A method for producing enzyme diluent for in vitro diagnostic reagents using bovine serum

Abstract

The application provides a method for producing enzyme diluent for in-vitro diagnostic reagent by using bovine serum, which comprises the following steps: collecting adult bovine serum; 37 DEG C water bath; ice water bath; primary centrifugation; secondary centrifugation, taking secondary supernatant; freezing preservation; thawing: the frozen primary supernatant in the negative pressure blood bag is thawed, and the thawing method is stepwise temperature control thawing, and the specific operation method is: placing at 0 DEG C-4 DEG C for 5-7 hours, placing at 4 DEG C-8 DEG C for 5-7 hours, and placing at 8 DEG C-16 DEG C for 5-7 hours; primary ultrafiltration; secondary ultrafiltration; tertiary ultrafiltration; the permeate after tertiary ultrafiltration is diluted in a phosphoric acid solution, and the bacteria are removed, and the bag is sealed and stored, the adult bovine serum is used instead of fetal bovine or calf blood to produce enzyme diluent for in-vitro diagnostic reagent, the cost of the adult bovine serum is about 30% of that of calf and fetal bovine, and meanwhile, the stability and sensitivity of the enzyme diluent for in-vitro diagnostic reagent are ensured.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagent technology, and relates to a method for producing a special enzyme diluent for in vitro diagnostic reagents using bovine serum. Background Technology

[0002] In vitro diagnostic reagents are chemical, biological, or immunological substances that can be used alone or in combination with instruments, appliances, equipment, or systems to examine human samples in vitro. They are used to detect or measure certain components in samples in in vitro diagnostic testing procedures. Stable and reliable in vitro diagnostic reagents can provide accurate clinical test data and play an important role in the prevention and control of diseases and the timely diagnosis and treatment of diseases.

[0003] Enzymes are essential raw materials for the in vitro diagnostics industry, core components of clinical chemistry and molecular diagnostics, and indispensable parts of enzyme-substrate signaling systems in immunodiagnostics. Enzymes are biological catalysts, proteins (a few being nucleic acids) synthesized by tissue cells with biocatalytic functions. They exhibit strong catalytic activity in chemical reactions and extremely high substrate selectivity (including selectivity for substrate type, region, site, and stereochemistry). Each enzyme can catalyze only one reaction with one substrate, resulting in high conversion and yield rates with virtually no side reactions. These characteristics give enzymes a significant advantage over other chemical methods in clinical testing, chemical analysis, and the production of specific products. Generally, enzyme activity and quantity remain relatively constant in a healthy human body. However, when certain organs and tissues become diseased, specific enzymes are released into the blood, urine, or body fluids, leading to corresponding changes in their quantity or activity.

[0004] Most existing enzyme diluents for in vitro diagnostic reagents are prepared by extracting serum from fetal calves or calves aged 3-6 months. These diluents offer high stability and sensitivity, but are also costly and do not benefit the growth of calves. Therefore, how to produce enzyme diluents for in vitro diagnostic reagents with high stability and sensitivity using adult bovine serum, which is less expensive, has become an urgent problem to be solved. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for producing a special enzyme diluent for in vitro diagnostic reagents using bovine serum, specifically including the following steps:

[0006] S1. Blood collection: Collect 600-800ml of blood from the neck of an adult cow. The blood collection method is a fully enclosed sterile bag for intravenous blood collection.

[0007] S2. Warm water bath: Transfer the fully sealed sterile blood bag filled with blood to a 37°C warm water bath and let it stand for 40-50 minutes, turning it upside down 2-3 times during the process. Standing will produce suspended matter. Turning it over is to prevent the formation of substances adhering to the wall and to ensure even heating. The warm water bath promotes the precipitation of some platelets, which are then removed.

[0008] S3, Ice water bath: Quickly transfer the fully enclosed sterile blood bag after S2 treatment to an ice water bath and let it stand for 50-70 minutes; the ice water bath can promote the rapid contraction of red blood cells and prevent them from rupturing. Afterwards, remove the red blood cells to avoid affecting the color of the serum later and introducing unnecessary impurities.

