CN115993460B - Alkaline phosphatase-labeled myoglobin antibody diluent and preparation method thereof - Google Patents
Alkaline phosphatase-labeled myoglobin antibody diluent and preparation method thereof Download PDFInfo
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Abstract
The application relates to the field of antibody stabilizers, and specifically discloses an alkaline phosphatase-labeled myoglobin antibody diluent which comprises the following components in concentration: 42-54mmol/L of trihydroxy aminomethane; 36-48mmol/L sulfamic acid; bovine serum albumin 8-12g/L; 50-80g/L of sodium chloride; sucrose 40-60g/L; polyethylene glycol 8-12g/L; zinc chloride 0.33-0.45mmol/L; magnesium chloride hexahydrate 0.7-1.5mmol/L; preservative 0.2-0.6mL/L; the balance being purified water. The application has the effects of effectively improving the long-term storage stability of the diluted enzyme at 2-8 ℃ and reducing the inactivation rate of the antibody.
Description
Technical Field
The present application relates to the field of antibody stabilizers, and more particularly, to an alkaline phosphatase-labeled myoglobin antibody diluent and a preparation method thereof.
Background
Alkaline phosphatase is an enzyme that is widely present in animals, plants, microorganisms, and the like. Alkaline phosphatase is often used to extract from calf intestinal mucosa and E.coli, and consists of a number of isozymes. In a suitable buffer, alkaline phosphatase may catalyze the hydrolysis of phosphate group-containing chemiluminescent substrates such as nitrophenyl phosphate (PNP), sodium beta-glycerophosphate, naphthalene phosphate, 3- (2-spiral adamantane) -4-methoxy-4- (3-phosphoryl) -phenyl-1, 2-dioxy-cyclohexane (AMPPD). Thus, alkaline phosphatase as a marker solution for alkaline phosphatase is one of the components of an in vitro diagnostic kit, and the stability of the enzyme activity of alkaline phosphatase becomes an important factor affecting the quality performance of the kit. When serum is assayed, the reaction microenvironment is almost entirely provided by the dilution of the enzyme label, i.e., the antigen-antibody reaction is performed in the enzyme label dilution.
The same marked enzyme is widely applied to quantitative diagnosis of clinical immunity, and as with other biological enzymes, alkaline phosphatase has short half-life and easy inactivation, and the change of acid, alkali, salt ions and temperature conditions can change and even completely deactivate the enzyme. Alkaline phosphatase is commercially available under the brand name Roche, sigma, etc. In order to achieve a quantitative diagnostic effect, it is important to ensure the activity of a stable alkaline phosphatase-labeled antigen or antibody. The enzyme is mainly horseradish peroxidase, which is used by factories taking the enzyme as an amplifying system, mainly because the enzyme is cheap and the labeled reagent is stored stably, but the sensitivity of the horseradish peroxidase labeled reagent is lower than that of alkaline phosphatase. Whereas alkaline phosphatase has not been widely used because of the stability of the phosphatase-labeled reagent.
At present, the conventional enzyme-labeled antibodies need to be diluted by tens to hundreds times in practical application, but the enzyme-labeled antibodies diluted by the conventional diluent, especially the antibodies with high concentration activity, are mostly easy to reduce in activity after being stored for a period of time. Thus, there is still room for improvement.
Disclosure of Invention
In order to effectively improve the long-term storage stability of the diluted enzyme at 2-8 ℃ and reduce the inactivation rate of the antibody, the application provides an alkaline phosphatase-labeled myoglobin antibody diluent and a preparation method thereof.
In a first aspect, the present application provides an alkaline phosphatase-labeled myoglobin antibody diluent, which adopts the following technical scheme:
an alkaline phosphatase-labeled myoglobin antibody diluent comprising the following concentration of components:
42-54mmol/L of trihydroxy aminomethane;
36-48mmol/L sulfamic acid;
bovine serum albumin 8-12g/L;
50-80g/L of sodium chloride;
sucrose 40-60g/L;
polyethylene glycol 8-12g/L;
zinc chloride 0.33-0.45mmol/L;
magnesium chloride hexahydrate 0.7-1.5mmol/L;
preservative 0.2-0.6mL/L;
the balance being purified water.
