CN101943698A - Dot immuno-gold filtration assay (DIGFA) quick detection card for leather hydrolyzate in milk and preparation method thereof - Google Patents
Dot immuno-gold filtration assay (DIGFA) quick detection card for leather hydrolyzate in milk and preparation method thereof Download PDFInfo
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- CN101943698A CN101943698A CN2010102739382A CN201010273938A CN101943698A CN 101943698 A CN101943698 A CN 101943698A CN 2010102739382 A CN2010102739382 A CN 2010102739382A CN 201010273938 A CN201010273938 A CN 201010273938A CN 101943698 A CN101943698 A CN 101943698A
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Abstract
The invention relates to a dot immuno-gold filtration assay (DIGFA) quick detection card for leather hydrolyzate in milk and a preparation method thereof. The detection card is composed of a sample layer, a colloidal gold labeled layer, a detection layer, a water absorption layer, a plastic lining plate and a plastic cover plate. The preparation method comprises the following steps: preparing a colloidal gold labeled leather hydrolyzed protein solution and a colloidal gold labeled layer; preparing a leather hydrolyzed protein multi-clone antibody solution and detection lines on the detection layer; and assembling the detection card. The detection card has the advantages of simple operation and accurate detection result, can be interpreted by naked eyes, does not need complex operation, special equipment and the like, is suitable for on-site casual detection of leather hydrolyzate in milk for milk product enterprises, and inspection and sanitary departments, and has wide application prospects.
Description
Technical field
The present invention relates to food safety detection, specifically belong to a kind of Ruzhong leather hydrolysate gold-labeled quick test card and preparation method thereof.
Background technology
Milk and milk products is popular food, and illegal operators is that the phenomenon of mingling to the Ruzhong of seeking exorbitant profit happens occasionally, and serious harm consumer's legitimate rights and interests have become puzzlement China dairy products industry healthy development, the key factor that the restriction quality of dairy products promotes.Common breast is mingled means to be had: convert water and gain in weight, add salt, nitrate and nitrite and increase proportion, mix microbiotic, alkaline matter and formaldehyde and delay corruption, add fatty powder and improve fat content; Add nitrogenous class materials such as urea, melamine for improving content of milk protein.Governments at all levels strengthen renovation dynamics for this reason, and illegal retailer begins to add a kind of novel adulterant---leather hydrolysate for escaping to hit.
The leather hydrolysate is the material that hydrolysis goes out from fur, and principal ingredient is a leather protein hydrolysate.It is that infusion behind process soaking and reducing, cleaning, interpolation complex enzyme and the activated charcoals such as the leftover bits and pieces of leather enterprise, fur is made.Contain potassium dichromate and sodium bichromate in the process hides waste material, produce leather protein hydrolysate with them, potassium dichromate and sodium bichromate will be brought in the product.Potassium dichromate and sodium bichromate are strong carcinogenic substances, and pungency and corrosivity are arranged, and can stimulate and corrode alimentary canal after eating, and be also toxic to the liver kidney.Even more serious is that they can't decompose in vivo, can slowly accumulate, and causes slow poisoning, makes the joint enlargement of loosening, even causes infant's death.
Leather hydrolysate and other adulterant difference are that the principal ingredient leather protein hydrolysate of leather hydrolysate is a kind of real protein, has the general physicochemical property of protein, therefore are difficult to it and the normal protein in Ruzhong are distinguished.The detection method of Ruzhong leather hydrolysate roughly has three kinds at present: the chromatography of ions of dichromate ion in L (-)-hydroxyproline method of leather protein hydrolysate, mercuric nitrate-picric acid method and the detection leather hydrolysate in the detection leather hydrolysate.Be described below respectively:
1, L (-)-hydroxyproline method
L (-)-hydroxyproline method is the standard detecting method that provides in " the non-edible material from soybeans list of the illegal interpolation of possibility in the food " (second batch).Its principle is that the content of leather protein hydrolysate hydroxyproline is higher, reaches more than 10%, and does not contain this composition in the normal milk protein.By measuring L (-)-hydroxyproline content in the milk, can extrapolate leather protein hydrolysate content, thereby determine to have or not interpolation leather hydrolysate.Sample discharges hydroxyproline through hydrochloric acid hydrolysis, through the toluene-sodium-sulfonchloramide oxidation, generates the oxide that contains pyrrole ring.Destroy excessive toluene-sodium-sulfonchloramide with perchloric acid.Hydroxyproline oxide and paradime thylaminobenzaldehyde reaction generate red compound, carry out colorimetric estimation at wavelength 558nm place.This method advantage is: detectability is low, judgement accurately, is not subject to disturb.Shortcoming is: 1, the reaction time long, only the acidolysis of sample just needs 16-24 hour.2, reagent corrosivity height uses corrosive substances such as hydrochloric acid, NaOH and phenol in a large number, requires the staff that special experimental skill is arranged.3, instrument requires height, needs specialized equipment such as spectrophotometer, water bath with thermostatic control and well heater.
