CN113698482A - Preparation method of beta 2-microglobulin multi-antiserum - Google Patents
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Abstract
The invention discloses a preparation method of beta 2-microglobulin polyclonal antiserum, which comprises the following steps: the beta 2-microglobulin antigen is used for immunizing an experimental rabbit not more than 4 times, and at least 12 subcutaneous injection points are selected for each immunization. The invention reduces the times of immunization and the dosage of immune antigen, and saves time cost, raw material cost and labor cost.
Description
Technical Field
The invention relates to the field of biological diagnosis and antibody raw material correlation. More specifically, the invention relates to a preparation method of beta 2-microglobulin polyclonal antiserum.
Background
The beta 2-microglobulin is a small molecular globulin produced by lymphocytes, platelets and polymorphonuclear leukocytes, the abnormal rise of the beta 2-microglobulin can indicate various related diseases, and the detection of the beta 2-microglobulin is considered as a simple, convenient, accurate and sensitive method for measuring the mild renal function decline and the observation of curative effect of diabetic patients. At present, animals for preparing the anti-human beta 2-microglobulin antibody mainly comprise sheep, rabbits, mice and the like, wherein the sheep-derived anti-human beta 2-microglobulin antibody is most applied, and is very suitable for clinical production requirements due to large sheep body size and large plasma collection amount, but the sheep-derived anti-human beta 2-microglobulin antibody has the problems of poor stability and low affinity. Moreover, the antigen prepared by the traditional immunization method is difficult to generate antibody with higher titer, and the immunization times and the antigen dosage are more. Therefore, it is desirable to design a technical solution that can overcome the above-mentioned drawbacks to a certain extent.
Disclosure of Invention
An object of the present invention is to provide a method for preparing beta 2-microglobulin polyclonal antiserum, which effectively avoids the interference of hyperlipidemia problem in the antiserum purification process, reduces the immunization times and the dosage of immune antigen, and saves time cost and raw material and labor cost.
To achieve these objects and other advantages of the present invention, according to one aspect of the present invention, there is provided a method for preparing a β 2-microglobulin antiserum, comprising: the beta 2-microglobulin antigen is used for immunizing an experimental rabbit not more than 4 times, and at least 12 subcutaneous injection points are selected for each immunization.
Further, the injection points are selected from two or three areas of the shoulder subcutaneous area, the groin subcutaneous area and the neck dorsal subcutaneous area, and at least 4 injection points are selected in each area.
Furthermore, the injection amount of the beta 2-microglobulin antigen at each injection point is 0.075-0.15 mg.
Further, the β 2-microglobulin antigen was made into an emulsion with 1xPBS buffer solution and freund's complete adjuvant or freund's incomplete adjuvant, and then used for injection.
Further, the PBS buffer solution contains an immunopotentiator, wherein the immunopotentiator is one or more of beta-glucan, mannose, lentinan, astragalus polysaccharide, codonopsis pilosula polysaccharide, ginseng polysaccharide, angelica polysaccharide, lycium barbarum polysaccharide, ganoderma lucidum polysaccharide, curcumin and hawthorn polysaccharide.
Furthermore, the interval time between two adjacent immunizations is 7-14 days.
Further, still include: after the last immunization, performing heart basal blood collection on the experimental rabbit, standing, sucking the supernatant, performing centrifugal precipitation, and separating the supernatant to obtain the beta 2-microglobulin polyclonal antiserum.
Furthermore, before the experiment rabbits are immunized, amino electrolytic multivitamins are added into the drinking water of the experiment rabbits.
Further, the experimental rabbit is a Japanese big ear rabbit.
