CN102590496A - Rapid snake wound identification method - Google Patents
Rapid snake wound identification method Download PDFInfo
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- CN102590496A CN102590496A CN2012100327550A CN201210032755A CN102590496A CN 102590496 A CN102590496 A CN 102590496A CN 2012100327550 A CN2012100327550 A CN 2012100327550A CN 201210032755 A CN201210032755 A CN 201210032755A CN 102590496 A CN102590496 A CN 102590496A
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Abstract
The invention discloses a rapid snake wound identification method using an enzyme mark strip as a carrier, which includes that an enzyme mark hole is coated by a snake venom species specific antibody, blood serum collected from a patient with a snake wound is dropped in the hole, and snake wound types can be judged in a short time. Screening of various specific antibodies and preparation of the enzyme mark strip and auxiliary reagents in the testing process are low in cost; a testing process is simple without the help of instruments, and testers do not need abundant clinical diagnosis experience; testing time is short, and the time from taking out the enzyme mark strip under low temperature to result developing is about 40-45 minutes; accuracy is high, an AB-double-antibody sandwich method can effectively guarantee combination of snake venom and specific antibodies, biotin-avidin amplifies signals effectively, and developing results can be observed directly through naked eyes.
Description
Affiliated technical field
The invention belongs to the snakebite discrimination method, be specially the method that enzyme mark bar that a kind of utilization is coated with the snake venom specific antibody is differentiated snakebite fast.
Background technology
Tradition snakebite type judges and relies on the snakebite analysis of clinical symptom to realize, because the doctor lacks experience, clinical symptoms is not obvious or diffused to and can't judge, will cause erroneous judgement, delays the snakebite treatment.Be the defective of avoiding traditional snakebite to judge; More existing countries carry out the research for preparing snakebite quick diagnosis product with the snake venom specific antibody in the world wide; The dependent diagnostic product utilization can be judged the snakebite type at short notice exactly by bite patient's body fluid of snake class, thereby ensures the accurate use of antivenin.Can both be used to differentiate snake venom technically although there are some researches show methods such as collaurum, latex agglutination, thereby confirm the snakebite type,, not have product to be able to practical application so far as yet in view of reasons such as the high perhaps complicated operations of cost.Enzyme-linked immune analytic method (ELISA) utilization in snake venom is differentiated is the most extensive, and wherein Australia's production snakebite quick diagnosis reagent kit is used for Clinical detection, for positive contribution has been made in the quick judgement and the treatment of snakebite.China is snake class resource big country, and areas to the south, the Changjiang river widespread distribution has all kinds of poisonous snakes, in the movable busy season in every year, the mankind's activity of locality has been caused serious harm.But up to now, domestic judgement to the snakebite type still relies on its clinical symptoms performance, and shortage is quick, simple, effective diagnostic method is used for clinical auxiliary judgment snakebite.
Summary of the invention
In view of the above problems, the method that the present invention provides a kind of snakebite to differentiate fast is characterized in that carrying out as follows:
1. prepare anti-snake venom immunoglobulin (Ig) (IgG).Choose 6 kinds of snake venom of required discriminating, after 20mM PBS dissolving, the centrifuging and taking supernatant, and measure protein content.Subcutaneous multi-point injection rat prepares 6 kinds of thick serum of the anti-snake venom of polyclone.Thick serum is removed non-protein component through ammonium sulfate precipitation earlier, through Hitrap protein IgG post purifying, obtains 6 kinds of anti-snake venom IgG again.
2. screen snake venom species specificity IgG.Snake venom is coupled on the CNBr-activated sepharose 4B affinity media of activation pipe in the centrifugal concentration tube of packing into after equivalent is appointed the moistening affinity media got after 5 kinds of snake venom couplings 0.3g is mixed altogether.Under the room temperature, the antiserum IgG of every kind of snake venom of 500 μ g respectively with all the other affinity medias of planting the snake venom couplings at horizontal shaking table mixing 2 hours leniently.Centrifugal subsequently, collect the eluate that does not combine component with centrifugal concentration tube outer tube, be the species specificity IgG that this kind snake venom is different from other 5 kinds of snake venom.
3. snake venom specific IgG biotinylation.After specific antibody and biotin weight ratio were mixed at 1: 5, the room temperature concussion combined 2 hours, and dialysis is subsequently removed and do not combined biotin, obtains the biotinylation specific IgG.
4. detecting enzyme mark bar makes.With 8 hole enzyme mark bars is carrier, is furnished with " T " shape support.6 sample detection holes are set, 1 positive and 1 negative control hole on the enzyme mark bar.Each aperture 2% cow's serum sealing, the rinsing control is done the back and is preserved at-20 degree.
5. snakebite is differentiated fast.During detection, gather snakebite patient wound site effluent, drop to and respectively detect the hole, pass through the combination of biotinylation specific antibody, Avidin HRP successively after, add the TMB colour developing, accurately judge the snakebite type according to the positive or negative findings that detects the hole.
Above-mentioned a kind of snakebite method for quick identification has following advantage:
1. cost is low.The preparation cost of the screening of each strain specific antibodies, test-strips and test process auxiliary reagent is low, need not expensive consumptive material and supports.