2, mercuric nitrate-picric acid method
Picric acid method is a detection method commonly used in the fresh milk purchase.Its principle is: the precipitable MC of mercuric nitrate, and leather protein hydrolysate can not be removed, the latter can with saturated picric acid generation precipitation reaction.In testing sample, add mercuric nitrate solution, with filter paper with the MC sedimentation and filtration after, slowly add saturated picric acid again.If normal breast, it is limpid to filter rear filtrate, adds surface of contact no change behind the frohde test solution; And mix the breast of leather protein hydrolysate, and to filter rear filtrate to be translucent, slightly newborn cyan adds behind the frohde test solution surface of contact ring-type that is white in color.This method advantage is: reagent is easy to get, and is with low cost.But distinct disadvantage is arranged also: used casein precipitation agent mercuric nitrate is a heavy metallic salt, and toxicity is big; And this method result is difficult for accurate interpretation, is subject to the interference of milk acidity and exotic, false positive occurs.
3, the chromatography of ions
The chromatography of ions ratio juris is the dichromate ion that contains trace in the leather hydrolysate, by measuring in the milk whether contain dichromate ion, just can judge whether added the leather hydrolysate in the milk.Plurality of advantages such as it is accurate that this method has assay, and highly sensitive, good stability, sample preparation are simple, but need expensive ion chromatograph, be difficult to promote the use of.Therefore press for a kind of method of inspection of easy, sensitive, reliable newborn leather hydrolysate.
Summary of the invention
The purpose of this invention is to provide a kind of Ruzhong leather hydrolysate gold-labeled quick test card and preparation method thereof.
A kind of Ruzhong provided by the invention leather hydrolysate gold-labeled quick test card is made up of sample layer, gold mark layer, detection layers, water accepting layer, plastics lining board and plastic cover plate; The stem of plastics lining board is the sample layer front end, and front end is stacked links to each other for the rear end of sample layer and gold mark layer, and a gold mark layer rear end links to each other with the detection layers front end is stacked, and the detection layers rear end links to each other with the water accepting layer front end is stacked, and the water accepting layer rear end is positioned at the afterbody of plastics lining board; The top of plastics lining board is coated with plastic cover plate, and plastic cover plate is provided with well and viewport, and well is over against sample layer, the detection line of viewport on the detection layers;
Described sample layer is made by glass fibre, water accepting layer is made by multilayer filter paper or glass fibre is made, detection layers is a nitrocellulose filter, be coated with the detection line that leather protein hydrolysate polyclonal antibody is made on it, gold mark layer is a glass fibre, is coated with the leather protein hydrolysate of colloid gold label on it.
