CN112375144A - Preparation method of rabbit anti-human ApoA1 polyclonal antiserum - Google Patents
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Abstract
The invention discloses a method for producing anti-human ApoA1 polyclonal antiserum by using rabbits, wherein 10-month-old male Japanese big-ear rabbits with strong physique and good immunity are selected as immune animals, low-dose multipoint immunization is performed under the subcutaneous area before shoulders, the subcutaneous area of inguinal region and the subcutaneous area of neck and back, and the immunization dose and the immunization interval are optimized according to the serum antibody titer of the animals in the immune period, so that the aim of activating multiple-part lymphocytes to improve the immunization titer and obtain the stable antibody level is fulfilled, and the stable performance of antibody raw materials is guaranteed.
Description
Technical Field
The invention relates to the field of biological diagnosis antibody raw materials, in particular to a preparation method of rabbit polyclonal antiserum resisting human ApoA 1.
Background
Apolipoprotein is an important apolipoprotein participating in the transportation of plasma lipid (cholesterol, triglyceride and phospholipid) in plasma, and the currently discovered apolipoproteins mainly comprise APOAI, APOAII, Apo IV, ApoB100, Apoa, ApoE, ApoH, ApoCII, ApoCIII and ApoB48, wherein ApoJA I is High in content and accounts for more than 70% of the total amount of High Density Lipoprotein (HDL) protein, ApoI is the main apolipoprotein of HDL, and HDL is plasma lipoprotein resisting atherosclerosis, so that the detection of the ApoAI level in plasma plays an important reference value for the evaluation of the condition of an atherosclerotic patient.
For the detection of ApoAI levels, biochemical turbidimetric methods are currently mainly used, which require the corresponding ApoAI biochemical reagents, of which ApoAI standards and anti-human ApoAI antibodies are the main constituents. Biochemical turbidimetry has strict requirements on repeatability of a reagent detection result, difference between batches (CV%), detection sensitivity, linear range and stability of a reagent, and the indexes are determined by antibody raw materials to a great extent.
At present, animals for preparing the anti-human ApoA I antibody mainly comprise sheep, rabbits, mice and the like, wherein the application of the sheep-derived anti-human ApoA1 antibody is the most, the sheep body is large in size, the amount of collected plasma is large, and the clinical production needs are very suitable for, but the sheep-derived anti-human ApoA1 antibody has the problems of poor stability and low affinity, and the amino acid sequence homology of the ApoA1 protein of the rabbit and the human is higher than that of the sheep, so that the affinity of the antibody obtained by immunization is higher than that of the sheep-derived anti-human ApoA1 antibody; the mouse mainly participates in the preparation of the monoclonal antibody, and the production period is long and the influence factors are more, so the application of the mouse in the preparation of the ApoA1 antibody is less. Compared with an immune animal sheep, the rabbit has lower serum fat content, is more suitable for the immunity of ApoA I, can effectively avoid the interference of blood fat in the collection of plasma antibodies, and has higher sensitivity of the obtained antibodies, wherein the Japanese big-ear rabbit has strong anti-stress capability, so the immune effect of an animal body on an antigen can be effectively ensured, and the immune titer is improved.
The traditional immunization method mainly adopts single-point large-dose injection, is influenced by emulsification operation and the functional difference of individual lymphatic systems of animals, is easy to reduce the capture probability of antigens and the absorption and diffusion capacity of local tissues, and finally reduces the immunization effect or causes immune tolerance.
Disclosure of Invention
The invention aims to improve the product performance of a biochemical reagent raw material antihuman ApoAI antibody and meet the production requirement, and provides a method for obtaining a high-titer and high-affinity antibody by using multi-site low-dose multipoint immunization of dominant animals.
To achieve this, a protocol for the preparation of rabbit anti-human ApoA1 polyclonal antiserum was carried out.
