Rabbit polyclonal antibody as well as preparation method and related application thereof

Abstract

The invention discloses a rabbit polyclonal antibody as well as a preparation method and related application thereof, and relates to the field of antibodies. It is found that when the novel adjuvant with the active ingredients including QS-21 and polyinosinic acid Poly I: C is applied to preparation of the rabbit polyclonal antibody, the immunogenicity of the antigen can be more effectively enhanced, generation of the antibody with higher affinity and specificity is promoted, and therefore the antibody with the higher level is induced in a rabbit body; and a new way is provided for efficient preparation of the high-titer rabbit polyclonal antibody.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and in particular to a rabbit polyclonal antibody and a preparation method and related applications thereof. Background Art

[0002] Polyclonal antibodies are a mixture of antibodies produced by multiple B lymphocyte clones that can bind to multiple antigen epitopes. They have broad application prospects in biomedical research, precision medicine, personalized therapy, and immunoassay technology. Polyclonal antibodies contain multiple different antibodies that can recognize multiple different epitopes of the same antigen molecule, which may provide higher sensitivity and overall antibody affinity. Polyclonal antibodies are relatively fast to produce and low in cost, and are usually the first commercialized antibodies against a specific target.

[0003] Rabbit polyclonal antibodies are groups of antibodies produced against specific antigens by immunizing rabbits. These antibodies can recognize multiple epitopes on the same antigen and are therefore more effective in certain applications. At the same time, rabbits are advantageous animals for the preparation of polyclonal antibodies due to their moderate size, good immune response and high antibody production. The preparation process of rabbit polyclonal antibodies includes antigen selection, animal immunization and antibody purification. In the process of animal immunization, the selection of appropriate immune adjuvants is conducive to the production of high titer rabbit polyclonal antibodies.

[0004] At present, Freund's adjuvant is mostly used in the field to prepare rabbit polyclonal antibodies. Freund's adjuvant needs to be emulsified before immunizing animals, which is a cumbersome operation and easily causes large batch differences. In addition, the antibody titer of rabbit polyclonal antibodies needs to be improved.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a rabbit polyclonal antibody and a preparation method and related applications thereof.

[0007] The present invention is achieved in that:

[0008] In a first aspect, an embodiment of the present invention provides use of a target adjuvant in preparing rabbit polyclonal antibodies, wherein the active ingredients of the target adjuvant include: QS-21 and polyinosinic acid (Poly I:C).

[0009] In a second aspect, an embodiment of the present invention provides a method for preparing rabbit polyclonal antibodies, comprising: immunizing rabbits with an immunogen to obtain rabbit polyclonal antibodies; wherein the immunogen comprises an antigen and the target adjuvant described in the aforementioned embodiment.

[0010] In a third aspect, an embodiment of the present invention provides a rabbit polyclonal antibody, which is prepared by the preparation method described in the above embodiment.

[0011] In a fourth aspect, an embodiment of the present invention provides a kit, comprising: the rabbit polyclonal antibody described in the above embodiment.

[0012] In a fifth aspect, embodiments of the present invention provide the use of the rabbit polyclonal antibodies described in the preceding embodiments in detecting relevant antigens for purposes other than disease diagnosis or treatment or in preparing products for detecting the relevant antigens.

[0013] The present invention has the following beneficial effects:

[0014] The present invention has found a new adjuvant that is beneficial for preparing high-titer rabbit polyclonal antibodies. Compared with other adjuvants, it can more effectively enhance the immunogenicity of antigens without the need for emulsification treatment, promote the production of antibodies with higher affinity and specificity, and thus induce higher levels of antibodies in rabbits. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 This is the fold chart of the serum RV-tG specific IgG antibody titer of each experimental group after immunization;

[0017] Figure 2 It is a bar graph of serum gE-specific IgG antibody titer after immunization in each experimental group;

[0018] Figure 3 The gE-specific CD4 + T cell immune response bar graph;

[0019] Figure 4 It is a bar graph of serum RSV-specific IgG antibody titer after immunization in each experimental group;

[0020] Figure 5 It is a bar graph of the neutralizing antibody titer against RSV A2 virus in the serum of each experimental group after immunization;

[0021] Figure 6 It is a bar graph of the serum hMPV-specific IgG antibody titer of each experimental group after immunization;

[0022] Figure 7 It is a bar graph of the neutralizing antibody titer against hMPV virus in the serum of each experimental group after immunization;

[0023] Figure 8It is a bar graph of the hPIV-specific IgG antibody titer in the serum of each experimental group after immunization;

[0024] Fig. 9 It is a bar graph of the neutralizing antibody titer against hPIV virus in the serum of each experimental group after immunization. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0026] The present invention finds that applying a new type of target adjuvant to the preparation of rabbit polyclonal antibodies has advantages. Compared with other adjuvants, the target adjuvant does not require additional emulsification treatment, and only needs to mix the target adjuvant and the antigen, which not only improves the convenience of operation, but also can induce higher antibody titers than other adjuvants.

[0027] On the one hand, an embodiment of the present invention provides use of a target adjuvant in preparing rabbit polyclonal antibodies, wherein the active ingredients of the target adjuvant include: QS-21 and polyinosinic acid (Poly I:C).

[0028] QS-21, CAS No. 141256-04-4, molecular formula: C 92 H 148 O 46 , molecular weight: 1990.14.

[0029] In some embodiments, in the target adjuvant, the final concentration of QS-21 is 35-300 μg / mL. The final concentration can be any one of 35, 40, 45, 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 and 300 μg / mL or a range between any two thereof.

[0030] Polyinosinic-polycytidylic acid (Poly I:C) is a toll-like receptor 3 (TLR3) agonist and a synthetic double-stranded RNA (dsRNA). CAS No.: 42524-50-0.

[0031] In some embodiments, in the target adjuvant, the final concentration of the polyinosinic acid Poly I:C is 400-3000 μg / mL. The final concentration can be any one of 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800 and 3000 μg / mL or a range between any two thereof.

[0032] In some embodiments, the target adjuvant further comprises a liposome carrier.