The preparation method of a kind of Ruzhong leather hydrolysate gold-labeled quick test card comprises the steps:
(1), the preparation of the leather protein hydrolysate solution of colloid gold label and gold mark layer
(a) preparation of colloidal gold solution: be equipped with colloidal gold solution by trisodium citrate-gold chloride legal system;
(b) preparation of the leather protein hydrolysate solution of colloid gold label: regulate colloidal gold solution pH to 7.0-8.0, the leather protein hydrolysate that in the 1L colloidal gold solution, adds 5-20mg, the vibration mixing, the bovine serum albumin(BSA) that adds 5g-20g again, 15, the centrifugal 15-30min of 000g, the gained precipitation is diluted with the phosphate buffer of the 10-50mmol/L pH 7.0-8.0 of the sodium azide of bovine serum albumin(BSA) that contains mass percentage concentration 0.5%-2% and mass percentage concentration 0.5%, gets the leather protein hydrolysate solution of colloid gold label;
(c) preparation of gold mark layer: glass fibre membrane is immersed the leather protein hydrolysate solution of colloid gold label, takes out, dry naturally golden mark layer;
(2), the preparation of detection line on leather protein hydrolysate polyclonal antibody solution and the detection layers
(a) preparation of leather protein hydrolysate polyclonal antibody solution: select rabbit, mouse or rat to carry out immunity, first immunisation is mixed the back injection with leather protein hydrolysate with isopyknic Freund's complete adjuvant; Carry out booster immunization after two weeks, mix the back injection with isopyknic incomplete Freund with leather protein hydrolysate; Two all booster immunizations are 3-5 time at interval, get blood, and separation of serum, purifying get leather protein hydrolysate polyclonal antibody; Described purification process is one or more in antigen affinity chromatography, Protein A affinity chromatography, the caprylic acid precipitation method and the ammonium sulfate precipitation method;
(b) preparation of detection line on the detection layers: in the nitrocellulose filter stage casing with the line of leather protein hydrolysate polyclonal antibody solution, dry naturally detection line;
(3), the assembling of test card: the rear and front end at plastics lining board is pasted sample layer and water accepting layer respectively; In the middle of sample layer and water accepting layer, paste detection layers, and the detection layers rear end is linked to each other with the water accepting layer front end is stacked; Paste gold mark layer between detection layers and sample layer, and make gold mark layer major part be pressed into sample layer, fraction is pressed into detection layers; Cover plastic cover plate then, make well on the plastic cover plate over against sample layer, viewport is over against detection layers; Test card and drying agent together are encapsulated in the aluminium foil bag, keep in Dark Place.
In order to guarantee the validity of test card, the present invention also can comprise positive control solution and negative controls, their preparation method: the leather protein hydrolysate of adding 20g-50g and the sodium azide of 5g obtain positive control solution in 1L milk; The sodium azide that adds 5g in 1L milk obtains negative controls.
The principle of work of test card of the present invention: utilize antigen and antibody can specific recognition, the characteristic of combination, with leather protein hydrolysate colloid gold label, be coated on the glass fibre membrane and mark layer, leather protein hydrolysate polyclonal antibody is coated on the nitrocellulose filter as detection line as gold.When fluid drips is added on the sample layer, immediately to the diffusion of water accepting layer direction, when arriving gold mark layer, gold mark leather protein hydrolysate is dissolved, together diffuses to detection line with liquid.If do not contain leather protein hydrolysate in the test sample product, gold mark leather protein hydrolysate will combine with the polyclonal antibody on the detection line, presents red stripes; If contain leather protein hydrolysate in the test sample product, the leather protein hydrolysate in gold mark leather protein hydrolysate and the sample will be competed with the polyclonal antibody on the detection line and combine, no band or present the pale red band.
The using method of Ruzhong leather hydrolysate gold mark test card: get positive control solution, negative controls and analyte sample fluid respectively with disposable dropper, splash into successively on the sample layer of three test card.Show detection line if drip the test card of negative controls, and the test card of dropping positive control solution does not show that detection line explanation test card is effective, otherwise be invalid.Then testing sample is negative if red detection line appears in dropping testing sample test card, does not contain the leather hydrolysate in the testing sample; Otherwise positive, contain the leather hydrolysate in the testing sample.
The present invention compared with prior art has following advantage:
1, simple operating steps only needs dropper to drip sample and gets final product.
2, but the visual result naked eyes are judged, need not special experimental apparatus.
3, the reaction time weak point detects omnidistance required time and is no more than 10 minutes.
4, with low cost, be fit to on-the-spot generaI investigation on a large scale, be easy to promote.
5, test card preparation method of the present invention is easy.
Description of drawings
Fig. 1 is the schematic appearance of test card of the present invention
Among the figure: 1-test card, 2-viewport, 3-well
Fig. 2 is the inner structure synoptic diagram of test card of the present invention
Among the figure: 2-viewport, 3-well, 4-plastics lining board, 5-water accepting layer, 6-detection layers, 7-detection line, 8-sample layer, 9-gold mark layer, 10-plastic cover plate
Embodiment
Content of the present invention can be described further by following example, but does not limit the scope of the invention.
