CN113533742A - Method for determining IgG absorption efficiency of newborn lambs - Google Patents
Method for determining IgG absorption efficiency of newborn lambs Download PDFInfo
- Publication number
- CN113533742A CN113533742A CN202110805622.1A CN202110805622A CN113533742A CN 113533742 A CN113533742 A CN 113533742A CN 202110805622 A CN202110805622 A CN 202110805622A CN 113533742 A CN113533742 A CN 113533742A
- Authority
- CN
- China
- Prior art keywords
- igg
- bovine
- bovine colostrum
- colostrum
- lambs
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000283903 Ovis aries Species 0.000 title claims abstract description 50
- 238000010521 absorption reaction Methods 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 27
- 241000283690 Bos taurus Species 0.000 claims abstract description 173
- 210000003022 colostrum Anatomy 0.000 claims abstract description 140
- 235000021277 colostrum Nutrition 0.000 claims abstract description 140
- 210000002966 serum Anatomy 0.000 claims abstract description 43
- 229920001817 Agar Polymers 0.000 claims description 50
- 239000008272 agar Substances 0.000 claims description 50
- 238000001556 precipitation Methods 0.000 claims description 44
- 239000012895 dilution Substances 0.000 claims description 40
- 238000010790 dilution Methods 0.000 claims description 40
- 239000000499 gel Substances 0.000 claims description 34
- 230000006651 lactation Effects 0.000 claims description 20
- 210000004369 blood Anatomy 0.000 claims description 19
- 239000008280 blood Substances 0.000 claims description 19
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 235000013336 milk Nutrition 0.000 claims description 10
- 239000008267 milk Substances 0.000 claims description 10
- 210000004080 milk Anatomy 0.000 claims description 10
- 230000010412 perfusion Effects 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 8
- 230000037396 body weight Effects 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 5
- 239000003085 diluting agent Substances 0.000 claims description 5
- 210000004731 jugular vein Anatomy 0.000 claims description 5
- 210000000214 mouth Anatomy 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000003805 procoagulant Substances 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- 230000010100 anticoagulation Effects 0.000 claims description 2
- 235000019687 Lamb Nutrition 0.000 abstract description 19
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 241001494479 Pecora Species 0.000 description 4
- 244000309466 calf Species 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 241000283707 Capra Species 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 230000003111 delayed effect Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000000474 nursing effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011895 specific detection Methods 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for determining newborn lamb IgG absorption efficiency, which specifically comprises the steps of preparing bovine colostrum, feeding the bovine colostrum to lambs which are suckled along with the mother, collecting serum samples, determining a standard curve of standard bovine IgG content, determining the IgG content of the serum samples, determining the IgG content of the bovine colostrum samples and calculating the newborn lamb IgG absorption efficiency.
Description
Technical Field
The invention relates to the technical field of lamb IgG absorption efficiency determination, in particular to a method for determining newborn lamb IgG absorption efficiency.
Background
IgG antibodies (immunoglobulin G) play a role in activating complement, neutralizing various toxins in immune response. Ruminant IgG antibodies cannot cross the placenta during pregnancy and they are secreted from the mammary gland into the colostrum, leaving the newborn baby protected with antibodies for the first time.
The newborn lamb has the capacity of completely absorbing macromolecular IgG in colostrum, so that passive immunity is formed, the immunity of the lamb in the lactation period is reduced due to the failure of the passive immunity caused by insufficient absorption of the newborn lamb IgG, and the newborn lamb is the main reason for the high incidence of illness and death in the lactation period of the lamb.
The newly born lamb IgG absorption capacity is rapidly reduced along with the prolongation of the birth time, generally, the IgG absorption capacity is only about 24 hours, then, intestinal closure is formed, meanwhile, the lamb IgG absorption capacity has huge animal individual difference, the lamb IgG absorption capacity is a main factor influencing the establishment of passive immunity, and the accurate determination of the lamb IgG absorption efficiency has great production and scientific research significance.
Foreigners typically used delayed colostrum feeding time for IgG absorption efficiency assays, but this approach has at least 3 problems: 1, after delaying the intestinal closure time delay caused by colostrum feeding, the measured IgG absorption efficiency does not reflect the normal state; 2, as the newborn lamb or calf does not eat for a long time in the delayed feeding process, the body of the newborn lamb or calf is already in a weak state, and the newborn lamb or calf has certain influence on the test result; 3, the feeding time of newborn lamb colostrums cannot be delayed for too long, otherwise, the survival of newborn lambs is difficult to guarantee.
