CN104360061A - Rabies virus IgG antibody immune gold-labeled test paper and preparation method thereof - Google Patents
Rabies virus IgG antibody immune gold-labeled test paper and preparation method thereof Download PDFInfo
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Abstract
The invention relates to rabies virus IgG antibody immune gold-labeled test paper. The test paper consists of a serum treatment pad, a gold-labeled release pad, a nitrocellulose membrane, an absorbent pad and a back plate, wherein the serum treatment pad, the gold-labeled release pad, the nitrocellulose membrane and the absorbent pad are sequentially superposed and adhered to the back plate, and the superposition distance between every two parts is 2-3mm; a purified rabies virus antigen-colloidal gold compound is coated on the gold-labeled release pad; a detection line and a quality control line are coated on the nitrocellulose membrane; the detection line is fixedly provided with SPA; and the quality control line is fixedly provided with anti-dog rabies virus positive IgG. The test paper disclosed by the invention is easy to operate, high in specificity and high in sensitivity, special instruments and equipment and professionals are not needed, whether vaccine immunity animals generate antibodies on a protection level can be rapidly screened, and a reference basis is provided for vaccine immunity and immune procedure formulation of rabies.
Description
Technical field
The present invention relates to rapid detection technical field, particularly relate to the immune-gold labeled Test paper of a kind of hydrophobin IgG antibody and preparation method.
Background technology
Rabies are a kind of important infectious diseases common to human beings and animals caused by hydrophobin (Rabies virus, RABV), and case fatality rate is almost 100%, are one of serious infectious diseases of current threat China population health and public health security.Human rabies more than 90% is by falling ill or being with the animal of poison to lick, sting and infected.According to incompletely statistics, the ratio of human rabies is caused to be respectively in China's various animals: dog 85%-95%, cat 4%, wild animal 3%.Current rabies there is no effective medicine, once morbidity, almost 100% is dead.The rabic key measure of prevention and control still propagates the immunity of animal and people for strengthening dog, cat etc., by dog, cat stings rear timely vaccinate or immunoglobulin (Ig) carries out exposing rear treatment.Since the nineties in last century; rabies vacciness is several times regenerated; quality has had large increase; but because the reasons such as individual difference, Vaccines classes and inoculation method may cause the neutralizing antibody not producing or only produce low level of protection, so that in treating after exposure, still have many people to be dead in vaccinate sequela.The immune effect of the animal rabies such as dog, cat vaccine how, can be assessed by the hydrophobin neutralizing antibody level measuring the animal blood serum such as dog, cat.The World Health Organization (WHO) points out: detect the Antibody agaianst rabies virus in dog serum, be the important references index evaluating immune effect at present, just have protection when the NAT of hydrophobin reaches 0.5IU/mL in serum, can resist the attack of strong poison.Therefore, the vaccine inoculation to dog should be paid attention to, also will pay attention to the detection of rabies virus antibodies after vaccine inoculation, like this could real antirabic generation.
What the detection of current Antibody against rabies virus mainly adopted is enzyme linked immunosorbent assay (ELISA), Mouse neutralization test (MNT), fluorescence antibody neutralization test (FAVNT) and rapid fluorescence stove inhibition test (RFFIT) etc., these methods need microplate reader, the valuable instrument and equipments such as fluorescent microscope and the condition such as more complicated operative technique, strict cell cultivation equipment, even some needs the experience relying on reviewer.In addition, because great majority test needs to operate live virus, therefore should carry out in the laboratory of higher biological safety level.Above feature seriously limits the application of these class methods, especially in the use of epidemic prevention unit of basic unit.Immunity colloidal gold test paper strip is a kind of novel microbial detection technique integrating immunoassay technology, immunochromatography technique, has the advantages such as simple to operate, quick, sensitive, and the family and the grass-roots unit that are applicable to very much scene and shortage test condition use.At present, immunity colloidal gold test paper strip has been widely used in the field such as medical science and food inspection, as: acquired immune deficiency syndrome (AIDS), hepatitis etc., but this technology is still in the starting stage in the application of animal medicine each side, at present also not used for the immune-gold labeled Test paper product detecting hydrophobin IgG antibody level in immune animal body.