2. simple to operate.Whole testing process required component is simple, volume is little, need not by instrument, the clinical diagnosis experience that the testing staff need not to enrich.
3. detection time is short.Take out down test-strips from low temperature and finish, time spent 40-45 minute, in snakebite scene or patient are taken place and transport in the process and just can accomplish to colour developing.
4. accuracy is high.By the antigen and antibody specific combination principle, can guarantee that effectively snake venom is attached on the specific antibody in the testing sample.By the biotin-avidin combination principle, double antibodies sandwich method that can the signal of final color developing effect is more traditional is amplified 4 times, guarantees that effectively positive test symbol can accurately be discerned by naked eyes.
The present invention has listed Hangzhou key lab of colleges and universities directly under the jurisdiction of a municipal government scientific and technical innovation project (contract No. of the project: 20080433T03) with Zhejiang Province science and technology bureau major scientific and technological project project (contract No. of the project: 2009C13045) in.
Description of drawings
Fig. 1 is the structural representation of 8 hole enzyme mark bars.
Fig. 2 is the vertical synoptic diagram that is attached with " T " shape support of enzyme mark bar.
Fig. 3 is the synoptic diagram that nonpoisonous snake is bitten among the embodiment.
Fig. 4 is the synoptic diagram that Bungarus multicinctus is bitten among the embodiment.
Fig. 5 is the synoptic diagram that cobra is bitten among the embodiment.
Fig. 6 is the synoptic diagram of agkistrodon acutus bite among the embodiment.
Fig. 7 is the synoptic diagram that pallas pit viper is bitten among the embodiment.
Fig. 8 is the synoptic diagram that green bamboo snake is bitten among the embodiment.
Fig. 9 is the synoptic diagram that solder horn bites among the embodiment.
Figure 10 is the synoptic diagram that test-strips lost efficacy among the embodiment.
1 is the positive control hole among Fig. 1,2; The 2nd, negative control hole; 3-8 is the sample detection hole.
Embodiment
Embodiment:
1. prepare anti-snake venom immunoglobulin (Ig) (IgG).Choose 6 kinds of snake venom (cobra, Bungarus multicinctus, long-noded pit viper, pallas pit viper, green bamboo snake and solder horn snake venom) of required discriminating, after 20mM PBS dissolving, the centrifuging and taking supernatant, and measure protein content.Subcutaneous multi-point injection rat prepares 6 kinds of thick serum of the anti-snake venom of polyclone.Thick serum is removed non-protein component through ammonium sulfate precipitation earlier, through Hitrap protein IgG post purifying, obtains 6 kinds of anti-snake venom IgG again.
2. screen snake venom species specificity IgG.Snake venom is coupled on the CNBr-activated sepharose 4B affinity media of activation pipe in the centrifugal concentration tube of packing into after equivalent is appointed the moistening affinity media got after 5 kinds of snake venom couplings 0.3g is mixed altogether.Under the room temperature, the antiserum IgG of every kind of snake venom of 500 μ g respectively with all the other affinity medias of planting the snake venom couplings at horizontal shaking table mixing 2 hours leniently.Centrifugal subsequently, collect the eluate that does not combine component with centrifugal concentration tube outer tube, be the species specificity IgG that this kind snake venom is different from other 5 kinds of snake venom.
3. snake venom specific IgG biotinylation.After specific antibody and biotin weight ratio were mixed at 1: 5, the room temperature concussion combined 2 hours, and dialysis is subsequently removed and do not combined biotin, obtains the biotinylation specific IgG.
4. detecting enzyme mark bar makes.With 8 hole enzyme mark bars is carrier, is furnished with " T " shape support.8 holes encapsulate 6 kinds of snake venom specific antibody mixed liquors (1), coating buffer (2), bungarotoxin specific IgG (3), cobra venom specific IgG (4), agkistrodon acutus venom specific IgG (5), short-tail pallas pit viper specific IgG (6), trimeresurus stejnegeri snake venom specific antibody (7) and solder horn snake venom specific antibody (8) successively; The antibody application of sample amount in every hole is 200ng, combines 2 hours at 37 degree.Each aperture 2% cow's serum sealing, the rinsing control is done the back and is preserved at-20 degree.