One embodiment of the present invention provides a method for preparing rabbit anti-human ApoA1 polyclonal antiserum, comprising the steps of:
(1) selection of immunized animals: the rabbit feed is strong in physique, good in anti-stress capability and low in blood fat level (namely, the triglyceride level (47 +/-5) mg/dl of the rabbit); wu Yin, Ester Carballo Jane, David G McLaren, et al, plasma Lipid profiling experiments for the identification of optimal animal models of human dyslipemia [ J ]. Journal of Lipid Research,2012,53(1):51-65] male Japanese big ear rabbits of 10 months of age were immunized animals;
(2) improvement of the anti-stress of the immunized animals: amino electrolysis multidimensional drinking water is given 3 days before immunization, so that the supply of electrolytes, amino acids and vitamins in the organism is maintained, and the anti-stress capability is improved;
(3) preparation of ApoA1 emulsified antigen: emulsifying purified human serum ApoA1 antigen 4mg 0.1ml, pH7.40.01M PBS 0.9ml and Freund's adjuvant 1ml by electric stirring to obtain 2ml total volume as primary immunity; the same antigen 8mg 0.1ml, pH7.40.01M PBS 0.9ml and incomplete Freund's adjuvant 1ml are stirred and emulsified electrically, the total volume is 2ml, as the second immunity; the total volume of 10mg 0.1ml of the same antigen, 0.9ml of pH7.40.01M PBS and 1ml of incomplete Freund's adjuvant after electric stirring and emulsification is 2ml, and the antigen is used as third immunity; the same antigen 10mg 0.1ml, pH7.40.01M PBS 0.9ml after even mixing the total volume is 2ml, as the fourth immunity; the same antigen 16mg 0.1ml, pH7.40.01M PBS 1.9ml after uniform mixing the total volume is 2ml, as the fifth immunity; the same antigen 16mg 0.1ml, pH7.40.01M PBS 1.9ml after uniform mixing the total volume is 2ml, as the sixth immunity;
(4) ApoA1 immunization program:
(a) primary immunization rabbits were subjected to a baoding kit for baoding, with 150 μ l of emulsified 4mg ApoA1 primary immunization antigen per injection site per average rabbit, at 4 injection sites subcutaneously in the anterior shoulder, 4 injection sites subcutaneously in the groin, and 4 injection sites subcutaneously in the back of the neck;
(b) the interval between the second immunization and the first immunization is 15 days, 4 injection points under the front skin of the shoulder, 4 injection points under the groin and 4 injection points under the back skin of the neck of the rabbit are injected, and 150 mu l of emulsified 8mg of ApoA1 second immunization antigen is injected into each injection point of each rabbit on average;
(c) separating the third immunization from the second immunization at 21d, taking blood from ear marginal veins of the immunized rabbits, separating out serum antibody titer and performing agar gel detection, determining the third immunization time according to the antibody level and the antigen absorption condition of the original immune part, and injecting 150 mu l of emulsified 10mg ApoA1 third immunization antigen into each injection point of each rabbit on average;
(d) separating the fourth immunization from the third immunization by 21d, collecting blood from ear marginal veins of immunized rabbits, separating out antibody titer of serum, performing agar detection, determining the fourth immunization time according to the antibody level and the antigen absorption condition of the original immune part, wherein the immune antigen is non-emulsified 10mg ApoA1 antigen aqua, and 150 mu l of non-emulsified 10mg ApoA1 fourth immune antigen is injected into each injection point of each rabbit on average;
(e) separating the fifth immunization from the fourth immunization by 21d, collecting blood from ear marginal veins of immunized rabbits, separating out serum antibody titer and linaloon detection, determining the fifth immunization time according to the antibody level and the antigen absorption condition of the original immune part, wherein the immune antigen is non-emulsified 16mg ApoA1 antigen aqua, and 150 mu l of non-emulsified 16mg ApoA1 fifth immune antigen is injected into each injection point of each rabbit on average;
(f) separating the sixth immunization from the fifth immunization by 21d, taking blood from ear marginal veins of immunized rabbits, separating out serum antibody titer and linaloon detection, determining the sixth immunization time according to the antibody level and the antigen absorption condition of the original immune part, wherein the immune antigen is non-emulsified 16mg ApoA1 antigen aqua, and 150 mu l of non-emulsified 16mg ApoA1 sixth immune antigen is injected into each injection point of each rabbit on average;
(g) collecting blood by ear vein 15d after 6 th immunization, separating out serum antibody titer, performing agar amplification detection, collecting blood by rabbit heart base after fasting for 8h the next day after the serum titer meets the target requirement, standing at 37 ℃ for 1h, placing at 4 ℃, sucking the upper layer serum by using a pipette gun after the serum is completely separated out, placing in a centrifuge tube, performing centrifugal precipitation separation at 3000rpm for 15min to obtain upper layer antiserum, taking the supernatant, adding thimerosal with the final concentration of 0.01%, and storing at 4 ℃ for a long time.