Disclosure of Invention
Technical problem to be solved
The invention can solve the problems that the conventional lamb IgG absorption efficiency measuring method cannot realize the efficiency measurement of lambs in a normal state, the normal lactation and production management are influenced in the measuring process, and the time limit is obvious.
(II) technical scheme
In order to achieve the purpose, the invention adopts the following technical scheme that the method for measuring the IgG absorption efficiency of the newborn lambs specifically comprises the following steps:
s1, preparing bovine colostrum: obtaining bovine colostrum, and storing at-18 deg.C to-22 deg.C;
s2, feeding lambs which are suckled along with the female with the bovine colostrum: weighing and recording the weight of the lambs, preheating the bovine colostrum to 38-40 ℃, feeding the bovine colostrum to the lambs nursing along with the mother through oral cavity perfusion, and determining the nursing amount of the perfused bovine colostrum;
s3, collecting serum samples: feeding bovine colostrum to lambs for 23-25 h, collecting jugular vein blood, collecting blood by using a procoagulant blood collection tube, standing for 1.5-2.5 h at room temperature, after serum is separated out, centrifuging for 14-16 min at 3400 Xg-3600 Xg by using a centrifuge at room temperature, and collecting serum samples, and storing the collected serum samples at-18-22 ℃;
s4, and measuring a bovine IgG content standard curve: diluting bovine IgG standard with 10 x phosphate buffer solution to multiple dilutions of 0.1mg/ml to 1.0mg/ml, each dilution having at least two replicates; adding 19-21 mul of standard bovine IgG diluent into each hole of the gel agar plate, placing the gel agar plate in a constant-temperature incubator at 36-38 ℃, and observing and recording the size of a precipitation ring until the precipitation ring does not continuously diffuse; measuring the diameter of each dilution precipitation ring by using a vernier caliper, taking the average value of the diameters of the precipitation rings with the same dilution as a measured value, taking the square of the measured value as a horizontal coordinate, taking the content of bovine IgG in a corresponding hole of a gel agar plate as a vertical coordinate, and drawing a standard curve;
s5, determination of IgG content of serum samples: selecting a new gel agar plate, adding 19-21 mul of diluted serum samples into each hole, and setting at least two repeat groups for each sample; measuring the diameter of each sample precipitation ring by using a vernier caliper, taking the average value of the diameters of the precipitation rings of the same sample as a measured value, and multiplying the bovine IgG content obtained by calculation according to a standard curve by the dilution multiple of the serum sample to obtain the content of the IgG of the serum sample;
s6, measuring the content of IgG in the bovine colostrum sample: selecting a new gel agar plate, adding 19-21 mul of diluted bovine colostrum sample into each hole, and setting at least two repeat groups for each sample; measuring the diameters of the precipitation rings of the samples by using a vernier caliper, taking the average value of the diameters of the precipitation rings of the same samples as a measured value, and calculating the bovine IgG content according to a standard curve and then multiplying the bovine IgG content by the dilution times of the bovine colostrum with the samples to obtain the content of the bovine colostrum sample IgG;
s7, calculation of IgG absorption efficiency of newborn lambs: IgG apparent absorption rate AEA (%) - [ serum bovine IgG content (g/L) × body weight (kg) × 7.5% ]/[ bovine colostrum IgG content (g/L) × lactation amount (L) ].
As a preferred technical scheme of the present invention, in S1, the obtained bovine colostrum is reduced bovine colostrum or fresh bovine colostrum, and the obtaining mode of the reduced bovine colostrum and the fresh bovine colostrum is as follows:
the method comprises the following steps of preparing reduced bovine colostrum by adding clean warm water into bovine colostrum powder, wherein the volume ratio of the bovine colostrum powder to the clean warm water is 1:4-1: 7;
collecting milk of cow within 3 days after calving to obtain fresh colostrum.
As a preferred technical solution of the present invention, in S2, the step of perfusing the amount of the colostrum fed to the mammal includes placing the colostrum into a perfusion container with scale lines in advance, recording the volume of the initial colostrum, and after perfusing the colostrum, recording the volume of the perfused colostrum, where the difference between the two is the amount of the perfused colostrum fed to the mammal.
In a preferred embodiment of the present invention, in S3, the amount of blood collected by the anticoagulation blood collection tube is 4ml to 6 ml.