[0009] S4. Centrifugation once: Place the fully enclosed sterile blood bag after S3 treatment into a centrifuge and centrifuge for 35-40 minutes, and take the supernatant once.

[0010] S5. Second centrifugation: Add the supernatant from the first treatment in S4 into a centrifuge and centrifuge for 35-40 minutes. Take the second supernatant.

[0011] S6. Cryopreservation: Transfer the supernatant to a negative pressure blood bag and freeze it at -15°C or below.

[0012] S7. Thawing: Thaw the supernatant stored in the negative pressure blood bag. The thawing method is segmented temperature-controlled thawing. The specific operation method is: place at 0℃-4℃ for 5-7 hours, place at 4℃-8℃ for 5-7 hours, and place at 8℃-16℃ for 5-7 hours.

[0013] S8. Primary ultrafiltration: The thawed secondary supernatant is subjected to primary ultrafiltration through a 1000kDa hollow fiber ultrafiltration membrane to obtain primary permeate and primary concentrate.

[0014] S9. Secondary ultrafiltration: The primary permeate is passed through a 100kDa hollow fiber ultrafiltration membrane for secondary ultrafiltration to obtain secondary permeate and secondary concentrate.

[0015] S10, Three-stage ultrafiltration: The secondary permeate is passed through a 100kDa hollow fiber ultrafiltration membrane for three-stage ultrafiltration to obtain a third-stage permeate and a third-stage concentrate;

[0016] S11. Finished product preparation: Dilute the three-stage permeate solution in phosphoric acid solution, sterilize, and then seal and store.

[0017] Moreover, blood is drawn from adult cattle 2-3 times a month.

[0018] Moreover, the centrifugal force of the centrifuge during a single centrifugation is 4800g-5000g.

[0019] Moreover, the centrifugal force of the centrifuge during the second centrifugation is 11000g-13000g.

[0020] Moreover, the feed rate of the supernatant added to the centrifuge is 0.5L / min-0.8L / min.

[0021] Furthermore, the sterilization method involves passing the mixture through a 0.22µm PVDF membrane.

[0022] Furthermore, the phosphoric acid solution has a concentration of 0.05 mol / L and a pH value of 7.5.

[0023] The beneficial effects of this invention are as follows:

[0024] 1. This invention uses adult bovine serum instead of fetal or calf blood to produce enzyme diluents for in vitro diagnostic reagents. The cost of adult bovine serum is about 30% of that of calf or fetal serum. This invention greatly saves the cost of enzyme diluents for in vitro diagnostic reagents, allowing for large-scale promotion, while also ensuring the stability and sensitivity of the enzyme diluents for in vitro diagnostic reagents.

[0025] 2. This invention employs a 37°C warm water bath and an ice water bath. The warm water bath causes platelets to release many substances, altering clotting factors. The ice water bath activates the clotting reaction, causing the blood to coagulate rapidly, forming a gel. Fibrinogen is converted into fibrin clots, effectively reducing platelet and red blood cell rupture, ensuring clear, non-reddish serum color, reducing fibrinogen, and increasing serum output. Attached Figure Description

[0026] Figure 1 This is a flowchart illustrating the process of producing a special enzyme diluent for in vitro diagnostic reagents according to the present invention. Detailed Implementation

[0027] Example 1

[0028] A method for producing a special enzyme diluent for in vitro diagnostic reagents using bovine serum includes the following steps:

[0029] S1. Blood collection: 600ml of blood is collected from the neck of an adult cow using a fully enclosed sterile bag for intravenous blood collection.

[0030] S2. Warm water bath: Transfer the fully sealed sterile blood bag filled with blood to a 37°C warm water bath and let it stand for 40 minutes, turning it upside down twice during the process.

[0031] S3, Ice water bath: Quickly transfer the fully enclosed sterile blood bag after S2 treatment to an ice water bath and let it stand for 50-70 minutes.

