Animal experiment method for evaluating effectiveness of histamine human immune globulin
By constructing a type I allergic reaction model and monitoring the IgE level in serum, the problem of the inability to continuously monitor the protective effect of histamine human immunoglobulin preparations in the prior art is solved, and an accurate assessment of its effectiveness is achieved.
Patent Information
- Application Number
- CN202411716316.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-03-14
AI Technical Summary
The existing methods for evaluating the effectiveness of histamine human immunoglobulin preparations cannot continuously monitor the protective effect of the preparations, and the observation and test results are not obvious.
A type I allergic reaction model was constructed. After mice were sensitized by injection of ovalbumin and adjuvant mixture, they were treated with histamine human immunoglobulin, and their effectiveness was evaluated by monitoring serum IgE levels.
Continuous observation and monitoring of the effectiveness of histamine human immunoglobulin is achieved, and the monitoring indicators are more reliable and intuitive, reducing experimental errors caused by human observation.
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Figure CN119619520A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and in particular to an animal experiment method for evaluating the effectiveness of histamine human immunoglobulin. Background Art
[0002] Histamine human immunoglobulin is a biological preparation made from healthy human plasma, purified by low-temperature ethanol protein separation or other approved separation methods, and then added with an appropriate amount of histamine phosphate or histamine hydrochloride, and a protective agent, and then treated with virus removal and inactivation. Histamine human immunoglobulin is mainly used to treat a wide range of allergic diseases, including bronchial asthma, allergic rhinitis and atopic dermatitis.
[0003] At present, the main methods for evaluating the effectiveness of histamine human immunoglobulin preparations are the New Zealand rabbit shock test and the guinea pig passive protection test. The New Zealand rabbit shock test and the guinea pig passive protection test cannot continuously examine the effectiveness of the product. They can only reflect the protective ability of the preparation on sensitized New Zealand rabbits or guinea pigs, and cannot continuously monitor the protective effect of the preparation on allergic animals. In addition, this method has the characteristic that the observation test results are not obvious. Summary of the invention
[0004] The present invention aims to provide an animal experimental method for evaluating the effectiveness of histamine human immunoglobulin, so as to achieve continuous observation and monitoring of the effectiveness of histamine human immunoglobulin.
[0005] To achieve the above objectives, the present invention adopts the following technical scheme: an animal experimental method for evaluating the effectiveness of histamine human immunoglobulin, after constructing a type I allergic reaction model, the effectiveness is evaluated by the IgE level in the serum after treatment with histamine human immunoglobulin.
[0006] Preferably, as an improvement, in the stage of constructing the type I allergic reaction model, the sensitizer is ovalbumin, and the injection concentration of ovalbumin is 200-1000ug / ml.
[0007] Preferably, as an improvement, the injection concentration of ovalbumin is 1000 ug / ml.
[0008] Preferably, as an improvement, the number of injections of ovalbumin is 8 times.
[0009] Preferably, as an improvement, during the treatment phase using histamine human immunoglobulin, the injection dosage of histamine human immunoglobulin is 6 to 12 mg, and the number of injections is 3 to 7 times.
[0010] Preferably, as an improvement, the injection dosage of histamine human immunoglobulin is 10 mg, and the number of injections is 7 times.
[0011] Preferably, as an improvement, an animal experimental method for evaluating the effectiveness of histamine human immunoglobulin comprises the following steps:
[0012] Step 1: Construct a type I allergic reaction model:
[0013] S1. Blood from the eyelids of BALB / c female mice was collected to separate the serum for later use, and then a mixture of ovalbumin and adjuvant was injected;
[0014] S2, injecting the mixture of ovalbumin and adjuvant into the experimental mice every week for 4 consecutive weeks, and then injecting the mice subcutaneously with ovalbumin at a concentration of 1000ug / ml for 4 consecutive days to obtain type I allergic reaction model mice;
[0015] Step 2: Histamine human immunoglobulin treatment:
[0016] I. Inject histamine human immunoglobulin into type I allergic reaction model mice for 3 to 7 consecutive immunizations, with one week interval between each injection;
[0017] II. One week and / or one month after the end of the experiment, blood was collected from the eyelids of the experimental mice, and the serum was separated by centrifugation and the IgE content was detected.