5. snakebite is differentiated fast.During detection, take out test-strips, room temperature left standstill 5 minutes.In control wells 1,2, add the mixed liquor of 6 kinds of snake venom, in detecting hole 3-8, evenly splash into snakebite patient body fluid (wound spillage, blood or urine), incubated at room 10 minutes.Remove liquid, do with control after the quick rinsing of clear water 3 times.The biotinylation specific IgG mixed liquor that in control wells, adds 6 kinds of snake venom subsequently; In detecting the hole, add biotinylated bungarotoxin specific IgG, cobra venom specific IgG, agkistrodon acutus venom specific IgG, cobra-venom specific IgG, trimeresurus stejnegeri snake venom specific IgG and solder horn snake venom specific IgG respectively, incubated at room 10 minutes.Remove liquid, do with control after the quick rinsing of clear water 3 times.The HRP that adds Avidinization, incubated at room 10 minutes.Remove liquid, with the quick rinsing of clear water 3 times.Add TMB colour developing liquid subsequently, color development at room temperature 5-10 minute, judge the snakebite type.Wherein, if only porose 1 colour developing then is nonpoisonous snake bite (Fig. 3); If develop the color simultaneously in hole 1,3, then be Bungarus multicinctus bite (Fig. 4); If develop the color simultaneously in hole 1,4, then be cobra bite (Fig. 5); If develop the color simultaneously in hole 1,5, then be agkistrodon acutus bite (Fig. 6); If develop the color simultaneously in hole 1,6, then be short-tail Pallas pit viper bite (Fig. 7); If develop the color simultaneously in hole 1,7, then be green bamboo snake bite (Fig. 8); If develop the color simultaneously in hole 1,8, then be solder horn bite (Fig. 9); If each Kong Jun does not develop the color, detector bar inefficacy (see figure 10) then should more renew test again after the test-strips.
Claims (3)
1. the method differentiated fast of a snakebite, its characterization step is to carry out as follows:
(1) preparation anti-snake venom immunoglobulin (Ig) (IgG).Choose 6 kinds of snake venom (cobra, Bungarus multicinctus, long-noded pit viper, pallas pit viper, green bamboo snake and solder horn snake venom) of required discriminating, subcutaneous multi-point injection rat prepares 6 kinds of thick serum of the anti-snake venom of polyclone.Thick serum is removed non-protein component through ammonium sulfate precipitation earlier, through Hitrap protein IgG post purifying, obtains anti-snake venom IgG again.
(2) screening snake venom species specificity IgG.Snake venom is coupled on the CNBr-activated sepharose 4B affinity media of activation pipe in the centrifugal concentration tube of packing into after equivalent is appointed the moistening affinity media got after 5 kinds of snake venom couplings 0.3g is mixed altogether.Under the room temperature, the antiserum IgG of every kind of snake venom of 500 μ g respectively with all the other affinity medias of planting the snake venom couplings at horizontal shaking table mixing 2 hours leniently.Centrifugal subsequently, collect the eluate that does not combine component with centrifugal concentration tube outer tube, be the species specificity IgG that this kind snake venom is different from other 5 kinds of snake venom.
(3) snake venom specific IgG biotinylation.After specific antibody and biotin weight ratio were mixed at 1: 5, the room temperature concussion combined 2 hours, and dialysis is subsequently removed and do not combined biotin, obtains the biotinylation specific IgG.
(4) detecting enzyme mark bar makes.With 8 hole enzyme mark bars is carrier, is furnished with " T " shape support.6 sample detection holes are set, 1 positive and 1 negative control hole on the enzyme mark bar.Each aperture 2% cow's serum sealing, the rinsing control is done the back and is preserved at-20 degree.
(5) snakebite is differentiated fast.During detection, gather snakebite patient wound site effluent, drop to and respectively detect the hole, pass through the combination of biotinylation specific antibody, Avidin HRP successively after, add the TMB colour developing, accurately judge the snakebite type according to the positive or negative findings that detects the hole.
2. the method for differentiating fast according to the said a kind of snakebite of claim 1; It is characterized in that: 8 holes in the said step (4) encapsulate 6 kinds of snake venom specific antibody mixed liquors (1), coating buffer (2), bungarotoxin specific IgG (3), cobra venom specific IgG (4), agkistrodon acutus venom specific IgG (5), short-tail pallas pit viper specific IgG (6), trimeresurus stejnegeri snake venom specific antibody (7) and solder horn snake venom specific antibody (8) respectively, and every hole antibody amount is 200ng.
3. the method for differentiating fast according to the said a kind of snakebite of claim 1 is characterized in that: in enzyme mark hole, encapsulate 6 kinds of positive contrasts of snake venom specific antibody mixed liquor (6) in the said step (4), in enzyme mark hole, add the negative contrast of coating buffer (5).
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| CN2012100327550A CN102590496A (en) | 2012-02-13 | 2012-02-13 | Rapid snake wound identification method |
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| CN2012100327550A CN102590496A (en) | 2012-02-13 | 2012-02-13 | Rapid snake wound identification method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113569985A (en) * | 2021-08-18 | 2021-10-29 | 梧州市中医医院 | Intelligent recognition system for bite of snake head or green bamboo snake |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
-
2012
- 2012-02-13 CN CN2012100327550A patent/CN102590496A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11609042B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11740019B2 (en) | 2019-03-14 | 2023-08-29 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11747082B2 (en) | 2019-03-14 | 2023-09-05 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11994343B2 (en) | 2019-03-14 | 2024-05-28 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| CN113569985A (en) * | 2021-08-18 | 2021-10-29 | 梧州市中医医院 | Intelligent recognition system for bite of snake head or green bamboo snake |
| CN113569985B (en) * | 2021-08-18 | 2023-08-22 | 梧州市中医医院 | Intelligent recognition system for bites of iron head snakes or bamboo leaf snakes |
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Application publication date: 20120718 |