[0032] S4. Centrifugation once: Place the fully enclosed sterile blood bag after S3 treatment into a centrifuge and centrifuge for 35 minutes, and take the supernatant once.

[0033] S5. Secondary centrifugation: Add the supernatant from the first treatment in S4 into a centrifuge and centrifuge for 35 minutes. Take the secondary supernatant.

[0034] S6. Cryopreservation: Transfer the supernatant to a negative pressure blood bag and freeze it at -15°C or below.

[0035] S7. Thawing: Thaw the supernatant stored frozen in the negative pressure blood bag. The thawing method is segmented temperature-controlled thawing. The specific operation method is: place at 0℃ for 5 hours, place at 4℃ for 5 hours, and place at 8℃ for 5 hours.

[0036] S8. Primary ultrafiltration: The thawed secondary supernatant is subjected to primary ultrafiltration through a 1000kDa hollow fiber ultrafiltration membrane to obtain primary permeate and primary concentrate.

[0037] S9. Secondary ultrafiltration: The primary permeate is passed through a 100kDa hollow fiber ultrafiltration membrane for secondary ultrafiltration to obtain secondary permeate and secondary concentrate.

[0038] S10, Three-stage ultrafiltration: The secondary permeate is passed through a 100kDa hollow fiber ultrafiltration membrane for three-stage ultrafiltration to obtain a third-stage permeate and a third-stage concentrate;

[0039] S11. Finished product preparation: Dilute the three-stage permeate solution in phosphoric acid solution, sterilize, and then seal and store.

[0040] Furthermore, blood is drawn from adult cattle twice a month.

[0041] Furthermore, the centrifugal force of the centrifuge during a single centrifugation is 4800g.

[0042] Furthermore, the centrifugal force of the centrifuge during the second centrifugation is 11000g.

[0043] Furthermore, the feed rate of the supernatant added to the centrifuge was 0.5 L / min.

[0044] Furthermore, the sterilization method is as follows: passing the mixture through a 0.22µm PVDF membrane.

[0045] Furthermore, the concentration of the phosphoric acid solution is 0.05 mol / L, and the pH value is 7.5.

[0046] Example 2

[0047] A method for producing a special enzyme diluent for in vitro diagnostic reagents using bovine serum includes the following steps:

[0048] S1. Blood collection: 800ml of blood is collected from the neck of an adult cow using a fully enclosed sterile bag for intravenous blood collection.

[0049] S2. Warm water bath: Transfer the fully sealed sterile blood bag filled with blood to a 37°C warm water bath and let it stand for 50 minutes, turning it up and down 3 times during the process.

[0050] S3, Ice water bath: Quickly transfer the fully enclosed sterile blood bag after S2 treatment to an ice water bath and let it stand for 50-70 minutes.

[0051] S4. Centrifugation once: Place the fully enclosed sterile blood bag after S3 treatment into a centrifuge and centrifuge for 40 minutes, then collect the supernatant once.

[0052] S5. Secondary centrifugation: Add the supernatant from the first treatment in S4 into a centrifuge and centrifuge for 40 minutes. Take the secondary supernatant.

[0053] S6. Cryopreservation: Transfer the supernatant to a negative pressure blood bag and freeze it at -15°C or below.

[0054] S7. Thawing: Thaw the supernatant stored in the negative pressure blood bag. The thawing method is segmented temperature-controlled thawing. The specific operation method is: place at 4℃ for 7 hours, place at 8℃ for 7 hours, and place at 16℃ for 7 hours.

[0055] S8. Primary ultrafiltration: The thawed secondary supernatant is subjected to primary ultrafiltration through a 1000kDa hollow fiber ultrafiltration membrane to obtain primary permeate and primary concentrate.

[0056] S9. Secondary ultrafiltration: The primary permeate is passed through a 100kDa hollow fiber ultrafiltration membrane for secondary ultrafiltration to obtain secondary permeate and secondary concentrate.