Therefore, be necessary that providing a kind of can detect fast rabies antibody level in immune animal body thus improve the immune-gold labeled Test paper of screening efficiency.
Summary of the invention
The object of this invention is to provide immune-gold labeled Test paper of a kind of hydrophobin IgG antibody and preparation method thereof.
In order to realize the object of the invention, head of the present invention provides a kind of hydrophobin IgG antibody immune-gold labeled Test paper, and described Test paper is made up of serum process pad, the release of gold mark pad, nitrocellulose filter, adsorptive pads and backboard; Serum process pad, gold mark release pad, nitrocellulose filter and adsorptive pads mutually superpose successively and are pasted onto on backboard, mutually superpose 2-3mm between each several part; Gold mark release pad is coated with the Rabies Virus Antigen-colloidal gold composite of purifying; Nitrocellulose filter is coated with detection line and nature controlling line, described detection line is fixed with SPA, and described nature controlling line is fixed with the positive IgG of dog anti-rabies virus.
In a preferred embodiment, described serum process pad is that bag is by the glass fibre of serum treating fluid element film.
In a preferred embodiment, described serum treating fluid is the tetraborate containing 0.05M, the bovine serum albumin(BSA) of 1%, the 20mM phosphate buffer of the Sodium azide of 0.04% and the Tween-20 of 0.2%.
In a preferred embodiment, Rabies Virus Antigen-colloidal gold composite gold mark conserving liquid used is for containing 2-3%BSA, 1-2% sucrose, 0.5-1% trehalose, the 20mM Tris-HCl damping fluid of the pH 8.0-8.5 of 0.2%Tween-20 and 0.04% Sodium azide.
In a preferred embodiment, the concentration of described SPA is 0.8-1.2mg/ml, and the spray film consumption on nitrocellulose filter is 1.0 μ L/cm.
In a preferred embodiment, the concentration of the positive IgG of described dog anti-rabies virus is 1.4-1.5mg/ml, and the spray film consumption on nitrocellulose filter is 1.0 μ L/cm.
In Test paper provided by the present invention, the material of described backboard can be tygon, and the material of described gold mark release pad is glass fibre membrane.
Present invention also offers the preparation method of the immune-gold labeled Test paper of a kind of immune animal hydrophobin IgG antibody, comprise the following steps:
(1) preparation of purifying Rabies Virus Antigen: hydrophobin is inoculated in the bsr cell growing up to individual layer by MOI=0.01, at 5%CO
2cultivate after 4-5 days, gathered in the crops cultured cell supernatant 2 times continuously every 2 days for 33 DEG C.By the vial supernatant of results in 1/4000 ratio add beta-propiolactone and carry out deactivation, cell fragment is removed in the centrifugal 30min of 3000rpm, supernatant adds 1M zinc acetate in 1/50 ratio, 4 DEG C precipitate 30 minutes-1 hour, 4 DEG C, the centrifugal 30min of 3000rpm, abandon supernatant, precipitate and suspend (100 times concentrate), to the transparent state of solution with saturated EDTA.Get viral concentration liquid and carry out purifying (being followed successively by 20%, 30%, 40%, 55% gradient) with sucrose density gradient centrifugation, virus band between collecting 40% and 55%, is the Rabies Virus Antigen of purifying.
(2) purifying Rabies Virus Antigen-colloidal gold composite is prepared: cut-off footpath is the collaurum 100ml of 20-30nm, with 0.2M K
2cO
3adjustment pH is 8.5-9.0, under agitation add the Rabies Virus Antigen 4.5mg of purifying fast, 25 DEG C of effect 30min, the BSA adding 10% makes its final concentration be 1%, 25 DEG C of effects are in conjunction with 15min, the centrifugal 30min of 8000-10000rpm removes unconjugated Rabies Virus Antigen, is the Rabies Virus Antigen-colloidal gold composite of purifying with the resuspended centrifugal gained precipitation of 10ml gold mark conserving liquid.