[0033] In some embodiments, the liposome carrier comprises: any one or more of a cationic liposome and a neutral liposome.

[0034] In some embodiments, the raw materials for preparing the cationic liposomes include any one or more of 1,2-dioleoyl-3-trimethylammonium chloride propane (DOTAP), dioleoylphosphatidylcholine (DOPC) and cholesterol.

[0035] In some embodiments, the mixing mass ratio of 1,2-dioleoyl-3-trimethylammonium chloride propane (DOTAP), dioleoylphosphatidylcholine (DOPC) and cholesterol can be any one of 1:1:1, 2:1:1, 3:1:1, 1:2:1, 1:3:1, 2:2:1, 3:3:1, 2:3:1, 3:2:1, or a range between any two of them.

[0036] In some embodiments, the method for preparing the cationic liposome comprises:

[0037] Mixing 1,2-dioleoyl-3-trimethylammonium chloride propane, dioleoylphosphatidylcholine and cholesterol in a mass ratio of 1 to 3:1 to 3:1 to obtain a first mixture;

[0038] The first mixture is used as a raw material, and cationic liposomes are prepared by using any one of an ethanol injection method, a thin film dispersion method, an ultrasonic dispersion method and a reverse evaporation method.

[0039] In some embodiments, the method for preparing the cationic liposome comprises:

[0040] The three lipid components (DOTAP, DOPC and cholesterol) are dissolved in ethanol and sheared online with the aqueous phase for 1 to 3 times; ultrafiltration replacement is performed using a sucrose-containing buffer solution, with a concentration multiple of 2 to 4 times and a filtration multiple of 6 to 8 times; extrusion is performed using a filter membrane with a pore size of 200 nm, and multiple cycles of extrusion are performed to control the uniformity of particle size; extrusion is performed using a filter membrane with a pore size of 100 nm, and multiple cycles of extrusion are performed to control the uniformity of particle size; and sterilization is performed by filtering using a sterile filter.

[0041] The physicochemical parameters of the cationic liposome are: median particle size (D50): 30-90nm; solution pH value: 4.8-7.0, preferably 4.8-6.0; Zeta potential: 40-72mV (pH6.5).

[0042] In some embodiments, the raw materials of the neutral liposomes include any one or more of dioleoylphosphatidylcholine (DOPC) and cholesterol.

[0043] In some embodiments, in the neutral liposomes, the mass ratio of dioleoylphosphatidylcholine to cholesterol is 3 to 5:1, specifically any one of 3:1, 4:1, 5:1 or a range between any two of them.

[0044] In some embodiments, the method for preparing the neutral liposome comprises:

[0045] Mixing dioleoylphosphatidylcholine and cholesterol according to a mass ratio to obtain a second mixture;

[0046] The second mixture is used as a raw material, and any one of an ethanol injection method, a thin film dispersion method, an ultrasonic dispersion method, and a reverse evaporation method is used to prepare neutral liposomes. Optionally, when the ethanol injection method is used for preparation, the preparation method of the neutral liposomes can be: after uniform dispersion, colostrum is prepared in an aqueous phase, and extrusion molding is performed using extrusion membranes with pore sizes of 200 nm and 100 nm in sequence, followed by ultrafiltration to replace the buffer, and filtration sterilization.

[0047] In some embodiments, the preparation steps of the neutral liposomes are as follows:

[0048] Dissolve dioleoylphosphatidylcholine and cholesterol in ethanol according to the mass ratio, and shear with the water phase online for 1 to 3 times; use a filter membrane with a pore size of 200 nm to filter, perform multiple cycles of extrusion, and control the uniformity of particle size; use a filter membrane with a pore size of 100 nm to filter, perform multiple cycles of extrusion, and control the uniformity of particle size; use a sucrose-containing buffer solution for ultrafiltration replacement, with a concentration multiple of 3 to 6 times and a filtration multiple of 6 to 8 times; use a sterile filter to filter and sterilize.

[0049] The physicochemical parameters of the neutral liposome are: median particle size (D50): 80-140 nm; solution pH value: 5.5-7.0, preferably 6.4-6.6.

[0050] In some embodiments, the final concentration of cholesterol in the liposome carrier in the immune adjuvant is 400-600 μg / mL, specifically any one of 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600 μg / mL or a range between any two of them.

[0051] In some embodiments, in the immune adjuvant, the mass ratio of QS-21, Poly I:C and cholesterol in the liposome carrier is 30-40:350-450:200-300, specifically it can be any one of 30:400:250, 35:400:250, 40:400:250, 30:350:250, 35:350:250, 40:350:250, 30:450:250, 35:450:250, 40:450:250, 30:400:200, 35:400:200, 40:400:200, 30:400:300, 35:400:300, and 40:400:300, or a range between any two of them.

[0052] In some embodiments, in the immune adjuvant, the mass ratio of cholesterol in neutral liposomes to cholesterol in cationic liposomes is 180-220:30-70, specifically it can be any one of 180:30, 180:40, 180:50, 180:60, 180:70, 200:30, 200:40, 200:50, 200:60, 200:70, 220:30, 220:40, 220:50, 220:60, 220:70 or the range between any two of them.

[0053] On the other hand, an embodiment of the present invention provides a method for preparing rabbit polyclonal antibodies, comprising: immunizing rabbits with immunogens to obtain rabbit polyclonal antibodies; wherein the immunogen comprises an antigen and the target adjuvant described in any of the aforementioned embodiments.

[0054] The embodiments of the present invention have no special restrictions on the specific source and type of antigens. The main invention of this application is to discover the advantages of a new target adjuvant in the preparation of rabbit polyclonal antibodies.

[0055] In some embodiments, the antigens include: any one or more combinations of human papillomavirus antigens, human herpesvirus antigens, human metapneumovirus antigens, human parainfluenza virus antigens, respiratory syncytial virus antigens, human immunodeficiency virus antigens, cytomegalovirus antigens, dengue virus antigens, enterovirus antigens, hepatitis A virus antigens, hepatitis B virus antigens, hepatitis C virus antigens, hepatitis D virus antigens, hepatitis E virus antigens, poliovirus antigens, rabies virus antigens, rotavirus antigens, herpes zoster virus antigens, SARS coronavirus antigens, and rhinovirus antigens.