By adopting the technical scheme, the enzyme-labeled antibody can better keep activity after the dilution of the diluent by adopting the trihydroxy aminomethane and the sulfamic acid as buffer solutions. Bovine serum albumin can act to stabilize alkaline phosphatase markers. Polyethylene glycol plays a role in reducing surface tension, thereby improving antigen-antibody reactivity. Zinc chloride and magnesium chloride act to stabilize alkaline phosphatase.
Therefore, the long-term storage stability of the diluted enzyme at 2-8 ℃ can be effectively improved by adopting the synergistic combination of the trihydroxy aminomethane, sulfamic acid, bovine serum albumin, sucrose, polyethylene glycol, zinc chloride, magnesium chloride hexahydrate, sodium chloride and the preservative in a specific proportion, so that the inactivation rate of the antibody is reduced, and meanwhile, myoglobin with lower concentration can be detected, thereby being beneficial to improving the sensitivity of the diluent.
Preferably, the alkaline phosphatase-labeled myoglobin antibody diluent comprises the following components in concentration:
46-51mmol/L of trihydroxy aminomethane;
41-46mmol/L sulfamic acid;
9-11g/L of bovine serum albumin;
55-65g/L of sodium chloride;
45-55g/L of sucrose;
9-11g/L of polyethylene glycol;
zinc chloride 0.35-0.40mmol/L;
magnesium chloride hexahydrate 0.9-1.2mmol/L;
0.4-0.5mL/L of preservative;
the balance being purified water.
By adopting the technical scheme, the sensitivity of the diluent is improved while the antibody after the diluent is diluted maintains higher activity, so that myoglobin with lower concentration can be detected.
Preferably, the mass ratio of the trihydroxy aminomethane, sulfamic acid, bovine serum albumin, polyethylene glycol, zinc chloride and magnesium chloride hexahydrate is 6:4:9:9:0.001:0.02.
by adopting the technical scheme, the specific proportion of the trihydroxy aminomethane, the sulfamic acid, the bovine serum albumin, the polyethylene glycol, the zinc chloride and the magnesium chloride hexahydrate are adopted for synergistic compounding, so that the diluted antibody has higher activity and higher sensitivity.
Preferably, the mass ratio of the trihydroxy aminomethane, sulfamic acid, bovine serum albumin, sodium chloride, sucrose, polyethylene glycol, zinc chloride and magnesium chloride hexahydrate is 6:4:10:60:50:10:0.001:0.02.
by adopting the technical scheme, the diluent is compounded by adopting the trihydroxy aminomethane, sulfamic acid, bovine serum albumin, sodium chloride, sucrose, polyethylene glycol, zinc chloride and magnesium chloride hexahydrate in a specific proportion, so that the diluent is not only suitable for detecting myoglobin, but also suitable for detecting beta-human chorionic gonadotropin (beta-HCG) and Alpha Fetoprotein (AFP).
Preferably, the preservative is one or more of sodium azide, proclin-300 and gentamicin.
By adopting the technical scheme, one or more of the substances are adopted as the preservative, so that the diluent can inhibit the growth of microorganisms such as bacteria, fungi and saccharomycetes more permanently, and the activity of the antibody is not easily affected.
Preferably, the preservative is a combination of sodium azide and Proclin-300.
Preferably, the volume ratio of the sodium azide to the Proclin-300 is 1: (1-1.5).
By adopting the technical scheme, the sodium azide and the Proclin-300 with specific proportions are adopted as the preservative, so that the diluent not only can better inhibit the growth of microorganisms, but also has good compatibility with alkaline phosphatase, and the activity of the enzyme in the system is better maintained, thereby being beneficial to improving the stability of the antibody.