According to another aspect of the present invention, there is provided a method for preparing a β 2-microglobulin antiserum, comprising: step one, mixing beta 2-microglobulin antigen, 1xPBS buffer solution and complete Freund's adjuvant to prepare first antigen emulsion, wherein the concentration of the beta 2-microglobulin antigen in the first antigen emulsion is 1 mg/mL; mixing beta 2-microglobulin antigen, 1xPBS buffer solution and incomplete Freund's adjuvant to prepare a second antigen emulsion, wherein the concentration of the beta 2-microglobulin antigen in the second antigen emulsion is 0.4-0.5 mg/mL; selecting 4 subcutaneous injection points in front of the shoulder, 4 subcutaneous injection points in the groin and 4 subcutaneous injection points in the back of the neck; secondly, performing primary immunization on the Japanese big ear rabbit by using a first antigen emulsion, wherein the injection amount of the first antigen emulsion at each injection point is 150-170 mu L; after 7-14 days, performing second, third and fourth immunizations on the Japanese big-ear rabbits by using a second antigen emulsion, wherein the interval between each immunization is 7-14 days, and the injection amount of the second antigen emulsion at each injection point is 150-170 mu L; and step four, after 7-14 days of the sixth immunization, performing auricular vein blood sampling, performing ELISA detection, performing heart basal blood sampling after meeting target requirements, standing, absorbing upper-layer serum after the serum is completely separated out, performing centrifugal precipitation separation to obtain upper-layer antiserum, taking the upper-layer antiserum, adding thimerosal into the upper-layer antiserum, and storing.
The invention at least comprises the following beneficial effects:
the invention selects the experimental rabbit with low blood lipid level and better anti-stress capability as the donor for producing the anti-human beta 2-microglobulin polyclonal antiserum, effectively avoids the interference of high blood lipid in the antiserum purification process, achieves higher level after 4 times of multi-site low-dose multi-point injection immunization, provides reliable guarantee for improving the stability of biological reagent products, greatly reduces the immunization times and the dosage of immune antigens, and saves time cost, raw materials and labor cost.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Embodiments of the present application provide methods for preparing a β 2-microglobulin polyclonal antiserum, comprising: the beta 2-microglobulin antigen is used for immunization experiments for no more than 4 times, optionally 4 times, and at least 12 subcutaneous injection points are selected for each immunization, optionally 12 times.
In other embodiments, the injection points are selected from two or three areas of the shoulder anterior subcutaneous area, the groin subcutaneous area and the neck back subcutaneous area, and at least 4 injection points are selected from each area, and the injection points can be selected from 4 injection points of the shoulder anterior subcutaneous area, the groin subcutaneous area and the neck back subcutaneous area.
In other embodiments, the amount of beta 2-microglobulin antigen injected per injection point is 0.075-0.15 mg.
In other embodiments, the β 2-microglobulin antigen is formulated in emulsion with 1xPBS buffer solution and freund's complete adjuvant or freund's incomplete adjuvant prior to injection, optionally in emulsion with 1xPBS buffer solution and freund's complete adjuvant for the first immunization, and in emulsion with 1xPBS buffer solution and freund's incomplete adjuvant for the subsequent immunization.
In other embodiments, the PBS buffer solution contains an immunopotentiator, which is one or more of β -glucan, mannose, lentinan, astragalus polysaccharide, codonopsis pilosula polysaccharide, ginseng polysaccharide, angelica polysaccharide, lycium polysaccharide, ganoderma lucidum polysaccharide, curcumin, and hawthorn polysaccharide, and is preferably added to the antigen-dissolving buffer solution at a concentration of 0.5mg/ml to 5 mg/ml.
In other embodiments, the interval time between two adjacent immunizations is 7-14 days, specifically, after one immunization is finished, blood is taken from the ear vein of an immunized rabbit, serum antibody titer is separated out for ELISA detection, and the next immunization time is determined according to the antibody level and the antigen absorption condition of the original immune part, preferably ganoderma lucidum polysaccharide, ginseng polysaccharide, angelica polysaccharide and lycium polysaccharide.