[0056] There is no particular limitation on the specific type of antigen, which can be routinely obtained based on existing technologies. For example, the respiratory syncytial virus antigen includes: respiratory syncytial virus PreF protein and / or inactivated RSV virus.

[0057] In some embodiments, in the immunogen, 60 to 240 μg of the antigen is added for every 17.5 to 60 μg of QS-21.

[0058] In some embodiments, in the immunogen, 60 to 240 μg of the antigen is added for every 17.5 to 60 μg of QS-21. Alternatively, for every 17.5, 20, 30, 35, 40, 50 or 60 μg of QS-21, the mass of the corresponding added antigen can be any one of 60, 80, 100, 120, 140, 160, 180, 200, 220, 240 μg or a range between any two thereof.

[0059] In some embodiments, the initial concentration of the antigen before mixing with the immune adjuvant is 100-300 μg / ml.

[0060] The initial concentration of the antigen can specifically be any one of 100 μg / ml, 120 μg / ml, 140 μg / ml, 160 μg / ml, 180 μg / ml, 200 μg / ml, 220 μg / ml, 240 μg / ml, 260 μg / ml, 280 μg / ml, 300 μg / ml, or a range between any two of them.

[0061] The preparation method provided in the present application has no special restrictions on the immunization method of the immunogen immunized rabbit, and the existing disclosed scheme can be used for immunization. In some embodiments, when performing the immunization, the inoculation amount of the antigen for each rabbit is: 50-200 μg / time, and the number of inoculations is 1-4 times. The inoculation amount of the antigen for each rabbit can specifically be 50μg / time, 60μg / time, 80μg / time, 100μg / time, 120μg / time, 140μg / time, 160μg / time, 180μg / time, 200μg / time. The number of inoculations can be any one of 1, 2, 3, 4 or a range between any two. Optionally, the inoculation method can be intramuscular injection on the outside of the rabbit thigh, with 50μg of antigen injected each time, 2 points on each side, and 25μg of antigen at each point.

[0062] In some embodiments, after immunizing the rabbit, the preparation method further comprises: isolating the serum of the immunized animal, and isolating and obtaining polyclonal antibodies from the serum. There are no special restrictions on the method of collecting serum and isolating polyclonal antibodies, and existing public methods can be used. Optionally, 14 days after the first and second immunizations, blood is collected from the rabbit's ear vein, and after the third immunization, terminal blood is collected by heart blood collection. Optionally, the collected rabbit blood is placed at room temperature for 2 hours, and the supernatant is collected by centrifugation at 2000g to obtain rabbit polyclonal antibody serum.

[0063] On the other hand, an embodiment of the present invention provides a rabbit polyclonal antibody, which is prepared by the preparation method described in any of the above embodiments.

[0064] On the other hand, an embodiment of the present invention provides a kit, comprising: the rabbit polyclonal antibody described in any of the above embodiments.

[0065] In addition, an embodiment of the present invention provides the use of the rabbit polyclonal antibody as described in any of the above embodiments in detecting related antigens of the rabbit polyclonal antibody for non-disease diagnosis or treatment purposes or in preparing a product for detecting the related antigens.

[0066] The embodiments of the present invention have no special restrictions on the specific sources and types of relevant antigens. The main invention of this application is to discover the advantages of a new target adjuvant in the preparation of rabbit polyclonal antibodies.

[0067] In some embodiments, the relevant antigens include: any one or more combinations of human papillomavirus antigens, human herpesvirus antigens, human metapneumovirus antigens, human parainfluenza virus antigens, respiratory syncytial virus antigens, human immunodeficiency virus antigens, cytomegalovirus antigens, dengue virus antigens, enterovirus antigens, hepatitis A virus antigens, hepatitis B virus antigens, hepatitis C virus antigens, hepatitis D virus antigens, hepatitis E virus antigens, poliovirus antigens, rabies virus antigens, rotavirus antigens, herpes zoster virus antigens, SARS coronavirus antigens and rhinovirus antigens.

[0068] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0069] The material information used in the following examples is as follows.

[0070] Table 1 Key materials and reagents information

[0071]

[0072]

[0073] Table 2 Cell line and virus strain information

[0074]

[0075] (2) Instruments

[0076] Table 3 Instrument and equipment information

[0077]

[0078] Example 1: Preparation of Adjuvant 1 and Adjuvant 2.

[0079] 1. Adjuvant 1

[0080] The preparation method of adjuvant 1 is as follows:

[0081] (a) Preparation of cationic liposomes:

[0082] The three lipid components (DOTAP, DOPC and cholesterol in a mass ratio of 2:2:1) were dissolved in ethanol and sheared online with the aqueous phase twice; ultrafiltration replacement was performed using a sucrose buffer solution, with a concentration multiple of 2 times and a filtration multiple of 8 times; 200nm liposomes were extruded using a filter membrane with a pore size of 200nm, and multiple cycles of extrusion were performed to control the uniformity of particle size; 100nm liposomes were extruded using a filter membrane with a pore size of 100nm, and multiple cycles of extrusion were performed to control the uniformity of particle size; and sterilization was performed by filtration using a sterile filter.

[0083] (b) Preparation of neutral liposomes:

[0084] DOPC and cholesterol were dissolved in ethanol at a mass ratio of 4:1 and sheared online with the aqueous phase twice; 200 nm liposomes were extruded, filtered using a filter membrane with a pore size of 200 nm, and extruded for multiple cycles to control the uniformity of particle size; 100 nm liposomes were extruded, filtered using a filter membrane with a pore size of 100 nm, and extruded for multiple cycles to control the uniformity of particle size; phosphate buffer was used for ultrafiltration replacement, with a concentration multiple of 2 times and a filtration multiple of 8 times; and sterilization was performed by filtration using a sterile filter.

[0085] (c) QS-21, Poly I:C, neutral liposomes and cationic liposomes were mixed at a mass ratio of QS-21, Poly I:C, cholesterol in neutral liposomes and cholesterol in cationic liposomes of 35:400:206.9:43.1 to prepare immune adjuvant 1.