In a second aspect, the present application provides a method for preparing an alkaline phosphatase-labeled myoglobin antibody diluent, which adopts the following technical scheme:
a method for preparing alkaline phosphatase-labeled myoglobin antibody diluent, which comprises the following steps:
s1: preparing magnesium chloride hexahydrate: adding magnesium chloride hexahydrate into purified water, uniformly stirring, and then fixing the volume to 100mL;
s2: preparing zinc chloride: adding zinc chloride into purified water, uniformly stirring, and then fixing the volume to 100mL;
s3: preparing a buffer solution: respectively weighing tris and sulfamic acid, adding the tris and sulfamic acid into purified water, and uniformly stirring to obtain a buffer solution;
s4: respectively weighing sodium chloride and sucrose according to a formula, sequentially adding the sodium chloride and the sucrose into a container, uniformly stirring, then adding bovine serum albumin, and uniformly stirring;
s5: sequentially adding the prepared magnesium chloride hexahydrate and zinc chloride into a container, and then adding polyethylene glycol and a preservative;
s6: finally, the volume is fixed to 1L, and the pH value is measured to be 7.4+/-0.2,2-8 ℃ and the mixture is preserved for standby.
Through adopting above-mentioned technical scheme, adopt above-mentioned method preparation diluent, easy operation is convenient to, still make the diluent can keep higher stability better, make the antibody be difficult to lose activity.
In summary, the present application includes at least one of the following beneficial technical effects:
1. by adopting the synergistic combination of the trihydroxy aminomethane, sulfamic acid, bovine serum albumin, sucrose, polyethylene glycol, zinc chloride, magnesium chloride hexahydrate, sodium chloride and preservative in a specific proportion, the long-term storage stability of the diluted enzyme at 2-8 ℃ can be effectively improved, the inactivation rate of the antibody is reduced, and meanwhile, myoglobin with lower concentration can be detected, so that the sensitivity of the diluent is improved. In addition, the diluent is also suitable for the detection of myoglobin, beta-HCG and alpha fetoprotein.
2. By adopting the synergistic combination of the trihydroxy aminomethane, the sulfamic acid, the bovine serum albumin, the polyethylene glycol, the zinc chloride and the magnesium chloride hexahydrate in a specific proportion, the antibody after the dilution of the diluent maintains higher activity, and the diluent has higher sensitivity.
3. By adopting sodium azide and Proclin-300 in specific proportions as preservatives, the diluent not only can inhibit the growth of microorganisms better, but also has good compatibility with alkaline phosphatase, and the activity of enzymes in a system is kept better, so that the stability of antibodies is improved.
Detailed Description
The present application is described in further detail below with reference to examples.
Example 1
The embodiment discloses an alkaline phosphatase-labeled myoglobin antibody diluent which comprises the following components:
trihydroxy aminomethane; sulfamic acid; bovine serum albumin; sodium chloride; sucrose; polyethylene glycol; zinc chloride; magnesium chloride hexahydrate; a preservative; purifying water;
wherein the preservative is gentamicin.
The embodiment also discloses a preparation method of the alkaline phosphatase-labeled myoglobin antibody diluent, which comprises the following steps:
s1: preparing magnesium chloride hexahydrate: weighing 14.23mg of magnesium chloride hexahydrate, adding the magnesium chloride hexahydrate into 50mL of purified water, uniformly stirring at the rotating speed of 600r/min, and then fixing the volume to 100mL, wherein the molar concentration of the magnesium chloride is 0.7mmol/L;
s2: preparing zinc chloride: 1.2mg of zinc chloride is weighed and added into 50mL of purified water, the mixture is stirred uniformly, the volume is fixed to 100mL, and the molar concentration of the zinc chloride is 0.33mmol/L;
s3: preparing a buffer solution: 5.09g of tris and 4.66g of sulfamic acid are respectively weighed and added into 600mL of purified water, and the mixture is stirred uniformly at the rotating speed of 700r/min to obtain a buffer solution, wherein the molar concentration of the tris is 42mmol/L and the molar concentration of the sulfamic acid is 48mmol/L;
s4: respectively weighing 80g of sodium chloride and 40g of sucrose, sequentially adding into a container (filled with the prepared buffer solution), uniformly stirring at a rotating speed of 600r/min, then adding 8g of bovine serum albumin, and uniformly stirring at a rotating speed of 600 r/min;
s5: sequentially adding 2mL of prepared magnesium chloride hexahydrate and 1mL of zinc chloride into a container, and then adding 8g of polyethylene glycol and 0.2mL of preservative;
s6: and finally, the volume is fixed to 1L, and the pH value is measured to be 7.2,2 ℃ and the mixture is preserved for standby.
