Homocysteine kit and preparation method thereof
阅读说明：本技术 一种同型半胱氨酸试剂盒及其制备方法 (Homocysteine kit and preparation method thereof ) 是由 张晓龙 李海燕 于 2021-08-04 设计创作，主要内容包括：本发明是关于一种同型半胱氨酸试剂盒及其制备方法,该同型半胱氨酸试剂盒包括：还原试剂、酶试剂、发光酶试剂及磁珠试剂,四者的体积比例为(0.5-10)：(0.5-10)：(0.5-10)：(0.5-25)；其中,所述还原试剂的组成为：三(2-羧乙基)膦0.01-0.55wt％、二硫苏糖醇0.05-0.08wt％、β-巯基乙醇0.1-0.2mol/L、异硫氰酸胍0.01-0.2mol/L、乙二胺四乙酸二钠0.02-0.8wt％、十二烷基硫酸钠0.01-0.05wt％,pH值为7.4-8.3；所述酶试剂为S-腺苷-同型半胱氨酸水解酶。本发明提供的试剂盒灵敏度较传统的同型半胱氨酸(HCY)测定试剂盒(酶循环法)灵敏度提高近50倍。(The invention relates to a homocysteine kit and a preparation method thereof, wherein the homocysteine kit comprises the following components: the volume ratio of the reduction reagent to the enzyme reagent to the luminescent enzyme reagent to the magnetic bead reagent is (0.5-10): (0.5-10): (0.5-10): (0.5-25); wherein, the reducing reagent comprises the following components: 0.01-0.55 wt% of tri (2-carboxyethyl) phosphine, 0.05-0.08 wt% of dithiothreitol, 0.1-0.2mol/L of beta-mercaptoethanol, 0.01-0.2mol/L of guanidine isothiocyanate, 0.02-0.8 wt% of disodium ethylenediamine tetraacetic acid, 0.01-0.05 wt% of sodium dodecyl sulfate and 7.4-8.3 of pH value; the enzyme reagent is S-adenosine-homocysteine hydrolase. Compared with the traditional Homocysteine (HCY) determination kit (enzyme cycling method), the sensitivity of the kit provided by the invention is improved by about 50 times.)
1. A homocysteine kit, comprising: the volume ratio of the reduction reagent to the enzyme reagent to the luminescent enzyme reagent to the magnetic bead reagent is (0.5-10): (0.5-10): (0.5-10): (0.5-25);
wherein the reducing agent comprises: 0.01-0.55 wt% of tri (2-carboxyethyl) phosphine, 0.05-0.08 wt% of dithiothreitol, 0.1-0.2mol/L of beta-mercaptoethanol, 0.01-0.2mol/L of guanidine isothiocyanate, 0.02-0.8 wt% of disodium ethylenediamine tetraacetic acid, 0.01-0.05 wt% of sodium dodecyl sulfate and 7.4-8.3 of pH value;
the enzyme reagent is S-adenosine-homocysteine hydrolase;
the luminous enzyme reagent is a homocysteine monoclonal antibody-alkaline phosphatase compound;
the magnetic bead reagent consists of streptavidin-coated magnetic beads and biotinylated homocysteine in the volume ratio of (0.1-5) to (0.5-10).
2. The homocysteine kit according to claim 1, whereby the reducing reagent comprises: 0.01 wt% of tris (2-carboxyethyl) phosphine, 0.05 wt% of dithiothreitol, 0.1mol/L of beta-mercaptoethanol, 0.2mol/L of guanidine isothiocyanate, 0.8 wt% of disodium ethylenediamine tetraacetic acid and 0.05 wt% of sodium dodecyl sulfate, and the pH value is 8.3.
3. The homocysteine kit according to claim 1, whereby the reducing reagent comprises: 0.05 wt% of tris (2-carboxyethyl) phosphine, 0.05 wt% of dithiothreitol, 0.2mol/L of beta-mercaptoethanol, 0.1mol/L of guanidine isothiocyanate, 0.2 wt% of disodium ethylene diamine tetraacetate, 0.25 wt% of sodium dodecyl sulfate and 8.0 of pH value.
4. The homocysteine kit according to claim 1, whereby the reducing reagent comprises: 0.55 wt% of tris (2-carboxyethyl) phosphine, 0.08 wt% of dithiothreitol, 0.1mol/L of beta-mercaptoethanol, 0.01mol/L of guanidine isothiocyanate, 0.02 wt% of disodium ethylene diamine tetraacetate, 0.01 wt% of sodium dodecyl sulfate and 7.4 of pH value.
5. The homocysteine kit according to claim 1, wherein the homocysteine kit further comprises a calibrator, a quality control material, a luminescent substrate reagent and a cleaning solution.
6. The homocysteine kit of claim 1, wherein the magnetic beads are 1-5 μm carboxyl magnetic beads.
7. A method of making a homocysteine kit according to any of claims 1-6 characterised in that it comprises the following steps:
1) preparation of a reducing agent: preparing a solution containing 0.01-0.55 wt% of tris (2-carboxyethyl) phosphine, 0.05-0.08 wt% of dithiothreitol, 0.1-0.2mol/L of beta-mercaptoethanol, 0.01-0.2mol/L of guanidinium isothiocyanate, 0.02-0.8 wt% of disodium ethylene diamine tetraacetate, 0.01-0.05 wt% of sodium dodecyl sulfate and the pH value of 7.4-8.3;
2) preparation of a luminescent enzyme reagent: coupling the anti-homocysteine monoclonal antibody with the alkaline phosphatase compound according to the weight ratio of (1-5) to (0.2-60) to obtain the homocysteine monoclonal antibody-alkaline phosphatase compound;
3) preparing a magnetic bead reagent: mixing the following components in percentage by weight (1-10): coupling the streptavidin (0.5-100) with the magnetic bead microspheres to obtain streptavidin-coated magnetic beads; mixing the following components in percentage by weight (1-10): (0.2-50) coupling biotin with homocysteine to obtain biotinylated homocysteine; mixing streptavidin-coated magnetic beads and biotinylated homocysteine according to the volume ratio of (0.1-5) to (0.5-10) to obtain the magnetic bead reagent.
8. The method of claim 7, wherein the step 2) of preparing the luciferase reagent comprises:
activating alkaline phosphatase, centrifuging, discarding supernatant, and repeatedly washing with buffer solution;
adding a coupling agent, incubating, centrifuging, and removing a supernatant;
③ adding the monoclonal antibody of anti-homocysteine into a buffer solution to pass through a purification column;
fifthly, passing the anti-homocysteine monoclonal antibody through a dialysis membrane and concentrating;
sixthly, adding the anti-homocysteine monoclonal antibody in the fifth step into the second step, uniformly mixing, incubating, centrifuging, discarding supernatant, and adding diluent;
seventhly, adding the sealing liquid, mixing uniformly, cleaning and centrifuging;
and eighthly, abandoning the supernatant, adding the preservative solution and preserving.
9. The method of claim 7, wherein the step of preparing streptavidin-coated magnetic beads in step 3) comprises:
magnetic bead activation;
adding coupling agent, mixing, incubating and centrifuging;
③ adding a buffer solution into the streptavidin and passing through a purification column;
fourthly, the streptavidin is filtered through a dialysis membrane and concentrated;
fifthly, adding the concentrated streptavidin in the step IV into the step II, incubating, centrifuging after uniformly mixing, discarding supernatant, and adding buffer solution;
sixthly, adding the sealing liquid, uniformly mixing and coupling, centrifuging after 8-24h, removing the supernatant, and adding the preservation liquid for suspension;
and seventhly, adding the preservation solution into the supernatant for suspension storage.
10. The method of claim 7, wherein the step 3) of preparing the biotinylated homocysteine specifically comprises:
washing homocysteine with washing buffer solution, centrifuging, discarding supernatant, and repeating for multiple times;
diluting the activated biotin by using a diluent;
thirdly, coupling the activated biotin with the homocysteine concentrated in the step I, incubating, uniformly mixing, centrifuging, discarding supernatant, and adding diluent;
adding stop solution to stop coupling;
adding a buffer solution for cleaning;
sixthly, adding the preservation solution for storage.
Technical Field
The invention relates to a homocysteine kit and a preparation method thereof, belonging to the technical field of immunoassay.