(3) preparation gold mark release pad: the purifying of above-mentioned preparation Rabies Virus Antigen-colloidal gold composite is evenly coated on glass fibre element film according to the spray film consumption of 10 μ l/cm, fully dry in 37 DEG C.
(4) nitrocellulose filter is prepared: SPA and the positive IgG of dog anti-rabies virus is diluted to final concentration for 1.0mg/ml and 1.5mg/ml with 20mM PBS respectively, detection line on nitrocellulose filter and nature controlling line is evenly coated on film spot injection system, spray film consumption is 1.0 μ L/cm, fully dry at 37 DEG C.
(5) by serum process pad, gold mark release pad, nitrocellulose filter and adsorptive pads successively mutually superposition be pasted onto on backboard, mutually superpose 2-3mm between each several part.
Test paper provided by the present invention uses and can carry out according to following steps with result decision method: use sample diluting liquid to carry out 100 times to blood serum sample to be checked and dilute.Test paper is kept flat on desktop, get blood serum sample 50 ~ 100 μ L diluted and add serum process pad.25 DEG C leave standstill after 15 minutes, according to following situation result of determination:
Test paper is effective: occur purplish red colo(u)r streak at nature controlling line (C), illustrate that this test paper is effective;
Negative: only at the upper appearance purplish red colo(u)r streak of nature controlling line (C), and colo(u)r streak does not appear in detection line (T) place;
Positive: respectively to occur a purplish red colo(u)r streak at nature controlling line (C) and detection line (T) district, and colo(u)r streak color depth is directly proportional to antibody titer height,
Antibody titer higher colo(u)r streak color is darker.
Wherein, described sample diluting liquid is the phosphate buffer of the pH 8.0 containing 0.2%Tween-20, the 20mM of 0.04% Sodium azide.
In the present invention, after utilizing Test paper to detect, positive findings illustrates that rabies antibody level is higher, need not carry out rabies vaccine for immunization inoculation again; Negative findings to illustrate in animal body without rabies antibody or antibody horizontal lower than the minimal protection level of resisting hydrophobin strong virus attack, if animal population is healthy, should carry out Rabies Vaccine in time.The immune-gold labeled Test paper of immune animal hydrophobin IgG antibody of this research development can carry out rapid screening, for the formulation of rabic vaccine immunity and immune programme for children provides reference frame to the antibody whether vaccine immunity animal has produced level of protection.
Test paper prepared by the present invention is simple to operate, high specificity, highly sensitive, without the need to special instruments and equipment and professional and technical personnel, hydrophobin IgG antibody content situation in sample can be detected fast, whether can produce enough antibody to vaccine immunity animal to differentiate fast, for rabic vaccine immunity provides foundation.
Accompanying drawing explanation
Fig. 1 is the structural representation of the immune-gold labeled Test paper of hydrophobin IgG antibody of the present invention; Wherein, 1 is serum process pad, and 2 is gold mark release pad, and 3 is nitrocellulose filter, and 4 is detection line, and 5 is nature controlling line, and 6 is adsorptive pads, and 7 is backboard.
Fig. 2 is the immune-gold labeled Test paper testing result of immune animal hydrophobin IgG antibody in the embodiment of the present invention 3.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition or the condition according to manufacturer's instructions suggestion all conveniently.
