CN102180967B - Method for extracting immunoglobulin A (IgA) of Przewalski's horse noninvasively - Google Patents
Method for extracting immunoglobulin A (IgA) of Przewalski's horse noninvasively Download PDFInfo
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Abstract
The invention discloses a method for extracting immunoglobulin A (IgA) of Przewalski's horse noninvasively. The method for extracting animal immunoglobulin comprises the following steps of: mixing animal excrement and extraction buffer to obtain a mixture; and centrifuging the mixture to obtain supernatant, namely immunoglobulin in the animal, wherein the extraction buffer consists of phosphate buffered saline (PBS) and Tween-20. The method for extracting the immunoglobulin of the Przewalski's horse noninvasively is simple and high-efficiency. In the method, the fresh excrement of the Przewalski's horse is collected and dissolved in the extraction buffer added with the Tween-20 at certain concentration, and the supernatant is obtained through two-step centrifugation, namely the high-concentration immunoglobulin of the Przewalski's horse can be extracted. The method is easy to operate and high in extraction efficiency.
Description
Technical field
The present invention relates to the method for a kind of non-damage extraction przhevalski's horse Immunoglobulin IgA in the animal immunology field.
Background technology
Przhevalski's horse (Equus przewalskii) once was widely distributed in area, grassland, Eurasia, open-air extinction of the middle of last century.Existing przhevalski's horse is the Western adventurer caught and transported to European stable breeding in Chinese and Mongolia western part in the 19th-century end offspring.China is in 1985 from drawing back these species abroad and having set up the center of breeding.Calendar year 2001, wilderness area in the OK a karaoke club wheat of Xinjiang implemented the Gui Yehua of putting of przhevalski's horse.The survival condition of species is heavily introduced in monitoring and assessment, and implements necessary manually managing and protecting, and be to guarantee to put the key link of returning the species long-term surviving and setting up wild stocks, but the work of these aspects is carried out seldom at present.Trace it to its cause, at first, the tissue samples that obtains the free living animal is very difficult, and regular or irregular collection research sample almost is impossible; Moreover catching and gathering animal blood sample itself is exactly a very strong stimulating factor, the orthobiosis of meeting severe jamming animal.Obviously, be subject to that sample obtains and the restriction of the aspect such as mensuration, there is not yet so far and put the immune physiological research of returning species.Therefore, as the physiological important indicator of monitoring animal body immunity, the extraction of immunoglobulin (Ig) and detection return species research particularly important to putting.
Obviously, for monitoring przhevalski's horse immunity physiological situation, traditional extraction blood sampling approach is no longer applicable, and urgent need will develop the method that immunoglobulin (Ig) was extracted and detected in a kind of simple, efficient, non-damage.Usually the non-damage sampling mainly is to gather target animal urine and ight soil, but its space for activities of przhevalski's horse is wide, and mostly be meadow or Sandy land, the urine of discharging will soon permeate the ground, be not easy to collect, and glomerular filtration membrane can not see through sphaeroprotein, so the immunoglobulin (Ig) that contains in the intact animal urine seldom.The gastrointestinal tract mucosa lamina propria can produce secretory immunoglobulin, enters also can carry part in the GI bile from the immunoglobulin (Ig) in the tissue juice.For these reasons, compare with gathering urine, the ight soil approach is the method that a desirable non-damage detects the przhevalski's horse immunoglobulin (Ig).
Summary of the invention
An object of the present invention is to provide a kind of method of extracting the animal immune sphaeroprotein.
A kind of method of extracting the animal immune sphaeroprotein provided by the present invention may further comprise the steps:
Animal excrement are mixed with Extraction buffer, obtain mixture; Described mixture is centrifugal, get supernatant liquor, namely obtain described animal immune sphaeroprotein; Described Extraction buffer is comprised of PBS damping fluid and tween 20.
In the described Extraction buffer, the concentration of volume percent of described tween 20 is 0.01%-0.5% or 0.03%-0.07% or 0.03% or 0.04% or 0.05% or 0.06% or 0.07%.