[0086] In adjuvant 1, the final concentration of cholesterol in liposomes (the total amount of cholesterol in neutral liposomes and positive liposomes) was 500 μg / ml, the final concentration of QS-21 was 70 μg / ml, and the final concentration of Poly I:C was 800 μg / ml.

[0087] 2. Adjuvant 2

[0088] (a) Preparation of neutral liposomes:

[0089] The preparation method of the neutral liposome specifically comprises: dissolving DOPC and cholesterol in ethanol at a mass ratio of 4:1, and shearing the mixture with the aqueous phase online twice; extruding 200 nm liposomes, filtering them with a filter membrane with a pore size of 200 nm, and performing multiple cycles of extrusion to control the uniformity of particle size; extruding 100 nm liposomes, filtering them with a filter membrane with a pore size of 100 nm, and performing multiple cycles of extrusion to control the uniformity of particle size; ultrafiltration replacement with phosphate buffer, with a concentration multiple of 2 times and a filtration multiple of 8 times; and filtering and sterilizing with a sterile filter.

[0090] (c) QS-21 and neutral liposomes were mixed at a mass ratio of QS-21 to cholesterol in the neutral liposomes of 35:250 and stirred evenly to prepare immune adjuvant 2; wherein the final concentration of cholesterol in the neutral liposomes was 500 μg / ml, and the final concentration of QS-21 was 70 μg / ml.

[0091] Example 2: Immunogenicity induced by adjuvant 1 and adjuvant 2 in mice.

[0092] (1) Immunization of Balb / c mice

[0093] The experiment used 5-week-old SPF female BALB / c mice, with 8 mice in the negative control group and 12 mice in the vaccine group. The experimental animal grouping and immunization information are shown in the table below.

[0094] Table 4 Experimental groups and immunization scheme

[0095]

[0096] Note: RV-tG is a recombinant protein antigen of rabies virus. The antigen and adjuvant are mixed at a volume ratio of 1:1. The antigen content is 100 μg / mL.

[0097] Immunization method: After the test sample is gently shaken before use, 1000μl of the test sample solution is drawn using a 1000μl disposable sterile syringe and needle and injected into the bilateral lateral thigh muscles of New Zealand rabbits.

[0098] Immunization time: two doses with an interval of 7 days.

[0099] Serum collection: Blood was collected from the experimental animals in each test group before immunization (D-1), D3, D6, D10 and D20, and the serum was separated and the serum-specific IgG antibody titer was determined by enzyme-linked immunosorbent assay (ELISA).

[0100] (2) ELISA detection of specific antibodies in serum

[0101] Dilute the RV-tG stock solution with coating solution to a protein concentration of 3μg / ml, and coat the ELISA plate with 100μl / well at 4℃ overnight. After washing 3 times with washing solution, add 200μl of blocking solution to each well and block at 25℃ for 1h. After blocking, pour out the blocking solution, dilute the rabbit antiserum in multiples and add the rabbit negative serum to the ELISA plate, 100μl / well, and incubate at 25℃ for 2h. After washing 3 times with washing solution, add HRP-labeled goat anti-rabbit IgG (1:5000), 100μl / well, and incubate at 25℃ for 1h. After washing 5 times with washing solution, add TMB substrate colorimetric solution, react in the dark for 15min at room temperature, and add 100μl stop solution to stop the reaction. Measure the OD of each well with an enzyme reader 450 The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is defined as the titer of the sample.

[0102] result:

[0103] The serum was separated and the specific IgG antibody titer in the rabbit serum was determined by enzyme-linked immunosorbent assay (ELISA). The bar graph of specific IgG antibody titer of each experimental group after immunization is shown in Figure 1 .

[0104] Six days after the first vaccination (D6), the serum IgG antibody titers of experimental animals in each test group increased. There was no statistical difference in the IgG antibody titers induced by RV-tG (adjuvant 2) and RV-tG (adjuvant 1), both of which were higher than the commercially available vaccines.

[0105] 3 days (D10) and 13 days (D20) after the second immunization, the serum IgG antibody titers of experimental animals in each test group were significantly higher than those in the first immunization. RV-tG (Adjuvant 2) and RV-tG (Adjuvant 1) can rapidly induce the production of high-level binding antibody IgG, and the antibody titer produced by RV-tG (Adjuvant 2) is slightly higher than that of RV-tG (Adjuvant 1). At the same time, the IgG antibody titers induced by the first two are higher than those of commercially available vaccines.

[0106] (3) Conclusion

[0107] In vivo experiments in mice showed that the immunogenicity induced by the vaccine prepared with adjuvant 2 + RV-tG stock solution in mice was better than that induced by the adjuvant 1 + RV-tG stock solution.

[0108] Example 3: Study on the immunogenicity of vaccines containing different doses of QS-21 in mice.

[0109] (1) Immunization of mice

[0110] The experiment used 7-week-old SPF female C57BL / 6J mice, with 5 mice in each negative control group and adjuvant control group, and 10 mice in each vaccine group. The experimental animal grouping and immunization information are shown in the table below.

[0111] Table 5 Experimental groups and immunization scheme

[0112]

[0113] Note: gE is a recombinant protein antigen of herpes zoster virus. The vaccine is a mixture of antigen and adjuvant in a volume ratio of 1:1. The antigen content is 100 μg / mL. For each QS-21 group in Table 5, only the QS-21 content is different from that of adjuvant 1. For details, see Table 5. The other components are consistent with adjuvant 1.

[0114] Immunization method: After the test product is gently shaken before use, 1000μl of the test product solution is drawn using a 1000μl disposable sterile syringe and needle and injected into the bilateral lateral thigh muscles of the mouse.

[0115] Immunization time: Two doses with an interval of 28 days.

[0116] Serum collection: Blood was collected from experimental animals in each test group on D28 and D42, and the serum was separated and the serum specific IgG antibody titer was determined by enzyme-linked immunosorbent assay (ELISA). Intracellular cytokine detection (ICS) was used to detect anti-gE antigen-specific CD4 + T cell immune response.