The purifying of embodiment 1 Rabies Virus Antigen
Hydrophobin is grown up to the bsr cell of individual layer by MOI=0.01 inoculation, 5%CO
2cultivate after 4-5 days, gathered in the crops cultured cell supernatant 2 times continuously every 2 days for 33 DEG C.Add beta-propiolactone in the 1:4000 ratio of viral volume, shake up, 4 DEG C of 24h, in order to deactivation hydrophobin.Be placed in 37 DEG C of 2h again, in order to be hydrolyzed beta-propiolactone.By the hydrophobin of deactivation at 4 DEG C with the centrifugal 30min of 3,000rpm, remove cell fragment, collect supernatant carry out zinc acetate precipitation.Supernatant is added 1M zinc acetate by 1/50 volume, and limit edged shakes up, 4 DEG C of standing 30-60min, 4 DEG C of centrifugal 30min of 3000rpm, supernatant discarded, and precipitate and suspend with the saturated EDTA of former supernatant volume 1/100,4 DEG C of dissolvings are spent the night.4 DEG C of centrifugal 30min of 3000rpm, remove non-dissolved substance, and supernatant is concentrated viral antigen liquid.
By Puncture needle sleeve on 2.5ml syringe, in ultracentrifugation pipe, configure saccharose gradient, sucrose concentration is from top to bottom followed successively by: 20%, 30%, 40%, 55%.First in pipe, add 20% sucrose, then stretch into bottom pipe by puncture needle, add 30% sucrose, the rest may be inferred.If ultracentrifugation pipe is 10ml pipe, each gradient adds 2ml.The superiors add concentrated virus liquid 2ml.Use ultracentrifuge 4 DEG C of centrifugal 90min of 21000rpm.Draw obvious white viral antigen bands between 40%-55%, add appropriate STE and mix.Ultracentrifuge 4 DEG C of 30,000rpm centrifugal 90min.Abandon supernatant, precipitate and suspend with STE, be the Rabies Virus Antigen liquid of purifying.The packing of EP pipe ,-70 DEG C of preservations.
The preparation of the immune-gold labeled Test paper of embodiment 2 immune animal hydrophobin IgG antibody
The preparation of serum process pad: glass fibre element film is fully immersed in tetraborate containing 0.05M, the bovine serum albumin(BSA) of 1%, the Sodium azide of 0.04%, in the phosphate buffer of the 20mM of the Tween-20 of 0.2%, take out rearmounted 37 DEG C fully dry.
Firing of 20-30nm collaurum: in fuming cupboard, immerses 250ml triangular flask in the chloroform silication liquid of 5% dichlorodimethylsilane, then slowly rotates vessel, makes the inwall of glassware can be silicified immersion bubble.Leave standstill 10 minutes, glassware is taken out, rinse well with for subsequent use after masking foil covering bottleneck with a large amount of distilled water.To learn from else's experience the triangular flask of silication, add 99mL distilled water and 1mL 1% gold chloride, put on magnetic force heating stirrer, be heated to boiling, add 1% trisodium citrate aqueous solution 1.6mL fast, continue heating 5 minutes until solution is vinicolor and no longer changes, after 25 DEG C of coolings, 4 DEG C of Refrigerator stores are for subsequent use.
The preparation of purifying Rabies Virus Antigen-colloidal gold composite: cut-off footpath is the collaurum 100ml of 20-30nm, with 0.2M K
2cO
3adjustment pH is 8.5-9.0, under agitation add the Rabies Virus Antigen 4.5mg of purifying fast, 25 DEG C of effect 30min, the BSA adding 10% makes its final concentration be 1%, 25 DEG C of effects are in conjunction with 15min, the centrifugal 30min of 8000-10000rpm removes unconjugated Rabies Virus Antigen, and be the Rabies Virus Antigen-colloidal gold composite of purifying with the resuspended centrifugal gained precipitation of 10ml gold mark conserving liquid, 4 DEG C save backup.Gold mark release pad preparation: by preparation Rabies Virus Antigen-colloidal gold composite with the spray film amount even application of 10 μ L/cm 300 × 5mm glass fibre element film on, 37 DEG C fully dry, be assemblnig gold mark release pad, after sealing, kept dry is for subsequent use.