In one embodiment, the rabbit from big ear of Japan with strong environmental adaptability and good immunity is selected as the antiserum donor against human ApoA1 in the step (1).
In one embodiment, the ApoA1 immunogen used in step (2) above is prepared by ultracentrifugation.
In one embodiment, step (3) above uses a low dose multiple spot immunization method near multiple subcutaneous superficial lymphomas.
In one embodiment, the method for determining the immune titer of step (4) above is an agarose gel assay.
In one embodiment, the purification step of the antigen apolipoprotein ai comprises: to normal human plasma [ HBV (-); HIV (-) adopts a Bechman L90k ultracentrifuge to carry out sequence ultracentrifugation, the centrifugal force is controlled at 4000 g-5000 g for 30-60min, the upper serum is separated after centrifugation, the serum concentration is adjusted to 1.0g/ml by PBS, 200 mu L of small sample is kept and is added with 0.01 percent sodium azide for preservation at 4 ℃, 1.0-1.4g/ml sodium bromide is added into the serum obtained by the rest separation in time for density gradient centrifugation, the sample adding sequence is that 1.0-1.3g/ml sodium bromide solution is added from low to high according to the concentration, after a gradient zone is formed by the low-speed centrifugation at 3000rpm, 20-30ml of 1.0g/ml serum is added into the wall of a slow-extension tube, and finally 1.4g/ml sodium bromide solution is added, and the total volume is 600 ml. After all samples are added, standing at room temperature for 20min, starting a centrifugal machine to adjust the speed to 48000rpm, centrifuging at 10 ℃ for 90min, separating to obtain an orange liquid layer HDL (high density lipoprotein) with the density of 1.063-1.210, filling the obtained HDL into a dialysis bag, putting the dialysis bag into osmotic pressure salt solutions with different concentrations, dialyzing for 48h by the dialysis bag, performing purity identification by 1% agarose gel electrophoresis and 3% PAGE (figure 1), degreasing according to a phosphotungstic acid method after identification, performing ion exchange purification by Sephadex CL-6B (figure 2), and collecting the obtained purified AI for enzyme-labeled detection.
In one embodiment, the apolipoprotein ai immunizing antigen is prepared as follows: 4mg of 0.1ml of apoA1 antigen, 0.9ml of pH7.40.01M PBS and 1ml of complete Freund's adjuvant which are extracted and purified by ultracentrifugation are emulsified by electric stirring, and the total volume is 2ml, which is used as primary immunity; the second immunization adopts 8mg of the same antigen emulsion for immunization; the third immunization adopts 10mg of the same antigen and 10mg of emulsion for immunization; the fourth immunization adopts 10mg of the same antigen aqua immunization; the fifth immunization adopts 16mg of the same antigen aqua for immunization; the sixth immunization was performed with 16mg of the same antigen in aqueous solution.
In one embodiment, the immunization program for apolipoprotein ai antigens is as follows:
(1) primary immunization rabbits were subjected to a baoding kit for baoding, with 150 μ l of emulsified 4mg ApoA1 primary immunization antigen per injection site per average rabbit, at 4 injection sites subcutaneously in the anterior shoulder, 4 injection sites subcutaneously in the groin, and 4 injection sites subcutaneously in the back of the neck;
(2) the interval between the second immunization and the first immunization is 15 days, 4 injection points under the front skin of the shoulder, 4 injection points under the groin and 4 injection points under the back skin of the neck of the rabbit are injected, and 150 mu l of emulsified 8mg of ApoA1 second immunization antigen is injected into each injection point of each rabbit on average;
(3) separating the third immunization from the second immunization at 21d, taking blood from ear marginal veins of the immunized rabbits, separating out serum antibody titer and performing agar gel detection, determining the third immunization time according to the antibody level and the antigen absorption condition of the original immune part, and injecting 150 mu l of emulsified 10mg ApoA1 third immunization antigen into each injection point of each rabbit on average; .
(4) And (3) separating the fourth immunization from the third immunization by 21d, collecting blood from ear marginal veins of the immunized rabbits, separating out serum antibody titer and performing Jojoba detection, determining the fourth immunization time according to the antibody level and the antigen absorption condition of the original immune part, wherein the immune antigen is non-emulsified 10mg ApoA1 antigen aqua, and 150 mu of non-emulsified 10mg ApoA1 fourth immune antigen is injected into each injection point of each rabbit on average.