Background
Homocysteine (HCY) is a sulfhydryl-containing amino acid, mainly derived from dietary methionine, an important intermediate in the metabolic process of methionine and cysteine, and does not participate in protein synthesis per se. In vivo, HCY and methyltetrahydrofolate of about 1/2 are reacted with Methionine Synthase (MS) to produce Methionine and tetrahydrofolate, and tetrahydrofolate is reacted with N5, N10-Methylenetetrahydrofolate (MTHFR) to produce methyltetrahydrofolate; the rest of Hcy of about 1/2 forms Cystathionine through a transsulfenyl pathway, i.e., Hcy and serine form Cystathionine under the action of Cystathionine beta-synthase (CBS), and a part of Hcy and serine form cysteine under the action of Cystathionine lyase to finally generate pyruvic acid, sulfuric acid and water, wherein vitamin B6 is needed in the process as coenzyme and serine hydroxymethyltransferase, and the other part of Hcy and serine form homoserine. When the former two metabolic pathways are obstructed for any reason, elevated HCY produces Homocysteine Thiolactone (HTL), which is a reaction product of HCY formed during editing or correction of aminoacyl-tRNA synthetase, as a cyclic thioester, under the action of aminoacyl-tRNA synthetase.
Relevant clinical data show that HCY level is closely related to cardiovascular diseases and is an important risk factor for the onset of cardiovascular diseases, and the increased HCY in blood stimulates the blood vessel wall to cause damage to arterial blood vessels, so that inflammation and plaque formation on the vessel wall are caused, and finally, the blood flow of the heart is blocked. Patients with hyperhomocysteinuria have hyperhcy blood syndrome due to severe genetic defects that affect HCY metabolism. Minor genetic defects or vitamin B nutritional deficiencies may be associated with moderate or mild elevations in HCY and may also increase the risk of heart disease. Elevated HCY can also cause birth defects such as neural tube malformation. Therefore, it is clinically important to determine the concentration of tHCY in blood. The Homocysteine (HCY) determination kit (enzyme circulation method) in the prior art, wherein oxidized homocysteine is reduced into free type HCY by triethylene glycol phosphonic acid (TCEP), and the free type HCY reacts with a substrate to be circularly amplified; the concentration of HCY in the substance to be detected can be calculated by detecting the rate of the reduction of the absorbance of NADH converted into NAD +; but the concentration sensitivity of HCY detected by the enzyme circulation method is low, the repeatability deviation of a test value is large near the clinical significance, and the relative deviation is more than 15% when the test value is less than or equal to 12 mu mol/L; the linear range is narrow, only 50 mu mol/L of samples can be detected at most, and the method can be only used in a biochemical analyzer which is a platform.
Disclosure of Invention
In view of the above, the main objective of the present invention is to provide a homocysteine kit and a preparation method thereof, and the technical problem to be solved is to prepare the homocysteine kit by a magnetic particle chemiluminescence method. The method is rapid, simple to operate and high in automation degree, and is suitable for most clinical laboratories.
The purpose of the invention and the technical problem to be solved are realized by adopting the following technical scheme. According to the present invention, a homocysteine kit is provided, which comprises: the volume ratio of the reduction reagent to the enzyme reagent to the luminescent enzyme reagent to the magnetic bead reagent is (0.5-10): (0.5-10): (0.5-10): (0.5-25);
wherein, the reducing reagent comprises the following components: 0.01-0.55 wt% of tris (2-carboxyethyl) phosphine (TCEP), 0.05-0.08 wt% of Dithiothreitol (DTT), 0.1-0.2mol/L of beta-mercaptoethanol, 0.01-0.2mol/L of guanidine isothiocyanate, 0.02-0.8 wt% of disodium ethylenediamine tetraacetic acid (EDTA-Na), 0.01-0.05 wt% of Sodium Dodecyl Sulfate (SDS) and the pH value is 7.4-8.3;
the enzyme reagent is recombinant S-adenosine-homocysteine hydrolase;
the luminous enzyme reagent is a homocysteine monoclonal antibody-alkaline phosphatase compound;
the magnetic bead reagent consists of streptavidin-coated magnetic beads and biotinylated homocysteine in the volume ratio of 0.1-5: 0.5-10.
Further, in the homocysteine kit described above, wherein the reducing reagent comprises: 0.01 wt% of tris (2-carboxyethyl) phosphine (TCEP), 0.05 wt% of Dithiothreitol (DTT), 0.1mol/L of beta-mercaptoethanol, 0.2mol/L of guanidine isothiocyanate, 0.8 wt% of disodium ethylenediaminetetraacetate (EDTA-Na), 0.05 wt% of Sodium Dodecyl Sulfate (SDS), and the pH value of the aqueous solution is 8.3.
Further, in the homocysteine kit described above, wherein the reducing reagent comprises: 0.05 wt% of tris (2-carboxyethyl) phosphine (TCEP), 0.05 wt% of Dithiothreitol (DTT), 0.2mol/L of beta-mercaptoethanol, 0.1mol/L of guanidine isothiocyanate, 0.2 wt% of disodium ethylenediaminetetraacetate (EDTA-Na), 0.25 wt% of Sodium Dodecyl Sulfate (SDS), and the pH value of the aqueous solution is 8.0.
Further, in the homocysteine kit described above, wherein the reducing reagent comprises: 0.55 wt% of tris (2-carboxyethyl) phosphine (TCEP), 0.08 wt% of Dithiothreitol (DTT), 0.1mol/L of beta-mercaptoethanol, 0.01mol/L of guanidine isothiocyanate, 0.02 wt% of disodium ethylenediaminetetraacetate (EDTA-Na), 0.01 wt% of Sodium Dodecyl Sulfate (SDS), and pH of 7.4.
The excessive dosage of a certain component can break the balance of the whole compatibility, and the excessive dosage does not play an original role, so that the dosages of all the components in the reducing reagent are empirical values. Multiple experiments show that the homocysteine treated under the three conditions is stable in acid property and can be used for experiments.
Further, in the homocysteine kit, the homocysteine kit further comprises a calibrator, a quality control material, a cleaning solution and a luminescent substrate reagent.
Further, in the homocysteine kit, the magnetic beads are 1-5 μm carboxyl magnetic beads.
The purpose of the invention and the technical problem to be solved can also be realized by adopting the following technical scheme. The preparation method of the homocysteine kit provided by the invention comprises the following steps:
1) preparation of a reducing agent: preparing a solution containing 0.01-0.55 wt% of tris (2-carboxyethyl) phosphine, 0.05-0.08 wt% of dithiothreitol, 0.1-0.2mol/L of beta-mercaptoethanol, 0.01-0.2mol/L of guanidinium isothiocyanate, 0.02-0.8 wt% of disodium ethylene diamine tetraacetate, 0.01-0.05 wt% of sodium dodecyl sulfate and the pH value of 7.4-8.3;
2) preparation of a luminescent enzyme reagent: coupling the anti-homocysteine monoclonal antibody with the alkaline phosphatase compound according to the weight ratio of (1-5) to (0.2-60) to obtain the homocysteine monoclonal antibody-alkaline phosphatase compound;
3) preparing a magnetic bead reagent: mixing the following components in percentage by weight (1-10): coupling the streptavidin (0.5-100) with the magnetic bead microspheres to obtain streptavidin-coated magnetic beads; mixing the following components in percentage by weight (1-10): (0.2-50) coupling biotin with homocysteine to obtain biotinylated homocysteine; mixing streptavidin-coated magnetic beads and biotinylated homocysteine according to the volume ratio of (0.1-5) to (0.5-10) to obtain the magnetic bead reagent.
Further, in the preparation method of the homocysteine kit, the preparation of the luminescent enzyme reagent in step 2) specifically comprises:
activating alkaline phosphatase, centrifuging, discarding supernatant, and repeatedly washing with buffer solution;
adding a coupling agent, incubating, centrifuging, and removing a supernatant;
③ adding the monoclonal antibody of anti-homocysteine into a buffer solution to pass through a purification column;
fifthly, passing the anti-homocysteine monoclonal antibody through a dialysis membrane and concentrating;
sixthly, adding the anti-homocysteine monoclonal antibody in the fifth step into the second step, uniformly mixing, incubating, centrifuging, discarding supernatant, and adding diluent;
seventhly, adding the sealing liquid, mixing uniformly, cleaning and centrifuging;
and eighthly, abandoning the supernatant, adding the preservative solution and preserving.
Further, in the preparation method of the homocysteine kit, the preparation of the streptavidin-coated magnetic beads in step 3) specifically comprises:
magnetic bead activation;
adding coupling agent, mixing, incubating and centrifuging;
③ adding a buffer solution into the streptavidin and passing through a purification column;
fourthly, the streptavidin is filtered through a dialysis membrane and concentrated;
fifthly, adding the concentrated streptavidin in the step IV into the step II, incubating, centrifuging after uniformly mixing, discarding supernatant, and adding buffer solution;
sixthly, adding the sealing liquid, uniformly mixing and coupling, centrifuging after 8-24h, removing the supernatant, and adding the preservation liquid for suspension;
and seventhly, adding the preservation solution into the supernatant for suspension storage.
