A method for producing calf serum for diagnostic reagent blocking solution

Abstract

The invention discloses a production method of calf serum for diagnostic reagent blocking solution, comprising the following steps: collecting 600-800 ml of blood from the neck of a calf; centrifuging once to obtain a supernatant; freezing and storing at below -15 DEG C; thawing the supernatant in sections with temperature control, and placing the supernatant at 0-4 DEG C, 4-8 DEG C, and 8-16 DEG C for 5-7 hours respectively; centrifuging twice, stirring the thawed supernatant uniformly, centrifuging twice, and obtaining a secondary supernatant; mixing the secondary supernatant uniformly, and then performing a first ultrafiltration through a 500 kDa hollow fiber ultrafiltration membrane to obtain a primary permeate and a primary concentrated solution; performing a second ultrafiltration through a 100 kDa hollow fiber ultrafiltration membrane to obtain a secondary permeate and a secondary concentrated solution; mixing the secondary permeate and the primary concentrated solution to obtain a mixed solution, sterilizing the mixed solution, and then filling to obtain a calf serum product. The diagnostic reagent blocking solution prepared by the invention has good blocking effect and low experimental cost.

Description

Technical Field

[0001] The invention relates to the technical field of immunology, and more specifically to a method for producing calf serum used as a diagnostic reagent blocking solution. Background Art

[0002] Diagnostic reagents refer to diagnostic reagents prepared by the principles or methods of immunology, microbiology, molecular biology, etc., and used in vitro for the diagnosis, detection and epidemiological investigation of human diseases. Commonly used blocking solutions for diagnostic reagents generally include normal serum, skimmed milk powder, BSA, gelatin, etc. The main active ingredients in skimmed milk powder are whey protein and casein, which are more complex than BSA and are prone to mold and deterioration. Insufficient skimming of skimmed milk powder will even cause more nonspecific protein binding; gelatin has low solubility in water and is a large molecule with poor blocking properties; the most commonly used in the prior art is BSA blocking solution, which is made from fetal bovine or newborn calf serum protein. It is expensive and has poor blocking effect, which seriously affects the cost of the experiment. In addition, fetal bovines need to undergo caesarean section on cows, which is not only more harmful to cows, but also has a low survival rate of fetal bovines, which violates the natural law of the biological life cycle of nature.

[0003] Therefore, how to provide a diagnostic reagent blocking solution dedicated to calf serum is a technical problem to be solved in this field. Summary of the invention

[0004] In view of this, the present invention provides a method for producing calf serum for diagnostic reagent blocking solution, wherein 3-6 month old calves are used for blood collection to prepare diagnostic reagent blocking solution, which has good blocking effect and low experimental cost, and does not affect the normal survival of the calves. The specific technical solution comprises the following steps:

[0005] S1. Blood collection: 600-800ml of blood is collected from the calf's neck using a fully enclosed blood bag venous blood collection method;

[0006] S2. Centrifugation once: Place the fully enclosed blood bag in a centrifuge for centrifugation for 35-40 minutes, and take the supernatant once;

[0007] S3. Cryopreservation: transfer the primary supernatant into a negative pressure blood bag and freeze it below -15°C;

[0008] S4, thawing: thawing the primary supernatant stored in the negative pressure blood bag by freezing, wherein the thawing method is segmented temperature controlled thawing, and the specific operation method is: placing at 0°C-4°C for 5-7 hours, placing at 4°C-8°C for 5-7 hours, and placing at 8°C-16°C for 5-7 hours. The treated primary supernatant can precipitate a large amount of fibrin;

[0009] S5, secondary centrifugation: add the thawed primary supernatant to the mixing tank (1), stir evenly, and then add it to the continuous flow butterfly centrifuge for secondary centrifugation to obtain the secondary supernatant;

[0010] S6, mixing and storing: adding the secondary supernatant into the mixing tank (2) and mixing evenly;

[0011] S7, primary ultrafiltration: the secondary supernatant in the mixing tank (2) is subjected to a primary ultrafiltration through a 500 kDa hollow fiber ultrafiltration membrane to obtain a primary permeate and a primary concentrate;

[0012] S8, secondary ultrafiltration: the primary permeate is passed through a 100 kDa hollow fiber ultrafiltration membrane for secondary ultrafiltration to obtain a secondary permeate and a secondary concentrate;

[0013] S9. Finished product preparation: The secondary permeate and the primary concentrated solution are mixed to obtain a mixed solution, and the mixed solution is sterilized and then filled to obtain a calf serum finished product.

[0014] Furthermore, the calves were healthy calves aged 3 to 6 months.

[0015] Furthermore, the frequency of blood sampling from calves is 1-3 times a month.

[0016] Moreover, the centrifugal force of the centrifuge is 3500-4000g.