In other embodiments, further comprising: after the last immunization, performing heart basal blood collection on the experimental rabbit, standing, sucking the supernatant, performing centrifugal precipitation, and separating the supernatant to obtain the beta 2-microglobulin polyclonal antiserum. Optionally standing at 37 deg.C for 1 hr, standing at 4 deg.C, sucking supernatant with pipette, centrifuging at 3000rpm for 15min, precipitating to obtain supernatant antiserum, adding thimerosal with final concentration of 0.01%, and storing at 4 deg.C for a long period.
In other embodiments, before the experimental rabbits are immunized, amino electrolytic multivitamins are added into the drinking water of the experimental rabbits to maintain the supply of electrolytes, amino acids and vitamins in the bodies of the experimental rabbits, so that the anti-stress capacity is improved.
In other embodiments, the experimental rabbit is a japanese big ear rabbit.
Embodiments of the present application also provide a method for preparing a β 2-microglobulin polyclonal antiserum, comprising: step one, mixing beta 2-microglobulin antigen, 1xPBS buffer solution and complete Freund's adjuvant to prepare first antigen emulsion, wherein the concentration of the beta 2-microglobulin antigen in the first antigen emulsion is 1 mg/mL; mixing beta 2-microglobulin antigen, 1xPBS buffer solution and incomplete Freund's adjuvant to prepare second antigen emulsion, wherein the concentration of the beta 2-microglobulin antigen in the second antigen emulsion is 0.4-0.5 mg/mL, and the immunopotentiator is added into the buffer solution for dissolving the antigen according to the concentration of 0.5-5 mg/mL; selecting 4 subcutaneous injection points in front of the shoulder, 4 subcutaneous injection points in the groin and 4 subcutaneous injection points in the back of the neck; secondly, performing primary immunization on the Japanese big ear rabbit by using a first antigen emulsion, wherein the injection amount of the first antigen emulsion at each injection point is 150-170 mu L; after 7-14 days, performing second, third and fourth immunizations on the Japanese big-ear rabbits by using a second antigen emulsion, wherein the interval between each immunization is 7-14 days, and the injection amount of the second antigen emulsion at each injection point is 150-170 mu L; and step four, after 7-14 days of the sixth immunization, performing auricular vein blood sampling, performing ELISA detection, performing heart basal blood sampling after meeting target requirements, standing, absorbing upper-layer serum after the serum is completely separated out, performing centrifugal precipitation separation to obtain upper-layer antiserum, taking the upper-layer antiserum, adding thimerosal into the upper-layer antiserum, and storing.
The following are illustrated by specific examples:
subject: 300 male Japanese big-ear rabbits with strong physique and vitality for 8 months are selected, the average weight (3.0 +/-0.5) kg of the male Japanese big-ear rabbits is obtained, all the rabbits are provided with ear tags, the ear numbers of the ear tags correspond to the immunity and production file information of each rabbit, the rabbits are divided into 5 groups, and each group comprises 60 rabbits. The breeding mode comprises cage culture, fixing drinking trough and tray, feeding granulated feed, automatic drinking water system, feeding feed three times a day (morning: middle: night: 3: 4), semi-open breeding mode, and adding amino electrolytic multivitamin into drinking water 3 days before immunization.
The experimental method comprises the following steps:
(1) preparing immune antigen: antigen demand was calculated according to the number of animals scheduled for immunization, the total volume of antigen emulsified in Freund's complete adjuvant or Freund's incomplete adjuvant was 620ml, 2 ml/mouse, and 150. mu.l was injected at 4 injection points subcutaneously in front of the shoulder, 4 injection points subcutaneously in the groin and 4 injection points subcutaneously in the back and neck.