Further, in the preparation method of the homocysteine kit, wherein the preparation of the biotinylated homocysteine in 3) specifically comprises:
washing homocysteine with washing buffer solution, centrifuging, discarding supernatant, and repeating for multiple times;
diluting the activated biotin by using a diluent;
thirdly, coupling the activated biotin with the homocysteine concentrated in the step I, incubating, uniformly mixing, centrifuging, discarding supernatant, and adding diluent;
adding stop solution to stop coupling;
adding a buffer solution for cleaning;
sixthly, adding the preservation solution for storage.
The purpose of the invention and the technical problem to be solved can also be realized by adopting the following technical scheme. The use method of the homocysteine kit provided by the invention comprises the following steps:
1) treating homocysteine in a sample by using a reducing reagent to obtain free homocysteine; the volume ratio of the reducing agent to the sample is 0.1-5): (0.5-10);
2) adding recombinant S-adenosine-homocysteine hydrolase (rSAHHase) into the free homocysteine obtained in the step 1) to obtain S-adenosine-homocysteine (SAH); the volume ratio of the S-adenosine-homocysteine hydrolase (rSAHHase) to the sample is (0.01-0.5) to (0.1-10);
3) diluting the streptavidin-coated magnetic beads by using a diluent in proportion; the weight ratio of the diluent to the streptavidin-coated magnetic beads is (10-1000) to (0.05-1);
4) diluting biotinylated homocysteine in proportion by using a diluent; the weight ratio of the diluent to the biotinylated homocysteine is (20-3000) to (0.02-10);
5) diluting the homocysteine monoclonal antibody-alkaline phosphatase complex by using a diluent in proportion; the weight ratio of the diluent to the homocysteine monoclonal antibody-alkaline phosphatase complex is (15-4000) to (0.06-18);
6) forming the reagents into a reagent boat, and placing the reagent boat into a full-automatic chemiluminescence detection analyzer for testing; one reagent boat is a box of reagent, and the specifications of the reagent box are 100 persons/box and 50 persons/box.
Further, in the preparation method of the homocysteine kit, the diluent comprises the following components: 10 to 100mM Tris-HCl, 0.1 to 2 wt% BSA, 0.1 to 0.5 wt% PC300, 0.02 to 0.5 wt% Tween 20, 0.1 to 2 wt% NaCl solution, and the pH value is 7.0 to 9.0.
The extra methylene group of homocysteine brings the thiol group closer to the carboxyl group, allowing it to initiate a chemical reaction to form a five-membered ring, called homocysteine thiolactone. This reaction occurs when an amino acid normally forms a peptide bond with its neighbour. Homocysteine in serum is therefore present in a bound state or dimerized (oxidized) form, and homocysteine is not suitable for mixing with proteins, since proteins containing homocysteine are subject to self-degradation. Therefore, the application carries out two-step pretreatment on homocysteine in serum by a reducing reagent and recombinant S-adenosyl-homocysteine hydrolase, so that the combined or dimerized homocysteine becomes a free reduced homocysteine, and is converted into S-adenosyl-homocysteine by the S-adenosyl-homocysteine hydrolase, and the preparation of magnetic particle chemiluminescence Homocysteine (HCY) becomes possible.
Compared with the prior art, the invention has the following beneficial effects:
compared with the traditional Homocysteine (HCY) determination kit (enzyme cycling method), the sensitivity of the kit provided by the invention is improved by about 50 times.
The lowest detection limit of the kit provided by the invention can reach 0.23 mu mol/L; meanwhile, the kit has strong specificity, and the measured values of 200 mmol/L-cysteine and 200 mmol/L-glutathione are lower than the minimum detection limit concentration (0.23 mu mol/L).
The kit provided by the invention has a wider linear range, the upper limit of linearity is wider than that of a traditional enzyme cycling method (the upper limit of the traditional enzyme method is 50 mu mol/L), the kit can be extended to 150 mu mol/L, and the HOOK effect cannot occur even if the test concentration of a test calibrator is up to 500 mu mol/L. The chemiluminescence linear range of the magnetic particles is 0.25-300 mu mol/L;
the development of the Homocysteine (HCY) determination kit (magnetic particle chemiluminescence) breaks through the history of testing homocysteine only on a biochemical platform, multiple platforms detect simultaneously, and one detection platform and one selection are provided for hospital clinical laboratories.
Drawings
FIG. 1 shows a standard curve of homocysteine in example 1 of the present invention;
FIG. 2 shows the linear range of the homocysteine kit in example 1 of the present invention;
FIG. 3 shows the correlation analysis of the homocysteine kit in example 1 of the present invention.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.
The materials or reagents mentioned below are commercially available unless otherwise specified, and the methods mentioned are conventional ones unless otherwise specified.
The invention provides a homocysteine kit, which comprises: the volume ratio of the reduction reagent to the enzyme reagent to the luminescent enzyme reagent to the magnetic bead reagent is (0.5-10): (0.5-10): (0.5-10): (0.5-25), preferably 3: 3: 3: 5, so that the lowest detection limit of the kit can reach 0.23 mu mol/L;
wherein the reducing agent comprises: 0.01-0.55 wt% of tris (2-carboxyethyl) phosphine (TCEP), 0.05-0.08 wt% of Dithiothreitol (DTT), 0.1-0.2mol/L of beta-mercaptoethanol, 0.01-0.2mol/L of guanidine isothiocyanate, 0.02-0.8 wt% of disodium ethylenediamine tetraacetic acid (EDTA-Na), 0.01-0.05 wt% of Sodium Dodecyl Sulfate (SDS) and the pH value is 7.4-8.3; preferably, the reducing agent comprises 0.02 percent of tris (2-carboxyethyl) phosphine, 0.06 percent of dithiothreitol, 0.15 percent of beta-mercaptoethanol, 0.2 percent of guanidine isothiocyanate, 0.8 percent of disodium ethylene diamine tetraacetate, 0.04 percent of sodium dodecyl sulfate and a solution with the pH value of 8.0, and the linear correlation coefficient r of the kit can be better after the solution is preferably prepared; if the deviation is smaller than the lower limit value or larger than the upper limit value, the deviation between the measured values of the high-value sample and the low-value sample and the target concentration value is large, so that the fitting correlation coefficient r value of the measured values and the target concentration value is poor.
The enzyme reagent is recombinant S-adenosine-homocysteine hydrolase; the volume ratio of the recombinant S-adenosyl-homocysteine hydrolase (rSAHHase) to the sample is (0.01-0.5): (0.1-10) which can convert homocysteine in free form into S-adenosyl-homocysteine (SAH); in order to make the test sample more reproducible and the deviation CV smaller, the volume ratio of the recombinant S-adenosyl-homocysteine hydrolase (rSAHHase) to the sample may preferably be 0.02: 0.5; if the value is less than the lower limit value or greater than the upper limit value, the stability of the enzyme reagent is affected, and the coefficient of variation CV is poor in the repeatability test.
The luminous enzyme reagent is a homocysteine monoclonal antibody-alkaline phosphatase compound; the weight ratio of the anti-homocysteine monoclonal antibody to the alkaline phosphatase is (1-5) to (0.2-60), preferably 2: 5, the HOOK effect does not occur after the test, the test concentration of the test calibrator can be up to 500 mu mol/L, and the HOOK effect does not occur; if the content of the homocysteine monoclonal antibody is less than the lower limit value or greater than the upper limit value, the HOOK effect exists, because the content of the homocysteine monoclonal antibody is too low, and a post-banding effect occurs due to the excessive homocysteine; the homocysteine monoclonal antibody has too high content, and relatively low homocysteine, so that a prozone effect occurs; in addition, the combination of the anti-homocysteine monoclonal antibody and alkaline phosphatase will generate a luminescence signal value, which is proportional to the antibody concentration.
The magnetic bead reagent comprises streptavidin-coated magnetic beads and biotinylated homocysteine in a volume ratio of (0.1-5) - (0.5-10), wherein the streptavidin on the magnetic beads can be spontaneously connected with biotin, the homocysteine can be spontaneously connected with a corresponding anti-homocysteine monoclonal antibody, after a substrate is added, the substrate reacts with alkaline phosphatase to generate luminescence, and a concentration value corresponding to the luminescence value can be deduced according to a calibrator, so that a concentration value in serum is indirectly obtained; the volume ratio of the streptavidin-coated magnetic beads to the biotinylated homocysteine is preferably 1:4, the streptavidin-coated magnetic beads and the biotinylated homocysteine are preferably fully combined, and the luminous value is very high; if the value is less than the lower limit value, the streptavidin-coated magnetic beads are less than biotinylated homocysteine, and the luminous value is very low; if the content of the streptavidin-coated magnetic beads is larger than the upper limit value, the streptavidin-coated magnetic beads are more than biotinylated homocysteine, so that the magnetic bead reagent is wasted.