[0017] Moreover, the liquid inlet rate of the supernatant added into the continuous flow butterfly centrifuge is 0.5-0.8 L / min.

[0018] Moreover, the centrifugal force of the continuous flow butterfly centrifuge is 8000-9000g.

[0019] Furthermore, the temperature of the mixing tank (2) is 4°C-8°C.

[0020] Furthermore, the sterilization method is: passing the mixed solution through a 0.22 um PVDF membrane.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. The serum of the present invention has a wide range of sources, and the calves used are 3-6 months old calves, which have more fetal cows and newborn cows and more blood volume than those in the prior art, and the cost is reduced to 1 / 3 of that of newborn cows and fetal cows.

[0023] 2. The present invention removes fibrin in serum to a greater extent through segmented temperature-controlled thawing, with hemoglobin below 100 mg / L, total protein below 50-55 g / L, high-density lipoprotein below 5 mmol / L, total proteoglycan below 10 μmol / L, total cholesterol below 10 mmol / L, triglyceride below 2 mmol / L, and gamma globulin below 1 g / L, thereby making full use of calf serum.

[0024] 3. The present invention undergoes two ultrafiltrations to effectively remove immunoglobulins and improve the efficacy of the blocking solution. DETAILED DESCRIPTION

[0025] In order to better illustrate the present invention and facilitate understanding of the technical solution of the present invention, the present invention is further described in detail below. However, the following embodiments are merely simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0026] Example 1

[0027] A method for producing calf serum for diagnostic reagent blocking solution comprises the following steps:

[0028] S1. Blood collection: 600 ml of blood is collected from the calf's neck using a fully enclosed blood bag venous blood collection method;

[0029] S2, one centrifugation: place the fully enclosed blood bag in a centrifuge for 35 minutes, and take the supernatant;

[0030] S3. Cryopreservation: transfer the primary supernatant into a negative pressure blood bag and freeze at -16°C;

[0031] S4, thawing: thawing the primary supernatant stored in the negative pressure blood bag by temperature-controlled thawing, wherein the thawing method is step-by-step temperature control thawing, and the specific operation method is: placing at 0°C for 5 hours, placing at 4°C for 5 hours, and placing at 8°C for 5 hours;

[0032] S5, secondary centrifugation: add the thawed primary supernatant to the mixing tank (1), stir evenly, and then add it to the continuous flow butterfly centrifuge for secondary centrifugation to obtain the secondary supernatant;

[0033] S6, mixing and storing: adding the secondary supernatant into the mixing tank (2) and mixing evenly;

[0034] S7, primary ultrafiltration: the secondary supernatant in the mixing tank (2) is subjected to a primary ultrafiltration through a 500 kDa hollow fiber ultrafiltration membrane to obtain a primary permeate and a primary concentrate;

[0035] S8, secondary ultrafiltration: the primary permeate is passed through a 100 kDa hollow fiber ultrafiltration membrane for secondary ultrafiltration to obtain a secondary permeate and a secondary concentrate;

[0036] S9. Finished product preparation: The secondary permeate and the primary concentrated solution are mixed to obtain a mixed solution, and the mixed solution is sterilized and then filled to obtain a calf serum finished product.

[0037] Furthermore, the calf is a healthy calf that is 3 months old.

[0038] Furthermore, the frequency of blood sampling from calves is once a month.

[0039] Furthermore, the centrifugal force of the centrifuge is 3500g.

[0040] Furthermore, the supernatant was added into the continuous flow butterfly centrifuge at a rate of 0.5 L / min.

[0041] Furthermore, the centrifugal force of the continuous flow butterfly centrifuge is 8000g.

[0042] Furthermore, the temperature of the mixing tank (2) is 4°C.

[0043] Furthermore, the sterilization method is: passing the mixed solution through a 0.22 um PVDF membrane.

[0044] Example 2

[0045] A method for producing calf serum for diagnostic reagent blocking solution comprises the following steps:

[0046] S1. Blood collection: 800 ml of blood is collected from the calf's neck using a fully enclosed blood bag venous blood collection method;

[0047] S2, one centrifugation: place the fully enclosed blood bag in a centrifuge for 40 minutes, and take the supernatant;

[0048] S3. Cryopreservation: transfer the primary supernatant into a negative pressure blood bag and freeze at -17°C;

[0049] S4, thawing: thawing the primary supernatant stored in the negative pressure blood bag by temperature-controlled thawing, wherein the thawing method is step-by-step temperature control thawing, and the specific operation method is: placing at 4°C for 7 hours, placing at 8°C for 7 hours, and placing at 16°C for 7 hours;

[0050] S5, secondary centrifugation: add the thawed primary supernatant to the mixing tank (1), stir evenly, and then add it to the continuous flow butterfly centrifuge for secondary centrifugation to obtain the secondary supernatant;