In a preferred embodiment of the present invention, the specific steps of preparing the gel agar plate in S4, S5 and S6 are as follows:
a0.9% sodium chloride solution is used for preparing a gel agar plate with the agar concentration of 1%, rabbit anti-bovine IgG antibody is added in the preparation process, and the ratio of the rabbit anti-bovine IgG antibody to the agar solution is 1: 30.
In a preferred embodiment of the present invention, in S4, six dilutions are provided, and the six dilutions are 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml and 1.0mg/ml, respectively.
In a preferred embodiment of the present invention, in S4, the standard curve is drawn by linear fitting
(III) advantageous effects
1. The method for determining the IgG absorption efficiency of the newborn lambs can realize the determination of the IgG absorption efficiency of the lambs which are suckled with the mother under the normal state;
2. the method for determining the IgG absorption efficiency of the newborn lambs does not influence the normal lactation and production management of the lambs;
3. the method for determining the IgG absorption efficiency of the newborn lambs breaks through the limitation of determining the postpartum time, and can determine the IgG absorption efficiency of the lambs at any time.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic flow diagram of the present invention;
FIG. 2 is a schematic diagram showing the results of antigen-specific detection of bovine IgG and ovine IgG of the present invention;
FIG. 3 is a schematic representation of a one-way immunodiffusion standard curve of the present invention;
FIG. 4 is a schematic diagram showing the dynamic change of the IgG absorption efficiency of the present invention.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.
In the description of the present invention, it is to be understood that the terms "longitudinal", "upper", "lower", "left", "right", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are used only for convenience in describing the present invention and for simplicity in description, and do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and thus, are not to be construed as limiting the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
Example 1
Referring to fig. 1, a method for determining IgG absorption efficiency of newborn lambs specifically includes the following steps:
s1, preparing bovine colostrum: obtaining bovine colostrum, and storing at-18 deg.C;
in this embodiment, in S1, the obtained bovine colostrum is reduced bovine colostrum, and the reduced bovine colostrum is obtained as follows: the reduced bovine colostrum is prepared by adding clean warm water into bovine colostrum powder, and the volume ratio of the bovine colostrum powder to the clean warm water is 1: 7.
S2, feeding lambs which are suckled along with the female with the bovine colostrum: weighing and recording the weight of the lambs, preheating the bovine colostrums to 38 ℃, feeding the bovine colostrums to the lambs along with the mother lactation through oral cavity perfusion, and determining the feeding amount of the perfused bovine colostrums;
in this embodiment, in S2, the specific step of perfusing the amount of milk fed by the bovine colostrum includes placing the bovine colostrum into a perfusion container with scale lines in advance, recording the volume of the initial bovine colostrum, and after perfusing the bovine colostrum, recording the volume of the perfused bovine colostrum, where the difference between the two is the amount of milk fed by the perfused bovine colostrum.
S3, collecting serum samples: collecting blood in jugular vein 24 hr after feeding calf colostrum, collecting 4ml blood with procoagulant blood collection tube, standing at room temperature for 1.5 hr, centrifuging at 3400 Xg for 16min at room temperature to collect serum sample, and storing at-18 deg.C;
s4, and measuring a bovine IgG content standard curve: diluting bovine IgG standard with 10 x phosphate buffer solution to multiple dilutions of 0.1mg/ml to 1.0mg/ml, each dilution having at least two replicates; adding 19 μ l of standard bovine IgG diluent into each well of the gel agar plate, placing in a constant temperature incubator at 36 ℃, and observing and recording the size of a precipitation ring until the precipitation ring does not continuously diffuse; measuring the diameter of each dilution precipitation ring by using a vernier caliper, taking the average value of the diameters of the precipitation rings with the same dilution as a measured value, taking the square of the measured value as an abscissa, taking the content of bovine IgG in a corresponding hole of a gel agar plate as an ordinate, and drawing a standard curve by adopting an Excel table;
in the present example, six dilutions were specifically set, and the six dilutions were 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml and 1.0mg/ml, respectively.