Described centrifugal method may further comprise the steps: it is centrifugal that described mixture is carried out the first step, gets supernatant liquor, is denoted as supernatant liquor I; It is centrifugal that described supernatant liquor I is carried out second step; The centrifugal rotating speed of the described the first step is that 2000rpm, temperature are that 25 ℃ and time are 20min; The centrifugal rotating speed of described second step is that 10000rpm, temperature are that 4 ℃ or 25 ℃ and time are 10min.
Before described mixture is centrifugal, comprise the step with described mixture mixing.
The method of described mixing for described mixture with the vortex instrument 10min that under 1200rpm, vibrates.
Described PBS damping fluid is comprised of sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic and water, and the concentration of each composition is respectively sodium-chlor 8.5g/L, SODIUM PHOSPHATE, MONOBASIC 0.3g/L and Sodium phosphate dibasic 2.2g/L.
The pH value of described Extraction buffer is 7.2.
The consumption of described Extraction buffer is: add the described Extraction buffer of 10mL in the described animal excrement of every 1g.
Described animal excrement are the animal excrement behind the mixing.
Described animal is przhevalski's horse;
Described immunoglobulin (Ig) is Immunoglobulin IgA.
The invention provides simple, the efficient method that a kind of non-damage extracts the przhevalski's horse immunoglobulin (Ig).By gathering the przhevalski's horse fresh excreta, fresh excreta is dissolved in the Extraction buffer that is added with finite concentration Tween-20, carry out two step centrifuging and taking supernatant liquors, can extract the przhevalski's horse immunoglobulin (Ig) that obtains higher concentration.This method is simple to operate and extraction efficiency is higher.
Description of drawings
Fig. 1 contains the Extraction buffer of different concns Tween-20 to the impact of IgA extracted amount in the przhevalski's horse ight soil.
Fig. 2 is that protamine is on the impact of IgA extracted amount in the przhevalski's horse ight soil.
Fig. 3 is that different centrifuging temperatures are on the impact of ight soil IgA extracted amount.
Fig. 4 is the typical curve of IgA concentration and the OD value in standard substance hole.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
One, tween concentration is on the impact of immunoglobulin (Ig) extracted amount in the przhevalski's horse ight soil
(1) non-damage extracts the przhevalski's horse immunoglobulin (Ig)
1, gathers the excrement sample
(the Xinjiang przhevalski's horse is bred research centre (88 ° of 45 ' E to object observing animal przhevalski's horse of same time period of every day (Equus przewalskii), 44 ° 10 ' N)), discharge fresh excreta in case find it, gather immediately, gather respectively the ight soil of female przhevalski's horse and male przhevalski's horse, sample objects is 10 adult wild horses individual (5
), gather for three days on end the fresh excreta of target individual, respectively gather in every morning and afternoon 1 time, be respectively charged in the valve bag female przhevalski's horse ight soil and the male przhevalski's horse ight soil that gathers and numbering, in time put into freezing preservation under-20 ℃ of conditions.
2, preparation Extraction buffer
(1) preparation PBS buffered soln
Take by weighing 8.5g sodium-chlor, 0.3g SODIUM PHOSPHATE, MONOBASIC and 2.2g Sodium phosphate dibasic constant volume in the 1000ml ultrapure water, the mixing that vibrates gently after dissolving, is mixed with the pH value and is 7.2 PBS buffered soln.The concentration of sodium-chlor, SODIUM PHOSPHATE, MONOBASIC and Sodium phosphate dibasic is respectively in the described PBS buffered soln: sodium-chlor 8.5g/L, SODIUM PHOSPHATE, MONOBASIC 0.3g/L and Sodium phosphate dibasic 2.2g/L.