Example 2
The difference from example 1 is that:
a method for preparing alkaline phosphatase-labeled myoglobin antibody diluent, which comprises the following steps:
s1: preparing magnesium chloride hexahydrate: weighing 30.50mg of magnesium chloride hexahydrate, adding the magnesium chloride hexahydrate into 50mL of purified water, uniformly stirring at the rotating speed of 600r/min, and then fixing the volume to 100mL, wherein the molar concentration of the magnesium chloride is 1.5mmol/L;
s2: preparing zinc chloride: 1.63mg of zinc chloride is weighed and added into 50mL of purified water, the mixture is stirred uniformly, the volume is fixed to 100mL, and the molar concentration of the zinc chloride is 0.45mmol/L;
s3: preparing a buffer solution: 6.54g of tris and 3.50g of sulfamic acid are respectively weighed and added into 600mL of purified water, and the mixture is stirred uniformly at the rotating speed of 700r/min to obtain a buffer solution, wherein the molar concentration of the tris is 54mmol/L and the molar concentration of the sulfamic acid is 36mmol/L;
s4: respectively weighing 50g of sodium chloride and 60g of sucrose, sequentially adding into a container, uniformly stirring at a rotating speed of 600r/min, then adding 12g of bovine serum albumin, and uniformly stirring at a rotating speed of 600 r/min;
s5: sequentially adding 2mL of prepared magnesium chloride hexahydrate and 1mL of zinc chloride into a container, and then adding 12g of polyethylene glycol and 0.6mL of preservative;
s6: and finally, the volume is fixed to 1L, and the pH value is measured to be 7.6,8 ℃ and the mixture is preserved for standby.
Example 3
The difference from example 2 is that: the amounts of the components are different and are shown in Table 1.
Wherein, the three hydroxyl amino methane 5.52g, sulfamic acid 4.43g, magnesium chloride hexahydrate 18.30mg, zinc chloride 1.27mg.
Example 4
The difference from example 2 is that: the amounts of the components are different and are shown in Table 1.
Wherein, the three hydroxyl amino methane 6.18g, sulfamic acid 3.96g, magnesium chloride hexahydrate 24.4mg, zinc chloride 1.45mg.
Example 5
The difference from example 4 is that:
a method for preparing alkaline phosphatase-labeled myoglobin antibody diluent, which comprises the following steps:
s1: preparing magnesium chloride hexahydrate: weighing 20.33mg of magnesium chloride hexahydrate, adding the magnesium chloride hexahydrate into 50mL of purified water, uniformly stirring at the rotating speed of 600r/min, and then fixing the volume to 100mL, wherein the molar concentration of the magnesium chloride is 1.0mmol/L;
s2: preparing zinc chloride: 1.45mg of zinc chloride is weighed and added into 50mL of purified water, the mixture is stirred uniformly, the volume is fixed to 100mL, and the molar concentration of the zinc chloride is 0.40mmol/L;
s3: preparing a buffer solution: 5.96g of tris and 4.19g of sulfamic acid are respectively weighed and added into 600mL of purified water, and the mixture is stirred uniformly at the rotating speed of 700r/min to obtain a buffer solution, wherein the molar concentration of the tris is 49mmol/L and the molar concentration of the sulfamic acid is 43mmol/L;
s4: respectively weighing 70g of sodium chloride and 50g of sucrose, sequentially adding into a container, uniformly stirring at a rotating speed of 600r/min, then adding 9g of bovine serum albumin, and uniformly stirring at a rotating speed of 600 r/min;
s5: sequentially adding 2mL of prepared magnesium chloride hexahydrate and 1mL of zinc chloride into a container, and adding 9g of polyethylene glycol and 0.3mL of preservative;
s6: and finally, the volume is fixed to 1L, and the pH value is measured to be 7.4,5 ℃ and the mixture is preserved for standby.