In some embodiments, the composition of the reducing agent is: 0.01 wt% of tris (2-carboxyethyl) phosphine (TCEP), 0.05 wt% of Dithiothreitol (DTT), 0.1mol/L of beta-mercaptoethanol, 0.2mol/L of guanidine isothiocyanate, 0.8 wt% of disodium ethylenediaminetetraacetate (EDTA-Na), 0.05 wt% of Sodium Dodecyl Sulfate (SDS), and the pH value of the aqueous solution is 8.3.
In other embodiments, the reducing agent consists of: 0.05 wt% of tris (2-carboxyethyl) phosphine (TCEP), 0.05 wt% of Dithiothreitol (DTT), 0.2mol/L of beta-mercaptoethanol, 0.1mol/L of guanidine isothiocyanate, 0.2 wt% of disodium ethylenediaminetetraacetate (EDTA-Na), 0.25 wt% of Sodium Dodecyl Sulfate (SDS), and the pH value of the aqueous solution is 8.0.
In other embodiments, the composition of the reducing agent is: 0.55 wt% of tris (2-carboxyethyl) phosphine (TCEP), 0.08 wt% of Dithiothreitol (DTT), 0.1mol/L of beta-mercaptoethanol, 0.01mol/L of guanidine isothiocyanate, 0.02 wt% of disodium ethylenediaminetetraacetate (EDTA-Na), 0.01 wt% of Sodium Dodecyl Sulfate (SDS), and the pH value of 7.4.
The excessive dosage of a certain component in the composition of the reducing reagent breaks the overall compatibility balance, and the excessively low dosage does not play an original role, so that the dosages of all the components in the reducing reagent are empirical values. Through multiple experiments, the proportion is found that the homocysteine treated by the three reducing reagents has stable performance and can be used for experiments.
Tris (2-carboxyethyl) phosphine (TCEP) is a very effective thiol reducing agent and also a highly effective disulfide reducing agent, is not afraid of oxidation in air, has good stability, has no unpleasant odor, can be selectively and quantitatively reduced in a wide PH range, and has good stability in acidic and alkaline solutions. The time for TCEP dissociation is usually controlled within 10 minutes, and the amount is selected to be 0.01 wt%.
Dithiothreitol (DTT) is a linear molecule in a reduction state, and is changed into a six-membered ring structure containing a disulfide bond after being oxidized, and the reduction force of DTT is influenced by pH value, because only deprotonated thiolate negative ions (-S-) have reaction activity, and can play a reduction role only in a slightly alkaline environment, such as a pH value of more than 7. The pKa of the thiol group is 8.3, so Dithiothreitol (DTT) has an optimum performance at pH 8.3.
Beta-mercaptoethanol can open disulfide bonds present in proteins and is often used to protect free cysteine thiols in proteins from misforming disulfide bonds. Meanwhile, the beta-mercaptoethanol can be dissolved in water, and the volatility of the solution is reduced. The defect is that the product is sensitive to air and easy to absorb moisture, and needs to be compatible with disodium ethylene diamine tetraacetate (EDTA-Na) and tris (2-carboxyethyl) phosphine (TCEP).
Guanidinium isothiocyanate is used to denature lysed cells and inhibit nucleases released by the cells. The guanidinium isothiocyanate solution is acidic, has pH of 4.5-7.0 in 4% water solution, and can be used together with disodium ethylenediaminetetraacetate (EDTA-Na) and Dithiothreitol (DTT) to improve cracking and denaturation ability.
Disodium ethylene diamine tetraacetate (EDTA-Na) is an important complexing agent, a pH regulator and an anticoagulant, and is used for controlling the reaction speed in experiments.
Sodium dodecyl sulfate is easily soluble in water, soluble in hot water, soluble in water, alkaline in water solution, pH of 7.5-9.5, and has special odor. The dodecyl sulfuric acid has good compatibility with tris (2-carboxyethyl) phosphine (TCEP), Dithiothreitol (DTT) and beta-mercaptoethanol, has good emulsifying, foaming, penetrating, decontamination and dispersing performances, and has strong degreasing capability, so the dodecyl sulfuric acid is often applied to in vitro diagnostic reagents.
Introducing tris (2-carboxyethyl) phosphine (TCEP), Dithiothreitol (DTT), beta-mercaptoethanol, guanidinium isothiocyanate, disodium ethylene diamine tetraacetate (EDTA-Na) and Sodium Dodecyl Sulfate (SDS) to treat homocysteine in serum, wherein the tris (2-carboxyethyl) phosphine (TCEP), Dithiothreitol (DTT), beta-mercaptoethanol, guanidinium isothiocyanate, disodium ethylene diamine tetraacetate (EDTA-Na) and Sodium Dodecyl Sulfate (SDS) synergistically prevent extra methylene of homocysteine from forming a five-membered ring, and simultaneously ensure that the homocysteine has a stable structural formula and cannot be automatically degraded.
In specific implementation, 100 parts of the homocysteine kit/kit may comprise: streptavidin-coated magnetic beads 5ml, biotinylated homocysteine 5ml, homocysteine monoclonal antibody-alkaline phosphatase complex 5 ml. Each part of the composition can comprise: 50. mu.l of diluted solution, 50. mu.l of streptavidin-coated magnetic beads, 50. mu.l of biotinylated homocysteine, and 50. mu.l of homocysteine monoclonal antibody-alkaline phosphatase complex.
The homocysteine kit further comprises a calibrator, a quality control product, a cleaning solution and a luminescent substrate reagent. The calibrator was 6 homocysteine (0. mu. mol/L, 7.5. mu. mol/L, 15. mu. mol/L, 30. mu. mol/L, 60. mu. mol/L, 150. mu. mol/L) at different concentrations, and the main purpose was to calibrate the kit. The quality control product is homocysteine with specified concentration (7.5 mu mol/L and 60 mu mol/L), is mainly used for quality control and is used for evaluating whether the test value of the system for evaluating the composition of the reagent is in a control state on the same day. The cleaning solution is a conventional cleaning solution and mainly used for cleaning a pipeline of the full-automatic immunochemistry luminescence analyzer and cleaning magnetic beads. The main component of the luminescent substrate reagent is a luminescent enzyme reagent (AMPPD, the concentration is 0.01 wt%) containing multiple benzene rings, and the effect is that the reagent in the kit can degrade the corresponding benzene rings of the luminescent substrate, and a luminescent substance can be generated in the degradation process.
The magnetic beads may be 1 to 5 μm carboxyl magnetic beads in consideration of suspension property; for better suspension, the particle size of the carboxyl magnetic beads is preferably 2 μm. If the value is larger than the upper limit value, the suspension property of the magnetic beads is influenced; if the value is less than the lower limit value, the magnetic properties of the magnetic beads are affected.
The invention also provides a preparation method of the homocysteine kit, which comprises the following steps:
1) preparation of a reducing agent: preparing a solution containing 0.01-0.55 wt% of tris (2-carboxyethyl) phosphine, 0.05-0.08 wt% of dithiothreitol, 0.1-0.2mol/L of beta-mercaptoethanol, 0.01-0.2mol/L of guanidinium isothiocyanate, 0.02-0.8 wt% of disodium ethylene diamine tetraacetate, 0.01-0.05 wt% of sodium dodecyl sulfate and the pH value of 7.4-8.3;
2) preparation of a luminescent enzyme reagent: coupling the anti-homocysteine monoclonal antibody with the alkaline phosphatase compound according to the weight ratio of (1-5) to (0.2-60) to obtain the homocysteine monoclonal antibody-alkaline phosphatase compound;
3) preparing a magnetic bead reagent: mixing the following components in percentage by weight (1-10): coupling (0.5-100) streptavidin with the magnetic beads to obtain streptavidin-coated magnetic beads; mixing the following components in percentage by weight (1-10): (0.2-50) coupling biotin with homocysteine to obtain biotinylated homocysteine; mixing streptavidin-coated magnetic beads and biotinylated homocysteine according to the volume ratio of (0.1-5) to (0.5-10) to obtain the magnetic bead reagent.