[0057] S10, Three-stage ultrafiltration: The secondary permeate is passed through a 100kDa hollow fiber ultrafiltration membrane for three-stage ultrafiltration to obtain a third-stage permeate and a third-stage concentrate;

[0058] S11. Finished product preparation: Dilute the three-stage permeate solution in phosphoric acid solution, sterilize, and then seal and store.

[0059] Furthermore, blood is drawn from adult cattle three times a month.

[0060] Furthermore, the centrifugal force of the centrifuge during a single centrifugation is 5000g.

[0061] Furthermore, the centrifugal force of the centrifuge during the second centrifugation is 13000g.

[0062] Furthermore, the feed rate of the supernatant added to the centrifuge was 0.8 L / min.

[0063] Furthermore, the sterilization method is as follows: passing the mixture through a 0.22µm PVDF membrane.

[0064] Furthermore, the concentration of the phosphoric acid solution is 0.05 mol / L, and the pH value is 7.5.

[0065] Example 3

[0066] A method for producing a special enzyme diluent for in vitro diagnostic reagents using bovine serum includes the following steps:

[0067] S1. Blood collection: 700ml of blood is collected from the neck of an adult cow using a fully enclosed sterile bag for intravenous blood collection.

[0068] S2. Warm water bath: Transfer the fully sealed sterile blood bag filled with blood to a 37°C warm water bath and let it stand for 45 minutes, turning it up and down 3 times during the process.

[0069] S3, Ice water bath: Quickly transfer the fully enclosed sterile blood bag after S2 treatment to an ice water bath and let it stand for 50-70 minutes; S4, Single centrifugation: Place the fully enclosed sterile blood bag after S3 treatment in a centrifuge for a single centrifugation for 38 minutes and collect the supernatant once.

[0070] S5. Second centrifugation: Add the supernatant from the first treatment in S4 into a centrifuge and centrifuge for 38 minutes. Take the second supernatant.

[0071] S6. Cryopreservation: Transfer the supernatant to a negative pressure blood bag and freeze it at -15°C or below.

[0072] S7. Thawing: Thaw the supernatant stored frozen in the negative pressure blood bag. The thawing method is segmented temperature-controlled thawing. The specific operation method is: place at 2℃ for 6 hours, place at 6℃ for 6 hours, and place at 12℃ for 6 hours.

[0073] S8. Primary ultrafiltration: The thawed secondary supernatant is subjected to primary ultrafiltration through a 1000kDa hollow fiber ultrafiltration membrane to obtain primary permeate and primary concentrate.

[0074] S9. Secondary ultrafiltration: The primary permeate is passed through a 100kDa hollow fiber ultrafiltration membrane for secondary ultrafiltration to obtain secondary permeate and secondary concentrate.

[0075] S10, Three-stage ultrafiltration: The secondary permeate is passed through a 100kDa hollow fiber ultrafiltration membrane for three-stage ultrafiltration to obtain a third-stage permeate and a third-stage concentrate;

[0076] S11. Finished product preparation: Dilute the three-stage permeate solution in phosphoric acid solution, sterilize, and then seal and store.

[0077] Furthermore, blood is drawn from adult cattle twice a month.

[0078] Furthermore, the centrifugal force of the centrifuge during a single centrifugation is 4900g.

[0079] Furthermore, the centrifugal force of the centrifuge during the second centrifugation is 12000g.

[0080] Furthermore, the feed rate of the supernatant added to the centrifuge was 0.6 L / min.

[0081] Furthermore, the sterilization method is as follows: passing the mixture through a 0.22µm PVDF membrane.

[0082] Furthermore, the concentration of the phosphoric acid solution is 0.05 mol / L, and the pH value is 7.5.

[0083] Comparative Example 1

[0084] This comparative example is identical to Example 3 except that it does not undergo three ultrafiltrations.

[0085] Comparative Example 2

[0086] This comparative example is the same as Example 3 except that it does not involve a warm water bath or an ice water bath.