Example 6
The difference from example 4 is that:
a method for preparing alkaline phosphatase-labeled myoglobin antibody diluent, which comprises the following steps:
s1: preparing magnesium chloride hexahydrate: weighing 20.33mg of magnesium chloride hexahydrate, adding the magnesium chloride hexahydrate into 50mL of purified water, uniformly stirring at the rotating speed of 600r/min, and then fixing the volume to 100mL, wherein the molar concentration of the magnesium chloride is 1.0mmol/L;
s2: preparing zinc chloride: 1.363mg of zinc chloride is weighed and added into 50mL of purified water, the mixture is stirred uniformly, the volume is then fixed to 100mL, and the molar concentration of the zinc chloride is 0.38mmol/L;
s3: preparing a buffer solution: 6.057g of trimethylol aminomethane and 4.250g of sulfamic acid are respectively weighed and added into 600mL of purified water, and the mixture is uniformly stirred at the rotating speed of 700r/min to obtain a buffer solution, wherein the molar concentration of the trimethylol aminomethane is 50mmol/L and the molar concentration of the sulfamic acid is 44mmol/L;
s4: respectively weighing 60g of sodium chloride and 50g of sucrose, sequentially adding into a container, uniformly stirring at a rotating speed of 600r/min, then adding 10g of bovine serum albumin, and uniformly stirring at a rotating speed of 600 r/min;
s5: sequentially adding 2mL of prepared magnesium chloride hexahydrate and 1mL of zinc chloride into a container, and adding 10g of polyethylene glycol and 0.5mL of preservative;
s6: and finally, the volume is fixed to 1L, and the pH value is measured to be 7.4,2 ℃ and the mixture is preserved for standby.
TABLE 1
Example 7
The difference from example 6 is that: the preservative is the combination of sodium azide and Proclin-300, and the volume ratio of the sodium azide to the Proclin-300 is 1:1, namely, the volumes of the sodium azide and the Proclin-300 are respectively 0.25mL and 0.25mL.
Example 8
The difference from example 6 is that: the preservative is the combination of sodium azide and Proclin-300, and the volume ratio of the sodium azide to the Proclin-300 is 1:1.5, namely, the volumes of sodium azide and Proclin-300 are 0.2mL and 0.3mL, respectively.
Example 9
The difference from example 8 is that: the preservative is the combination of sodium azide and Proclin-300, and the volume ratio of the sodium azide to the Proclin-300 is 1.5:1, namely, the volumes of the sodium azide and the Proclin-300 are respectively 0.3mL and 0.2mL.
Example 10
The difference from example 8 is that: the preservative is Proclin-300.
Example 11
The difference from example 8 is that: the preservative is sodium azide.
Comparative example 1
The difference from example 1 is that: the polyvinyl alcohol was replaced with an equal amount of purified water.
Comparative example 2
The difference from example 1 is that: equal amounts of phosphate buffer were used to replace the trihydroxy aminomethane and sulfamic acid.
Comparative example 3
The difference from example 1 is that: the same amount of purified water was used instead of the trihydroxy aminomethane.
Comparative example 4
The difference from example 1 is that: the sulfamic acid was replaced with an equal amount of purified water.
Comparative example 5
The difference from example 1 is that: equal amounts of purified water were used to replace zinc chloride and magnesium chloride hexahydrate.
Comparative example 6
The difference from example 1 is that:
an alkaline phosphatase-labeled myoglobin antibody diluent comprises the following components in mass:
30mmol (3.63 g) of trihydroxyaminomethane; sulfamic acid 54mmol (5.24 g); bovine serum albumin 6g; 40g of sodium chloride; 70g of sucrose; 5g of polyethylene glycol; zinc chloride 0.2mmol (0.73 mg); magnesium chloride hexahydrate 2mmol (40.66 mg); preservative 1mL; purified water was fixed to a volume of 1L.