[0117] (2) ELISA detection of specific antibodies in serum

[0118] Dilute the gE stock solution with coating solution to a concentration of 3μg / ml, and coat the ELISA plate with 100μl / well at 4℃ overnight. After washing 3 times with washing solution, add 200μl of blocking solution to each well and block at 25℃ for 1h. After blocking, pour out the blocking solution, add the sample to the ELISA plate, 100μl / well, and incubate at 25℃ for 1.5h. After washing 3 times with washing solution, add HRP-labeled goat anti-mouse IgG (1:2500), 100μl / well, and incubate at 25℃ for 1.5h. After washing 5 times with washing solution, add TMB substrate colorimetric solution, react at 25℃ in the dark for 15min, and add 100μl of stop solution to stop the reaction. Measure the OD of each well with an enzyme reader 450 The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is defined as the titer of the sample.

[0119] Results Figure 2 : After the second immunization, within a certain range, the titer of mouse serum binding antibody IgG was positively correlated with the content of QS-21, but there was no significant difference among various types.

[0120] (3) Splenic cytokine detection

[0121] After collecting the mouse spleen, store it in an EP tube containing 4 mL of maintenance culture medium, grind and collect spleen cells in a biosafety cabinet, and centrifuge at room temperature (531g, 5min); add 5 mL of ACK lysis buffer to the collected cells for 3 min, add cell washing solution, and centrifuge at room temperature (531g, 5min); centrifuge the collected cells again at room temperature (531g, 5min), discard the supernatant, resuspend the collected cells and count them; after counting, centrifuge at room temperature (531g, 5min) to collect the cells and adjust the cell density to 2×107cell / mL; after adding cell stimulants, 100μl / well was placed in a 96-well U-shaped cell culture plate in a 37℃5% CO2 incubator for stimulation for 1h; after stimulation, 10μl / well of Golgi plug working solution was added to adjust the final concentration to 1μl / mL and continue to incubate at 37℃5% The cells were cultured in a CO2 incubator for 5 h; after centrifugation at room temperature (604 g, 5 min), 200 μl / well D-PBS was added to resuspend the cells; after centrifugation at room temperature (604 g, 5 min), the cells were stained for live and dead cells and 150 μl / well D-PBS was added to resuspend the cells; after centrifugation at room temperature (604 g, 5 min), the cells were collected and blocked with 50 μl / well FACS block, and incubated at 4°C in the dark for 5 min; after blocking, 50 μl / well surface antibody mixture was added to stain the cell surface, and incubated at 4°C in the dark for 30 min; 100 μl / well FACS Buffer, collect cells by centrifugation at room temperature (604g, 5min), add 100μl / well Cytofix / cytoperm, and incubate at 4℃ in dark for 20min; add 100μl / well Perm / wash and centrifuge at room temperature (604g, 5min) to collect cells, then add 50μl / well cytokine antibody mixture for staining, and incubate at 4℃ in dark for 30min; add 150μl / well Perm / wash and centrifuge at room temperature (604g, 5min) to collect cells, add 200μl / well Perm / wash and centrifuge at room temperature (750g, 3min) to collect cells again, and finally add 150μl / well fixative to fix the cells and detect them by flow cytometer.

[0122] Results Figure 3 : After the second immunization, the cellular immune response level induced by QS-21 at a dose of 25 μg was the best, but the difference among the groups was not significant.

[0123] Example 4: Preparation of rabbit anti-respiratory syncytial virus polyclonal antibodies.

[0124] (1) Immunization of New Zealand rabbits

[0125] The experiment used 2.5kg ordinary-grade female New Zealand rabbits, with 5 rabbits in each negative control group and 8 rabbits in each vaccine group. The experimental animal grouping and immunization information are shown in the table below.

[0126] Table 6 Experimental groups and immunization scheme

[0127]

[0128] Note: 1. The first immunization of the Freund's adjuvant group used Freund's complete adjuvant, and the second and third immunizations used Freund's incomplete adjuvant. 2. RSVPreF3 is the respiratory syncytial virus PreF protein antigen. The antigen and adjuvant are mixed in a volume ratio of 1:1. The antigen content is 200μg / mL.

[0129] Immunization method: After the test sample is gently shaken before use, 1000μl of the test sample solution is drawn using a 1000μl disposable sterile syringe and needle and injected into the bilateral lateral thigh muscles of New Zealand rabbits.

[0130] Immunization time: three doses with 14-day intervals.

[0131] Serum collection: Blood was collected from experimental animals in each test group on D14, D28 and D39, and the serum was separated and the serum-specific IgG antibody titer was determined by enzyme-linked immunosorbent assay (ELISA). The rapid fluorescent focus inhibition test (RFFIT) was used to detect the anti-RSV A2 virus neutralizing antibody titer in the serum. The neutralizing antibody titer was expressed as the highest dilution factor of the sample that inhibited 90% virus growth (the number of fluorescent foci in the sample well ≤ the neutralization judgment critical value). Each well was judged separately, and the geometric mean titer of 2 duplicate wells was taken as the final result.

[0132] (2) ELISA detection of specific antibodies in serum

[0133] The RSVPreF3 stock solution was diluted with coating solution to a RSVPreF3 concentration of 4μg / ml, and 100μl / well was coated on the ELISA plate at 4℃ overnight. After washing 3 times with washing solution, 200μl of blocking solution was added to each well and blocked at 25℃ for 1h. After blocking, the blocking solution was poured out, and the rabbit antiserum was diluted in multiples and added to the ELISA plate with rabbit negative serum, 100μl / well, and incubated at 25℃ for 2h. After washing 3 times with washing solution, HRP-labeled goat anti-rabbit IgG (1:2500) was added, 100μl / well, and incubated at 25℃ for 1h. After washing 5 times with washing solution, TMB substrate colorimetric solution was added, and the reaction was carried out at 25℃ in the dark for 15min. 100μl of stop solution was added to stop the reaction. The OD of each well was measured with an enzyme reader 450 The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is defined as the titer of the sample.