(2) preparation Extraction buffer
Add respectively the tween 20 (Tween-20) of different amounts (see Table 1 and table 2) in the above-mentioned PBS buffered soln of every 1000ml, vibrate gently behind the mixing, preparation obtains PBST (PBS+Tween-20) aqueous solution of 18 different Tween-20 concentration (see Table 1 and table 2), as the experimental group Extraction buffer.Simultaneously organize in contrast Extraction buffer with above-mentioned PBS buffered soln.
The addition of Tween-20 and concentration (0.05%-0.50%) in the table 1 experimental group Extraction buffer
The addition of Tween-20 and concentration (0.01%-0.09%) in the table 2 experimental group Extraction buffer
3, extract the przhevalski's horse immunoglobulin (Ig)
Female przhevalski's horse ight soil and male przhevalski's horse ight soil difference mixing (mixing method: take by weighing each 100g of excrement sample that gather every day in each individual three days with step 1 collection, pulverizer (23000r) is pulverized 10min, divide pouch to save backup in the sample of mixing) after, take by weighing respectively female przhevalski's horse ight soil and male przhevalski's horse ight soil 1g in the 15ml plastic centrifuge tube, add respectively the experimental group Extraction buffer and the control group Extraction buffer that prepare in the 10ml step 2, at 1200rpm vibration 10min mixing, obtain mixture with the vortex instrument; Be that 2000rpm and temperature be 25 ℃ condition under centrifugal 20min with low speed centrifuge (LDZ5-2 type table-type low-speed whizzer) at rotating speed with described mixture, get supernatant liquor I1ml in the 2ml plastic centrifuge tube, be that 10000rpm and temperature be 4 ℃ lower centrifugal 10mins with high speed freezing centrifuge (Heal ForceNeofuge 15R table-type high-speed refrigerated centrifuge) at rotating speed with described supernatant liquor I, get supernatant liquor II, namely obtain immunoglobulin (Ig).Get supernatant liquor II in the 2ml plastic centrifuge tube, to be measured in-20 ℃ of freezing preservations.Each sample is established three repetitions.
(2) detect
Purchase is strictly pressed the operation of test kit specification sheets for the single-minded enzyme-linked immunosorbent assay kit of the immunoglobulin (Ig) that will detect.
Used kit is that the Holg IgA enzyme-linked immunosorbent assay kit that bio tech ltd is produced is more ground in Shanghai.The significant parameter of test kit is:
(1) measurement range: 5 μ g/ml-100 μ g/ml;
(2) sensitivity:＜2.5 μ g/ml;
(3) average recovery rate: 96-101%;
(4) variation coefficient: in batch＜6%;
(5) specificity: IgA and IgM, IgG and IgE cross reaction are all＜0.01%.
Contain following reagent in this test kit: coating antigen is that horse IgA antibody, the standard substance of purifying are that horse IgA, ELIAS secondary antibody are IgA antibody, washings (the 50mM Tris of HRP mark, 0.14M NaCl, 0.05%Tween-20, pH8.0) and stop buffer (0.18M H
2SO
4) and sample diluting liquid (50mM Tris, 0.14M NaCl, 1%BSA, 0.05%Tween-20).
Enzyme plate is carried by test kit.
Concrete detecting step is as follows:
1, the dilution of standard substance and application of sample
Establish 10 holes, standard substance hole at the coated plate of enzyme mark, in first, second hole, add respectively standard substance 100 μ l, then in first, second hole, add standard substance diluent 50 μ l, mixing; Then from the first hole, the second hole, respectively get 100 μ l and be added to respectively the 3rd hole and the 4th hole, add respectively again standard substance diluent 50 μ l, mixing in the 3rd, the 4th hole; Then in the 3rd hole and the 4th hole, respectively get 50 μ l first and discard, respectively get again 50 μ l and be added to respectively in the 5th, the 6th hole, in the 5th, the 6th hole, add respectively again standard substance diluent 50ul, mixing; Get respectively from the 5th, the 6th hole behind the mixing that 50 μ l are added to respectively the 7th, in the octal, again the 7th, add respectively standard substance diluent 50 μ l in the octal, behind the mixing from the 7th, get respectively 50 μ l the octal and be added in the 9th, the tenth hole, add respectively again standard substance diluent 50 μ l in the 90 hole, from the 90 hole, respectively get 50 μ l behind the mixing and discard.(each hole application of sample amount all is 50 μ l after the dilution, and concentration is respectively 90 μ g/ml, 60 μ g/ml, 30 μ g/ml, 15 μ g/ml and 7.5 μ g/ml).