[0018] Preferably, as an improvement, in step one, the mass ratio of ovalbumin to adjuvant is 1:1, and the mixture is emulsified at room temperature for 5 to 10 minutes before injection.
[0019] Preferably, as an improvement, in step 1, the adjuvant is Freund's complete adjuvant.
[0020] Preferably, as an improvement, after blood is collected from the mouse eyelids, the serum is separated by centrifugation, the centrifugation condition is 2000g, and the serum storage temperature is -20°C.
[0021] The principle and advantages of this scheme are: in actual application, in this technical scheme, in the study of the construction of the effectiveness evaluation method of histamine immunoglobulin, based on the use of histamine human immunoglobulin mainly for the treatment of allergic diseases caused by type I allergic reactions such as bronchial asthma, allergic rhinitis and atopic dermatitis, a type I allergic model mouse was constructed using ovalbumin. This model is stable and can continuously monitor the protective effect of the drug; at the same time, combined with the characteristics of type I allergic reactions--the levels of IgE, IL-4, etc. in serum are increased, mast cell degranulation involves the release of histamine and other allergic mediators, and type I allergic reactions are mainly IgE-mediated immune reactions. The inventor of this case first proposed to continuously evaluate the protective ability of histamine immunoglobulin products by monitoring the changes in IgE levels in the serum of the sensitized model. Compared with traditional shock experiments and guinea pig passive protection experiments, this method has more reliable and intuitive monitoring indicators and reduces the experimental errors caused by human observation. During the technical research and development stage, the inventors found that OVA concentration, histamine human immunoglobulin concentration, and the number of histamine immunoglobulin immunizations are the key factors affecting the effectiveness evaluation of the present technical solution. Through targeted research on the above key points, the optimal OVA concentration, the optimal histamine human immunoglobulin concentration, and the number of histamine immunoglobulin immunizations were determined, so that the obtained IgE changes are the most obvious, which can best reflect the effect of histamine immunoglobulin and can more effectively evaluate histamine immunoglobulin.
[0022] During the research and development process, the sensitized mouse model was constructed using the conditional parameters constructed by sensitized mice. The determination of the adjuvant type and injection strategy is a major research and development difficulty of this technical solution: Freund's complete adjuvant and Freund's incomplete adjuvant are commonly used adjuvants. The existing operation method usually uses one of them. However, after the complete adjuvant stimulates the immune system of the model animal, it will bias the Th1 / Th2 in the mouse body and turn to the Th1-mediated immune response, resulting in a relatively weakened sensitization reaction (Th2). On the contrary, the incomplete adjuvant has less irritation to the mouse immune system and cannot cause a Th2 reaction, resulting in a poor sensitization effect. Secondly, the subcutaneous tissue contains relatively more Trge cells, which is more conducive to Th2 reaction. Through some preliminary experiments, a scheme of 4 consecutive subcutaneous stimulations was determined, and Freund's complete adjuvant was used for the first sensitization, and then incomplete adjuvant was used for three consecutive times to strengthen immunity. Finally, pure ovalbumin was subcutaneously injected for 4 consecutive times, so that the Th1 / Th2 ratio of the mouse was increased at the same time, and the Th2-mediated reaction was also increased accordingly, achieving a sensitization effect.
[0023] In summary, this technical solution proposed for the first time to use sensitized model mice to evaluate the effectiveness of histamine human immunoglobulin. On this basis, through the overall optimization of the adjuvant type, injection strategy, OVA concentration, histamine human immunoglobulin concentration, and histamine immunoglobulin immunization times in the model construction of the solution, an accurate evaluation of the effectiveness of histamine human immunoglobulin was achieved. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 This is a graph showing changes in IgE in the serum of BALB / c mice in each group during the sensitization model establishment stage in Example 1 of the present invention.