(2) Immunization program: 5 groups of one or more different combinations in the immunopotentiator, wherein the first group is beta-glucan and mannose, the second group is codonopsis pilosula polysaccharide, ginseng polysaccharide, angelica polysaccharide and medlar polysaccharide, the third group is codonopsis pilosula polysaccharide, ginseng polysaccharide, angelica polysaccharide and medlar polysaccharide, the fourth group is ganoderma polysaccharide, ginseng polysaccharide, angelica polysaccharide and medlar polysaccharide, the fifth group is hawthorn polysaccharide, medlar polysaccharide, lentinan and astragalus polysaccharide, 60 groups are mixed with purified serum beta 2-microglobulin antigen 2mg 0.1mL and 1x PBS 0.9mL according to the concentration of 0.5mg/mL-5mg/mL, and the total volume is 2mL after the mixture is electrically stirred and emulsified with complete Freund's adjuvant 1mL, and the mixture is used as primary immunity; the total volume of 1mg of the same antigen is 2ml, 0.1ml of 1x PBS and 1ml of incomplete Freund's adjuvant are emulsified by electric stirring, and the mixture is used as a second immunity; 1mg of the same antigen is 0.1ml, 1x PBS is 0.9ml and 1ml of incomplete Freund's adjuvant is emulsified by electric stirring, and the total volume is 2ml as the third, fourth and fifth times of immunity; the same antigen 0.8mg 0.1ml, 1x PBS 1.9ml after uniform mixing total volume is 2ml, as the sixth, seven times immunity.
(3) Rabbit anti-human beta 2-microglobulin serum collection
According to the requirement of an immunization program, rabbit marginal vein blood collection is carried out for 2-3 ml/rabbit 5 days after the fifth immunization, after standing for 1h at room temperature (37 ℃), serum is separated out, centrifugation is carried out for 15min at 3000rpm, supernatant is taken and added with 0.01% thimerosal, and the rabbit marginal vein blood collection is preserved at 4 ℃.
After reaching the target titer by serum detection, blood is collected from the heart 7-14 days after the sixth immunization. After intravenous injection anesthesia, supine fixation is carried out, hair is cut and skin is disinfected conventionally at the position close to the heart of the xiphoid process of the sternum, the heart is pushed to the left chest gently by using a left middle finger, an index finger and a thumb until the heart is fixed, a 16-gauge needle is selected to be connected with a hemagglutination tube to be pierced at the heart base with stronger heart beat, 20-25ml of blood is collected, blood collection is stopped, and 250ml of 125-plus-blood-containing normal saline containing chlorphenamine is intravenously dripped to assist fluid infusion and hemostasis. The collected blood was gently mixed in reverse, then coagulated at room temperature for 1 hour or more, centrifuged at 3000rpm for 20min, then serum was separated and numbered and stored at 4 ℃.
The results of the comparison were determined with a beta 2-microglobulin assay kit (ELISA method) for rabbit anti-human serum.
The kit is used for determining the level of the beta 2-microglobulin in a sample by using a double-antibody sandwich method. Coating a microporous plate with purified beta 2-microglobulin antigen to prepare solid phase antigen, sequentially adding beta 2 microglobulin into the micropores coated with the monoclonal antibody, combining with beta 2-microglobulin antigen marked by HRP to form an antibody-antigen-enzyme-labeled antibody compound, and adding substrate TMB for developing after thorough washing. TMB is converted to blue by the catalysis of HRP enzyme and to the final yellow by the action of acid. The shade of the color is positively correlated with the beta 2-microglobulin in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using a microplate reader, and the concentration of β 2-microglobulin in the sample was calculated from the standard curve.
Tables 1-5 show the serum titer levels and the number of rabbits after the four-immunization in different groups.