Wherein the preparation of the luminescent enzyme reagent in the step 2) specifically comprises:
washing alkaline phosphatase with a MES buffer solution having a pH of 6.5 and 90-120mM (for washing purposes, preferably 100mM MES, having a strong ionic strength and giving a better luminescence signal; if the pH is less than the lower limit or greater than the upper limit, the luminescence signal will be affected), centrifuging 5000g for 5min, discarding the supernatant, and washing with a MES buffer solution having a pH of 6.5 and 10-1000mM (for precipitating, preferably 100mM MES buffer solution, having a strong ionic strength and giving a better luminescence signal; if the pH is less than the lower limit or greater than the upper limit, the luminescence value will be affected);
adding a coupling agent into the solution obtained in the step I, adding 100mM MES buffer solution with the pH value of 6.5, washing, centrifuging at 500-15000g for 1-25min (preferably, centrifuging at 5000g for 5min in consideration of efficiency and safety, so that the experimental requirement can be met, if the rotating speed is too low, the time is prolonged, the service life of the centrifuge is influenced by increasing the rotating speed, and the danger is also brought), and discarding the supernatant; the coupling agent is carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a weight ratio of 1 (1-5);
thirdly, adding 10-500mM potassium carbonate buffer solution (preferably 50mM, if the pH value is less than the lower limit, the pH value is influenced, and if the pH value is greater than the upper limit, the antibody is influenced) with the pH value of 8.0 into the anti-homocysteine monoclonal antibody for dilution, and enabling the diluted anti-homocysteine monoclonal antibody to have the concentration of 0.01-30mg/ml (preferably 0.1mg/ml, the loss of the antibody in the concentration is less, the protein purity is high, if the concentration is less than the lower limit, the subsequent concentration step is influenced, and if the concentration is greater than the upper limit, the protein purity is influenced), and passing through a purification column;
fifthly, loading the purified anti-homocysteine monoclonal antibody into a dialysis membrane (the retention molecule is 3kd), adding 1-200g of PEG2000 powder (preferably 5g, the dialysis effect is just less than the lower limit value, the water cannot be dialyzed, the dialysis is overlarge if the water is more than the upper limit value) outside the dialysis membrane, and concentrating to 0.1-10mg/mL (preferably 1mg/mL, the antibody effect of the concentration is better if the antibody effect is less than the lower limit value or more than the upper limit value, and the HOOK effect is generated);
sixthly, adding the anti-homocysteine monoclonal antibody solution obtained in the fifth step into the fifth step, uniformly mixing, carrying out incubation for 2-48h at 25-48 ℃, carrying out centrifugation for 500-15000g for 1-25min (preferably incubation for 12h at 37 ℃, uniformly mixing, carrying out centrifugation for 5min at 5000g, bringing the temperature closer to the body temperature of a human body and the protein activity is highest, carrying out 12h and overnight time without influencing working hours, and testing the luminous value to be higher), abandoning the supernatant, adding a diluent (MES buffer solution with the concentration of 10-1000mM, the pH value of 6.5, preferably 100mM, the ionic strength is just right at this moment, the luminous signal is better, and if the ionic strength is smaller than the lower limit value or larger than the upper limit value, the luminous value is influenced);
seventhly, adding sealing liquid (1-2 wt% of bovine serum albumin solution, preferably 0.5 wt% of bovine serum albumin solution, wherein the luminous signal is better, and if the luminous signal is smaller than a lower limit value or larger than an upper limit value, the luminous value is influenced), and uniformly mixing;
eighthly, discarding the supernatant, adding a preserving fluid for preservation, wherein the preserving fluid comprises the following components: 10 to 100mM Tris-HCl, 0.1 to 2 wt% BSA, 0.1 to 0.5 wt% PC300, 0.02 to 0.5 wt% Tween 20, 0.1 to 2 wt% (preferably 20mM Tris-HCl, 0.12 wt% BSA, 0.15 wt% PC300, 0.025 wt% Tween 20, 0.12 wt% NaCl solution, pH 8.0, preferably better after stability), NaCl solution, pH 7.0 to 9.0 (preferably 8.0, better after stability).
Wherein the preparation of the streptavidin-coated magnetic beads in the step 3) specifically comprises:
firstly, taking 1-100mL of 1-200mg/mL magnetic bead solution (containing 1-200mg of magnetic beads), centrifuging for 1-25min at 500-15000g, removing the supernatant, adding 1-210mL of 10-100mM MES buffer solution (preferably 10mL of 100mM MES buffer solution (containing 100mg of magnetic beads) with the pH value of 4-7, centrifuging for 5min at 5000g, removing the supernatant, adding 10m of 100mM MES buffer solution with the pH value of 6.5 and the luminescence value is preferably higher), placing the mixture on a blood mixing instrument, and mixing for 5min, wherein the cleaning process is the magnetic bead activation process.
Adding a coupling agent into the step I, uniformly mixing, incubating at 25-48 ℃ for 2-48h, centrifuging at 500-15000g for 1-25min (preferably incubating at 37 ℃ for 12h, uniformly mixing, centrifuging at 5000g for 5min, wherein the temperature at 37 ℃ is closer to the body temperature of a human body, the protein activity is highest, the working hours are not influenced by 12h and the overnight time, and the test luminescence value is higher); the coupling agent is carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a weight ratio of (0.1-5) to (0.5-10), preferably 0.5:2, and the luminescent signal is better; the weight ratio of the carbodiimide (EDC) to the magnetic bead microspheres is (0.1-5) - (0.5-10), preferably 0.5:2, and the luminescent signal is better at the moment; too much coupling agent will cause waste of raw material, and too low amount of coupling agent will not achieve coupling effect.
Thirdly, re-dissolving streptavidin by using potassium carbonate buffer solution with the pH value of 8.0 and 10-500mM (preferably 50mM, the pH value is influenced by the lower limit value or the antibody is influenced by the upper limit value), wherein the concentration after re-dissolving is 0.1-10mg/ml (preferably 1mg/ml, the antibody effect of the concentration is better, if the concentration is smaller than the lower limit value or larger than the upper limit value, the luminous value to be tested is very low), and passing through a purification column by an upper purifier;
fourthly, filling the purified streptavidin obtained in the third step into a dialysis membrane (the interception aperture is 30kd), adding 1-200g of PEG2000 powder (preferably 5g, the dialysis effect is just right, if the dialysis effect is smaller than the lower limit value, the water cannot be dialyzed, if the dialysis effect is larger than the upper limit value, the dialysis is too large), the PEG2000 powder can separate out the water in the dialysis membrane, and the concentration treatment is carried out to 0.1-20mg/ml (preferably 1mg/ml, the antibody effect of the concentration is better, if the concentration is smaller than the lower limit value or larger than the upper limit value, the luminous value of the test is very low);
fifthly, adding the concentrated streptavidin solution obtained in the step (four) into the step (two), incubating for 2-48h at 25-48 ℃, centrifuging for 1-25min at 500-15000g (preferably incubating for 12h at 37 ℃, uniformly mixing, centrifuging for 5min at 5000g, wherein the 37 ℃ is closer to the body temperature of a human body, the protein activity is highest, the 12h is the time after night without influencing working hours, and the test luminous value is higher), discarding the supernatant, adding 10-1000mM (preferably 100mM MES buffer solution, the ionic strength is just right at this time, the luminous signal is better, if the ionic strength is less than the lower limit value or greater than the upper limit value, the luminous value is influenced), and adding 1-20ml (preferably 2.5 ml) of MES buffer solution with the pH value of 6.5, wherein the ionic strength is just right at this time, the luminous signal is better, if the cleaning is less than the lower limit value, the cleaning is not thorough, and the waste is caused if the cleaning is greater than the upper limit value;
sixthly, adding a sealing liquid (1-2 wt% of bovine serum albumin solution, preferably 0.5 wt% of bovine serum albumin, wherein the luminescent signal is better, if the luminescent signal is less than a lower limit value or greater than an upper limit value, the luminescent value is influenced), uniformly mixing and coupling, and after 8-24h, centrifuging for 1-25min at 500-15000g (preferably 5min at 5000 g), thus improving the efficiency and the safety);
and storage: removing the supernatant of the solution obtained in the step (c) by using a pipette, and adding a preservation solution, wherein the preservation solution comprises the following components: 10 to 100mM Tris-HCl, 0.1 to 2 wt% BSA (bovine serum albumin), 0.1 to 0.5 wt% PC300, 0.02 to 0.5 wt% Tween 20, 0.1 to 2 wt% NaCl solution, and a pH value of 7.0 to 9.0 (preferably 20mM Tris-HCl, 0.12 wt% BSA, 0.15 wt% PC300, 0.025 wt% Tween 20, 0.12 wt% NaCl solution, and a pH value of 8.0, preferably, the post-stability is better).