Comparative example 7
The difference from example 1 is that:
an alkaline phosphatase antigen antibody diluent comprises the following components in mass:
phosphate buffer: 8.0g of NaCl; KCl 0.2g; na (Na) 2 HP0 4 ﹒12H 2 0 2.9g；KH 2 P0 4 0.2g；
Adjusting the pH to 7.4, adding DDW to a volume of 1000mL;
polyhydroxy organic compounds: 30.0g of mannitol;
amino acid and protein compounds: 10.0g of BSA (bovine serum albumin); 20.0g of glycine;
metal ions: mgCl 2 1mM；ZnC1 2 0.1mM；
Nonionic surfactant: tweenX-20.5 ml;
preservative: naN (NaN) 3 (sodium azide) 0.90g.
Experiment 1
The alkaline phosphatase marked myoglobin antibody diluent prepared by the method is prepared according to the following ratio of 1:600 to obtain enzyme-labeled working solution, which is used for researching the titer of different enzyme dilutions on the enzyme label of the project, and the inactivation rate is expressed by 7 days and 14 days. The method for calculating the inactivation rate comprises the following steps:
deactivation rate = [ (B-ase:Sub>A)/ase:Sub>A ×100% ]/n
Wherein: a: enzyme-labeled test results placed at 2-8 ℃;
b: the enzyme-labeled test result is placed at 37 ℃ for 7 days or 14 days;
n: number of test samples.
The specific experimental steps are as follows:
(1) 18 enzyme-labeled working solutions are prepared in different projects: the enzyme dilutions prepared in examples 1-11 and comparative examples 1-7 were used to dilute alkaline phosphatase-labeled MYO antibody, beta-HCG antibody and AFP antibody, respectively, at a concentration of 0.5ug/mL for the enzyme-labeled working concentration solution. The 18 enzyme-labeled working solutions obtained by diluting each item are respectively split into 3 groups in equal quantity, and are marked as A/B/C. Wherein group A is stored at 2-8deg.C, group B is stored at 37deg.C for 7 days, and group C is stored at 37deg.C for 14 days.
(2) And (3) testing:
MYO: adding 10 mu L of sample, 50 mu L of MYO magnetic bead solution and 50 mu L of MYO enzyme labeling solution into a reaction hole, mixing, incubating for 20min at 37 ℃, magnetically adsorbing, washing for 3 times, adding 100 mu L of substrate, and detecting a result.
beta-HCG: adding 10 mu L of sample, 50 mu L of beta-HCG magnetic bead solution and 50 mu L of beta-HCG enzyme labeling solution into a reaction hole, mixing, incubating for 20min at 37 ℃, magnetically adsorbing, washing for 3 times, adding 100 mu L of substrate, and detecting a result.
AFP: adding 10 mu L of sample, 50 mu LAFP magnetic bead solution and 50 mu L AFP enzyme-labeled solution into a reaction hole, mixing, incubating for 20min at 37 ℃, magnetically adsorbing, washing for 3 times, adding 100 mu L of substrate, and detecting a result. The experimental results are shown in Table 2.
Experiment 2 sensitivity of the diluent
By adopting a magnetic particle chemiluminescence immunoassay method, and applying a double-antibody sandwich method principle, mixing a sample to be detected (calibrator or quality control material), MYO/beta-HCG/AFP coated magnetic particles and MYO/beta-HCG/AFP-alkaline phosphatase (ALP) markers, and combining MYO/beta-HCG/AFP in the sample (calibrator or quality control material) with a double antibody through incubation reaction to form a magnetic particle-coated antibody-sample to be detected-antibody enzyme marker immune complex. Introducing a magnetic field, and washing unbound components by a series of processes of magnetic adsorption, washing and separation; and adding chemiluminescent substrate liquid into the immune complex, performing catalytic cleavage on the substrate liquid by alkaline phosphatase to form an unstable excited state intermediate, releasing photons when the excited state intermediate returns to a ground state, and detecting the luminous intensity by using a luminometer. In the detection range, the luminous intensity is positively correlated with the concentration of MYO/beta-HCG/AFP in the sample, so that the concentration of MYO/beta-HCG/AFP in the sample to be detected is calculated.