[0051] S6, mixing and storing: adding the secondary supernatant into the mixing tank (2) and mixing evenly;

[0052] S7, primary ultrafiltration: the secondary supernatant in the mixing tank (2) is subjected to a primary ultrafiltration through a 500 kDa hollow fiber ultrafiltration membrane to obtain a primary permeate and a primary concentrate;

[0053] S8, secondary ultrafiltration: the primary permeate is passed through a 100 kDa hollow fiber ultrafiltration membrane for secondary ultrafiltration to obtain a secondary permeate and a secondary concentrate;

[0054] S9. Finished product preparation: The secondary permeate and the primary concentrated solution are mixed to obtain a mixed solution, and the mixed solution is sterilized and then filled to obtain a calf serum finished product.

[0055] Furthermore, the calf is a healthy calf that is 6 months old.

[0056] Furthermore, the frequency of blood sampling from calves is 3 times a month.

[0057] Furthermore, the centrifugal force of the centrifuge is 4000g.

[0058] Furthermore, the supernatant was added into the continuous flow butterfly centrifuge at a rate of 0.8 L / min.

[0059] Furthermore, the centrifugal force of the continuous flow butterfly centrifuge is 9000g.

[0060] Furthermore, the temperature of the mixing tank (2) is -8°C.

[0061] Furthermore, the sterilization method is: passing the mixed solution through a 0.22 um PVDF membrane.

[0062] Example 3

[0063] A method for producing calf serum for diagnostic reagent blocking solution comprises the following steps:

[0064] S1. Blood collection: 700 ml of blood is collected from the calf's neck using a fully enclosed blood bag venous blood collection method;

[0065] S2, one centrifugation: place the fully enclosed blood bag in a centrifuge for 35-40 minutes, and take the supernatant;

[0066] S3. Cryopreservation: transfer the primary supernatant into a negative pressure blood bag and freeze at -18°C;

[0067] S4, thawing: thawing the primary supernatant stored in the negative pressure blood bag by freezing, wherein the thawing method is segmented temperature controlled thawing, and the specific operation method is: placing at 2°C for 6 hours, placing at 6°C for 6 hours, and placing at 12°C for 6 hours;

[0068] S5, secondary centrifugation: add the thawed primary supernatant to the mixing tank (1), stir evenly, and then add it to the continuous flow butterfly centrifuge for secondary centrifugation to obtain the secondary supernatant;

[0069] S6, mixing and storing: adding the secondary supernatant into the mixing tank (2) and mixing evenly;

[0070] S7, primary ultrafiltration: the secondary supernatant in the mixing tank (2) is subjected to a primary ultrafiltration through a 500 kDa hollow fiber ultrafiltration membrane to obtain a primary permeate and a primary concentrate;

[0071] S8, secondary ultrafiltration: the primary permeate is passed through a 100 kDa hollow fiber ultrafiltration membrane for secondary ultrafiltration to obtain a secondary permeate and a secondary concentrate;

[0072] S9. Finished product preparation: The secondary permeate and the primary concentrated solution are mixed to obtain a mixed solution, and the mixed solution is sterilized and then filled to obtain a calf serum finished product.

[0073] Furthermore, the calf is a healthy calf that is 4 months old.

[0074] Furthermore, the frequency of blood sampling from calves is twice a month.

[0075] Furthermore, the centrifugal force of the centrifuge is 3800g.

[0076] Furthermore, the supernatant was added into the continuous flow butterfly centrifuge at a rate of 0.7 L / min.

[0077] Furthermore, the centrifugal force of the continuous flow butterfly centrifuge is 8500g.

[0078] Furthermore, the temperature of the mixing tank (2) is 6°C.

[0079] Furthermore, the sterilization method is: passing the mixed solution through a 0.22 um PVDF membrane.

[0080] Comparative Example 1

[0081] Except that the thawing method of this comparative example is different from that of Example 3, other technical schemes are the same as those of Example 3. The thawing method of this comparative example is: thawing the primary supernatant stored frozen in the negative pressure blood bag. The thawing method is: placing the primary supernatant stored frozen in the negative pressure blood bag at 10°C for 18 hours for thawing.

[0082] Comparative Example 2

[0083] Except that the thawing method of this comparative example is different from that of Example 3, other technical schemes are the same as those of Example 3. The thawing method of this comparative example is: thawing the primary supernatant stored frozen in the negative pressure blood bag, and the thawing method is segmented temperature-controlled thawing, and the specific operation method is: placing at 0°C-4°C for 4 hours, placing at 4°C-8°C for 4 hours, and placing at 8°C-16°C for 4 hours.