TABLE 1 first group
| Serum antibody titer | Number of rabbits (only) | The rabbit accounts for a certain percentage |
| >200 ten thousand | 30 | 50％ |
| 72-200 ten thousand | 18 | 30％ |
| 24-72 million | 2 | 3.3％ |
| <24 ten thousand | 10 | 16.7％ |
TABLE 2 second group
| Serum antibody titer | Number of rabbits (only) | The rabbit accounts for a certain percentage |
| >200 ten thousand | 21 | 35％ |
| 72-200 ten thousand | 15 | 25％ |
| 24-72 million | 10 | 16.7％ |
| <24 ten thousand | 14 | 23.3％ |
TABLE 3 third group
| Serum antibody titer | Number of rabbits (only) | The rabbit accounts for a certain percentage |
| >200 ten thousand | 30 | 50％ |
| 72-200 ten thousand | 21 | 35％ |
| 24-72 million | 3 | 5％ |
| <24 ten thousand | 6 | 10％ |
TABLE 4 fourth group
| Serum antibody titer | Number of rabbits (only) | The rabbit accounts for a certain percentage |
| >200 ten thousand | 36 | 60％ |
| 72-200 ten thousand | 18 | 30％ |
| 24-72 million | 1 | 1.7％ |
| <24 ten thousand | 5 | 8.3％ |
TABLE 5 fifth group
| Serum antibody titer | Number of rabbits (only) | The rabbit accounts for a certain percentage |
| >200 ten thousand | 24 | 40％ |
| 72-200 ten thousand | 18 | 30％ |
| 24-72 million | 3 | 5％ |
| <24 ten thousand | 15 | 25％ |
Tables 6-10 show the serum titer levels and the number of rabbits after the six-immunization in different groups.
TABLE 6 first group
| Serum antibody titer | Number of rabbits (only) | The rabbit accounts for a certain percentage |
| >200 ten thousand | 27 | 45％ |
| 72-200 ten thousand | 12 | 20％ |
| 24-72 million | 9 | 15％ |
| <24 ten thousand | 12 | 20％ |
TABLE 7 second group
| Serum antibody titer | Number of rabbits (only) | The rabbit accounts for a certain percentage |
| >200 ten thousand | 24 | 40％ |
| 72-200 ten thousand | 9 | 15％ |
| 24-72 million | 9 | 15％ |
| <24 ten thousand | 18 | 30％ |
TABLE 8 third group
| Serum antibody titer | Number of rabbits (only) | The rabbit accounts for a certain percentage |
| >200 ten thousand | 33 | 55％ |
| 72-200 ten thousand | 21 | 35％ |
| 24-72 million | 0 | 0％ |
| <24 ten thousand | 6 | 10％ |
TABLE 9 fourth group
| Serum antibody titer | Number of rabbits (only) | The rabbit accounts for a certain percentage |
| >200 ten thousand | 36 | 60％ |
| 72-200 ten thousand | 21 | 35％ |
| 24-72 million | 0 | 0％ |
| <24 ten thousand | 3 | 5％ |
Fifth group of watch 10
Serum titer evaluation results: in the example, the fourth group of immune rabbit serum titers obtained after the fourth immunization has better effect; the rabbit anti-human beta 2-microglobulin serum antibody titer range is greater than or equal to 72 ten thousand and is qualified (table 1-5);
serum titer evaluation results: in the embodiment, the fourth group of immune rabbit serum titers obtained after the sixth immunization has better effect; the rabbit anti-human beta 2-microglobulin serum antibody titer range is greater than or equal to 72 ten thousand and is qualified (table 6-10);
the results of the potency assay are summarized as follows: the application fully proves that the rabbit anti-human beta 2-microglobulin polyclonal antibody with high efficiency and stability can be prepared by immunizing the Japanese big ear rabbit for 4 times by multi-part low-dose multi-point injection immunization (generally, 6-8 times are needed), reliable guarantee is provided for improving the stability of a biological reagent product, the immunization times and the dosage of an immune antigen are greatly reduced, and the time cost, the raw material cost and the labor cost are saved.
The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. The application, modification and variation of the method for preparing the beta 2-microglobulin antiserum of the invention will be apparent to those skilled in the art.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details given herein and to the embodiments shown and described without departing from the generic concept as defined by the claims and their equivalents.