[0025] Figure 2 This is a graph showing changes in serum IgE in each group of BALB / c mice after histamine human immunoglobulin immunotherapy during the treatment phase in Example 2 of the present invention.
[0026] Figure 3 This is a graph showing changes in serum IgE in each group of BALB / c mice after histamine human immunoglobulin immunotherapy according to Example 3 of the present invention.
[0027] Figure 4 This is a graph showing changes in IgE in the serum of BALB / c mice in each group during the sensitization model establishment stage in Example 4 of the present invention.
[0028] Figure 5 This is a graph showing changes in serum IgE in each group of BALB / c mice after treatment with histamine human immunoglobulin in Example 4 of the present invention.
[0029] Figure 6 This is a graph showing changes in IgE in the serum of BALB / c mice in each group during the sensitization model establishment stage in Example 5 of the present invention.
[0030] Figure 7 This is a graph showing changes in serum IgE in each group of BALB / c mice after treatment with histamine human immunoglobulin in Example 5 of the present invention. DETAILED DESCRIPTION
[0031] The following is further described in detail through specific implementations, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following implementations are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.
[0032] Program Overview:
[0033] An animal experimental method for evaluating the effectiveness of histamine human immunoglobulin comprises the following steps:
[0034] (1) Randomly select 24 BALB / c female mice. Before the experiment, blood is collected from the eyelids of the mice and centrifuged to separate the serum for later use. The centrifugation condition is 2000g, and the serum is stored in a -20℃ refrigerator. 0.1ml of the mixture of ovalbumin and adjuvant is injected intraperitoneally, and the mixing ratio of ovalbumin and adjuvant is 1:1 (the same below); the concentration of ovalbumin (OVA) is 200ug / ml, 400ug / ml, and 1000ug / ml, and the adjuvant is Freund's complete adjuvant. The mixture needs to be emulsified at room temperature for 5-10min.
[0035] (2) After an interval of one week, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 200 to 1000 ug / ml and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5 to 10 minutes.
[0036] (3) After an interval of two weeks, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 200-1000 ug / ml and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5-10 minutes.
[0037] (4) After an interval of three weeks, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and an adjuvant, wherein the concentration of ovalbumin was 200 to 1000 ug / ml and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5 to 10 minutes.
[0038] (5) After the experiment in step (4), ovalbumin at a concentration of 1000ug / ml was subcutaneously injected into the mice 4 times for 4 consecutive days. Blood was collected from the eyelids of the experimental mice for one week, and the serum was separated by centrifugation for later use. The centrifugation condition was 2000g, and the serum was stored in a -20°C refrigerator.
[0039] (6) The serum treated in step (5) was tested for IgE content, and the detection kit was provided by Shanghai Biotechnology.
[0040] (7) After the experiment in step (4), the experimental mice were subcutaneously injected with 0.1 ml of histamine human immunoglobulin, with a protein dose of 6 to 12 mg, for 3 or 7 consecutive immunization injections, with an interval of one week between each injection.
[0041] (8) One week after the end of step (7), blood was collected from the eyelids of the experimental mice and centrifuged to separate the serum for later use. The centrifugation condition was 2000g, and the serum was stored in a -20°C refrigerator.
[0042] (9) The serum treated in step (8) was used to detect IgE content, and the detection kit was provided by Shanghai Biotechnology.
[0043] (10) After the experiment in step (7), the experimental mice were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 200-1000 ug / ml and the adjuvant was Freund's complete adjuvant. The mixture was emulsified at room temperature for 5-10 minutes.
[0044] (11) After one week, the experimental mice after the experiment in step (10) were intraperitoneally injected with 0.1 ml of the mixture of ovalbumin and adjuvant, and the immunization injections were repeated twice, each time with one week interval. The concentration of ovalbumin was 200-1000 ug / ml, the adjuvant was Freund's incomplete adjuvant, and the mixture was emulsified at room temperature for 5-10 minutes.