And (3) taking a sample without myoglobin as a sample to be detected, and repeatedly measuring the myoglobin content in the sample to be detected for 20 times by using a full-automatic chemiluminescence immunoassay. And (3) obtaining the RLU value (relative luminescence value) of 20 measurement results, calculating the average value (M) and Standard Deviation (SD) of the RLU value, obtaining M+2SD, performing two-point regression fitting according to the concentration-RLU value result between the blank reference and the MYO calibrator 1 to obtain a primary equation, substituting the RLU value of M+2SD into the equation, and obtaining the corresponding concentration value, namely the blank. The blank is the lowest concentration that the kit can detect.
TABLE 2
According to the comparison of the data in Table 2 with the data of the comparative examples, the examples all use the synergistic combination of the trihydroxymethane, sulfamic acid, bovine serum albumin, sucrose, polyethylene glycol, zinc chloride, magnesium chloride hexahydrate, sodium chloride and preservative, the inactivation rate of the antibody is far lower than that of the comparative examples, and the sensitivity of the diluent is higher than that of the comparative examples, which shows that the synergistic combination of the trihydroxymethane, sulfamic acid, bovine serum albumin, sucrose, polyethylene glycol, zinc chloride, magnesium chloride hexahydrate, sodium chloride and preservative can effectively improve the long-term storage stability of the enzyme after dilution at 2-8 ℃, so that the inactivation rate of the antibody is reduced, and simultaneously, the sensitivity of the diluent can be improved, thereby the myoglobin with lower concentration can be detected. In addition, the diluent is not only suitable for detecting MYO antibodies, but also suitable for detecting beta-HCG antibodies and AFP antibodies, and is beneficial to expanding the application range of the diluent.
However, no polyvinyl alcohol was added in comparative example 1, phosphate buffer solution was used instead of the trihydroxymethane and sulfamic acid in comparative example 2, trihydroxymethane was not added in comparative example 3, sulfamic acid was not added in comparative example 4, zinc chloride and magnesium chloride hexahydrate were not added in comparative example 5, the inactivation rates of the MYO antibodies, β -HCG antibodies, AFP antibodies were much higher in comparative examples 1-5 than in example 1, and the sensitivity of the dilutions in comparative examples 1-5 was less than in example 1, indicating that the absence of any one or more of trihydroxymethane, sulfamic acid, bovine serum albumin, sucrose, polyethylene glycol, zinc chloride, magnesium chloride hexahydrate, sodium chloride could not achieve the results of the present application.
According to the comparison of comparative example 6 with example 1 in Table 2, the amounts of the trihydroxymethane, sulfamic acid, bovine serum albumin, sucrose, polyethylene glycol, zinc chloride, magnesium chloride hexahydrate, sodium chloride and preservative in comparative example 6 were not within the scope of the present application, the inactivation rate of the antibody in comparative example 6 was much higher than that in example 1, and the sensitivity of the diluent was also lower than that in example 1, indicating that the amounts of the components had an effect on the activity of the antibody. Therefore, the proportion of each component is only within the protection scope of the application, so that the activity of the antibody is not easy to lose.
According to the comparison of comparative example 7 with example 1 in Table 2, the inactivation rates of comparative example 7 after being left at 37℃for 7 days and 14 days were 15% or more, and the activity retention effect on the antibodies was small, indicating that the dilution of comparative example 7 was not suitable for dilution of MYO antibodies, β -HCG antibodies and AFP antibodies.
From a comparison of example 5 with example 4 in table 2, the mass ratio of the trihydroxymethane, sulfamic acid, bovine serum albumin, polyethylene glycol, zinc chloride, magnesium chloride hexahydrate in example 5 is 6:4:9:9:0.001: the inactivation rate of the antibody in the embodiment 5 is reduced to a certain extent, and the sensitivity of the diluent is increased to a certain extent, which means that the specific proportion of the trihydroxy aminomethane, sulfamic acid, bovine serum albumin, polyethylene glycol, zinc chloride and magnesium chloride hexahydrate are adopted for synergistic compounding, so that the activity of the antibody after the diluent is diluted is improved, and meanwhile, the diluent has higher sensitivity.