[0087] Comparative Example 3

[0088] This comparative example is identical to Example 3 except that it does not use segmented temperature-controlled thawing and instead thaws at 12°C for 10 hours.

[0089] Experimental Section

[0090] Experiment 1:

[0091] The following steps are used to detect HAV in serum or plasma samples using HAV-IgM enzyme conjugate solution (1:2000):

[0092] (1) Add 50 μL of the sample to the microplate coated with HAV-IgM antigen, and set up two negative control wells, one positive control well, and one blank control well. Then add 50 μL of anti-HAV-IgM enzyme conjugate solution of fetal calf, calf, and adult calf to each well (except for the blank control well) and incubate at 37°C for 30 minutes.

[0093] (2) Discard the liquid in the hole and wash the plate 5 times with washing solution.

[0094] (3) Add 50 μL of substrate solution A and 50 μL of substrate solution B to each well and incubate at 37°C for 15 minutes.

[0095] (4) Add 50 μL of stop solution to each well to terminate the reaction.

[0096] (5) Place the reaction plate at the 450nm wavelength of the microplate reader colorimeter and zero it with a blank well, then read the OD value of each well.

[0097] (6) Calculate the CutOff value as follows: (average OD value of negative control + average OD value of positive control) x 0.5.

[0098] (7) Result interpretation: A specimen OD value less than CutOff is considered positive, and a value greater than or equal to CutOff is considered negative.

[0099] Serially diluted antibodies were added to microplates coated with HAV-IgM antigen, and the experiment was performed according to the standard reaction procedure. The traditional thyroxine conjugate and the enzyme conjugate of this invention were compared. A chromogenic reagent was added for color development, and the reaction was terminated with 2M sulfuric acid solution. The results were then read on a microplate reader. The results are as follows:

[0100]

[0101]

[0102] The above data indicate that the stability and sensitivity of the enzyme diluent in the adult bovine serum enzyme-linked immunosorbent assay (ELISA) are not significantly different from those in fetal bovines and calves.

[0103] Experiment 2:

[0104] The stability of enzyme conjugate diluents for bovine serum HAV antigen detection kits (ELSA) at different ages is shown below. The diluent numbers and contents for each serum enzyme conjugate diluent are as follows:

[0105] ①The kit containing PBST buffer and enzyme conjugate dilution stabilized with fetal bovine serum-irrelevant proteins is the first combination;

[0106] ②The kit containing PBST buffer and enzyme conjugate dilution stabilized with fetal serum-irrelevant proteins is the second combination;

[0107] ③The kit containing PBST buffer and enzyme conjugate dilution stabilized with bovine serum-irrelevant proteins is the third combination;

[0108] Three different combinations of HAV antibody enzyme-linked immunosorbent assay (ELISA) kits were used to test the quality control serum. The destructive stability of the kits was tested according to Chinese biological product regulations, specifically for those placed at 40°C and 37°C for 3 days.

[0109] Example 1:

[0110] The components and concentrations of the enzyme conjugate dilution solution are as follows:

[0111] Component 1 (PBST buffer)

[0112]

[0113] Adjust the pH of the solution to 7.4 with HCl, add distilled water to 1000 mL, and store at 4°C; Component 2 (stabilizer)

[0114] 0.1g fetal bovine serum

[0115] Add washing buffer to 100 mL and store at 4°C for later use.

[0116] Example 2:

[0117] The components and concentrations of the enzyme conjugate dilution solution are as follows:

[0118] Component 1 (PBST buffer)

[0119]

[0120] Adjust the pH of the solution to 7.4 with HCl, add distilled water to 1000 mL, and store at 4°C; Component 2 (stabilizer)

[0121] 0.1g calf serum

[0122] Add washing buffer to 100 mL and store at 4°C for later use.