2, application of sample
Establish respectively blank well (the blank hole does not add sample and enzyme marking reagent, and all the other each step operations are identical), testing sample hole.On the coated plate of enzyme mark, add first sample diluent (50mM Tris in the testing sample hole, 0.14M NaCl, 1%BSA, 0.05%Tween-20) 40 μ l, and then add testing sample (being that above-mentioned steps () is extracted experimental group and the control group immunoglobulin (Ig) sample that obtains) 10 μ l (the final extent of dilution of sample is 5 times).Sample is added on bottom, enzyme plate hole, does not touch hole wall as far as possible, rock gently mixing.
3, incubation
With rearmounted 37 ℃ of incubations of shrouding film shrouding 30 minutes.
4, dosing
With 30 times of concentrated cleaning solutions (0.05%Tween-20, pH 8.0 for 50mM Tris, 0.14M NaCl) with for subsequent use after 30 times of dilutions of distilled water.
5, washing
Carefully take the shrouding film off, discard liquid, dry, washings is filled it up with in every hole, leaves standstill after 30 seconds to discard, and so repeats 5 times, pats dry.
6, enzyme-added
Every hole adds enzyme marking reagent 50 μ l, except the blank well.
7, incubation
Operation is with step 3.
8, washing
Operation is with step 5.
9, colour developing
Every hole adds first developer A 50 μ l, adds developer B50 μ l again, the light shaking mixing, and 37 ℃ of lucifuges developed the color 15 minutes.
10, stop
Every hole adds stop buffer 50 μ l, termination reaction (this moment blueness immediately yellowing).
11, measure
With the blank well zeroing, the 450nm wavelength sequentially detects the absorbancy (OD value) in each hole.Mensuration should be after adding stop buffer be carried out with interior in 15 minutes.
According to the OD value (table 3) that application of sample concentration value (μ g/ml) and each hole in standard substance hole records, production standard curve (Fig. 4) is according to typical curve equation y=-0.45186+0.11139X-0.00079X
2(R
2=0.97963), obtain the concentration (μ g/ml) of IgA in the sample well, again according to formula: concentration (μ g/ml) the * 5*10ml/ excrement sample of IgA heavy (g) in the amount of IgA (μ g/g)=sample well in every gram ight soil, converting obtains the amount (μ g/g) of IgA in every gram ight soil.
The IgA application of sample concentration value in table 3 standard substance hole and the OD value in hole
| IgA concentration (μ g/m1) | The OD value |
| 7.5 | 0.4160 |
| 15 | 0.8065 |
| 30 | 2.4315 |
| 60 | 3.2475 |
| 90 | 3.1985 |
The result is as follows:
The concentration that step () is extracted the experimental group obtain and control group immunoglobulin (Ig) sees Table 4 and Fig. 1.The result gets the mean value that all female przhevalski's horse immunoglobulin (Ig)s extract immunoglobulin (Ig) amount in the product, gets the mean value that all male przhevalski's horse immunoglobulin (Ig)s extract immunoglobulin (Ig) amount in the product.
The extracted amount of IgA in the every gram ight soil of table 4 experimental group and control group przhevalski's horse
From Fig. 1 A as seen, when Tween-20 concentration was 0.05% in the used Extraction buffer of experimental group, the Immunoglobulin IgA extracted amount was the highest, is significantly higher than the Extraction buffer of other Tween-20 concentration and the extracted amount of control group Extraction buffer.0 to 0.1%Tween-20 concentration gradient section is encrypted, from Fig. 1 B as seen, Tween-20 concentration is in 0.03%-0.07% (v/v) scope in the Extraction buffer, the extracted amount of IgA is higher.Therefore, the optimal concentration scope of determining Tween-20 in the Extraction buffer is 0.03%-0.07% (v/v).Female przhevalski's horse immunoglobulin (Ig) concentration and male przhevalski's horse immunoglobulin (Ig) concentration all do not have significant difference in each group.