S5, determination of IgG content of serum samples: selecting a new gel agar plate, adding 19 μ l of diluted serum sample into each well, and setting at least two repeat groups for each sample; measuring the diameter of each sample precipitation ring by using a vernier caliper, taking the average value of the diameters of the precipitation rings of the same sample as a measured value, and multiplying the bovine IgG content obtained by calculation according to a standard curve by the dilution multiple of the serum sample to obtain the content of the IgG of the serum sample;
s6, measuring the content of IgG in the bovine colostrum sample: selecting a new gel agar plate, adding 19 μ l diluted bovine colostrum sample into each well, and setting at least two repeat groups for each sample; measuring the diameters of the precipitation rings of the samples by using a vernier caliper, taking the average value of the diameters of the precipitation rings of the same samples as a measured value, and calculating the bovine IgG content according to a standard curve and then multiplying the bovine IgG content by the dilution times of the bovine colostrum with the samples to obtain the content of the bovine colostrum sample IgG;
in this embodiment, in S4, S5 and S6, the specific steps of preparing the gel agar plate are: a0.9% sodium chloride solution is used for preparing a gel agar plate with the agar concentration of 1%, rabbit anti-bovine IgG antibody is added in the preparation process, and the ratio of the rabbit anti-bovine IgG antibody to the agar solution is 1: 30.
S7, calculation of IgG absorption efficiency of newborn lambs: IgG apparent absorption rate AEA (%) - [ serum bovine IgG content (g/L) × body weight (kg) × 7.5% ]/[ bovine colostrum IgG content (g/L) × lactation amount (L) ].
Example 2
Referring to fig. 1, a method for determining IgG absorption efficiency of newborn lambs specifically includes the following steps:
s1, preparing bovine colostrum: obtaining bovine colostrum, and storing at-22 deg.C;
in this embodiment, in S1, the obtained bovine colostrum is fresh bovine colostrum, and the fresh bovine colostrum is obtained in the following manner: collecting milk of cow within 3 days after calving to obtain fresh colostrum.
S2, feeding lambs which are suckled along with the female with the bovine colostrum: weighing and recording the weight of the lambs, preheating the bovine colostrums to 40 ℃, feeding the bovine colostrums to the lambs along with the mother lactation through oral cavity perfusion, and determining the feeding amount of the perfused bovine colostrums;
in this embodiment, in S2, the specific step of perfusing the amount of milk fed by the bovine colostrum includes placing the bovine colostrum into a perfusion container with scale lines in advance, recording the volume of the initial bovine colostrum, and after perfusing the bovine colostrum, recording the volume of the perfused bovine colostrum, where the difference between the two is the amount of milk fed by the perfused bovine colostrum.
S3, collecting serum samples: collecting blood in jugular vein 24 hr after feeding bovine colostrum, collecting 6ml blood with procoagulant blood collection tube, standing at room temperature for 2.5 hr, centrifuging at 3600 × g for 14min at room temperature with centrifuge, collecting serum sample, and storing at-22 deg.C;
s4, and measuring a bovine IgG content standard curve: diluting bovine IgG standard with 10 x phosphate buffer solution to multiple dilutions of 0.1mg/ml to 1.0mg/ml, each dilution having at least two replicates; adding 21 mul of standard bovine IgG diluent into each hole of the gel agar plate, placing the gel agar plate in a constant-temperature incubator at 38 ℃, and observing and recording the size of a precipitation ring until the gel agar plate does not continuously diffuse; measuring the diameter of each dilution precipitation ring by using a vernier caliper, taking the average value of the diameters of the precipitation rings with the same dilution as a measured value, taking the square of the measured value as an abscissa, taking the content of bovine IgG in a corresponding hole of a gel agar plate as an ordinate, and drawing a standard curve by adopting an Excel table;
in the present example, six dilutions were specifically set, and the six dilutions were 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml and 1.0mg/ml, respectively.
S5, determination of IgG content of serum samples: selecting a new gel agar plate, adding 21 mul of diluted serum samples into each well, and setting at least two repeat groups for each sample; measuring the diameter of each sample precipitation ring by using a vernier caliper, taking the average value of the diameters of the precipitation rings of the same sample as a measured value, and multiplying the bovine IgG content obtained by calculation according to a standard curve by the dilution multiple of the serum sample to obtain the content of the IgG of the serum sample;
s6, measuring the content of IgG in the bovine colostrum sample: selecting a new gel agar plate, adding 21 mul diluted bovine colostrum sample into each hole, and setting at least two repeat groups for each sample; measuring the diameters of the precipitation rings of the samples by using a vernier caliper, taking the average value of the diameters of the precipitation rings of the same samples as a measured value, and calculating the bovine IgG content according to a standard curve and then multiplying the bovine IgG content by the dilution times of the bovine colostrum with the samples to obtain the content of the bovine colostrum sample IgG;
in this embodiment, in S4, S5 and S6, the specific steps of preparing the gel agar plate are: a0.9% sodium chloride solution is used for preparing a gel agar plate with the agar concentration of 1%, rabbit anti-bovine IgG antibody is added in the preparation process, and the ratio of the rabbit anti-bovine IgG antibody to the agar solution is 1: 30.