Embodiment 1, a kind of Ruzhong leather hydrolysate gold-labeled quick test card as shown in Figure 1 and Figure 2, are made up of sample layer 8, gold mark layer 9, detection layers 6, water accepting layer 5, plastics lining board 4 and plastic cover plate 10; The stem of plastics lining board 4 is sample layer 8 front ends, 9 front end is stacked links to each other for the rear end of sample layer 8 and gold mark layer, gold mark layer 9 rear end and stacked linking to each other of detection layers 6 front ends, detection layers 6 rear ends are positioned at the afterbody of plastics lining board 4 with stacked linking to each other of water accepting layer 5 front ends, water accepting layer 5 rear ends; The top of plastics lining board 4 is coated with plastic cover plate 10, and plastic cover plate 10 is provided with well 3 and viewport 2, and well 3 is over against sample layer 8, the detection line 7 of viewport 2 on the detection layers 6; Described sample layer 8 is made by glass fibre, water accepting layer 5 is made by multilayer filter paper, also can make with glass fibre, detection layers 6 is nitrocellulose filters, be coated with the detection line 7 that leather protein hydrolysate polyclonal antibody is made on it, gold mark layer 9 is glass fibre, is coated with the leather protein hydrolysate of colloid gold label on it.
The preparation method of embodiment 2, Ruzhong leather hydrolysate gold-labeled quick test card, step is as follows:
(1), the preparation of the leather protein hydrolysate solution of colloid gold label and gold mark layer
(a) preparation of colloidal gold solution: press trisodium citrate---gold chloride method operation; The gold chloride of getting 100mL mass percentage concentration 0.01% is heated with stirring to boiling, adds the trisodium citrate 1.5mL of mass percentage concentration 0.1%, continues agitating heating 15min, obtains colloidal gold solution;
(b) preparation of the leather protein hydrolysate solution of colloid gold label: regulate colloidal gold solution pH to 7.5, the leather protein hydrolysate that in the 1L colloidal gold solution, adds 10mg, the vibration mixing, the bovine serum albumin(BSA) that adds 10g again, 15, the centrifugal 20min of 000g, the gained precipitation is diluted with the phosphate buffer of the 20mmol/L pH 7.2 of the sodium azide of bovine serum albumin(BSA) that contains mass percentage concentration 1% and mass percentage concentration 0.5%, gets the leather protein hydrolysate solution of colloid gold label;
(c) preparation of gold mark layer: glass fibre membrane is immersed the leather protein hydrolysate solution of colloid gold label, takes out, dry naturally golden mark layer 9;
(2), the preparation of detection line on leather protein hydrolysate polyclonal antibody solution and the detection layers
(a) preparation of leather protein hydrolysate polyclonal antibody solution: select rabbit to carry out immunity.After first immunisation is used 2mg leather protein hydrolysate and isopyknic Freund's complete adjuvant is mixed, carry out subcutaneous multi-point injection; Carry out booster immunization after two weeks, mix hypodermic injection with isopyknic incomplete Freund with 1mg leather protein hydrolysate.Two all booster immunizations are 5 times at interval, and tame rabbit ear vein is got blood, leave standstill back separation clot and get serum.Every 5mL serum is diluted to 5 times of volumes with sodium-acetate buffer 60mmol/L pH 4.5, the 0.625mL caprylic acid is dropwise stirred to add in the dilute serum again, continues to stir 30min, 10, the centrifugal 30min of 000g.Supernatant is through filter paper filtering, the bag filter of packing into, with 7.5,4 ℃ of dialysed overnight of phosphate buffer pH of 50mmol/L, during change liquid 4 times.Stir adding solid ammonium sulfate to 45% saturation degree in the dislysate, continue to stir 30min, 10, the centrifugal 30min of 000g abandons supernatant.After phosphate buffer pH 7.5 dissolvings of precipitation with 50mmol/L, do not detect to there being the ammonium radical ion in 4 ℃ of dialysis with 50mmol/L phosphate buffer pH7.5.Dislysate is through 10, and the centrifugal 30min of 000g, gained supernatant are the leather protein hydrolysate polyclonal antibody solution behind the purifying.
(b) preparation of detection line on the detection layers: in the nitrocellulose filter stage casing with the line of leather protein hydrolysate polyclonal antibody solution, dry naturally detection line 7;
(3), the assembling of test card: paste sample layer 8 and water accepting layer 5 respectively in the rear and front end of plastics lining board 4; In the middle of sample layer 8 and water accepting layer 5, paste detection layers 6, make detection layers 6 rear ends and stacked linking to each other of water accepting layer 5 front ends.Folder is gilded and is marked layer 9 between detection layers 6 and sample layer 8, and 4/5 part of gold mark layer 9 sandwiches sample layer 8,1/5 parts and is pressed on the detection layers 6; Cover plastic cover plate 10 then, make well 3 on the plastic cover plate 10, the detection line 7 of viewport 2 on the detection layers 6 over against sample layer 8; Test card and drying agent together are encapsulated in the aluminium foil bag, keep in Dark Place.