From a comparison of example 6 with example 4 in table 2, the mass ratio of tris aminomethane, sulfamic acid, bovine serum albumin, sodium chloride, sucrose, polyethylene glycol, zinc chloride, magnesium chloride hexahydrate in example 6 was 6:4:10:60:50:10:0.001:0.02, example 6 has improved activity of the diluted antibody compared with example 4, and the sensitivity of the diluted solution is also improved, which shows that the diluted antibody has higher activity and higher sensitivity when the specific proportion of trihydroxymethane, sulfamic acid, bovine serum albumin, sodium chloride, sucrose, polyethylene glycol, zinc chloride and magnesium chloride hexahydrate are adopted for synergistic compounding.
According to the comparison of examples 7-8 in Table 2 with example 6, respectively, the preservative in examples 7-8 is compounded by adopting sodium azide and Proclin-300, and the ratio between the sodium azide and the Proclin-300 is limited, so that the inactivation rate of the antibody is reduced, and the diluent can better inhibit the growth of microorganisms, has good compatibility with alkaline phosphatase, and better maintains the activity of enzymes in the system, thereby ensuring that the antibody maintains higher activity in the diluent.
According to the comparison of examples 9-11 in Table 2 with example 8, respectively, the volume ratio of sodium azide to Proclin-300 in example 9 is 1.5: in example 10, proclin-300 was used as the preservative, in example 11, sodium azide was used as the preservative, and in examples 9-11, the inactivation rate of the antibody was higher than that of example 8, which indicated that the dilution could not only better inhibit the growth of microorganisms, but also maintain the activity of the enzyme in the system, increase the activity of antibody preservation, and further decrease the inactivation rate of the antibody, only by using sodium azide and Proclin-300 in specific proportions as the preservative.
The foregoing are all preferred embodiments of the present application, and are not intended to limit the scope of the present application in any way, therefore: all equivalent changes in structure, shape and principle of this application should be covered in the protection scope of this application.
Claims (2)
1. Use of an alkaline phosphatase-labeled myoglobin antibody diluent in the preparation of a myoglobin detection reagent, characterized in that: the diluent comprises the following components in concentration:
46-51mmol/L of trihydroxy aminomethane;
41-46mmol/L sulfamic acid;
9-11g/L of bovine serum albumin;
55-65g/L of sodium chloride;
45-55g/L of sucrose;
9-11g/L of polyethylene glycol;
zinc chloride 0.35-0.40mmol/L;
magnesium chloride hexahydrate 0.9-1.2mmol/L;
0.4-0.5mL/L of preservative;
the balance being purified water;
the mass ratio of the trihydroxy aminomethane to the sulfamic acid to the bovine serum albumin to the polyethylene glycol to the zinc chloride to the magnesium chloride hexahydrate is 6:4:9:9:0.001:0.02;
the preservative is the compound of sodium azide and Proclin-300;
the volume ratio of the sodium azide to the Proclin-300 is 1: (1-1.5).
2. A method of preparing the alkaline phosphatase-labeled myoglobin antibody diluent of claim 1, wherein: the method comprises the following steps:
s1: preparing magnesium chloride hexahydrate: adding magnesium chloride hexahydrate into purified water, uniformly stirring, and then fixing the volume to 100mL;
s2: preparing zinc chloride: adding zinc chloride into purified water, uniformly stirring, and then fixing the volume to 100mL;
s3: preparing a buffer solution: respectively weighing tris and sulfamic acid, adding the tris and sulfamic acid into purified water, and uniformly stirring to obtain a buffer solution;
s4: respectively weighing sodium chloride and sucrose according to a formula, sequentially adding the sodium chloride and the sucrose into a container, uniformly stirring, then adding bovine serum albumin, and uniformly stirring;
s5: sequentially adding the prepared magnesium chloride hexahydrate and zinc chloride into a container, and then adding polyethylene glycol and a preservative;
s6: finally, the volume is fixed to 1L, and the pH value is measured to be 7.4+/-0.2,2-8 ℃ and the mixture is preserved for standby.
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