S7, calculation of IgG absorption efficiency of newborn lambs: IgG apparent absorption rate AEA (%) - [ serum bovine IgG content (g/L) × body weight (kg) × 7.5% ]/[ bovine colostrum IgG content (g/L) × lactation amount (L) ].
Example 3
Referring to fig. 1-4, a method for determining IgG uptake efficiency of newborn lambs includes the following steps:
s1, preparing bovine colostrum: obtaining bovine colostrum, and storing at-20 deg.C;
in this embodiment, in S1, the obtained bovine colostrum is reduced bovine colostrum or fresh bovine colostrum, and the reduced bovine colostrum and the fresh bovine colostrum are obtained in the following specific manner:
the method comprises the following steps of (1) preparing reduced bovine colostrum by adding clean warm water into bovine colostrum powder, wherein the volume ratio of the bovine colostrum powder to the clean warm water is 1: 4;
collecting milk of cow within 3 days after calving to obtain fresh colostrum.
S2, feeding lambs which are suckled along with the female with the bovine colostrum: weighing and recording the weight of the lambs, preheating the bovine colostrums to 39 ℃, feeding the bovine colostrums to the lambs along with the mother lactation through oral cavity perfusion, and determining the feeding amount of the perfused bovine colostrums;
in this embodiment, in S2, the specific step of perfusing the amount of milk fed by the bovine colostrum includes placing the bovine colostrum into a perfusion container with scale lines in advance, recording the volume of the initial bovine colostrum, and after perfusing the bovine colostrum, recording the volume of the perfused bovine colostrum, where the difference between the two is the amount of milk fed by the perfused bovine colostrum.
S3, collecting serum samples: collecting blood in jugular vein 24h after feeding bovine colostrum to lamb, collecting 5ml blood by using procoagulant blood collection tube, standing for 2h at room temperature, after separating out serum, centrifuging at 3500 Xg for 15min at room temperature by using a centrifuge, collecting serum sample, and storing the collected serum sample at-20 ℃;
s4, and measuring a bovine IgG content standard curve: diluting bovine IgG standard with 10 x phosphate buffer solution to multiple dilutions of 0.1mg/ml to 1.0mg/ml, each dilution having at least two replicates; adding 20 μ l of standard bovine IgG diluent into each well of the gel agar plate, placing in a constant temperature incubator at 37 ℃, and observing and recording the size of a precipitation ring until the precipitation ring does not continuously diffuse; measuring the diameter of each dilution precipitation ring by using a vernier caliper, taking the average value of the diameters of the precipitation rings with the same dilution as a measured value, taking the square of the measured value as an abscissa, taking the content of bovine IgG in a corresponding hole of a gel agar plate as an ordinate, and drawing a standard curve by adopting an Excel table;
in the present example, six dilutions were specifically set, and the six dilutions were 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml and 1.0mg/ml, respectively.
S5, determination of IgG content of serum samples: selecting a new gel agar plate, adding 20 μ l diluted serum sample into each well, and setting at least two repeat groups for each sample; measuring the diameter of each sample precipitation ring by using a vernier caliper, taking the average value of the diameters of the precipitation rings of the same sample as a measured value, and multiplying the bovine IgG content obtained by calculation according to a standard curve by the dilution multiple of the serum sample to obtain the content of the IgG of the serum sample;
s6, measuring the content of IgG in the bovine colostrum sample: selecting a new gel agar plate, adding 20 μ l diluted bovine colostrum sample into each well, and setting at least two repeat groups for each sample; measuring the diameters of the precipitation rings of the samples by using a vernier caliper, taking the average value of the diameters of the precipitation rings of the same samples as a measured value, and calculating the bovine IgG content according to a standard curve and then multiplying the bovine IgG content by the dilution times of the bovine colostrum with the samples to obtain the content of the bovine colostrum sample IgG;
in this embodiment, in S4, S5 and S6, the specific steps of preparing the gel agar plate are: a0.9% sodium chloride solution is used for preparing a gel agar plate with the agar concentration of 1%, rabbit anti-bovine IgG antibody is added in the preparation process, and the ratio of the rabbit anti-bovine IgG antibody to the agar solution is 1: 30.