Detection line and nature controlling line preparation: to take and the height of separating immune hydrophobin exempts from dog serum, in 50ml centrifuge tube, carry out 2 times of dilutions with physiological saline.Add isopyknic saturated ammonium sulfate (final concentration is 50%), mix rear 4 DEG C of standing 30min.The centrifugal 30min of 3000r/min, gets precipitation and dissolves with the physiological saline of 2 times of former serum volumes.Add the saturated ammonium sulfate (final concentration is 33.3%) of 1 times of former serum volume, mix rear 4 DEG C of standing 30min.The centrifugal 30min of 3000r/min, gets precipitation and dissolves with the physiological saline of 2 times of former serum volumes.Carry out dialysis treatment with the PBS of 0.02M pH7.0, be the positive IgG of dog anti-rabies virus of purifying, packing after mensuration protein concentration, saves backup in-20 DEG C.
The NC film of 30cm will be cut into, be put on Membrane jetter platform and flatten, and put press strip, form detection line (T line) and nature controlling line (C line) by diluting for the dog anti-rabies virus IgG of SPA and 1.5mg/ml of concentration 1.0mg/ml is sprayed on NC film according to the spray film consumption of 1.0 μ L/cm respectively.After 25 DEG C of (humidity less than 40%) natural drying 24h, airtightly to save backup.
The assembling of immune-gold labeled Test paper: by smooth for the NC film central authorities in support plate, apart from upper end 2cm, lower end 1.5cm.Notice that stretching, extension is smooth, after subsides, space or bubble can not be had.The release of gold mark is leveled up the top being affixed on NC film detection line, overlapping NC film 0.2cm; Then even concora crush.Serum process is leveled up the top being affixed on the release of gold mark and padding, overlapping gold mark pad 0.2cm, then even concora crush.By smooth for the thieving paper below in NC film nature controlling line, overlapping NC film 0.2cm, then even concora crush (Fig. 1).Test strips sample end after stickup should be that edge is neat, open and flat, tight, is semi-manufacture test paper.Start CM4000 cutting machine, select test strips width (4mm) and cutting speed, semi-manufacture test paper is put into groove, cuts.The immune-gold labeled Test paper semi-manufacture of preparation are fixed in a plastic clip, reinstate packaging of aluminium foil bag sealing together with drying agent, suction pipe one.
The evaluation of the immune-gold labeled Test paper of embodiment 3 immune animal hydrophobin IgG antibody
Immune colloidal gold detection test paper uses and judges with result: use sample diluting liquid (containing 0.2%Tween-20, the phosphate buffer of the pH 8.0 of the 20mM of 0.04% Sodium azide) 100 times of dilutions are carried out (wherein to blood serum sample to be checked, blood serum sample 1 is the Healthy Dogs class serum confirming to have high rabies IgG antibody level through fluorescence antibody virus neutralization tests (FAVNT), blood serum sample 2 is the Healthy Dogs class serum confirming to have low rabies IgG antibody level through FAVNT test, blood serum sample 3 is through the Healthy Dogs class serum of FAVNT test confirmation without rabies IgG antibody).Test paper is kept flat on desktop, get the blood serum sample 50 μ L diluted and add serum process pad.25 DEG C leave standstill after 15 minutes, according to following situation result of determination: test paper is effective: occur purplish red colo(u)r streak at nature controlling line (C), illustrate that this test paper is effective; Negative: only at the upper appearance purplish red colo(u)r streak of nature controlling line (C), and colo(u)r streak does not appear in detection line (T) place; Positive: respectively to occur a purplish red colo(u)r streak at nature controlling line (C) and detection line (T) district, and colo(u)r streak color depth is directly proportional to antibody titer height, and antibody titer higher colo(u)r streak color is darker.The result display blood serum sample 1 of Fig. 2 is positive (test paper on the left of Fig. 2, all there is purplish red colo(u)r streak in nature controlling line (C) and detection line (T)), blood serum sample 2 is weak positive (the middle test paper of Fig. 2, there is purplish red colo(u)r streak in nature controlling line (C), there is more shallow purplish red colo(u)r streak in detection line (T)), blood serum sample 3 is negative sample (on the right side of Fig. 2, test paper, only has nature controlling line (C) to occur purplish red colo(u)r streak).