In the step 3), the weight ratio of the streptavidin to the magnetic bead microspheres is (1-10): 100, preferably 1:10, and if the weight ratio is more than (1-10): 100, namely the dosage of the streptavidin is continuously increased on the basis, the carboxyl dosage on the magnetic bead microspheres is saturated, the streptavidin raw material is wasted, and the economic cost is (1-10): a weight ratio of 100 has met the experimental requirements.
Wherein the preparation of the biotinylated homocysteine in 3) specifically comprises the following steps:
washing homocysteine by 10ml to 1000mM MES buffer solution (preferably 100mM MES buffer solution, the ionic strength is just right at the moment, the luminous signal is better, if the ionic strength is smaller than the lower limit value or larger than the upper limit value, the luminous value is influenced)), centrifuging for 1 min to 25min by 500 plus 15000g (preferably 5000g centrifuging for 5min in consideration of efficiency and safety, so that the experimental requirement can be met, if the rotating speed is too low, the time is prolonged, the service life of the centrifuge is influenced by increasing the rotating speed, and the danger is brought), discarding the supernatant, precipitating, re-dissolving by 1 ml to 10ml of 10 mM MES buffer solution (preferably 2.5ml of 100mM MES buffer solution, the ionic strength is just right at the moment, and the luminous signal is better; if the washing is not thorough below the lower limit value and the washing is not thorough and the washing is wasted above the upper limit value), repeating the steps for many times, so that the concentration after redissolution is 0.02-20mg/mL (preferably 1mg/mL, the concentration is the optimal concentration for coupling, the concentration is too dilute and is not beneficial to coupling when the concentration is below the lower limit value, and the coupling is low when the concentration is above the upper limit value); MES buffer was added for dilution and for coupling assistance;
adding 1-100mM potassium carbonate solution with pH value of 7.0-9.0 into biotin, centrifuging for 2-25min at 500-15000g (preferably adding 100mM potassium carbonate solution with pH value of 8.5 at 10ml, centrifuging for 5min at 5000g for removing impurities); removing supernatant, adding 100mM potassium carbonate solution (pH 8.5) 10ml (since potassium carbonate solution is slightly alkaline, it is used to dilute biotin, making the system weakly alkaline, and facilitating coupling);
thirdly, coupling the activated biotin with the concentrated homocysteine, incubating for 2-48h at 25-48 ℃, mixing uniformly, centrifuging for 1-25min at 500-15000g (preferably incubating for 12h at 37 ℃, mixing uniformly, centrifuging for 5min at 5000g, wherein the temperature at 37 ℃ is closer to the body temperature of a human body, the protein activity is highest, 12h is the time of overnight without influencing the working hours, and the test luminous value is higher), adding diluent (potassium carbonate buffer system with the pH value of 8.5 and 100 mM) into the supernatant, and suspending;
fourthly, terminating: adding stop solution (1-2 wt% bovine serum albumin solution, preferably 1 wt% bovine serum albumin solution, wherein the luminescence signal is better, and if the light signal is less than the lower limit value or greater than the upper limit value, the luminescence value is influenced) to stop coupling;
cleaning: washing with 1-10ml of pH 8.5, 10-500mM potassium carbonate buffer (preferably 2.5ml of pH 8.5, 50mM potassium carbonate buffer, wherein pH is affected when the buffer is lower than the lower limit, and antibody is affected when the buffer is higher than the upper limit);
sixthly, storage: adding a preservation solution (10-100 mM Tris-HCl, 0.1-2 wt% BSA, 0.1-0.5 wt% PC300, 0.02-0.5 wt% Tween 20, 0.1-2 wt% NaCl solution, pH value of 7.0-9.0, preferably 20mM Tris-HCl, 0.12 wt% BSA, 0.15 wt% PC300, 0.025 wt% Tween 20, 0.12 wt% NaCl solution, pH value of 8.0, preferably better after-stability) for storage.
The invention also provides a using method of the homocysteine kit, which comprises the following steps:
1) treating homocysteine in a sample by using a reducing reagent to obtain free homocysteine; the volume ratio of the reducing agent to the sample is (0.1-5): (0.5-10); preferably 1:2, preferably such that the test signal value is significantly increased; if the dosage is less than the lower limit, the dosage of the reducing agent is insufficient, and the signal value is obviously reduced; if the dosage of the reducing agent is more than the upper limit, the dosage of the reducing agent exceeds the standard, the stability of the reagent is reduced, and the repeatability of the test is influenced;
2) adding recombinant S-adenosine-homocysteine hydrolase (rSAHHase) into the free homocysteine obtained in the step 1) to obtain S-adenosine-homocysteine (SAH); the volume ratio of the S-adenosine-homocysteine hydrolase (rSAHHase) to the sample is (0.01-0.5) to (0.1-10); preferably 0.02: 0.5; the test repeatability coefficient of variation CV is good after optimization; if the value is less than the lower limit, the stability of the reagent is impaired, and if the value is more than the upper limit, the coefficient of variation CV is poor.
3) Diluting the streptavidin-coated magnetic beads by using a diluent in proportion; the weight ratio of the diluent to the streptavidin-coated magnetic beads is (10-1000) to (0.05-1); preferably 1000: 0.5, preferably, the concentration of the reagent R1 composed of streptavidin-magnetic beads is the optimal concentration for sufficiently binding with biotin in the reagent R2, and if the concentration is less than the lower limit value, the concentration of streptavidin-magnetic beads is too low, and the light-emitting signal value is reduced as a whole; if the value is larger than the upper limit value, the streptavidin-magnetic bead reagent is wasted;
4) diluting biotinylated homocysteine in proportion by using a diluent; the weight ratio of the diluent to the biotinylated homocysteine is (20-3000) to (0.02-10); preferably 3000:0.5, preferably so that the concentration of reagent R2 consisting of biotinylated homocysteine is the optimal concentration for sufficient binding to the concentration of streptavidin-magnetic beads in reagent R1, and if the concentration of biotinylated homocysteine is lower than the lower limit value, the luminescence signal value is reduced as a whole; if the concentration is higher than the upper limit, the biotinylated homocysteine is wasted.
5) Diluting the homocysteine monoclonal antibody-alkaline phosphatase complex by using a diluent in proportion; the weight ratio of the diluent to the homocysteine monoclonal antibody-alkaline phosphatase complex is (15-4000) to (0.06-18); preferably 4000: 0.25, preferably so that the concentration of reagent R3 consisting of homocysteine monoclonal antibody-alkaline phosphatase complex is the optimal concentration for fully binding with biotin in reagent R2, and if the concentration is less than the lower limit value, the homocysteine monoclonal antibody-alkaline phosphatase complex concentration is too low, and the luminescence signal value is reduced as a whole; if the content is more than the upper limit, the homocysteine monoclonal antibody-alkaline phosphatase complex is wasted.
6) Forming the reagents into a reagent boat, and placing the reagent boat into a full-automatic chemiluminescence detection analyzer for testing; one reagent boat is a box of reagent, and the specifications of the reagent box are 100 persons/box and 50 persons/box.
Further, in the preparation method of the homocysteine kit, the diluent comprises the following components: 10-100mM Tris-HCl, 0.1-2 wt% BSA, 0.1-0.5 wt% PC300, 0.02-0.5 wt% Tween 20, 0.1-2 wt% NaCl solution, and the pH value is 7.0-9.0; preferably 20mM Tris-HCl, 0.12 wt% BSA, 0.15 wt% PC300, 0.025 wt% Tween 20, 0.12 wt% NaCl solution, pH 8.0, preferably better post stability.
Because Homocysteine (HCY) is easy to form disulfide bonds, and plasma contains free or unbound HCY (1-2 wt%), homocysteine-cysteine or homocysteine dimer (10-20 wt%) or protein-bound HCY (>80 wt%), homocysteine in serum is subjected to two-step pretreatment by a reducing agent and recombinant S-adenosyl-homocysteine hydrolase, so that the bound or dimerized homocysteine becomes free reduced homocysteine and is converted into S-adenosyl-homocysteine by the S-adenosyl-homocysteine hydrolase, and the preparation of magnetic particle chemiluminescence Homocysteine (HCY) is possible; the two-step pretreatment mainly reduces the HCY of the dimer or the binding protein into free HCY with reducibility, and simultaneously ensures that the HCY in the free state is stable, does not polymerize by self and is not degraded.
The following is a further description with reference to specific examples.