S7, calculation of IgG absorption efficiency of newborn lambs: IgG apparent absorption rate AEA (%) - [ serum bovine IgG content (g/L) × body weight (kg) × 7.5% ]/[ bovine colostrum IgG content (g/L) × lactation amount (L) ].
In this example, as shown in fig. 2, the results of antigen-specific detection of bovine IgG and ovine IgG are shown in fig. 1, and it can be seen that when standard bovine IgG is detected using a rabbit anti-bovine IgG antibody, a clear milky precipitation ring is visible in the agar plate (fig. 2A), and when IgG in bovine colostrum is detected using a rabbit anti-ovine IgG antibody, no precipitation ring is present in the agar plate (fig. 2B); when rabbit anti-bovine IgG antibody is used for detecting IgG in goat colostrum, no precipitation ring appears in the agar plate (figure 2C), and when rabbit anti-goat IgG antibody is used for detecting standard goat IgG, clear milky precipitation ring can be seen in the agar plate (figure 2D); when the rabbit anti-bovine IgG antibody is used for detecting the mixed IgG of the cattle and the sheep, clear milky precipitation rings can be seen in the agar plate (figure 2E); when the mixed IgG of cattle and sheep was detected with rabbit anti-sheep IgG antibody, clear milky white precipitate rings were visible on the agar plates (FIG. 2F). The method shows that the sheep IgG and the cattle IgG have antigen specificity, and can also be used for detecting the mixed IgG of cattle and sheep.
As can be seen from FIG. 3A, the linear relationship between bovine IgG concentration and concentration is good at 0.1-1.0 mg/mL, and the linear correlation coefficient is 0.99 or more. As can be seen from FIG. 3B, the linear relationship of the goat IgG concentration is good in the range of 0.1-1.0 mg/mL, and the linear correlation coefficient is more than 0.95.
As can be seen from fig. 4, the overall difference in IgG uptake efficiency was very significant (P <0.01) with the change in colostrum feeding time, with an IgG uptake efficiency of 33% at 2h postpartum feeding of colostrum followed by a tendency to decrease rapidly and to decrease to 0% at 24h postpartum feeding of colostrum.
In conclusion, the method can realize the measurement of the IgG absorption efficiency of the lambs in the normal state along with the lactation of the mother, does not influence the normal lactation and production management of the lambs, breaks through the limitation of the measurement of the postpartum time, and can measure the IgG absorption efficiency of the lambs at any time.
In conclusion, the method can realize the measurement of the IgG absorption efficiency of the lambs in the normal state along with the lactation of the mother, does not influence the normal lactation and production management of the lambs, breaks through the limitation of the measurement of the postpartum time, and can measure the IgG absorption efficiency of the lambs at any time.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A method for measuring IgG absorption efficiency of newborn lambs is characterized by comprising the following steps:
s1, preparing bovine colostrum: obtaining bovine colostrum, and storing at-18 deg.C to-22 deg.C;
s2, feeding lambs which are suckled along with the female with the bovine colostrum: weighing and recording the weight of the lambs, preheating the bovine colostrums to 38-40 ℃ in a water bath, feeding the bovine colostrums to the lambs along with the lactation of the mother through oral cavity perfusion, and determining the lactation amount of the perfused bovine colostrums;
s3, collecting serum samples: feeding bovine colostrum to lambs for 23-25 h, collecting jugular vein blood, collecting blood by using a procoagulant blood collection tube, standing for 1.5-2.5 h at room temperature, after serum is separated out, centrifuging for 14-16 min at 3400 Xg-3600 Xg by using a centrifuge at room temperature, and collecting serum samples, and storing the collected serum samples at-18-22 ℃;