(5) And (3) taking blood from the ear marginal vein of the immunized rabbits at an interval of 21d between the fifth immunization and the fourth immunization, separating out serum antibody titer and agar detection, determining the fifth immunization time according to the antibody level and the antigen absorption condition of the original immune part, wherein the immune antigen is non-emulsified 16mg ApoA1 antigen aqua, and 150 mu of non-emulsified 16mg ApoA1 fifth immune antigen is injected into each injection point of each rabbit on average.
(6) And (3) carrying out ear marginal vein blood collection on the immunized rabbits at an interval of 21d between the sixth immunization and the fifth immunization, separating out serum antibody titer and Roqiong diffusion detection, determining the sixth immunization time according to the antibody level and the antigen absorption condition of the original immune part, wherein the immune antigen is a non-emulsified 16mg ApoA1 antigen aqua, and each injection point of each rabbit is averagely injected with 150 mu of non-emulsified 16mg ApoA1 sixth immune antigen.
(7) Collecting blood by ear vein 15d after 6 th immunization, separating out serum antibody titer, performing agar amplification detection, collecting blood by rabbit heart base after fasting for 8h the next day after the serum titer meets the target requirement, standing at 37 ℃ for 1h, placing at 4 ℃, sucking the upper layer serum by using a pipette gun after the serum is completely separated out, placing in a centrifuge tube, performing centrifugal precipitation separation at 3000rpm for 15min to obtain upper layer antiserum, taking the supernatant, adding thimerosal with the final concentration of 0.01%, and storing at 4 ℃ for a long time.
The invention has the beneficial effects that: according to the invention, the dominant animal, namely the Japanese big ear rabbit, is selected as a donor for producing anti-human ApoA1 polyclonal antiserum, and the interference of hyperlipidemia in the antiserum purification process is effectively avoided based on the low blood lipid level and the better anti-stress capability of the dominant animal; meanwhile, subcutaneous multi-point low-dose immunization near superficial lymph is beneficial to promoting local antigen diffusion and enhancing immune response of multiple lymphocyte, so that an organism is guaranteed to obtain more memory immune cells, the antibody titer and stability are improved, and the stability of a biological reagent and the accuracy of product detection are improved.
Drawings
The following figures are further illustrative of the results of the present invention, wherein:
FIG. 1 shows purity determination by 1% agarose gel electrophoresis and 3% PAGE.
FIG. 2 is a chromatogram of ion exchange purification of Sephadex CL-6B, in which the abscissa is time and the ordinate is absorbance A.
FIG. 3 shows the serum titer of anti-human ApoA1 from different rabbits.
FIG. 4 shows the agarose gel electrophoresis assay of the rabbit anti-human ApoA1 mixed serum titer.
Detailed Description
Subject: 50 healthy and vigorous 8-month-old male Japanese big-ear rabbits with average weight (5.0 +/-0.54) kg are selected, all the rabbits are provided with ear tags, and the ear numbers of the ear tags correspond to the immunity and production archive information of each rabbit. The breeding mode comprises cage culture, fixing drinking trough and tray, feeding granulated feed, automatic drinking water system, feeding three times a day (morning: middle: night: 3: 4), semi-open breeding mode, and adding amino electrolytic multivitamin into drinking water 3d before immunization.
The experimental method comprises the following steps:
(1) preparing immune antigen: calculating the antigen demand according to the number of animals to be immunized according to a plan, wherein the total amount of 50 Japanese big-ear rabbits subjected to primary immunization needs 200mg of purified human serum ApoA1 antigen, the total volume of the antigen after emulsification by Freund's complete adjuvant or Freund's incomplete adjuvant is 100ml, 2 ml/rabbit, and 150 mul is injected into 4 injection points of the subcutaneous site in front of the shoulder, 4 injection points of the subcutaneous site in the groin and 4 injection points of the subcutaneous site in the back of the neck on average; the water aqua antigen without emulsification operation is prepared through diluting purified human serum ApoA1 antigen 200mg with 0.01MPBS (in the ratio of 1 to 9) and has the same injection dosage, injection site and injection point as those of emulsifying adjuvant.
(2) Immunization program, wherein the primary immunization is carried out after 4mg of purified human serum ApoA1 antigen and Freund's complete adjuvant are evenly emulsified; the second immunization adopts 8mg of the same antigen emulsion for immunization; the third immunization adopts 10mg of the same antigen and 10mg of emulsion for immunization; the fourth immunization adopts 10mg of the same antigen aqua immunization; the fifth immunization adopts 16mg of the same antigen aqua for immunization; the sixth immunization was performed with 16mg of the same antigen in aqueous solution.