Specific detection: according to the immune-gold labeled Test paper operation instruction of immune animal hydrophobin IgG antibody, detect known hydrophobin positive serum, be positive.Detect hydrophobin (RABV) negative serum, CDV (CDV) positive serum, canine parvovirus (CPV) positive serum, canine coronavirus (CCV) positive serum, hepatitis infectiosa canis virus (CAV) positive serum, canine parainfluenza virus (CPIV) positive serum, canine infectious hepatitis virus (ICHV) positive serum, Feline Panleukopenia Virus (FPV) positive serum, is all negative (table 1).
Table 1
Sensitivity Detection: according to the immune-gold labeled Test paper operation instruction of hydrophobin IgG antibody, detects the serum of known hydrophobin neutralizing antibody (VNA >=1IU/mL), and testing result is positive.The critical serum of hydrophobin neutralizing antibody (0.5IU/mL≤VNA<1IU/mL), testing result is that positive ratio is greater than 75%.Detect the serum of hydrophobin neutralizing antibody (VNA<0.5IU/mL), testing result is negative (table 2).
Table 2
Although above done at large to describe to the present invention with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (7)
1. the immune-gold labeled Test paper of hydrophobin IgG antibody, is characterized in that, described Test paper is made up of serum process pad, the release of gold mark pad, nitrocellulose filter, adsorptive pads and backboard; Serum process pad, gold mark release pad, nitrocellulose filter and adsorptive pads mutually superpose successively and are pasted onto on backboard, mutually superpose 2-3mm between each several part; Gold mark release pad is coated with the Rabies Virus Antigen-colloidal gold composite of purifying; Nitrocellulose filter is coated with detection line and nature controlling line, described detection line is fixed with SPA, and described nature controlling line is fixed with the positive IgG of dog anti-rabies virus.
2. Test paper according to claim 1, is characterized in that, described serum process pad is that bag is by the glass fibre of serum treating fluid element film.
3. Test paper according to claim 2, is characterized in that, described serum treating fluid is the tetraborate containing 0.05M, the bovine serum albumin(BSA) of 1%, the 20mM phosphate buffer of the Sodium azide of 0.04% and the Tween-20 of 0.2%.
4. according to the Test paper in claim 1-3 described in any one, it is characterized in that, Rabies Virus Antigen-colloidal gold composite gold mark conserving liquid used is for containing 2-3%BSA, 1-2% sucrose, 0.5-1% trehalose, the 20mM Tris-HCl damping fluid of the pH 8.0-8.5 of 0.2%Tween-20 and 0.04% Sodium azide.
5. Test paper according to claim 4, is characterized in that, the concentration of described SPA is 0.8-1.2mg/ml, and the spray film consumption on nitrocellulose filter is 1.0 μ L/cm.
6. Test paper according to claim 5, is characterized in that, the concentration of the positive IgG of described dog anti-rabies virus is 1.4-1.5mg/ml, and the spray film consumption on nitrocellulose filter is 1.0 μ L/cm.
7. prepare a method for the Test paper in claim 1-6 described in any one, comprise the following steps:
(1) preparation and purification of Rabies Virus Antigen: the Rabies Virus Antigen being obtained purifying after utilizing zinc acetate to precipitate viral antigen by sucrose density gradient centrifugation; (2) Rabies Virus Antigen-colloidal gold composite is prepared; (3) preparation gold mark release pad; (4) on nitrocellulose filter, tested survey line and nature controlling line is wrapped; (5) by serum process pad, gold mark release pad, nitrocellulose filter and adsorptive pads successively mutually superposition be pasted onto on backboard, mutually superpose 2-3mm between each several part.
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| CN111562367A (en) * | 2020-06-15 | 2020-08-21 | 郑州方欣生物科技有限责任公司 | Novel urine coronavirus rapid detection kit, preparation method and use method |
| CN112051400A (en) * | 2020-09-03 | 2020-12-08 | 江苏美克医学技术有限公司 | Immunochromatography kit for detecting novel coronavirus neutralizing antibody and detection method |
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