Claims (10)
1. The preparation method of the beta 2-microglobulin polyclonal antiserum is characterized by comprising the following steps:
the beta 2-microglobulin antigen is used for immunizing an experimental rabbit not more than 4 times, and at least 12 subcutaneous injection points are selected for each immunization.
2. The method of claim 1, wherein the injection site is selected from two or three regions selected from the group consisting of the subcutaneous region before shoulder, the subcutaneous region in groin, and the subcutaneous region at back of neck, and at least 4 injection sites are selected from each region.
3. The method of claim 1, wherein the amount of β 2-microglobulin antigen injected at each injection site is from 0.075 to 0.15 mg.
4. The method for preparing β 2-microglobulin antiserum according to claim 3, wherein β 2-microglobulin antigen is prepared into an emulsion using 1xPBS buffer solution and Freund's complete adjuvant or Freund's incomplete adjuvant, and then used for injection.
5. The method for preparing the beta 2-microglobulin polyclonal antiserum of claim 3, wherein the PBS buffer solution contains an immunopotentiator, wherein the immunopotentiator is one or more of beta-glucan, mannose, lentinan, astragalus polysaccharide, codonopsis pilosula polysaccharide, ginseng polysaccharide, angelica polysaccharide, lycium barbarum polysaccharide, ganoderma lucidum polysaccharide, curcumin and hawthorn polysaccharide.
6. The method for preparing beta-2-microglobulin polyclonal antiserum according to claim 1, wherein the interval between two adjacent immunizations is 7-14 days.
7. The method of preparing a β 2-microglobulin polyclonal antiserum according to claim 1, further comprising:
after the last immunization, performing heart basal blood collection on the experimental rabbit, standing, sucking the supernatant, performing centrifugal precipitation, and separating the supernatant to obtain the beta 2-microglobulin polyclonal antiserum.
8. The method of claim 1, wherein amino electrolytic multivitamins are added to the drinking water of the experimental rabbit before the experimental rabbit is immunized.
9. The method for producing a β 2-microglobulin polyclonal antiserum according to claim 1, wherein said experimental rabbit is a japanese big ear rabbit.
10. The preparation method of the beta 2-microglobulin polyclonal antiserum is characterized by comprising the following steps:
step one, mixing beta 2-microglobulin antigen, 1xPBS buffer solution and complete Freund's adjuvant to prepare first antigen emulsion, wherein the concentration of the beta 2-microglobulin antigen in the first antigen emulsion is 1 mg/mL; mixing beta 2-microglobulin antigen, 1xPBS buffer solution and incomplete Freund's adjuvant to prepare a second antigen emulsion, wherein the concentration of the beta 2-microglobulin antigen in the second antigen emulsion is 0.4-0.5 mg/mL; selecting 4 subcutaneous injection points in front of the shoulder, 4 subcutaneous injection points in the groin and 4 subcutaneous injection points in the back of the neck;
secondly, performing primary immunization on the Japanese big ear rabbit by using a first antigen emulsion, wherein the injection amount of the first antigen emulsion at each injection point is 150-170 mu L;
after 7-14 days, performing second, third and fourth immunizations on the Japanese big-ear rabbits by using a second antigen emulsion, wherein the interval between each immunization is 7-14 days, and the injection amount of the second antigen emulsion at each injection point is 150-170 mu L;
and step four, after 7-14 days of the sixth immunization, performing auricular vein blood sampling, performing ELISA detection, performing heart basal blood sampling after meeting target requirements, standing, absorbing upper-layer serum after the serum is completely separated out, performing centrifugal precipitation separation to obtain upper-layer antiserum, taking the upper-layer antiserum, adding thimerosal into the upper-layer antiserum, and storing.
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| US20120213796A1 (en) * | 2011-02-22 | 2012-08-23 | Fox Barbara S | Polyclonal antibody compositions |
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