[0045] (12) One week after the end of step (11), blood was collected from the eyelids of the experimental mice and centrifuged to separate the serum for later use. The centrifugation condition was 2000g, and the serum was stored in a -20°C refrigerator.
[0046] (13) The serum treated in step (12) was used to detect the IgE content, and the detection kit was provided by Shanghai Biotechnology.
[0047] Example 1
[0048] Establishment of type I allergic sensitization model: BALB / c mice were sensitized four times with ovalbumin at different concentrations, and the changes in IgE levels in mouse serum were detected to determine the range of ovalbumin injection doses. The specific steps are as follows:
[0049] (1) Eighteen BALB / c female mice were randomly selected. Before the experiment, blood was collected from the eyelids of the mice and the serum was separated by centrifugation. The centrifugation condition was 2000g and the serum was stored in a -20℃ refrigerator. 0.1ml of a mixture of ovalbumin and adjuvant was intraperitoneally injected, where the concentration of ovalbumin was 200ug / ml, 400ug / ml, and 1000ug / ml, and the adjuvant was Freund's complete adjuvant. The mixture was emulsified at room temperature for 5-10min.
[0050] (2) After an interval of one week, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 200 ug / ml, 400 ug / ml, and 1000 ug / ml, and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5-10 min.
[0051] (3) After an interval of two weeks, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 200 ug / ml, 400 ug / ml, and 1000 ug / ml, and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5-10 min.
[0052] (4) After an interval of three weeks, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 200 ug / ml, 400 ug / ml, and 1000 ug / ml.
[0053] (5) After the experiment in step (4), ovalbumin at concentrations of 200ug / ml, 400ug / ml, and 1000ug / ml was subcutaneously injected into mice 4 times for 4 consecutive days. One week later, blood was collected from the eyelids and centrifuged to separate the serum for later use. The centrifugation condition was 2000g, and the serum was stored in a -20°C refrigerator.
[0054] (6) The serum treated in step (5) was tested for IgE content, and the detection kit was provided by Shanghai Biotechnology.
[0055] The changes of IgE in the serum of BALB / c mice in each group were detected. The test results were as follows Figure 1 As shown: BALB / c mice were sensitized with ovalbumin at concentrations of 200ug / ml, 400ug / ml, and 1000ug / ml. The IgE level in the mouse serum at each stage was significantly higher than that in the blank control, and the IgE in the serum also increased significantly after four sensitizations. The sensitization model was established, and the optimal concentration of the allergen (ovalbumin) was 1000ug / ml.
[0056] Example 2
[0057] Immunotherapy stage: After the sensitized model animals are established, histamine immunoglobulin treatment is carried out, and finally sensitization is carried out again to evaluate its protective effect on sensitized mice.
[0058] (1) After the sensitization model animals were established, 30 sensitized mice were randomly divided into 5 groups, 6 mice in each group, and a blank control group was set up. The experimental groups were subcutaneously injected with 0.1 ml of histamine human immunoglobulin, with protein doses of 6 mg, 8 mg, 10 mg, and 12 mg, and the blank control group was replaced with normal saline. The immunization injections were repeated 3 times, with an interval of one week between each.
[0059] (2) Step (1) One week after the end of the experiment, blood was collected from the eyelids of the experimental mice and centrifuged to separate the serum for later use. The centrifugation condition was 2000g, and the serum was stored in a -20°C refrigerator.
[0060] (3) The serum treated in step (2) was used to detect IgE content, and the detection kit was provided by Shanghai Biotechnology.
[0061] (4) After the experiment in step (2), the experimental mice were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 1000 ug / ml and the adjuvant was Freund's complete adjuvant. The mixture was emulsified at room temperature for 5-10 minutes.
[0062] (5) After one week, the experimental mice after the experiment in step (4) were intraperitoneally injected with 0.1 ml of the mixture of ovalbumin and adjuvant, and the immunization injections were repeated twice, each time with one week interval. The concentration of ovalbumin was 1000 ug / ml, the adjuvant was Freund's incomplete adjuvant, and the mixture was emulsified at room temperature for 5-10 minutes.