[0084] Comparative Example 3

[0085] Except that the thawing method of this comparative example is different from that of Example 3, other technical schemes are the same as those of Example 3. The thawing method of this comparative example is: thawing the primary supernatant stored frozen in the negative pressure blood bag, and the thawing method is segmented temperature-controlled thawing, and the specific operation method is: placing at 0°C-4°C for 8 hours, placing at 4°C-8°C for 8 hours, and placing at 8°C-16°C for 8 hours.

[0086] Comparative Example 4

[0087] In this comparative example, except that the cattle used for blood sampling are adult cattle, other technical schemes are the same as those in Example 3.

[0088] Experimental Section

[0089] The calf serum products obtained in the above examples and comparative examples were subjected to serum index determination, and the specific experimental methods are shown in Table 1 below:

[0090] Table 1 Determination methods of serum indicators

[0091]

[0092]

[0093] The specific experimental data measured are shown in Table 2:

[0094] Table 2 Calf serum finished product indicators

[0095]

[0096] It can be concluded from the above experimental data that since Comparative Example 1 did not perform segmented temperature-controlled thawing but adopted constant temperature thawing, high-density lipoprotein, total bilirubin, total cholesterol, triglycerides, and globulin were significantly increased compared with Examples 1-3, and although Comparative Examples 2-3 adopted segmented temperature-controlled thawing, the solution time was one hour less and one hour more than the examples, respectively, and the measured high-density lipoprotein, total bilirubin, total cholesterol, triglycerides, and γ-globulin values ​​were also increased compared with Examples 1-3, which shows that the thawing method and thawing time of the present invention are the best technical solutions; and Comparative Example 4 selected adult cattle for blood collection, and the measured total protein, hemoglobin, high-density lipoprotein, total bilirubin, total cholesterol, triglycerides, and γ-globulin were significantly increased compared with Examples 1-3, and other indicators also increased to varying degrees.

Claims

1. A method for producing calf serum for use as a diagnostic reagent blocking solution, characterized in that: The steps include: S1. Blood collection: 600-800ml of blood is collected from the calf's neck using a fully enclosed blood bag venous blood collection method; S2. Centrifugation once: Place the fully enclosed blood bag in a centrifuge for centrifugation for 35-40 minutes, and take the supernatant once; S3. Cryopreservation: transfer the primary supernatant into a negative pressure blood bag and freeze it below -15°C; S4, thawing: thawing the primary supernatant stored in the negative pressure blood bag by freezing, wherein the thawing method is segmented temperature controlled thawing, and the specific operation method is: placing at 0°C-4°C for 5-7 hours, placing at 4°C-8°C for 5-7 hours, and placing at 8°C-16°C for 5-7 hours; S5, secondary centrifugation: add the thawed primary supernatant into the mixing tank 1, stir evenly, add into the continuous flow butterfly centrifuge for secondary centrifugation, and take the secondary supernatant; S6, mixing and storing: adding the secondary supernatant into the mixing tank 2 and mixing evenly; S7, primary ultrafiltration: the secondary supernatant in the mixing tank 2 is subjected to a primary ultrafiltration through a 500 kDa hollow fiber ultrafiltration membrane to obtain a primary permeate and a primary concentrate; S8, secondary ultrafiltration: the primary permeate is passed through a 100 kDa hollow fiber ultrafiltration membrane for secondary ultrafiltration to obtain a secondary permeate and a secondary concentrate; S9. Finished product preparation: The secondary permeate and the primary concentrated solution are mixed to obtain a mixed solution, and the mixed solution is sterilized and then filled to obtain a calf serum finished product.

2. The method for producing a special calf serum for diagnostic reagent blocking solution according to claim 1, characterized in that: The calf is a healthy calf aged 3-6 months.

3. The method for producing a special calf serum for diagnostic reagent blocking solution according to claim 1, characterized in that: The frequency of blood sampling for calves is 1-3 times a month.

4. The method for producing a special calf serum for diagnostic reagent blocking solution according to claim 1, characterized in that: The centrifugal force of the centrifuge is 3500-4000g.

5. The method for producing a special calf serum for diagnostic reagent blocking solution according to claim 1, characterized in that: The supernatant is added into the continuous flow butterfly centrifuge at a rate of 0.5-0.8 L / min.

6. The method for producing calf serum for use as a diagnostic reagent blocking solution according to claim 5, characterized in that: The centrifugal force of the continuous flow butterfly centrifuge is 8000-9000g.

7. The method for producing a special calf serum for diagnostic reagent blocking solution according to claim 5, characterized in that: The temperature of the mixing tank 2 is 4°C-8°C.

8. The method for producing a special calf serum for diagnostic reagent blocking solution according to claim 1, characterized in that: The sterilization method is: passing the mixed solution through a 0.22 um PVDF membrane.

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