s4, and measuring a bovine IgG content standard curve: diluting bovine IgG standard with 10 x phosphate buffer solution to multiple dilutions of 0.1mg/ml to 1.0mg/ml, each dilution having at least two replicates; adding 19-21 mul of standard bovine IgG diluent into each hole of the gel agar plate, placing the gel agar plate in a constant-temperature incubator at 36-38 ℃, and observing and recording the size of a precipitation ring until the precipitation ring does not continuously diffuse; measuring the diameter of each dilution precipitation ring by using a vernier caliper, taking the average value of the diameters of the precipitation rings with the same dilution as a measured value, taking the square of the measured value as a horizontal coordinate, taking the content of bovine IgG in a corresponding hole of a gel agar plate as a vertical coordinate, and drawing a standard curve;
s5, determination of IgG content of serum samples: selecting a new gel agar plate, adding 19-21 mul of diluted serum samples into each hole, and setting at least two repeat groups for each sample; measuring the diameter of each sample precipitation ring by using a vernier caliper, taking the average value of the diameters of the precipitation rings of the same sample as a measured value, and multiplying the bovine IgG content obtained by calculation according to a standard curve by the dilution multiple of the serum sample to obtain the content of the IgG of the serum sample;
s6, measuring the content of IgG in the bovine colostrum sample: selecting a new gel agar plate, adding 19-21 mul of diluted bovine colostrum sample into each hole, and setting at least two repeat groups for each sample; measuring the diameters of the precipitation rings of the samples by using a vernier caliper, taking the average value of the diameters of the precipitation rings of the same samples as a measured value, and calculating the bovine IgG content according to a standard curve and then multiplying the bovine IgG content by the dilution times of the bovine colostrum with the samples to obtain the content of the bovine colostrum sample IgG;
s7, calculation of IgG absorption efficiency of newborn lambs: IgG apparent absorption rate AEA (%) - [ serum bovine IgG content (g/L) × body weight (kg) × 7.5% ]/[ bovine colostrum IgG content (g/L) × lactation amount (L) ].
2. The method of claim 1 for determining IgG uptake efficiency of newborn lambs, wherein: in the S1, the obtained bovine colostrum is reduced bovine colostrum or fresh bovine colostrum, and the obtaining mode of the reduced bovine colostrum and the fresh bovine colostrum is as follows:
the method comprises the following steps of preparing reduced bovine colostrum by adding clean warm water into bovine colostrum powder, wherein the volume ratio of the bovine colostrum powder to the clean warm water is 1:4-1: 7;
collecting milk of cow within 3 days after calving to obtain fresh colostrum.
3. The method of claim 1 for determining IgG uptake efficiency of newborn lambs, wherein: in S2, the concrete step of pouring into bovine colostrum lactation volume is that, put into the pouring container that has the scale mark with bovine colostrum in advance, record the volume of initial bovine colostrum, pour into bovine colostrum after, record the volume of post-pouring bovine colostrum, the difference between them is the lactation volume of pouring into bovine colostrum.
4. The method of claim 1 for determining IgG uptake efficiency of newborn lambs, wherein: in the S3, the amount of blood collected by the anticoagulation blood collection tube is 4ml to 6 ml.
5. The method of claim 1 for determining IgG uptake efficiency of newborn lambs, wherein: in the S4, the S5 and the S6, the gel agar plate is prepared by the following steps:
a0.9% sodium chloride solution is used for preparing a gel agar plate with the agar concentration of 1%, rabbit anti-bovine IgG antibody is added in the preparation process, and the ratio of the rabbit anti-bovine IgG antibody to the agar solution is 1: 30.
6. The method of claim 1 for determining IgG uptake efficiency of newborn lambs, wherein: in the S4, the number of the dilutions is six, and the six dilutions are respectively 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml and 1.0 mg/ml.