[0134] Results: The specific IgG antibody titer in rabbit serum was detected by ELISA. The bar graph of specific IgG antibody titer in each experimental group after immunization is shown in Figure 4 .

[0135] After the first, second and third immunizations, the binding antibody levels induced by adjuvant 1+RSVPreF3 stock solution group were better than those induced by adjuvant 2 and Freund's adjuvant+RSVPreF3 stock solution groups, and the effects of adjuvant 2 and Freund's adjuvant were basically equivalent.

[0136] (3) Serum anti-RSV virus neutralizing antibody detection

[0137] The day before the experiment, Hep-2 cells were plated and cultured in 96-well plates. The serum samples to be tested were diluted in multiples with diluent, and the RSV A2 virus diluted to the appropriate multiple was added. After shaking and mixing, the cells were incubated in a 37°C incubator for neutralization for 1 hour. The cell culture medium in the 96-well plate with monolayer Hep-2 cells was discarded, the residual liquid in the well plate was washed with DPBS, and 100 μL / well serum-virus neutralization solution was added to the 96-well plate, and cultured at 37°C, 5% CO2 for 18 hours. The supernatant in the cell culture plate was discarded, 100 μL / well of 80% acetone solution was added, and incubated at 120°C in the dark for 20 minutes; the acetone solution was discarded and air-dried in a fume hood for about 20 minutes until all wells were completely dry. 50 μL PBST was added to each well, moistened at room temperature for 5 minutes, dried, and the RSV A2 primary antibody solution diluted to the appropriate multiple was added, and incubated at 37°C for 1 hour. Discard the primary antibody, wash the plate 3 times with PBST at 150 μL / well, add the secondary antibody solution diluted to an appropriate multiple, and incubate at 37°C for 1 hour. Discard the secondary antibody, wash the plate 3 times with PBST at 150 μL / well, then observe and count under a fluorescent microscope to calculate the neutralization titer.

[0138] Results: The neutralizing activity of animal sera against RSV virus was detected by rapid fluorescent focus inhibition test (RFFIT). The titer of neutralizing antibodies against RSV A2 virus in sera of each experimental group is shown in the bar graph. Figure 5 .

[0139] After the first, second and third immunizations, the level of anti-RSV A2 neutralizing antibodies induced by adjuvant 1+RSVPreF3 stock solution group was better than that of adjuvant 2 and Freund's adjuvant+RSVPreF3 stock solution group, and the effects of adjuvant 2 and Freund's adjuvant were basically equivalent.

[0140] At the same time, the experiment of using MF59 / aluminum / MPL adjuvant to prepare rabbit anti-respiratory syncytial virus polyclonal antibodies was simultaneously verified in this experiment. The results showed that the effects of several adjuvants were inferior to adjuvant 1.

[0141] (4) Conclusion

[0142] Adjuvant 1 + RSVPreF3 stock solution immunized New Zealand rabbits and induced high titer rabbit anti-RSV polyclonal antibody serum, and the induced antibody level was superior to that of traditional Freund's adjuvant and adjuvant 2.

[0143] Example 5: Preparation of rabbit anti-human metapneumovirus polyclonal antibodies.

[0144] (1) Immunization of New Zealand rabbits

[0145] The experiment used 2.5kg ordinary-grade female New Zealand rabbits, with 5 rabbits in each negative control group and 8 rabbits in each vaccine group. The experimental animal grouping and immunization information are shown in the table below.

[0146] Table 7 Experimental groups and immunization scheme

[0147]

[0148] Note: 1. The first immunization of the Freund's adjuvant group used Freund's complete adjuvant, and the second and third immunizations used Freund's incomplete adjuvant. 2. hMPVPreF3 is the human metapneumovirus PreF protein antigen. The antigen and adjuvant are mixed in a volume ratio of 1:1. The antigen content is 200ug / mL.

[0149] Immunization method: After the test sample is gently shaken before use, 1000μl of the test sample solution is drawn using a 1000μl disposable sterile syringe and needle and injected into the bilateral lateral thigh muscles of New Zealand rabbits.

[0150] Immunization time: three doses with 14-day intervals.

[0151] Serum collection: Blood was collected from experimental animals in each test group on D14, D28 and D42, and the serum was separated and the serum-specific IgG antibody titer was determined by enzyme-linked immunosorbent assay (ELISA). The rapid fluorescent focus inhibition test (RFFIT) was used to detect the anti-hMPV virus neutralizing antibody titer in the serum. The neutralizing antibody titer was expressed as the highest dilution factor of the sample that inhibited 90% virus growth (the number of fluorescent foci in the sample well ≤ the neutralization judgment critical value). Each well was judged separately, and the geometric mean titer of 2 duplicate wells was taken as the final result.

[0152] (2) ELISA detection of specific antibodies in serum

[0153] The hMPVPreF protein stock solution was diluted with coating solution to a protein concentration of 1μg / ml, and 100μl / well was coated on the ELISA plate at 4°C overnight. After washing 3 times with washing solution, 200μl of blocking solution was added to each well and blocked at 25°C for 1h. After blocking, the blocking solution was poured out, and the rabbit antiserum was diluted in multiples and added to the ELISA plate with rabbit negative serum, 100μl / well, and incubated at 25°C for 2h. After washing 3 times with washing solution, HRP-labeled goat anti-rabbit IgG (1:500) was added, 100μl / well, and incubated at 25°C for 1h. After washing 5 times with washing solution, TMB substrate colorimetric solution was added, and the reaction was protected from light for 15min at room temperature, and 100μl of stop solution was added to stop the reaction. The OD of each well was measured using an enzyme reader. 450The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is defined as the titer of the sample.

[0154] Results: The specific IgG antibody titer in rabbit serum was detected by ELISA. The bar graph of specific IgG antibody titer in each experimental group after immunization is shown in Figure 6 .

[0155] After the first, second and third immunizations, the binding antibody levels induced by adjuvant 1+hMPVPreF3 stock solution group were better than those of adjuvant 2 and Freund's adjuvant+hMPVPreF3 stock solution groups, and the effects of adjuvant 2 and Freund's adjuvant were basically equivalent.

