cultivate, got the supernatant hybridoma on the 15th day; C, the screening of positive anti-salmonella H antigen monoclonal antibody: bag quilt: with the carbonate bag of 0.05M pH9.6 be cushioned liquid with salmonella H antigen diluent to 1-10 μ g/ml, bag is by 96 hole ELISA Plate, 100 μ L/ holes, 4 ℃ are spent the night, discard liquid physiological saline one tween lavation buffer solution 3 times in the hole then, pat dry, application of sample: will have the culture supernatant of clonal growth to be sequentially added into, 37 ℃ of incubations in 100 μ L/ holes 1 hour wash 3 times, dry, adding two resists: with tween cleansing solution dilution horseradish enzyme mark sheep anti mouse, put 37 ℃ of incubation 10-30 minutes in 100 μ L/ holes, and cessation reaction: every hole adds 2M H
2SO
4Stop buffer 50 μ L, the result judges that negative control hole is colourless, clearly develop the color in the positive control hole, measure the OD value, with the blank zeroing, the OD value is promptly positive more than or equal to 2.1 times of negative controls, wherein positive supernatant is remake the test of free salmonella, filter out special positive cell, in time enlarge, frozen, transferred species and carry out the limiting dilution assay purifying, can obtain the hybridoma cell strain of anti-salmonella monoclonal antibody.
(2) inoculation hybridoma: in 1 week before the assorted oncocyte of inoculation, give Balb/C mouse peritoneal injection 0.5ml whiteruss earlier; Anti-salmonella monoclonal antibody hybridoma cell number is transferred to 1 * 10
6-2 * 10
6/ ml, every mouse peritoneal injection 0.5ml.
(3) gather ascites: behind the inoculation hybridoma the 10th day, extract ascites with syringe, centrifugal 15 minutes of 300g collects supernatant, promptly gets the anti-salmonella monoclonal antibody.
3. anti-salmonella H antigen polyclonal antibody is preparation like this: with about 6 months, the healthy rabbits of body weight 2.5Kg, the antigen of every foot injection 0.1-1ml bovine serum albumin(BSA) compound and the emulsification of Fu Shi Freund's complete adjuvant, booster immunization after two weeks, with carry out with dosage bovine serum albumin(BSA) compound and the emulsification of Fu Shi Freund's complete adjuvant second and third, four booster immunizations, the 5th day ear vein behind each booster immunization got blood, detect the immune response effect with agar double immunodiffusion method, when titre reaches 1: 64 when above, the arteria carotis bloodletting, centrifugal, collect serum.
4. immunoaffinity purification polyclonal antibody and monoclonal antibody, and intersect that the purifying polyclonal antibody is performed such:
(1) pre-service of salmonella H antigen monoclonal antibody and polyclonal antibody: with 1 times of phosphate buffer dilution, through 0.22 μ m micro-pore-film filtration, 4 ℃, 10000g are centrifugal with 1-10ml serum or ascites; (2) sad-ammonium sulfate method purifying: in pretreated antiserum of 1-10ml milliliter or ascites, add 2-10ml 0.06M PH4.0 acetate buffer, transfer PH to 4.8, slowly add sad 33-300 μ L in following 30 minutes in stirring, 4 ℃ left standstill 2 hours, 15000g, 4 ℃ centrifugal 30 minutes, abandon precipitation, supernatant adds the 0.1M PH7.4 phosphate buffer of 1/10 volume, transfers PH to 7.4, add saturated ammonium sulfate in 30 minutes, make into 45% saturation degree, left standstill 1 hour, centrifugal 30 minutes of 4 ℃ of following 10000g, precipitation is dissolved in the 1/2 amount PH7.4 phosphate buffer, to the dialysis of above-mentioned damping fluid, centrifugal 30 minutes of 4 ℃ of following 10000g ,-20 ℃ of preservations
5. colloid gold label anti-salmonella H antigen monoclonal antibody and polyclonal antibody compound are preparations like this: get 0.01% tetra chlorauric acid aqueous solution 200ml, be heated to boiling, add 8ml 1% trisodium citrate aqueous solution, boiled 5 minutes, and orange red colloid gold particle diameter occurred and be determined as 200nm by Electronic Speculum.Get 50ml collaurum portion, transfer PH to 8.0 with 0.1mol/L sal tartari.Stir down colloidal gold solution is mixed with the anti-salmonella H antigen monoclonal antibody or the polyclonal antibody of immunoaffinity purification respectively, add Macrogol 2000 0 aqueous solution again, making ultimate density is 0.05%; With centrifugal 45 minutes of semifinished product 6000g, centrifugal after, sediment physiological saline is suspended to 1.5ml, 4 ℃ of preservations;
6. the polyclonal antibody and the monoclonal antibody with mixing in 1: 5 of the colloid gold label that will prepare as stated above spray respectively on three all-glass papers of thickness 1mm, and the freeze-drying sealing is preserved.
Characteristics of the present invention are: make the pick-up unit of fast detecting salmonella, shortened detection time greatly, after increasing the bacterium cultivation, its testing process only needs about 10 minutes, and running program is simple, and is easy to use.
Description of drawings
Fig. 1 is a structural representation of the present invention.