Two, protamine is on the impact of immunoglobulin (Ig) extracted amount in the przhevalski's horse ight soil
(1) non-damage extracts the przhevalski's horse immunoglobulin (Ig)
1, gathers the excrement sample
Identical with the collection excrement quadrat method of step 1.
2, preparation Extraction buffer
(1) preparation PBS buffered soln
Identical with the compound method of step 1.
(2) preparation Extraction buffer
Add 0.5mL Tween-20 in the above-mentioned PBS buffered soln of every 1000ml, behind the mixing that vibrates gently, preparation obtains Extraction buffer.Tween-20 concentration is 0.05% (v/v) in this Extraction buffer.
3, preparation protamine solution
Take by weighing 0.8g protamine powder and be dissolved in the 10ml ultrapure water, preparation obtain the protamine aqueous solution.
4, extract the przhevalski's horse immunoglobulin (Ig)
Extracting method is with basic identical described in the experiment one, and different is: (1) at the centrifugal front adding protamine aqueous solution, (2) Extraction buffer forms, and is specific as follows:
(1) adds the protamine aqueous solution that above-mentioned steps 3 is prepared
The add-on of the protamine aqueous solution is: add 100ul before the first step is centrifugal, add 10ul before second step is centrifugal.Experiment is divided into following 4 groups: the 1st group, namely control group does not add the protamine aqueous solution before two steps are centrifugal; The 2nd group, add the protamine aqueous solution in the centrifugal forward direction supernatant liquor of second step; The 3rd group, all add the protamine aqueous solution before two steps are centrifugal, namely add in the centrifugal forward direction mixture of the first step and add the protamine aqueous solution in the protamine aqueous solution and the centrifugal forward direction supernatant liquor of second step; The 4th group, add the protamine aqueous solution in the centrifugal forward direction mixture of the first step.
(2) Extraction buffer is that the Tween-20 concentration for preparing in the step 2 is the Extraction buffer of 0.05% (v/v).
Each sample is established three repetitions.
(2) detect
Identical with the detection method of experiment one.
The result is as follows:
Step (one) is extracted the concentration of 4 groups of immunoglobulin (Ig)s that obtain and is seen Fig. 2, and 1,2,3 and 4 represent respectively the 1st group, the 2nd group, the 3rd group and the 4th group of female przhevalski's horse immunoglobulin (Ig) and male przhevalski's horse immunoglobulin (Ig) that extracts among Fig. 2.
As seen from Figure 2, the 1st group (being control group), the 2nd group and the 4th group extract the immunoglobulin (Ig) concentration that obtains does not have significant difference, and the immunoglobulin (Ig) concentration that the 3rd group (all adding the protamine aqueous solution before two steps are centrifugal) obtains significantly is lower than other three groups.This result shows, can not increase the extracted amount of immunoglobulin (Ig) at the leaching process adding protamine of ight soil immunoglobulin (Ig).Female przhevalski's horse immunoglobulin (Ig) concentration and male przhevalski's horse immunoglobulin (Ig) concentration all do not have significant difference in each group.
Three, different centrifugation are on the impact of ight soil IgA extracted amount
(1) non-damage extracts the przhevalski's horse immunoglobulin (Ig)
1, gathers the excrement sample
Identical with the collection excrement quadrat method of experiment one.
2, preparation Extraction buffer
(1) preparation PBS buffered soln
Identical with the compound method of experiment one.
(2) preparation Extraction buffer
Identical with the compound method of experiment two, obtain the Extraction buffer that Tween-20 concentration is 0.05% (v/v).