[0156] (3) Serum anti-RSV virus neutralizing antibody detection

[0157] LLC-MK2 cells were plated and cultured in 96-well plates the day before the experiment. The serum samples to be tested were diluted in multiples with diluent, and the hMPV virus diluted to the appropriate multiple was added. After shaking and mixing, the cells were incubated in a 37°C incubator for neutralization for 1 hour. The cell culture medium in the 96-well plate with Hep-2 cells grown in a monolayer was discarded, the residual liquid in the well plate was washed with DPBS, and 100 μL / well serum-virus neutralization solution was added to the 96-well plate, and cultured at 37°C, 5% CO2 for 19 hours. The supernatant in the cell culture plate was discarded, 100 μL / well of 80% acetone solution was added, and incubated at 120°C in the dark for 20 minutes; the acetone solution was discarded and air-dried in a fume hood for about 20 minutes until all wells were completely dry. 50 μL PBST was added to each well, moistened at room temperature for 5 minutes, dried, and the hMPV primary antibody solution diluted to the appropriate multiple was added, and incubated at 37°C for 1 hour. Discard the primary antibody, wash the plate 3 times with PBST at 150 μL / well, add the secondary antibody solution diluted to an appropriate multiple, and incubate at 37°C for 1 hour. Discard the secondary antibody, wash the plate 3 times with PBST at 150 μL / well, then observe and count under a fluorescent microscope to calculate the neutralization titer.

[0158] Results: The neutralizing activity of animal sera against hMPV virus was detected by rapid fluorescent focus inhibition test (RFFIT). The titer of neutralizing antibodies against hMPV virus in sera of each experimental group after immunization is shown in the bar graph. Figure 7 .

[0159] After the first, second and third immunizations, the level of anti-hMPV virus neutralizing antibodies induced by adjuvant 1+hMPVPreF3 stock solution group was better than that of adjuvant 2 and Freund's adjuvant+hMPVreF3 stock solution group, and the effects of adjuvant 2 and Freund's adjuvant were basically equivalent.

[0160] At the same time, the experiment of using MF59 / aluminum / MPL adjuvant to prepare rabbit anti-parainfluenza virus polyclonal antibodies was simultaneously verified in this experiment. The results showed that the effects of several adjuvants were inferior to adjuvant 1.

[0161] (4) Conclusion

[0162] Adjuvant 1 + hMPVPreF3 stock solution immunized New Zealand rabbits and induced high titer rabbit anti-parainfluenza virus polyclonal antibody serum, and the induced antibody level was superior to that of traditional Freund's adjuvant and adjuvant 2.

[0163] Example 6: Preparation of rabbit anti-human parainfluenza virus polyclonal antibodies.

[0164] (1) Immunization of New Zealand rabbits

[0165] The experiment used 2.5kg ordinary-grade female New Zealand rabbits, with 5 rabbits in each negative control group and 8 rabbits in each vaccine group. The experimental animal grouping and immunization information are shown in the table below.

[0166] Table 8 Experimental groups and immunization scheme

[0167]

[0168] Note: 1. The first immunization of the Freund's adjuvant group used Freund's complete adjuvant, and the second and third immunizations used Freund's incomplete adjuvant. 2. hPIVPreF3 is the human parainfluenza virus PreF protein antigen. The antigen and adjuvant are mixed in a volume ratio of 1:1. The antigen content is 200ug / mL.

[0169] Immunization method: After the test sample is gently shaken before use, 1000μl of the test sample solution is drawn using a 1000μl disposable sterile syringe and needle and injected into the bilateral lateral thigh muscles of New Zealand rabbits.

[0170] Immunization time: three doses with 14-day intervals.

[0171] Serum collection: Blood was collected from experimental animals in each test group on D14, D28 and D42, and the serum was separated and the serum-specific IgG antibody titer was determined by enzyme-linked immunosorbent assay (ELISA). The rapid fluorescent focus inhibition test (RFFIT) was used to detect the anti-hPIV virus neutralizing antibody titer in the serum. The neutralizing antibody titer was expressed as the highest dilution factor of the sample that inhibited 90% virus growth (the number of fluorescent foci in the sample well ≤ the neutralization judgment critical value). Each well was judged separately, and the geometric mean titer of 2 duplicate wells was taken as the final result.

[0172] (2) ELISA detection of specific antibodies in serum

[0173] The hPIVPreF protein stock solution was diluted with coating solution to a protein concentration of 1μg / ml, and 100μl / well was coated on the ELISA plate at 4°C overnight. After washing 3 times with washing solution, 200μl of blocking solution was added to each well and blocked at 25°C for 1h. After blocking, the blocking solution was poured out, and the rabbit antiserum was diluted in multiples and added to the ELISA plate with rabbit negative serum, 100μl / well, and incubated at 25°C for 2h. After washing 3 times with washing solution, HRP-labeled goat anti-rabbit IgG (1:500) was added, 100μl / well, and incubated at 25°C for 1h. After washing 5 times with washing solution, TMB substrate colorimetric solution was added, and the reaction was protected from light for 15min at room temperature, and 100μl of stop solution was added to stop the reaction. The OD of each well was measured using an enzyme reader. 450 The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is defined as the titer of the sample.

[0174] Results: The specific IgG antibody titer in rabbit serum was detected by ELISA. The bar graph of specific IgG antibody titer in each experimental group after immunization is shown in Figure 8 .

[0175] After the first, second and third immunizations, the binding antibody levels induced by adjuvant 1 + hPIVPreF3 stock solution group were better than those of adjuvant 2 and Freund's adjuvant + hPIVPreF3 stock solution groups, and the effects of adjuvant 2 and Freund's adjuvant were basically equivalent.