Embodiment
Embodiment 1 as shown in Figure 1, pick-up unit of the present invention is to be made of the box body 1 of outside and the carrier 2 that is positioned at box body, box body is provided with testing sample inlet 3 and checks result's form 4 and label symbol T, the C in detection line district, nature controlling line district, and the support sector of carrier is divided into nitrocellulose membrane.Carrier is divided into three zones, and two ends are respectively sample area and suction zones, and the middle part is experiment reaction zone (containing detection line T district and nature controlling line C district).The experiment reaction zone has three bands that have the biopreparate mark, contains specific anti-salmonella IgG on three bands, anti-salmonella H antigen monoclonal antibody IgG, the Quality Control antigen of colloid gold label and the antibody of corresponding Quality Control antigen of colloid gold label.The biopreparate of above-mentioned four kinds of compositions, the use amount of its every kind composition are 70 μ l, and the concentration of every kind of composition is 1: 70u.Absorbent material covers above the monoclonal antibody IgG of anti-salmonella H antigen of colloid gold label.After the mark of each band is finished, with 1% bSA sealing nitrocellulose membrane, then carrier is put into box, at last with the dry encapsulation of aluminium foil bag.
Every kind of composition use amount of embodiment 2 biopreparates is 100 μ l, and concentration is 1: 200u.Other content is identical with embodiment 1 all.
Every kind of composition use amount of embodiment 3 biopreparates is 55 μ l, and concentration is 1: 55u.Other content is identical with embodiment 1 all.
Every kind of composition use amount of embodiment 4 biopreparates is 80 μ l, and concentration is 1: 100u.Other content is identical with embodiment 1 all.
When using this pick-up unit, at first adopt nutrient solution that impaired or complete salmonella is repaired, survives and the recovery breeding.Processing through nutrient solution can impel the salmonella selective enrichment to grow into the detectable level of this pick-up unit.During detection, getting 120ul enrichment culture liquid is added in the circular filling orifice on the pick-up unit box body, sample moves along carrier by capillarity, if have antigen in the sample then can be attached on the golden labelled antibody and form antigen antibody complex, and present a purplish red colour band line in detection line T district, positive result, remaining sample continues to move to the carrier end and finally is deposited in the waste liquid pool.As there not being determined antigen in the sample, then no any vitta occurs in detection line T district, is negative findings.Under two kinds of situations, purplish red colour band line all appears in nature controlling line C district, and expression detects effectively.Do not have the band line as the C district, no matter the T district has or not purplish red colour band line, and it is invalid all to represent to detect.
Claims (1)
1, salmonella device for fast detecting, comprise carrier, it is characterized in that preparation has biopreparate on the carrier, the composition of biopreparate is specific anti-salmonella IgG, colloid gold label anti-salmonella H antigen monoclonal antibody IgG, the Quality Control antigen of colloid gold label and the antibody of corresponding Quality Control antigen, the content of each composition is at least 55 microlitres, working concentration is 1: 55--1: 200u, form thus can the fast detecting salmonella pick-up unit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031337988A CN100357736C (en) | 2003-07-25 | 2003-07-25 | Fast detection device for salmonella |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031337988A CN100357736C (en) | 2003-07-25 | 2003-07-25 | Fast detection device for salmonella |
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| Publication Number | Publication Date |
|---|---|
| CN1570637A CN1570637A (en) | 2005-01-26 |
| CN100357736C true CN100357736C (en) | 2007-12-26 |
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| CNB031337988A Expired - Fee Related CN100357736C (en) | 2003-07-25 | 2003-07-25 | Fast detection device for salmonella |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102135539A (en) * | 2010-09-03 | 2011-07-27 | 李克生 | Method for detecting salmonella, rapid gold-labeled diagnostic kit of salmonella and preparation method of kit |
| CN103941006B (en) * | 2014-02-25 | 2015-12-30 | 上海理工大学 | Salmonella choleraesuls immune colloid gold Rapid detection test strip |
| CN106018799A (en) * | 2016-05-12 | 2016-10-12 | 山东省农业科学院畜牧兽医研究所 | Test paper for salmonella and preparation method of test paper |
| CN106226536B (en) * | 2016-08-01 | 2017-11-10 | 福建省产品质量检验研究院 | A kind of position phase polymorphism test method of salmonella H antigens |
| CN106771183A (en) * | 2016-11-29 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | The detection card of Bacterium enteritidis in a kind of detection tableware |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US538872A (en) * | 1895-05-07 | Ferrule for umbrella-sheaths | ||
| US5538872A (en) * | 1988-12-21 | 1996-07-23 | Ortho Diagnostic Systems, Inc. | Method of preparing nucleotide probes using a bridging complement |
| CN1318152A (en) * | 1998-09-17 | 2001-10-17 | 莱比锡试验诊断有限公司 | Salmonella antigen formulation and kit for detecting salmonella antibodies |
-
2003
- 2003-07-25 CN CNB031337988A patent/CN100357736C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US538872A (en) * | 1895-05-07 | Ferrule for umbrella-sheaths | ||
| US5538872A (en) * | 1988-12-21 | 1996-07-23 | Ortho Diagnostic Systems, Inc. | Method of preparing nucleotide probes using a bridging complement |
| CN1318152A (en) * | 1998-09-17 | 2001-10-17 | 莱比锡试验诊断有限公司 | Salmonella antigen formulation and kit for detecting salmonella antibodies |
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| CN1570637A (en) | 2005-01-26 |
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