EXAMPLE 1 preparation of homocysteine kit
1.1 preparation of reducing reagent
Taking a container with the capacity of 1L, and adding about 800g of hot purified water (50-60 ℃); 0.1g of tris (2-carboxyethyl) phosphine (TCEP) was added; stirring until the mixture is completely dissolved; adding 0.5g Dithiothreitol (DTT), and stirring to completely dissolve; adding 10ml of 10mol/L beta-mercaptoethanol (DTT), and stirring until the mixture is completely dissolved; adding 20ml of 10mol/L guanidine isothiocyanate, and stirring until the guanidine isothiocyanate is completely dissolved; adding 8g of disodium ethylene diamine tetraacetate (EDTA-Na), and stirring until the disodium ethylene diamine tetraacetate is completely dissolved; adding 0.5g of Sodium Dodecyl Sulfate (SDS), and stirring until the SDS is completely dissolved; adjusting the pH value of the solution to 8.3 +/-0.01 by using 4mol/L sodium hydroxide solution; diluting to 1kg with purified water to obtain a solution with pH of 8.3 containing 0.01 wt% tris (2-carboxyethyl) phosphine (TCEP), 0.05 wt% Dithiothreitol (DTT), 0.1mol/L beta-mercaptoethanol, 0.2mol/L guanidinium isothiocyanate, 0.8 wt% disodium ethylenediaminetetraacetate (EDTA-Na), and 0.05 wt% Sodium Dodecyl Sulfate (SDS); then filtering it with 0.22 μm filter to remove impurities and bacteria; the storage temperature was +4 ℃.
1.2 preparation of enzymatic reagents
1) Treating homocysteine in a sample by using a reducing agent, wherein the volume ratio of the reducing agent to the sample is 1:1, so as to obtain free homocysteine;
2) adding recombinant S-adenosine-homocysteine hydrolase (rSAHHase) into the free homocysteine obtained in the step 1) to convert the free homocysteine into S-adenosine-homocysteine (SAH); the volume ratio of the recombinant S-adenosine-homocysteine hydrolase (rSAHHase) to the sample is 1: 10.
the dosage of the recombined S-adenosine-homocysteine hydrolase (rSAHHase) is in positive correlation with the reducing agent in the step 1) within 0-0.1 mu mol/L, and the increasing trend still exists when the dosage is more than 0.1 mu mol/L, so that the raw material of 0.1 mu mol/L can meet the requirement of experiments without increasing the concentration dosage of the recombined S-adenosine-homocysteine hydrolase (rSAHHase) from the viewpoint of saving raw materials.
1.3 preparation of the luminogenic enzyme reagent
Washing alkaline phosphatase with 100mM MES buffer solution (for washing), pH 6.5, centrifuging for 5min at 5000g, discarding the supernatant, and washing with 100mM MES buffer solution (for precipitation);
adding a coupling agent into the solution obtained in the step one, adding 100mM MES buffer solution with the pH value of 6.5, washing, centrifuging for 5min at 5000g, and discarding the supernatant; the coupling agent is carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a weight ratio of 1 (1-5);
③ adding 50mM potassium carbonate buffer solution with the pH value of 8.0 into the anti-homocysteine monoclonal antibody for dilution, leading the diluted solution to have the concentration of 1mg/ml, and passing the diluted solution through a purification column;
fifthly, filling the purified anti-homocysteine monoclonal antibody into a dialysis membrane (the retention molecule is 3kd), adding 5g of PEG20000 powder outside the dialysis membrane, and concentrating to 1 mg/mL;
sixthly, adding the anti-homocysteine monoclonal antibody solution obtained in the fifth step, incubating for 12h at 37 ℃, mixing uniformly, centrifuging for 5min at 5000g, discarding supernatant, and adding diluent (MES buffer solution with the concentration of 100mM and the pH value of 6.5);
seventhly, adding sealing liquid (1.5 wt% bovine serum albumin solution) and uniformly mixing;
eighthly, discarding the supernatant, adding a preserving fluid for preservation, wherein the preserving fluid comprises the following components: 20mM Tris-HCl, 0.12 wt% BSA, 0.15 wt% PC300, 0.025 wt% Tween 20, 0.12 wt% NaCl solution, pH 8.0.
1.4 preparation of magnetic bead reagent
1.4.1 preparation of streptavidin-coated magnetic beads:
taking 10mL of 100mg/mL carboxyl magnetic beads (containing 100mg of magnetic beads and having the granularity of 1.5 mu m), centrifuging 5000g for 5min, removing original supernatant, adding 10mL of 100mM MES buffer solution with the pH value of 6.5, and uniformly mixing for 5min on a blood uniformly-mixing instrument;
adding a coupling agent into the solution obtained in the step one, uniformly mixing, incubating at 37 ℃, and centrifuging for 5min at 5000 g; the coupling agent is carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a weight ratio of 1: 2; the weight ratio of the carbodiimide (EDC) to the magnetic bead microspheres is 1: 1.
③ redissolving the streptavidin by using 50mM potassium carbonate buffer solution with the pH value of 8.0, the concentration after redissolving is 1mg/ml, and passing through a purification column by a purifier;
fourthly, filling the purified streptavidin in the third step into a dialysis membrane (the interception aperture is 30kd), adding 5g of PEG20000 powder outside the dialysis membrane, separating out water in the dialysis membrane by the PEG20000 powder, and concentrating to 10 mg/ml;
fifthly, adding the concentrated streptavidin solution obtained in the step (iv) into the step (ii), keeping the temperature constant at 37 ℃ for 12 hours, centrifuging 5000g for 5min, discarding the supernatant, and adding 2.5ml of MES buffer solution with the pH value of 6.5 and the diameter of 100 Mm;
sixthly, adding 1.5 wt% bovine serum albumin solution into the solution obtained in the fifth step, and centrifuging for 5min at 5000g after 12 h;
and storage: removing the supernatant of the solution obtained in the step (c) by using a pipette and adding a preservation solution, wherein the preservation solution comprises the following components: 20mM Tris-HCl, 0.12 wt% BSA, 0.15 wt% PC300, 0.025 wt% Tween 20, 0.12 wt% NaCl solution, pH 8.0.
1.4.2 preparation of biotinylated homocysteine:
washing homocysteine with 10mL of 100mM MES buffer solution with the pH value of 6.5, centrifuging for 5min at 5000g, discarding supernatant, re-dissolving precipitate with 10mL of 100mM MES buffer solution with the pH value of 6.5, wherein the concentration of re-dissolved precipitate is 1 mg/mL;
② adding 100mM potassium carbonate solution with pH value of 8.5 into biotin reagent 10ml, centrifuging for 5min (for removing impurity); removing supernatant, adding 100mM potassium carbonate solution with pH of 8.5 (the potassium carbonate solution is slightly alkaline, so that the system is weakly alkaline and coupling is facilitated);
③ coupling the activated biotin with the concentrated homocysteine, incubating and uniformly mixing at 37 ℃ for 12h, centrifuging (5000g, 5min), abandoning the supernatant, adding a diluent (a potassium carbonate buffer system with the pH value of 8.5 and the concentration of 100 mM), and suspending.
Adding stop solution (1 wt% bovine serum albumin solution) to stop coupling;
adding 5ml of 100mM potassium carbonate buffer solution with the pH value of 8.5 for cleaning;
sixthly, adding a preservation solution (20mM Tris-HCl, 0.12 wt% BSA, 0.15 wt% PC300, 0.025 wt% Tween 20, 0.12 wt% NaCl solution, pH value of 8.0) for storage.
1.5 diluting streptavidin-coated magnetic beads by using a diluent in proportion; the weight ratio of the diluent to the streptavidin-coated magnetic beads is 500: 0.2;
1.6 diluting biotinylated homocysteine in proportion by using a diluent; the weight ratio of the diluent to the biotinylated homocysteine is 2000: 1;
1.7 diluting the homocysteine monoclonal antibody-alkaline phosphatase compound by using a diluent in proportion; the weight ratio of the diluent to the homocysteine monoclonal antibody-alkaline phosphatase complex is 3000: 0.5;
1.8 make up the above-mentioned various reagents into a reagent ship.
1.9 preparation of calibrator and quality control:
preparation of a calibrator: 500ml of preservation solution (20mM Tris-HCl, 0.12 wt% BSA, 0.15 wt% PC300, 0.025 wt% Tween 20, 0.12 wt% NaCl solution, pH 8.0) is measured in a 1000ml measuring cylinder and added into a glass bottle, then 75 mu mol of homocysteine is sucked by a sample gun and added into the glass bottle, then the glass bottle is placed on a hemomixer and mixed for 30min at room temperature to obtain 150 mu mol/L homocysteine, which is prepared into 5 types of homocysteine (0 mu mol/L, 7.5 mu mol/L, 15 mu mol/L, 30 mu mol/L, 60 mu mol/L) with diluent (20mM Tris-HCl, 0.12 wt% BSA, 0.15 wt% PC300, 0.025 wt% Tween 20, 0.12 wt% NaCl solution, pH 8.0) with different concentrations.