[0123] Example 3

[0124] The components and concentrations of the enzyme conjugate dilution solution are as follows:

[0125] Component 1 (PBST buffer)

[0126]

[0127] Adjust the pH of the solution to 7.4 with HCl, add distilled water to 1000 mL, and store at 4°C; Component 2 (stabilizer)

[0128] 0.1g of adult bovine serum

[0129] Add washing buffer to 100 mL and store at 4°C for later use.

[0130] To ensure that the enzyme conjugate dilutions were prepared under the same conditions for comparison, the same batch of HAV antigen detection reagents (ELISA) was used. Simultaneously, anti-HAV enzyme conjugates with the above three different enzyme dilutions were prepared to form three sets of kits, and parallel comparison experiments were performed on these three sets of kits.

[0131] During the experiment, the procedures were strictly followed according to the kit instructions. For each combination, 5 wells were added for a negative control and 5 wells for a positive control. 10 wells were added for each combination of 1 Ncu / ml and 2 Ncu / ml HAV serum. One positive control serum sample (denoted as P) was added to 10 wells, and 30 HAV negative sera were added to one well each, for a total of 70 samples. The average S / CO values ​​for the negative control, positive control, 1 Ncu / ml, 2 Ncu / ml, and positive control serum (P), and the average S / CO value for the 30 anti-HAV negative sera were calculated.

[0132] Enzyme-labeled antibody activity verification:

[0133] Table 1. Average S / CO values ​​and experimental results of four reagent kits when placed at 4℃ to detect quality control serum.

[0134] Reagent Name negative control Positive control 1 Ncu / ml 2 Ncu / ml Quality control P negative serum ① Liquid reagent kit 0.027 4.346 1.684 3.313 4.013 0.045 ② Liquid reagent kit 0.028 4.331 1.577 3.279 4.022 0.046 ③ Liquid reagent kit 0.026 4.296 1.631 3.512 4.337 0.156

[0135] Table 1 shows that the average S / CO values ​​of the four kits placed at 4℃ for sample detection results did not differ significantly, and the negative and positive results were clearly distinguishable.

[0136] Table 2. Average S / CO values ​​and experimental results of four reagent kits after 3 days of incubation at 37℃ in quality control serum.

[0137] Reagent Name negative control Positive control 1 Ncu / ml 2 Ncu / ml Quality control P negative serum ① Liquid reagent kit 0.029 4.278 1.784 3.321 4.003 0.055 ② Liquid reagent kit 0.025 4.325 1.546 3.3369 4.182 0.047 ③ Liquid reagent kit 0.026 4.256 1.644 3.407 4.037 0.044

[0138] Table 2 shows that the average S / CO values ​​of the four kits after being placed at 37℃ for 3 days showed significant differences.

[0139] Analysis of the results of the quality control serum test using three kits: From the numbers and contents of the three enzyme conjugate diluents, it can be seen that the fetal bovine serum ① enzyme conjugate diluent uses the least amount of enzyme, while ② and ③ enzyme conjugate diluents use slightly more. Table 1 shows that the experimental results of the three different enzyme diluent combinations are basically the same, with no significant differences. Table 2 shows that the experimental results of kits ①, ②, and ③ have good stability; the S / CO values ​​for both negative and positive samples show very little change compared to the S / CO value at 4℃, indicating good stability and low background.

[0140] The experimental results above show that solution ③ exhibits very slight turbidity, and after being placed at 4℃ for a period of time (1 m), virtually no precipitation occurred, indicating that solution ③ is stable. Moreover, it is less expensive than solutions ① and ②. Therefore, solution ③, a diluted enzyme solution, can be used for the mass production of HAV core antigen detection kits (ELISA).

[0141] The bovine serum enzyme conjugate diluent provided by this invention can be stably stored for a long time in environments of 2-8℃ and 37℃, exhibiting good stability and long-term application. It also saves costs, making it both economical and convenient to use.

[0142] Experiment 3

[0143] Serum markers were measured using the in vitro diagnostic reagent-specific enzyme dilution solutions obtained from the above examples and comparative examples. The specific experimental methods are shown in Table 3 below.