[0063] (6) One week after the end of step (5), the experimental mice were subcutaneously injected with ovalbumin at a concentration of 1000ug / ml for 4 consecutive days. One week later, blood was collected from the eyelids and centrifuged to separate the serum for later use. The centrifugation condition was 2000g, and the serum was stored in a -20°C refrigerator.
[0064] (7) The serum treated in step (6) was tested for IgE content, and the detection kit was provided by Shanghai Biotechnology.
[0065] The changes of IgE in the serum of BALB / c mice in each group were detected. Figure 2 As shown: 6mg, 8mg, 10mg, and 12mg of histamine human immunoglobulin were used to treat sensitized BALB / c mice. The IgE levels in the serum of the mice in the histamine treatment group were significantly higher than those in the blank control group, but significantly lower than those in the saline group. After three histamine immunotherapy treatments, the IgE in the 10mg group was lower than that in the other dose groups. Therefore, the optimal immunotherapy dose is 10mg.
[0066] Example 3
[0067] An animal experimental method for evaluating the effectiveness of histamine human immunoglobulin comprises the following steps:
[0068] (1) 30 BALB / c female mice were randomly selected. Before the experiment, blood was collected from the eyelids of the mice and the serum was separated by centrifugation. The centrifugation condition was 2000g and the serum was stored in a -20℃ refrigerator. 0.1ml of a mixture of ovalbumin and adjuvant was intraperitoneally injected, where the concentration of ovalbumin was 1000ug / ml and the adjuvant was Freund's complete adjuvant. The mixture was emulsified at room temperature for 5-10min.
[0069] (2) After one week, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 1000 ug / ml and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5-10 min.
[0070] (3) After an interval of two weeks, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 1000 ug / ml and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5-10 min.
[0071] (4) After an interval of three weeks, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 1000 ug / ml and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5-10 min.
[0072] (5) One week after the end of step (4), the experimental mice were subcutaneously injected with ovalbumin at a concentration of 1000ug / ml for 4 consecutive days. One week later, blood was collected from the eyelids and centrifuged to separate the serum for later use. The centrifugation condition was 2000g, and the serum was stored in a -20°C refrigerator.
[0073] (6) The serum treated in step (5) was tested for IgE content, and the detection kit was provided by Shanghai Biotechnology.
[0074] (7) After the experiment in step (4), the experimental mice were subcutaneously injected with 0.1 ml of histamine human immunoglobulin, with a protein dose of 10 mg, for 7 consecutive immunization injections, each with an interval of one week.
[0075] (8) One week after the end of step (7), blood was collected from the eyelids of the experimental mice and centrifuged to separate the serum for later use. The centrifugation condition was 2000g, and the serum was stored in a -20°C refrigerator.
[0076] (9) The serum treated in step (8) was used to detect IgE content, and the detection kit was provided by Shanghai Biotechnology.
[0077] (10) After the experiment in step (7), the experimental mice were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 1000 ug / ml and the adjuvant was Freund's complete adjuvant. The mixture was emulsified at room temperature for 5-10 min.
[0078] (11) After one week, the experimental mice after the experiment in step (10) were intraperitoneally injected with 0.1 ml of the mixture of ovalbumin and adjuvant, and the immunization injections were repeated twice, each time with one week interval. The concentration of ovalbumin was 1000 ug / ml, the adjuvant was Freund's incomplete adjuvant, and the mixture was emulsified at room temperature for 5-10 minutes.
[0079] (12) Step (11) One week and one month after the end of the experiment, blood was collected from the eyelids of the experimental mice, and the serum was separated by centrifugation for later use. The centrifugation condition was 2000g, and the serum was stored in a -20°C refrigerator.
[0080] (13) The serum treated in step (12) was used to detect the IgE content, and the detection kit was provided by Shanghai Biotechnology.