7. The method of claim 1 for determining IgG uptake efficiency of newborn lambs, wherein: in S4, the standard curve is drawn by linear fitting.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110805622.1A CN113533742A (en) | 2021-07-16 | 2021-07-16 | Method for determining IgG absorption efficiency of newborn lambs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110805622.1A CN113533742A (en) | 2021-07-16 | 2021-07-16 | Method for determining IgG absorption efficiency of newborn lambs |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113533742A true CN113533742A (en) | 2021-10-22 |
Family
ID=78128400
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110805622.1A Pending CN113533742A (en) | 2021-07-16 | 2021-07-16 | Method for determining IgG absorption efficiency of newborn lambs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113533742A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4757002A (en) * | 1983-06-13 | 1988-07-12 | Regents Of The University Of Minnesota | Method and kit for the estimation of serum immunoglobulin |
| CN1295667A (en) * | 1998-04-01 | 2001-05-16 | 尤西·哈勒恩 | Method for measuring bone resorption rate |
| CN101210922A (en) * | 2006-12-29 | 2008-07-02 | 河南农业大学 | Method for detecting SPA FC fragment bound mammalian blood serum total IgG by dolloidal gold marker protein A |
| CN101271107A (en) * | 2008-04-09 | 2008-09-24 | 陕西科技大学 | Fast immunology detecting method for goat milk and its milk product doped with cow's milk |
| CN105348385A (en) * | 2015-10-23 | 2016-02-24 | 照日格图 | Sheep blood IgG extract product and application thereof |
-
2021
- 2021-07-16 CN CN202110805622.1A patent/CN113533742A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4757002A (en) * | 1983-06-13 | 1988-07-12 | Regents Of The University Of Minnesota | Method and kit for the estimation of serum immunoglobulin |
| CN1295667A (en) * | 1998-04-01 | 2001-05-16 | 尤西·哈勒恩 | Method for measuring bone resorption rate |
| CN101210922A (en) * | 2006-12-29 | 2008-07-02 | 河南农业大学 | Method for detecting SPA FC fragment bound mammalian blood serum total IgG by dolloidal gold marker protein A |
| CN101271107A (en) * | 2008-04-09 | 2008-09-24 | 陕西科技大学 | Fast immunology detecting method for goat milk and its milk product doped with cow's milk |
| CN105348385A (en) * | 2015-10-23 | 2016-02-24 | 照日格图 | Sheep blood IgG extract product and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 刘珍 等: "绵羊IgG粗品的应用效果研究", 《内蒙古科技与经济》, no. 16, pages 183 - 185 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Isabel de Moraes-Pinto et al. | Placental transfer and maternally acquired neonatal IgG immunity in human immunodeficiency virus infection | |
| Robertson et al. | Development and clinical application of a new method for the radioimmunoassay of arginine vasopressin in human plasma | |
| Evans et al. | C-reactive protein as a predictor of infectious morbidity with premature rupture of membranes | |
| US3911096A (en) | Radioimmunoassay for measurement of thyroxine (T{HD 4{B ) and triiodothyonine (T{HD 3{B ) in blood serum | |
| Trostle et al. | High antibody levels in cord blood from pregnant women vaccinated against COVID-19 | |
| Buttle et al. | Plasma prolactin measured by radioimmunoassay and bioassay in pregnant and lactating goats and the occurrence of a placental lactogen | |
| Miotti et al. | Prevalence of serum and milk antibodies to Giardia lamblia in different populations of lactating women | |
| CN101581726A (en) | New-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit | |
| US4554256A (en) | Antigen associated with early detection of mammalian pregnancy | |
| OSBORN et al. | Studies of the immunology of the newborn infant: III. Permeability of the placenta to maternal antibody during fetal life | |
| Wiener et al. | PERMEABILITY OF THE HUMAN PLACENTA TO ANTIBODIES: A Quantitative Study | |
| Marshall et al. | SARS-CoV-2 vaccine booster elicits robust prolonged maternal antibody responses and passive transfer to the offspring via the placenta and breastmilk | |
| CN113533742A (en) | Method for determining IgG absorption efficiency of newborn lambs | |
| Pittard et al. | Longitudinal changes in the bone mineral content of term and premature infants | |
| GUSDON Jr | Improved hemagglutination-inhibition assay: Clinical application to measurement of human placental lactogen | |
| CN101819210A (en) | Beta-human chorionic gonadotrophin test kit (time-resolved fluoroimmunoassay) for prenatal screening and preparation method thereof | |
| Oreiby et al. | Diagnosis of ovine caseous lymphadenitis by blood and milk gamma interferon assays | |
| El Amiri et al. | Measurement of ovine pregnancy‐associated glycoprotein (PAG) during early pregnancy in Lacaune sheep | |
| US4705748A (en) | Antigen associated with early detection of mammalian pregnancy | |
| Ree | C-reactive protein in Gambian Africans with special reference to P. falciparum malaria | |
| Odendaal et al. | Effects of alcohol, cigarettes, methamphetamine and marijuana exposure during pregnancy on maternal serum alpha-fetoprotein levels at 20-24 weeks’ gestation | |
| Obiekwe et al. | Circulating levels of placental protein 5 in the mother: relation to birth weight | |
| Johansson et al. | Determination of α-fetoprotein by a new paper disc radioimmunoassay of the sandwich type | |
| Sharma et al. | Iron profile in term small for gestational age infants at 10 weeks of age and correlation with maternal iron profile: a prospective cohort study | |
| CEDERQVIST et al. | The fetal serum alpha-fetoprotein and its relationship to immunoglobulins and birth weight at term |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |