CN103760354A - Immune colloidal gold quick detection test strip of staphylococcal enterotoxin D, and preparation method of test strip - Google Patents
Immune colloidal gold quick detection test strip of staphylococcal enterotoxin D, and preparation method of test strip Download PDFInfo
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Abstract
The invention relates to an immune colloidal gold quick detection test strip of staphylococcal enterotoxin D (SED), and a preparation method of the immune colloidal gold test strip, and belongs to the technical field of immunodetection. The test strip consists of a sample pad, a gold labeling pad coated with a gold labeled SED antibody 5F2 (CGMCCNo.7212 (China General Microbiological Culture Collection Center Number 7212)), and a nitrocellulose membrane coated with an SED antibody 10F1 (CGMCCNo.7213) and goat anti mouse IgG (Immunoglobulin G) at a detection line and a quality control line respectively, and the sample pad, the gold labeling pad, the nitrocellulose membrane and a water absorption pad are sequentially adhered to a PVC (Polyvinyl Chloride) bottom plate. The test strip is easy, simple, quick and accurate to operate, good in specificity and high in detection sensitivity, and is free from interference of an environmental condition; the whole detection process only requires 5min; and the minimum detection limit is 0.25ng/mL. The test strip is suitable for a custom, an enterprise, a hospital, inspection and quarantine units and the like, and can achieve quick detection of the SED in food or a clinic sample.
Description
Technical field
The present invention relates to a kind of immune colloid gold Rapid detection test strip of Enterotoxin D, staphylococcal, and the preparation method of this immunity colloidal gold test paper strip, can realize the fast detecting diagnosis of Enterotoxin D, staphylococcal, be applicable to the Site Detection of a large amount of samples, the fast detecting of food poisoning and emergency processing, belong to technical field of immunoassay.
Background technology
Staphylococcus aureus (Staphylococcus aureus) is a kind of common food-borne pathogens, although constantly there is in recent years novel Staphylococcus aureus enterotoxin (Staphylococcal enterotoxin, SE) be found, but poisoning 95% of the staphylococcus aureus food poisoning that accounts for that enterotoxin SEA, SEB, SEC, SED, SEE cause, the food poisoning that staphylococcal enterotoxin causes is in the majority with A type, and D type takes second place.From raw milk, raw meat and cheese, separated staphylococcus aureus be take generation D type enterotoxin as main.
In food processing process, through bakingout process, can kill staphylococcus aureus, once yet thalline produces exocrine enterotoxin, the enterotoxin being present in so in food is highly stable, 100 ℃ of 30min are not destroyed, after taking in human body, can resist the decomposition of proteinase in stomach liquid, and cause the symptoms such as vomiting, diarrhoea of human body.Within 2011, China announces quick-frozen face rice made products new national standard GB19295-2011, from original must not detecting, becoming limits the quantity of staphylococcus aureus detects, therefore in order to stop the qualified and situation that enterotoxin exceeds standard of Staphylococcus aureus in food viable count, the fast detecting of enterotoxin has more importantly meaning for ensuring food safety.
In recent years, ELISA relies on that it is sensitive, quick, specificity is good, be easy to the feature promoted more and more for the detection of enterotoxin, but that colloidal gold strip has advantages of is simple to operate, stability is high, need to not be by specialized equipment, be applicable to field quick detection, therefore for the Site Detection of a large amount of samples, the fast detecting of food poisoning have great importance.
Summary of the invention
The object of the invention is to provide a kind of immune colloid gold Rapid detection test strip that detects Enterotoxin D, staphylococcal, simple to operate, quick and convenient, highly sensitive, and good stability is with low cost.
Another object of the present invention is to provide a kind of preparation method of immunity colloidal gold test paper strip, comprises the preparation of anti-SED specific antibody, and SED double antibody detects the screening of antibody, the preparation of SED immune colloidal gold detection test paper strip.This test strips is easy and simple to handle, quick, accurate, detects overall process and only needs 5 minutes, is not subject to the interference of environmental baseline, and specificity is good, and detection sensitivity is high, and lowest detection is limited to 0.25ng/mL.The present invention is applicable to customs, enterprise, hospital, inspection and quarantine unit etc., can realize the fast detecting of Enterotoxin D, staphylococcal in food or clinical sample.
Technical scheme of the present invention, a kind of immune colloid gold Rapid detection test strip of Enterotoxin D, staphylococcal, comprises PVC base plate, at PVC base plate two ends, is respectively equipped with sample pad and adsorptive pads;
PVC base plate middle part is provided with nitrocellulose filter detection layers, is provided with gold mark pad between nitrocellulose filter detection layers and sample pad; Described gold mark pad one end is connected and stacks with sample pad, and the other end is stacked in nitrocellulose filter detection layers;
In described nitrocellulose filter detection layers, be disposed with detection line and nature controlling line.
Described gold mark pad is coated with the anti-SED antibody 5F2(CGMCC No.7212 of gold mark mark).
On described detection line, be coated with anti-SED antibody 10F1(CGMCC No.7213).
On described nature controlling line, be coated with sheep anti-mouse igg.
The preparation method of the immune colloid gold Rapid detection test strip of described Enterotoxin D, staphylococcal, Enterotoxin D, staphylococcal is abbreviated as SED, and step is as follows:
(1) preparation of anti-SED monoclonal antibody specific:
The recombinant expressed SED that Beijing Military Medical Science Institute provides of take is immunogene, and the BALB/c mouse in 8 week age of immunity, through immunity, Fusion of Cells, screening, screens 10 cell strain of monoclonal antibody altogether;
Immune programme for children is as follows: within first week, carries out head and exempts from, and 10 μ g/, subcutaneous multi-point injection after Freund's complete adjuvant emulsification; 4th week is carried out two and is exempted from, 8 μ g/ only, subcutaneous multi-point injection after incomplete Freund's adjuvant emulsification; Within the 6th week, carry out three and exempt from, 6 μ g/, subcutaneous multi-point injection after incomplete Freund's adjuvant emulsification; Afterbody blood sampling in the 7th week is surveyed and is tired, and selects to tire the highest mouse; Within the 8th week, make a spurt immune, 4 μ g/, physiological saline solution, lumbar injection; After spurt immunity posterior orbit blood sampling in 3 days, merge.Screening adopts indirect ELISA to carry out, and screens altogether 10 cell lines.
(2) specificity SED double antibody detects the screening of antibody:
The 10 strain cell strain of monoclonal antibody difference mark horseradish peroxidases (HRP) by after step (1) purifying, carry out double antibody sandwich method pairing after the success of direct method identification marking, determine the required anti-SED antibody of specific immunity colloidal gold fast detecting test paper strip; Wherein 5F2 is golden labeling antibody, and 10F1 is coated antibody;
(3) preparation of Enterotoxin D, staphylococcal immune colloid gold Rapid detection test strip:
The preparation of a, blank collaurum:
Adopt citrate reducing process to prepare colloid gold particle, in conical flask, add the gold chloride that 200mL deionized water and 2mL mass concentration are 1%, be heated with stirring to boiling, adding fast 4mL mass concentration is 1% sodium citrate, continue heating 15 ~ 20 minutes, to being shiny red, then cooling in room temperature, 4 ℃ of stored refrigerated;
The preparation of b, colloidal gold labeled monoclonal antibody:
The K of 0.1M for blank colloidal gold solution
2cO
3regulate pH value to 7.4, drip antibody prepared by appropriate concentration step (2) in stirring, every milliliter of blank colloidal gold solution adds 30 μ g antibody 5F2(CGMCC No.7212), continue to stir 30 minutes, centrifugal, draw supernatant, with golden labeling antibody, preserve liquid and heavily revolve;
The preparation of c, gold mark pad:
Debugging three-dimensional planar point film gold spraying instrument instrument, by the anti-SED antibody 5F2(CGMCC No.7212 of the good collaurum of mark) be sprayed on uniformly on glass fibre membrane, spouting liquid 0.6 μ L/cm, 37 ℃ of oven dry are spent the night, and envelope is standby;
The preparation of d, coated film:
Debugging three-dimensional planar point film gold spraying instrument instrument, by the anti-SED antibody 10F1(CGMCC No.7213 having diluted) be evenly sprayed on nitrocellulose filter, obtain detection line; The sheep anti-mouse igg having diluted is evenly sprayed on nitrocellulose filter, obtains nature controlling line, spouting liquid is 0.6 μ L/cm, and 37 ℃ of oven dry are spent the night, and envelope is standby;
By PVC base plate, nitrocellulose filter detection layers, sample pad, gold mark pad, detection line, nature controlling line and adsorptive pads combination, obtain the immune colloid gold Rapid detection test strip of product Enterotoxin D, staphylococcal.
It is applicable to customs, enterprise, hospital, inspection and quarantine unit, can realize the fast detecting of Enterotoxin D, staphylococcal in food or clinical sample, and lowest detection is limited to 0.25ng/mL.
The principle of work of test strips of the present invention: adopt colloidal gold immunochromatographimethod technology, select SED specific antibody as solid formation, utilize double antibody sandwich method principle to detect in sample whether contain SED.While containing SED in sample to be checked, anti-SED antibody (5F2) combination of antigen elder generation and colloid gold label, because chromatography effect compound moves forward along coated film, during anti-SED antibody (10F1) on running into detection line, form antibody-antigen-Jin labeling antibody compound, enrichment on detection line, forms red precipitate line.
The present invention compared with prior art tool has the following advantages:
1, detection speed is fast, and overall process only needs 5 minutes, can test the fast detecting of batch samples;
2, highly sensitive, lowest detection is limited to 0.25ng/mL;
3, easy and simple to handle, without through professional training, be easy to promote;
4, do not need instrument, be applicable to Site Detection;
5, process is simple, direct sample detection, and sample size is few.
Biological material specimens preservation:
1, a strain monoclonal cell strain, No. 12, cell line, strain number is 5F2, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, accession designation number is CGMCC No. 7212, and it is on January 23rd, 2013 that the phase is said in preservation.
2, a strain monoclonal cell strain, No. 13, cell line, strain number is 10F1, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, accession designation number is CGMCC No. 7213, and it is on January 23rd, 2013 that the phase is said in preservation.
Accompanying drawing explanation
Fig. 1 is test strips structural front view of the present invention.
Fig. 2 is test strips structure vertical view of the present invention.
Description of reference numerals: 1, PVC base plate; 2, nitrocellulose filter detection layers; 3, sample pad; 4, gold mark pad; 5, detection line (T line); 6, nature controlling line (C line); 7, adsorptive pads.
Embodiment
Embodiment 1
As shown in Figure 1-2, a kind of immune colloid gold Rapid detection test strip of Enterotoxin D, staphylococcal, comprises PVC base plate 1, at PVC base plate 1 two ends, is respectively equipped with sample pad 3 and adsorptive pads 7;
PVC base plate 1 middle part is provided with nitrocellulose filter detection layers 2, is provided with gold mark pad 4 between nitrocellulose filter detection layers 2 and sample pad 3; Described gold mark pad 4 one end are connected and stack with sample pad 3, and the other end is stacked in nitrocellulose filter detection layers 2;
In described nitrocellulose filter detection layers 2, be disposed with detection line (T line) 5 and nature controlling line (C line) 6.
Described gold mark pad 4 is coated with the anti-SED antibody 5F2 of gold mark mark.On described detection line (T line) 5, be coated with anti-SED antibody 10F1.On described nature controlling line (C line) 6, be coated with sheep anti-mouse igg.
The preparation method of the immune colloid gold Rapid detection test strip of described Enterotoxin D, staphylococcal, Enterotoxin D, staphylococcal is abbreviated as SED, the steps include:
(1) preparation of anti-SED monoclonal antibody specific:
Take recombinant expressed SED(Beijing Military Medical Science Institute) be immunogene, the BALB/c mouse in 8 weeks age of immunity, immune programme for children is as follows: within first week, carries out head and exempts from, 10 μ g/, subcutaneous multi-point injection after Freund's complete adjuvant emulsification; 4th week is carried out two and is exempted from, 8 μ g/only, subcutaneous multi-point injection after incomplete Freund's adjuvant emulsification; Within the 6th week, carry out three and exempt from, 6 μ g/only, subcutaneous multi-point injection after incomplete Freund's adjuvant emulsification; Afterbody blood sampling in the 7th week is surveyed and is tired, and selects to tire the highest mouse; Within the 8th week, make a spurt immune, 4 μ g/only, physiological saline solution, lumbar injection; After spurt immunity posterior orbit blood sampling in 3 days, merge.Screening adopts indirect ELISA to carry out, and screens altogether 10 cell lines.
(2) specificity SED double antibody detects the screening of antibody
The 10 strain monoclonal antibodies difference mark horseradish peroxidases (HRP) by after purifying, carry out double antibody sandwich method pairing after the success of direct method identification marking, determine the required anti-SED antibody of specific immunity colloidal gold fast detecting test paper strip.Wherein 5F2 is golden labeling antibody, and 10F1 is coated antibody.
(3) preparation of Enterotoxin D, staphylococcal immune colloid gold Rapid detection test strip:
The preparation of a, blank collaurum:
Adopt citrate reducing process to prepare colloid gold particle, in conical flask, add 200mL deionized water and 2mL 1% gold chloride, be heated with stirring to boiling, add fast 4mL 1% sodium citrate, continue heating 15 ~ 20 minutes, to being shiny red, then cooling in room temperature, 4 ℃ of stored refrigerated;
The preparation of b, colloidal gold labeled monoclonal antibody:
Blank colloidal gold solution 0.1M K
2cO
3regulate pH value to 7.4, drip appropriate concentration antibody in stirring, every milliliter of collaurum adds 30 μ g antibody 5F2, continues to stir 30 minutes, centrifugal, draws supernatant, preserves liquid heavily revolve with golden labeling antibody;
The preparation of c, gold mark pad:
Debugging three-dimensional planar point film gold spraying instrument instrument (HM3055), is sprayed on the anti-SED antibody 5F2 of the good collaurum of mark on glass fibre membrane uniformly, spouting liquid 0.6 μ L/cm, and 37 ℃ of oven dry are spent the night, and envelope is standby;
The preparation of d, coated film:
Debugging three-dimensional planar point film gold spraying instrument instrument (HM3055), is evenly sprayed on the anti-SED antibody 10F1 having diluted on nitrocellulose filter, obtains detection line; The sheep anti-mouse igg having diluted is evenly sprayed on nitrocellulose filter, obtains nature controlling line, spouting liquid is 0.6 μ L/cm, and 37 ℃ of oven dry are spent the night, and envelope is standby.
By PVC base plate 1, nitrocellulose filter detection layers 2, sample pad 3, gold mark pad 4, detection line 5, nature controlling line 6 and adsorptive pads 7 combinations, obtain the immune colloid gold Rapid detection test strip of product Enterotoxin D, staphylococcal.
The principle of work of test strips of the present invention: adopt colloidal gold immunochromatographimethod technology, select SED specific antibody as solid formation, utilize double antibody sandwich method principle to detect in sample whether contain SED.While containing SED in sample to be checked, anti-SED antibody (5F2) combination of antigen elder generation and colloid gold label, because chromatography effect compound moves forward along coated film, during anti-SED antibody (10F1) on running into detection line, form antibody-antigen-Jin labeling antibody compound, enrichment on detection line, forms red precipitate line.
During detection, as sample is negative, test strips only has the colour developing of nature controlling line C line; If sample is positive, enterotoxin content value is greater than 0.25ng/mL, and test strips nature controlling line C line, detection line T line all develop the color.If test strips C line does not develop the color, illustrate that test strips quality goes wrong.
Claims (6)
1. an immune colloid gold Rapid detection test strip for Enterotoxin D, staphylococcal, is characterized in that: comprise PVC base plate (1), at PVC base plate (1) two ends, be respectively equipped with sample pad (3) and adsorptive pads (7);
PVC base plate (1) middle part is provided with nitrocellulose filter detection layers (2), is provided with gold mark pad (4) between nitrocellulose filter detection layers (2) and sample pad (3); Described gold mark pad (4) one end is connected and stacks with sample pad (3), and the other end is stacked in nitrocellulose filter detection layers (2);
In described nitrocellulose filter detection layers (2), be disposed with detection line (5) and nature controlling line (6).
2. the immune colloid gold Rapid detection test strip of Enterotoxin D, staphylococcal according to claim 1, is characterized in that: described gold mark pad (4) is coated with the anti-SED antibody 5F2 of gold mark mark.
3. the immune colloid gold Rapid detection test strip of Enterotoxin D, staphylococcal according to claim 1, is characterized in that: on described detection line (5), be coated with anti-SED antibody 10F1.
4. the immune colloid gold Rapid detection test strip of Enterotoxin D, staphylococcal according to claim 1, is characterized in that: described nature controlling line is coated with sheep anti-mouse igg on (6).
5. the preparation method of the immune colloid gold Rapid detection test strip of Enterotoxin D, staphylococcal described in claim 1, Enterotoxin D, staphylococcal is abbreviated as SED, it is characterized in that step is as follows:
(1) preparation of anti-SED monoclonal antibody specific:
The recombinant expressed SED that Beijing Military Medical Science Institute provides of take is immunogene, and the BALB/c mouse in 8 week age of immunity, through immunity, Fusion of Cells, screening, screens 10 cell strain of monoclonal antibody altogether;
(2) specificity SED double antibody detects the screening of antibody:
The 10 strain cell strain of monoclonal antibody difference mark horseradish peroxidase HRP by after step (1) purifying, carry out double antibody sandwich method pairing after the success of direct method identification marking, determine the required anti-SED antibody of specific immunity colloidal gold fast detecting test paper strip; Wherein 5F2 is golden labeling antibody, and 10F1 is coated antibody;
(3) preparation of Enterotoxin D, staphylococcal immune colloid gold Rapid detection test strip:
The preparation of a, blank collaurum:
Adopt citrate reducing process to prepare colloid gold particle, in conical flask, add the gold chloride that 200mL deionized water and 2mL mass concentration are 1%, be heated with stirring to boiling, adding fast 4mL mass concentration is 1% sodium citrate, continue heating 15 ~ 20 minutes, to being shiny red, then cooling in room temperature, 4 ℃ of stored refrigerated;
The preparation of b, colloidal gold labeled monoclonal antibody:
The K of 0.1M for blank colloidal gold solution
2cO
3regulate pH value to 7.4, drip antibody prepared by appropriate concentration step (2) in stirring, every milliliter of blank colloidal gold solution adds 30 μ g antibody 5F2, continues to stir 30 minutes, centrifugal, draws supernatant, preserves liquid heavily revolve with golden labeling antibody;
The preparation of c, gold mark pad:
Debugging three-dimensional planar point film gold spraying instrument instrument, is sprayed on the anti-SED antibody 5F2 of the good collaurum of mark on glass fibre membrane uniformly, spouting liquid 0.6 μ L/cm, and 37 ℃ of oven dry are spent the night, and envelope is standby;
The preparation of d, coated film:
Debugging three-dimensional planar point film gold spraying instrument instrument, is evenly sprayed on the anti-SED antibody 10F1 having diluted on nitrocellulose filter, obtains detection line; The sheep anti-mouse igg having diluted is evenly sprayed on nitrocellulose filter, obtains nature controlling line, spouting liquid is 0.6 μ L/cm, and 37 ℃ of oven dry are spent the night, and envelope is standby;
By PVC base plate (1), nitrocellulose filter detection layers (2), sample pad (3), gold mark pad (4), detection line (5), nature controlling line (6) and adsorptive pads (7) combination, obtain the immune colloid gold Rapid detection test strip of product Enterotoxin D, staphylococcal.
6. the application of the immune colloid gold Rapid detection test strip of Enterotoxin D, staphylococcal described in claim 1, it is characterized in that: it is applicable to customs, enterprise, hospital, inspection and quarantine unit, the fast detecting that can realize Enterotoxin D, staphylococcal in food or clinical sample, lowest detection is limited to 0.25ng/mL.
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| CN110940810A (en) * | 2019-11-29 | 2020-03-31 | 扬州大学 | Plastic package colloidal gold detection card for detecting salmonella toxin |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105353128A (en) * | 2015-10-16 | 2016-02-24 | 江南大学 | Colloidal gold test strip for simultaneous detection of five staphylococcus aureus enterotoxins and preparation method thereof |
| CN105353128B (en) * | 2015-10-16 | 2017-01-25 | 江南大学 | Colloidal gold test strip for simultaneous detection of five staphylococcus aureus enterotoxins and preparation method thereof |
| CN110940810A (en) * | 2019-11-29 | 2020-03-31 | 扬州大学 | Plastic package colloidal gold detection card for detecting salmonella toxin |
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