[0081] The changes of IgE in the serum of BALB / c mice in each group were detected. Figure 3As shown: 1000ug / ml ovalbumin was used as the sensitizer and injected intraperitoneally four times to successfully establish the sensitization model, and then histamine immunoglobulin was subcutaneously injected for 7 times, each dose was 10mg, and 1000ug / ml ovalbumin was used again for attack. The IgE level of the histamine immunoglobulin group was lower than that of the saline control group. The modified product can maintain a low level of IgE for one month, and the protective effect is long-lasting; and the optimal number of immunotherapy times is 7 times.
[0082] Example 4
[0083] An animal experimental method for evaluating the effectiveness of histamine human immunoglobulin comprises the following steps:
[0084] (1) 30 BALB / c female mice were randomly selected. Before the experiment, blood was collected from the eyelids of the mice and the serum was separated by centrifugation. The centrifugation condition was 2000g and the serum was stored in a -20℃ refrigerator. 0.1ml of a mixture of ovalbumin and adjuvant was intraperitoneally injected, where the concentration of ovalbumin was 1000ug / ml and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5-10min.
[0085] (2) After one week, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 1000 ug / ml and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5-10 min.
[0086] (3) After an interval of two weeks, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 1000 ug / ml and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5-10 min.
[0087] (4) After an interval of three weeks, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 1000 ug / ml and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5-10 min.
[0088] (5) Step (4) One week after the end of the experiment, blood was collected from the eyelids of the experimental mice and centrifuged to separate the serum for later use. The centrifugation condition was 2000g, and the serum was stored in a -20°C refrigerator.
[0089] (6) The serum treated in step (5) was tested for IgE content, and the detection kit was provided by Shanghai Biotechnology.
[0090] The changes of IgE in the serum of BALB / c mice in each group were detected. Figure 4As shown: 1000ug / ml ovalbumin was used as the allergen, mixed with Freund's incomplete adjuvant and emulsified, and then injected intraperitoneally four times, but the sensitization model could not be successfully established.
[0091] (7) One week after the end of step (4), the experimental mice were subcutaneously injected with ovalbumin at a concentration of 1000ug / ml for 4 consecutive days. One week later, blood was collected from the eyelids and centrifuged to separate the serum for later use. The centrifugation condition was 2000g, and the serum was stored in a -20°C refrigerator.
[0092] (8) The serum treated in step (7) was used to detect IgE content, and the detection kit was provided by Shanghai Biotechnology.
[0093] The changes of IgE in the serum of BALB / c mice in each group were detected. Figure 5 As shown: 1000ug / ml ovalbumin was used as the allergen and mixed with Freund's complete adjuvant for emulsification and then injected intraperitoneally four times to construct a sensitized model. The degree of IgE increase was low and the sensitized mouse model was not successfully constructed.
[0094] Example 5
[0095] An animal experimental method for evaluating the effectiveness of histamine human immunoglobulin comprises the following steps:
[0096] (1) 30 BALB / c female mice were randomly selected. Before the experiment, blood was collected from the eyelids of the mice and the serum was separated by centrifugation. The centrifugation condition was 2000g and the serum was stored in a -20℃ refrigerator. 0.1ml of a mixture of ovalbumin and adjuvant was intraperitoneally injected, where the concentration of ovalbumin was 1000ug / ml and the adjuvant was Freund's complete adjuvant. The mixture was emulsified at room temperature for 5-10min.
[0097] (2) After one week, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 1000 ug / ml and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5-10 min.
[0098] (3) After an interval of two weeks, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 1000 ug / ml and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5-10 min.
[0099] (4) After an interval of three weeks, the experimental mice in step (1) were intraperitoneally injected with 0.1 ml of a mixture of ovalbumin and adjuvant, wherein the concentration of ovalbumin was 1000 ug / ml and the adjuvant was Freund's incomplete adjuvant. The mixture was emulsified at room temperature for 5-10 min.
[0100] (5) Step (4) One week after the end of the experiment, blood was collected from the eyelids of the experimental mice and centrifuged to separate the serum for later use. The centrifugation condition was 2000g, and the serum was stored in a -20°C refrigerator.
[0101] (6) The serum treated in step (5) was tested for IgE content, and the detection kit was provided by Shanghai Biotechnology.