3, extract the przhevalski's horse immunoglobulin (Ig)
Extracting method is basic identical with the extracting method in the experiment one, and different is that centrifugation is different, specific as follows:
(1) the female przhevalski's horse immunoglobulin (Ig) that gathers in the extraction step 1.
(2) different according to the centrifugal centrifugation of second step, be divided into two groups: first group: second step is centrifugal to be common high speed centrifugation, uses Hunan instrument TG16-WS table model high speed centrifuge, is room temperature (25 ℃) at centrifuging temperature, rotating speed is under the condition of 10000rpm, centrifugal 10min; Second group: second step is centrifugal to be the high speed frozen centrifugation, uses Heal ForceNeofuge 15R table-type high-speed refrigerated centrifuge, is 4 ℃ at centrifuging temperature, and rotating speed is under the condition of 10000rpm, centrifugal 10min.
Every kind of centrifugation is established five repetitions, results averaged.
(2) detect
Identical with the detection method of experiment one.
The result is as follows:
Step (one) is extracted the concentration of two groups of immunoglobulin (Ig)s that obtain and is seen Fig. 3, and A represents the female przhevalski's horse immunoglobulin (Ig) that first group of common high speed centrifugation extracts among Fig. 3; B represents the female przhevalski's horse immunoglobulin (Ig) that second group of high speed frozen centrifugation extracts.
As seen from Figure 3, first group and second group extract the immunoglobulin (Ig) concentration that obtains does not have significant difference.This result shows that the centrifugal high speed frozen centrifugation of second step and common high speed centrifugation do not have significant difference to the impact of the extraction effect of immunoglobulin (Ig).Therefore, the high speed centrifugation step can be used common supercentrifuge, compares with the cryogenic freezing whizzer, and the former has low price, little, the easy to operate advantage of volume, and mini portable high-speed whizzer is also very general, more convenient experimental implementation.
Claims (5)
1. method of extracting the przhevalski's horse Immunoglobulin IgA may further comprise the steps:
Przhevalski's horse ight soil is mixed with Extraction buffer, obtain mixture; Described mixture is centrifugal, get supernatant liquor, namely obtain described przhevalski's horse Immunoglobulin IgA; Described Extraction buffer is comprised of PBS damping fluid and tween 20;
In the described Extraction buffer, the concentration of volume percent of described tween 20 is 0.03%-0.07%;
Described centrifugal method may further comprise the steps: it is centrifugal that described mixture is carried out the first step, gets supernatant liquor, is denoted as the supernatant liquor I; It is centrifugal that described supernatant liquor I is carried out second step; The centrifugal rotating speed of the described the first step is that 2000rpm, temperature are that 25 ℃ and time are 20min; The centrifugal rotating speed of described second step is that 10000rpm, temperature are that 4 ℃ or 25 ℃ and time are 10min;
The consumption of described Extraction buffer is: add the described Extraction buffer of 10mL in the described przhevalski's horse ight soil of every 1g;
Described PBS damping fluid is comprised of sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic and water, and the concentration of each composition is respectively sodium-chlor 8.5g/L, SODIUM PHOSPHATE, MONOBASIC 0.3g/L and Sodium phosphate dibasic 2.2g/L;
The pH value of described PBS buffered soln is 7.2.
2. method according to claim 1, it is characterized in that: in the described Extraction buffer, the concentration of volume percent of described tween 20 is 0.03% or 0.04% or 0.05% or 0.06% or 0.07%.
3. method according to claim 1 and 2 is characterized in that: before described mixture is centrifugal, comprise the step with described mixture mixing.
4. method according to claim 3 is characterized in that: the method for described mixing for described mixture with the vortex instrument 10min that under 1200rpm, vibrates.
5. method according to claim 1, it is characterized in that: described przhevalski's horse ight soil is the przhevalski's horse ight soil behind the mixing.
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| CN1401077A (en) * | 2000-02-16 | 2003-03-05 | 国际试药株式会社 | Method for examination of feces ocoult blood |
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