(3) Rabbit anti-human ApoA I serum collection
According to the requirement of an immunization program, performing rabbit marginal vein blood collection for 2-3 ml/rabbit at 3w after the second immunization, the third immunization, the fourth immunization and the fifth immunization, standing for 1h at room temperature (37 ℃), separating out serum, centrifuging at 3000rpm for 15min, taking supernatant, adding 0.01% thimerosal, and preserving at 4 ℃.
After reaching the target titer by serum detection, blood was collected from the heart 15 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 ℃. Rabbit anti-human serum ApoA1 serum titer agar-agar assay
Preparing agar: weighing 1g of agar powder, adding into 100ml of physiological saline, heating and boiling by an induction cooker until the agar solution is completely dissolved for about 20min, turning off a heating power supply, cooling the agar solution to 70 ℃, uniformly adding the agar solution onto a clean glass dish by using a glass suction pipe, controlling the thickness to be 2-3mm, after the agar solution is completely solidified by natural cooling, punching by using a plum blossom puncher, and slightly picking out the agar.
Experiment design: firstly, carrying out agar-agar amplification detection on the serum antibody titer of a single animal and recording; next, sera from all immunized animals were run according to 1: 1, after uniformly mixing, carrying out titer detection and recording on the agar-agar antibody.
And (3) detection of agar-agar and agar-agar: the agar-agar gel of preparation # was prepared by adding normal human serum as antigen to the middle well, adding gradiently diluted rabbit antiserum to be tested (0.01M PBS (pH7.4) to the peripheral well), adding 20. mu.l of antiserum to each well, and adding pBS to the negative well as control. After the sample adding is finished, the agar plate is placed into a wet box to be kept overnight at 37 ℃, the position and the definition of a precipitation line between a peripheral hole and a central hole are observed the next day, and the antiserum titer of rabbits with different numbers is recorded according to the condition of the maximum dilution multiple of the hole in which the precipitation line is positioned.
TABLE 1 comparison of Rabbit number ratios and protein concentrations at different serum titer levels
Serum titer evaluation results: the serum titer and rabbit specific distribution of the immune rabbit obtained by the invention are 1: 16 (0.00%), 1:32 (22.00%), 1: 64 (78.00%) and 1: 128 (0.00%); the rabbit anti-human ApoA1 serum titer range is 1: 32-1: 64 (fig. 3), the centrifugally obtained rabbit anti-human ApoA1 serum antibody was raised at a rate of 1: 1, mixing, and detecting the titer of the mixed serum to be 1: 64 (fig. 4).
The results of the potency assay are summarized as follows: the rabbit of Japanese big ear is used as an immune animal, after 6 times of multi-site low-dose multi-point injection immunization, the antibody titer of rabbit anti-human ApoA1 serum can reach more than 1:32, and the antibody titer level of mixed serum reaches nearly 1: 64. the method fully proves that the rabbit anti-human ApoA1 polyclonal antibody with high efficiency and stability can be prepared by immunizing the Japanese tremella rabbit with a plurality of parts of low dose, and provides favorable guarantee for stabilizing the raw material conditions of the biological reagent and improving the stability of the biological reagent product.