[0144] Table 3. Methods for measuring serum markers

[0145]

[0146]

[0147] The specific experimental data obtained are shown in Table 4:

[0148] Table 4. Finished Product Specifications of Enzyme Diluent for In Vitro Diagnostic Reagents

[0149]

[0150] As can be seen from Table 4, the contents of total protein, hemoglobin, high-density lipoprotein, total bilirubin, total cholesterol, triglycerides, and γ-globulin in the finished products obtained by the embodiments of the present invention are all lower than those in the comparative example, indicating that the technical solution adopted in the present invention is a better technical solution.

Claims

1. A method for producing a special enzyme diluent for in vitro diagnostic reagents using bovine serum, characterized in that, Includes the following steps: S1. Blood collection: Collect 600-800ml of blood from the neck of an adult cow. The blood collection method is a fully enclosed sterile bag for intravenous blood collection. S2. Warm water bath: Transfer the fully sealed sterile blood bag filled with blood to a 37℃ warm water bath and let it stand for 40-50 minutes, turning it up and down 2-3 times during the process. S3, Ice water bath: Quickly transfer the fully enclosed sterile blood bag after S2 treatment to an ice water bath and let it stand for 50-70 minutes. S4. Centrifugation once: Place the fully enclosed sterile blood bag after S3 treatment into a centrifuge and centrifuge for 35-40 minutes, and take the supernatant once. S5. Second centrifugation: Add the supernatant from the first treatment in S4 into a centrifuge and centrifuge for 35-40 minutes. Take the second supernatant. S6. Cryopreservation: Transfer the secondary supernatant to a negative pressure blood bag and freeze it at -15°C or below. S7. Thawing: Thaw the supernatant stored in the negative pressure blood bag. The thawing method is segmented temperature-controlled thawing. The specific operation method is: place at 0℃-4℃ for 5-7 hours, place at 4℃-8℃ for 5-7 hours, and place at 8℃-16℃ for 5-7 hours. S8. Primary ultrafiltration: The thawed secondary supernatant is subjected to primary ultrafiltration through a 1000kDa hollow fiber ultrafiltration membrane to obtain primary permeate and primary concentrate. S9. Secondary ultrafiltration: The primary permeate is passed through a 100kDa hollow fiber ultrafiltration membrane for secondary ultrafiltration to obtain secondary permeate and secondary concentrate. S10, Three-stage ultrafiltration: The secondary permeate is passed through a 100kDa hollow fiber ultrafiltration membrane for three-stage ultrafiltration to obtain a third-stage permeate and a third-stage concentrate; S11. Finished product preparation: Dilute the three-stage permeate solution in phosphoric acid solution, sterilize, and then seal and store.

2. The method for producing a special enzyme diluent for in vitro diagnostic reagents using bovine serum as described in claim 1, characterized in that, Blood is drawn from adult cattle 2-3 times a month.

3. The method for producing a special enzyme diluent for in vitro diagnostic reagents using bovine serum as described in claim 1, characterized in that, The centrifugal force of the centrifuge during a single centrifugation is 4800g-5000g.

4. The method for producing a special enzyme diluent for in vitro diagnostic reagents using bovine serum as described in claim 1, characterized in that, The centrifugal force of the centrifuge during the second centrifugation is 11000g-13000g.

5. The method for producing a special enzyme diluent for in vitro diagnostic reagents using bovine serum as described in claim 1, characterized in that, The feed rate of the supernatant added to the centrifuge is 0.5L / min-0.8L / min.

6. The method for producing a special enzyme diluent for in vitro diagnostic reagents using bovine serum as described in claim 1, characterized in that, The sterilization method is as follows: passing the mixture through a 0.22µm PVDF membrane.

7. The method for producing a special enzyme diluent for in vitro diagnostic reagents using bovine serum as described in claim 1, characterized in that, The phosphoric acid solution has a concentration of 0.05 mol / L and a pH value of 7.5.

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