Claims (4)
1. a Ruzhong leather hydrolysate gold-labeled quick test card is characterized in that being made up of sample layer (8), gold mark layer (9), detection layers (6), water accepting layer (5), plastics lining board (4) and plastic cover plate (10); The stem of plastics lining board (4) is sample layer (a 8) front end, the rear end of sample layer (8) and gold mark layer stacked linking to each other of (9) front end, gold mark layer (9) rear end and detection layers (6) stacked linking to each other of front end, detection layers (6) rear end is positioned at the afterbody of plastics lining board (4) with water accepting layer (5) stacked linking to each other of front end, water accepting layer (5) rear end; The top of plastics lining board (4) is coated with plastic cover plate (10), and plastic cover plate (10) is provided with well (3) and viewport (2), and well (3) is over against sample layer (8), the detection line (7) of viewport (2) on the detection layers (6);
Described sample layer (8) is made by glass fibre, water accepting layer (5) is made by multilayer filter paper, detection layers (6) is a nitrocellulose filter, be coated with the detection line (7) that leather protein hydrolysate polyclonal antibody is made on it, gold mark layer (9) is a glass fibre, is coated with the leather protein hydrolysate of colloid gold label on it.
2. a kind of Ruzhong as claimed in claim 1 leather hydrolysate gold-labeled quick test card is characterized in that described water accepting layer (5) made by glass fibre.
3. the preparation method of a kind of Ruzhong as claimed in claim 1 leather hydrolysate gold-labeled quick test card is characterized in that comprising the steps:
(1), the preparation of the leather protein hydrolysate solution of colloid gold label and gold mark layer
(a) preparation of colloidal gold solution;
(b) preparation of the leather protein hydrolysate solution of colloid gold label: regulate colloidal gold solution pH to 7.0-8.0, the leather protein hydrolysate that in the 1L colloidal gold solution, adds 5-20mg, the vibration mixing, the bovine serum albumin(BSA) that adds 5g-20g again, 15, the centrifugal 15-30min of 000g, the gained precipitation is diluted with the phosphate buffer of the 10-50mmol/L pH 7.0-8.0 of the sodium azide of bovine serum albumin(BSA) that contains mass percentage concentration 0.5%-2% and mass percentage concentration 0.5%, gets the leather protein hydrolysate solution of colloid gold label;
(c) preparation of gold mark layer: glass fibre membrane is immersed the leather protein hydrolysate solution of colloid gold label, takes out, dry naturally golden mark layer (9);
(2), the preparation of detection line on leather protein hydrolysate polyclonal antibody solution and the detection layers
(a) preparation of leather protein hydrolysate polyclonal antibody solution: select rabbit, mouse or rat to carry out immunity, first immunisation is mixed the back injection with leather protein hydrolysate with isopyknic Freund's complete adjuvant; Carry out booster immunization after two weeks, mix the back injection with isopyknic incomplete Freund with leather protein hydrolysate; Two all booster immunizations are 3-5 time at interval, get blood, and separation of serum, purifying get leather protein hydrolysate polyclonal antibody solution;
(b) preparation of detection line on the detection layers: in the nitrocellulose filter stage casing with the line of leather protein hydrolysate polyclonal antibody solution, dry naturally detection line (7);
(3), the assembling of test card: paste sample layer (8) and water accepting layer (5) in the rear and front end of plastics lining board (4) respectively; In the middle of sample layer (8) and water accepting layer (5), paste detection layers (6), and make detection layers (6) rear end and water accepting layer (5) stacked linking to each other of front end; Paste gold mark layer (9) between detection layers (6) and sample layer (8), and make gold mark layer (9) major part be pressed into sample layer (8), fraction is pressed into detection layers (6); Cover plastic cover plate (10) then, make well (3) on the plastic cover plate (10) over against sample layer (8), viewport (2) makes test card (1) over against detection layers (6).
4. the preparation method of a kind of Ruzhong as claimed in claim 3 leather hydrolysate gold-labeled quick test card, it is characterized in that described purification process is one or more in antigen affinity chromatography, Protein A affinity chromatography, the caprylic acid precipitation method and the ammonium sulfate precipitation method.
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Application publication date: 20110112 |