Claims (5)
1. A method for preparing rabbit anti-human ApoA1 polyclonal antiserum, comprising the steps of:
(1) selection of immunized animals: selecting 10-month-old male Japanese big-ear rabbits as immune animals, wherein the male Japanese big-ear rabbits are strong in physique, good in anti-stress capability and good in triglyceride level (47 +/-5) mg/dl;
(2) improvement of the anti-stress of the immunized animals: amino electrolysis multidimensional drinking water is given 3 days before immunization, so that the supply of electrolytes, amino acids and vitamins in the organism is maintained, and the anti-stress capability is improved;
(3) preparation of ApoA1 emulsified antigen: emulsifying purified human serum ApoA1 antigen 4mg 0.1ml, pH7.40.01M PBS 0.9ml and Freund's adjuvant 1ml by electric stirring to obtain 2ml total volume as primary immunity; the same antigen 8mg 0.1ml, pH7.40.01M PBS 0.9ml and incomplete Freund's adjuvant 1ml are stirred and emulsified electrically, the total volume is 2ml, as the second immunity; the total volume of 10mg 0.1ml of the same antigen, 0.9ml of pH7.40.01M PBS and 1ml of incomplete Freund's adjuvant after electric stirring and emulsification is 2ml, and the antigen is used as third immunity; the same antigen 10mg 0.1ml, pH7.40.01M PBS 0.9ml after even mixing the total volume is 2ml, as the fourth immunity; the same antigen 16mg 0.1ml, pH7.40.01M PBS 1.9ml after uniform mixing the total volume is 2ml, as the fifth immunity; the same antigen 16mg 0.1ml, pH7.40.01M PBS 1.9ml after uniform mixing the total volume is 2ml, as the sixth immunity;
(4) ApoA1 immunization program:
(a) primary immunization rabbits were subjected to a baoding kit for baoding, with 150 μ l of emulsified 4mg ApoA1 primary immunization antigen per injection site per average rabbit, at 4 injection sites subcutaneously in the anterior shoulder, 4 injection sites subcutaneously in the groin, and 4 injection sites subcutaneously in the back of the neck;
(b) the interval between the second immunization and the first immunization is 15 days, 4 injection points under the front skin of the shoulder, 4 injection points under the groin and 4 injection points under the back skin of the neck of the rabbit are injected, and 150 mu l of emulsified 8mg of ApoA1 second immunization antigen is injected into each injection point of each rabbit on average;
(c) separating the third immunization from the second immunization at 21d, taking blood from ear marginal veins of the immunized rabbits, separating out serum antibody titer and performing agar gel detection, determining the third immunization time according to the antibody level and the antigen absorption condition of the original immune part, and injecting 150 mu l of emulsified 10mg ApoA1 third immunization antigen into each injection point of each rabbit on average;
(d) separating the fourth immunization from the third immunization by 21d, collecting blood from ear marginal veins of immunized rabbits, separating out antibody titer of serum, performing agar detection, determining the fourth immunization time according to the antibody level and the antigen absorption condition of the original immune part, wherein the immune antigen is non-emulsified 10mg ApoA1 antigen aqua, and 150 mu l of non-emulsified 10mg ApoA1 fourth immune antigen is injected into each injection point of each rabbit on average;
(e) separating the fifth immunization from the fourth immunization by 21d, collecting blood from ear marginal veins of immunized rabbits, separating out serum antibody titer and linaloon detection, determining the fifth immunization time according to the antibody level and the antigen absorption condition of the original immune part, wherein the immune antigen is non-emulsified 16mg ApoA1 antigen aqua, and 150 mu l of non-emulsified 16mg ApoA1 fifth immune antigen is injected into each injection point of each rabbit on average;
(f) separating the sixth immunization from the fifth immunization by 21d, taking blood from ear marginal veins of immunized rabbits, separating out serum antibody titer and linaloon detection, determining the sixth immunization time according to the antibody level and the antigen absorption condition of the original immune part, wherein the immune antigen is non-emulsified 16mg ApoA1 antigen aqua, and 150 mu l of non-emulsified 16mg ApoA1 sixth immune antigen is injected into each injection point of each rabbit on average;
(g) collecting blood by ear vein 15d after 6 th immunization, separating out serum antibody titer, performing agar amplification detection, collecting blood by rabbit heart base after fasting for 8h the next day after the serum titer meets the target requirement, standing at 37 ℃ for 1h, placing at 4 ℃, sucking the upper layer serum by using a pipette gun after the serum is completely separated out, placing in a centrifuge tube, performing centrifugal precipitation separation at 3000rpm for 15min to obtain upper layer antiserum, taking the supernatant, adding thimerosal with the final concentration of 0.01%, and storing at 4 ℃ for a long time.
2. The method according to claim 1, wherein step (1) selects the Japanese big-ear rabbit with strong environmental adaptability and good immunity as an antiserum donor against human ApoA 1.
3. The method of claim 1 wherein the ApoA1 immunogen used in step (2) is prepared by ultracentrifugation.
4. The method of claim 1, wherein step (3) uses a low dose multiple spot immunization method near multiple subcutaneous superficial lymphomas.
5. The method of claim 1, wherein the step (4) immunotiter assay is an agarose detection.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113698482A (en) * | 2021-08-30 | 2021-11-26 | 北京达成生物科技有限公司 | Preparation method of beta 2-microglobulin multi-antiserum |
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