[0102] The changes of IgE in the serum of BALB / c mice in each group were detected. Figure 6 As shown: 1000ug / ml ovalbumin was used as the sensitizer and mixed with Freund's complete adjuvant for emulsification and then injected intraperitoneally four times, but the sensitization model could not be successfully established.
[0103] (7) One week after the end of step (4), the experimental mice were subcutaneously injected with ovalbumin at a concentration of 1000ug / ml for 4 consecutive days. One week later, blood was collected from the eyelids and centrifuged to separate the serum for later use. The centrifugation condition was 2000g, and the serum was stored in a -20°C refrigerator.
[0104] (8) The serum treated in step (7) was used to detect IgE content, and the detection kit was provided by Shanghai Biotechnology.
[0105] The changes of IgE in the serum of BALB / c mice in each group were detected. Figure 7 As shown: 1000ug / ml ovalbumin was used as the allergen and mixed with Freund's complete adjuvant for emulsification and then injected intraperitoneally four times to successfully establish a sensitization model.
[0106] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.
Claims
1. An animal experimental method for evaluating the effectiveness of histamine human immunoglobulin, characterized in that: After constructing a type Ⅰ allergic reaction model, the effectiveness was evaluated by the serum IgE level after treatment with histamine human immunoglobulin.
2. The animal experimental method for evaluating the effectiveness of histamine human immunoglobulin according to claim 1, characterized in that: In the stage of constructing a type I allergic reaction model, the sensitizer is ovalbumin, and the injection concentration of ovalbumin is 200-1000ug / ml.
3. An animal experimental method for evaluating the effectiveness of histamine human immunoglobulin according to claim 2, characterized in that: The injection concentration of ovalbumin was 1000ug / ml.
4. The animal experimental method for evaluating the effectiveness of histamine human immunoglobulin according to claim 3, characterized in that: The number of ovalbumin injections was 8.
5. The animal experimental method for evaluating the effectiveness of histamine human immunoglobulin according to claim 4, characterized in that: During the treatment phase using histamine human immunoglobulin, the injection dose of histamine human immunoglobulin is 6 to 12 mg, and the number of injections is 3 to 7 times.
6. The animal experimental method for evaluating the effectiveness of histamine human immunoglobulin according to claim 5, characterized in that: The injection dosage of histamine human immunoglobulin is 10 mg, and the number of injections is 7 times.
7. An animal experimental method for evaluating the effectiveness of histamine human immunoglobulin according to claim 6, characterized in that: The steps include: Step 1: Construct a type I allergic reaction model: S1. Blood from the eyelids of BALB / c female mice was collected to separate the serum for later use, and then a mixture of ovalbumin and adjuvant was injected; S2, injecting the mixture of ovalbumin and adjuvant into the experimental mice every week for 4 consecutive weeks, and then injecting the mice subcutaneously with ovalbumin at a concentration of 1000ug / ml for 4 consecutive days to obtain type I allergic reaction model mice; Step 2: Histamine human immunoglobulin treatment: I. Inject histamine human immunoglobulin into type I allergic reaction model mice for 3 to 7 consecutive immunizations, with one week interval between each injection; II. One week and / or one month after the end of the experiment, blood was collected from the eyelids of the experimental mice, and the serum was separated by centrifugation and the IgE content was detected.
8. The animal experimental method for evaluating the effectiveness of histamine human immunoglobulin according to claim 7, characterized in that: In step 1, the mass ratio of ovalbumin to adjuvant is 1:1, and the mixture is emulsified at room temperature for 5 to 10 minutes before injection.
9. An animal experimental method for evaluating the effectiveness of histamine human immunoglobulin according to claim 8, characterized in that: In step 1, the adjuvant is Freund's complete adjuvant.
10. An animal experimental method for evaluating the effectiveness of histamine human immunoglobulin according to claim 9, characterized in that: After blood was collected from the mouse eyelids, serum was separated by centrifugation at 2000 g and the serum was stored at -20°C.