[0176] (3) Serum anti-RSV virus neutralizing antibody detection

[0177] LLC-MK2 cells were plated and cultured in 96-well plates the day before the experiment. The serum samples to be tested were diluted in multiples with diluent, and the hPIV virus diluted to an appropriate multiple was added. After oscillation and mixing, the cells were incubated in a 37°C incubator for neutralization for 1 hour. The cell culture medium in the 96-well plate with Hep-2 cells grown in a monolayer was discarded, the residual liquid in the well plate was washed with DPBS, and 100 μL / well serum-virus neutralization solution was added to the 96-well plate, and cultured at 37°C, 5% CO2 for 18 hours. The supernatant in the cell culture plate was discarded, 100 μL / well of 80% acetone solution was added, and incubated at 120°C in the dark for 20 minutes; the acetone solution was discarded and air-dried in a fume hood for about 20 minutes until all wells were completely dry. 50 μL PBST was added to each well, moistened at room temperature for 5 minutes, dried, and the hPIV primary antibody solution diluted to an appropriate multiple was added, and incubated at 37°C for 1 hour. Discard the primary antibody, wash the plate 3 times with PBST at 150 μL / well, add the secondary antibody solution diluted to an appropriate multiple, and incubate at 37°C for 1 hour. Discard the secondary antibody, wash the plate 3 times with PBST at 150 μL / well, then observe and count under a fluorescent microscope to calculate the neutralization titer.

[0178] Results: The neutralizing activity of animal sera against hPIV virus was detected by rapid fluorescent focus inhibition test (RFFIT). The titer of neutralizing antibodies against hPIV virus in sera of each experimental group after immunization is shown in the bar graph. Fig. 9 .

[0179] After the first, second and third immunizations, the level of anti-hPIV virus neutralizing antibodies induced by adjuvant 1+hPIVPreF3 stock solution group was better than that of adjuvant 2 and Freund's adjuvant+hPIVreF3 stock solution group, and the effects of adjuvant 2 and Freund's adjuvant were basically equivalent.

[0180] At the same time, the study on the use of MF59 / aluminum / MPL adjuvant to prepare rabbit anti-parainfluenza virus polyclonal antibodies was simultaneously verified in this experiment. The results showed that the effects of several adjuvants were inferior to adjuvant 1.

[0181] (4) Conclusion

[0182] Adjuvant 1+hPIVPreF3 stock solution immunized New Zealand rabbits and induced high titer rabbit anti-parainfluenza virus polyclonal antibody serum, and the induced antibody level was superior to that of traditional Freund's adjuvant and adjuvant 2.

[0183] In addition, the present invention also simultaneously conducted experiments on rabbit polyclonal antibodies to human papillomavirus type 16 E1 protein in rabbits, and the experimental results were basically consistent with the results of respiratory syncytial virus protein, human metapneumovirus protein and parainfluenza virus protein, that is, the level of polyclonal antibodies induced by New Zealand rabbits immunized with adjuvant 1 + HPV 16E1 stock solution was significantly better than that of traditional Freund's adjuvant and adjuvant 2.

[0184] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a target adjuvant in preparing rabbit polyclonal antibodies, characterized in that: The active ingredients of the target adjuvant include: QS-21 and polyinosinic acid (PolyI:C).

2. The use according to claim 1, characterized in that: In the target adjuvant, the final concentration of QS-21 is 35-300 μg / mL; Optionally, in the target adjuvant, the final concentration of the polyinosinic-acid Poly I:C is 400-3000 μg / mL.

3. The use according to claim 1, characterized in that: The target adjuvant also includes a liposome carrier; Optionally, the liposome carrier comprises: any one or more of cationic liposomes and neutral liposomes; Optionally, the raw materials for preparing the cationic liposomes include: any one or more of 1,2-dioleoyl-3-trimethylammonium chloride propane, dioleoylphosphatidylcholine and cholesterol; Optionally, the preparation method of the cationic liposome comprises: Mixing 1,2-dioleoyl-3-trimethylammonium chloride propane, dioleoylphosphatidylcholine and cholesterol in a mass ratio of 1 to 3:1 to 3:1 to obtain a first mixture; Using the first mixture as a raw material, preparing cationic liposomes by any one of an ethanol injection method, a thin film dispersion method, an ultrasonic dispersion method and a reverse evaporation method; Optionally, the raw materials of the neutral liposome include: any one or more of dioleoylphosphatidylcholine and cholesterol; Optionally, in the neutral liposome, the mass ratio of dioleoylphosphatidylcholine to cholesterol is 3 to 5:1; Optionally, the preparation method of the neutral liposome comprises: Mixing dioleoylphosphatidylcholine and cholesterol according to a mass ratio to obtain a second mixture; The second mixture is used as a raw material, and neutral liposomes are prepared by any one of an ethanol injection method, a thin film dispersion method, an ultrasonic dispersion method and a reverse evaporation method.

4. A method for preparing a rabbit polyclonal antibody, characterized in that: It includes: Immunize rabbits with immunogens to obtain rabbit polyclonal antibodies; wherein the immunogens include antigens and the target adjuvant described in any one of claims 1 to 3.

5. The preparation method according to claim 4, characterized in that: The antigens include: any one or more combinations of human papillomavirus antigens, human herpesvirus antigens, human metapneumovirus antigens, human parainfluenza virus antigens, respiratory syncytial virus antigens, human immunodeficiency virus antigens, cytomegalovirus antigens, dengue virus antigens, enterovirus antigens, hepatitis A virus antigens, hepatitis B virus antigens, hepatitis C virus antigens, hepatitis D virus antigens, hepatitis E virus antigens, poliovirus antigens, rabies virus antigens, rotavirus antigens, herpes zoster virus antigens, SARS coronavirus antigens and rhinovirus antigens.

6. The preparation method according to claim 4, characterized in that: In the immunogen, 60 to 240 μg of the antigen was added for every 17.5 to 60 μg of QS-21.

7. The preparation method according to any one of claims 4 to 6, characterized in that: During the immunization, the inoculation amount of the antigen for each rabbit is 50-200 μg / time, and the number of inoculations is 1-4 times.

8. A rabbit polyclonal antibody, characterized in that The compound is prepared by the preparation method according to any one of claims 4 to 7.

9. A kit, characterized in that: It includes: The rabbit polyclonal antibody according to claim 8.

10. Use of the rabbit polyclonal antibody according to claim 8 in detecting relevant antigens for purposes other than disease diagnosis or treatment or in preparing products for detecting the relevant antigens.

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