Preparing a quality control product: 100ml of a preservation solution (20mM Tris-HCl, 0.12 wt% BSA, 0.15 wt% PC300, 0.025 wt% Tween 20, 0.12 wt% NaCl solution, pH 8.0) is measured by using a 100ml measuring cylinder and added into a glass bottle, 6 mu mol of homocysteine is absorbed by a sample adding gun and added into the glass bottle, then the glass bottle is placed on a hemomixer to be mixed for 30min at room temperature, 60 mu mol/L homocysteine is obtained, and the homocysteine is prepared into 7.5 mu mol/L homocysteine (60 mu mol/L) by using a diluent (20mM Tris-HCl, 0.12 wt% BSA, 0.15 wt% PC300, 0.025 wt% Tween 20, 0.12 wt% NaCl solution, pH 8.0) and subpackaged.
1.11 preparation of washing solution and luminescent substrate reagent:
taking a container with the capacity of 1L, firstly adding about 800g of hot purified water (50-60 ℃); adding 12.14g Tris buffer solution (Tris), 0.5ml concentrated hydrochloric acid of 12mol/L and 2g Tween 20 in sequence, adding 200g purified water again, placing the container on a blood mixing instrument, mixing at room temperature for 30min to obtain a solution containing 100mM Tris and 0.2 wt% Tween 20, and subpackaging.
Luminogenic substrate reagents were purchased from apies.
1.12 establishment of HCY Standard Curve
Mixing 30 mu l of reducing reagent with 60 mu l of calibrator, incubating at 37 ℃ for 3min, adding 30 mu l of enzyme reagent, incubating at 37 ℃ for 10min, adding 50 mu l of magnetic bead reagent, incubating at 37 ℃ for 5min again, washing with cleaning solution for 3 times, adding 200 mu l of luminescent enzyme reagent, testing with a full-automatic chemiluminescence immunoassay analyzer, recording luminescence value (RLU), averaging to obtain luminescence average value, wherein the specific data are shown in Table 1. A standard curve can be established according to theoretical concentration and a luminescence mean value. FIG. 1 is a standard curve established in example 1.
Table 1 results of testing HCY standard curve for luminescence values in example 1
Example 2 detection of homocysteine kit
2.1 minimum detection Limit
The test was carried out using a diluent (20mM Tris-HCl, 0.12 wt% BSA, 0.15 wt% PC300, 0.025 wt% Tween 20, 0.12 wt% NaCl solution, pH 8.0) as a sample, and the measurement was repeated 20 times to obtain RLU values (relative luminescence values) for 20 measurements, and the average value (Ave) and Standard Deviation (SD) thereof were calculated to obtain a concentration value corresponding to Ave +2 XSD as the lowest detection limit, as shown in Table 2 below. From Table 2, it can be obtained that the homocysteine kit of the present invention can detect the minimum value sample of 0.23 (mu mol/L), and compared with Homocysteine (HCY) determination kit (enzyme cycling method), the homocysteine kit of the present invention only detects 12 (mu mol/L), and the sensitivity is improved by about 50 times, which indicates that the homocysteine kit of the present invention is very sensitive.
TABLE 2 dilution of the calibrator luminescence values
Number of tests
Luminous value
Concentration value
1
51756052
0.11
2
51715947
0.11
3
50581632
0.19
4
52507593
0.05
5
51819772
0.10
6
52304052
0.07
7
51291974
0.14
8
51983931
0.09
9
51401704
0.13
10
52045064
0.09
11
51147751
0.15
12
51279926
0.14
13
50441391
0.20
14
51075896
0.16
15
52285923
0.07
16
50593237
0.19
17
51553052
0.12
18
50601547
0.19
19
50497743
0.20
20
52017783
0.09
Ave
51445099
0.13
2SD
1317023
0.10
Detection limit of 0.23
2.2 repeatability analysis
And (3) repeatedly testing 10 times by using a low-value sample (the concentration of homocysteine is 5 mu mol/L) and a medium-high value sample (the concentration of homocysteine is 20 mu mol/L), calculating the average value (M) and the Standard Deviation (SD) of 10 detection concentrations, and calculating the Coefficient of Variation (CV) according to the formula (2), wherein the Coefficient of Variation (CV) is not more than 10.0%.
CV＝SD/M×100％……………………(2)
In the formula: CV-coefficient of variation;
SD-standard deviation of detection results;
m is the average value of the detection results.
TABLE 3 repeatability test for Low value test
Table 4 high value test repeatability
Number of tests
Luminous value
Concentration value
1
5528701
19.59
2
5541924
19.55
3
5388369
20.11
4
5450883
19.87
5
5418848
19.99
6
5391873
20.09
7
5582201
19.40
8
5638680
19.21
9
5575463
19.43
10
5599129
19.35
Ave
5511607
19.66
SD
91831
0.33
CV
1.7％
1.7％
As can be seen from the data in tables 3 and 4, the test was repeated 10 times using the low value sample (5 μmol/L) and the medium and high value sample (20 μmol/L), the average value (M) and the Standard Deviation (SD) of the test concentrations were calculated 10 times, and the Coefficient of Variation (CV) was calculated according to the formula (2), and the Coefficients of Variation (CV) were 1.6% and 1.7%, respectively, and were both less than 10.0%, indicating that the kit of example 1 of the present invention meets the kit standard YY/T1258-2015.
2.3 Linear analysis
The samples with high values close to the upper limit of the linear range were diluted to at least 5 concentrations of 150. mu. mol/L, 60. mu. mol/L, 30. mu. mol/L, 15. mu. mol/L, 7.5. mu. mol/L, 0.5. mu. mol/L, respectively, and the samples at each concentration were repeatedly tested 3 times, and the mean value was calculated to find the deviation CV, and the results are shown in Table 5. The average of the three test values and the prepared target concentration (the diluted sample concentration) are logarithmized, then straight line fitting is carried out by using a least square method, and a linear correlation coefficient R is calculated. From the data in Table 5, it can be seen that the reproducibility of each gradient is less than 10%, the mean of the triplicate test values deviates less than 10% from the target concentration of the formulation, and the correlation coefficient R is2≧ 0.9900 (see FIG. 2). This indicates that the linear range of the HCY kit of the invention is wide enough to fully meet clinical requirements.
TABLE 5
2.4 specific assay
The results of direct tests using the HCY kit of example 1 of the present invention with concentrations of L-cysteine of not less than 100mmol/L and L-glutathione of 100mmol/L are shown in Table 6, and it can be seen from Table 6 that the test results of tests 1-5 are lower than the lowest detection limit level of the HCY kit. This demonstrates the specificity of the HCY kit of example 1 of the invention.
TABLE 6
2.5 correlation analysis
We collected 50Clinical serum samples were tested using the kit of example 1 of the present invention, and statistical analyses such as correlation regression were performed on the test results and the test results of the conventional Homocysteine (HCY) assay kit (enzyme cycling method, test using roche biochemical analyzer), as shown in table 7 below, and as shown in fig. 3. As can be seen from fig. 3, the result of the correlation line fitting shows that the fitting equation is 0.9864x +0.5378, and the correlation coefficient is R20.9772, it shows that the HCY kit of example 1 of the present invention has high correlation and accuracy with the conventional Homocysteine (HCY) assay kit (enzyme cycling method), and meets the kit standard YY/T1258-2015.
TABLE 7
Test sequence number
Hospital value
Test value
Test sequence number
Hospital value
Test value
μmol/L
μmol/L
μmol/L
μmol/L
μmol/L
1
6.43
6.93
26
19.29
15.49
2
5.23
6.22
27
13.14
15.74
3
6.22
7.99
28
19.37
18.85
4
8.99
6.54
29
13.87
17.20
5
9.45
10.12
30
4.99
4.56
6
9.54
10.03
31
30.28
33.07
7
6.43
6.09
32
39.09
41.27
8
7.99
8.06
33
59.94
56.83
9
6.78
7.01
34
28.28
21.50
10
9.98
9.32
35
23.05
28.87
11
6.42
5.34
36
26.36
21.78
12
7.74
9.01
37
19.86
20.13
13
7.04
6.06
38
19.23
20.23
14
15.37
15.60
39
20.43
19.67
15
10.92
11.23
40
29.78
30.24
16
15.92
16.78
41
35.23
36.14
17
45.74
46.39
42
48.75
49.34
18
32.56
33.12
43
40.23
41.25
19
40.32
40.17
44
33.23
34.14
20
10.91
14.61
45
32.39
32.28
21
12.9
13.20
46
26.85
26.87
22
11.8
12.07
47
3.6
2.55
23
6.06
7.04
48
20.39
22.29
24
12.28
13.61
49
17.69
18.17
25
24.06
25.17
50
14.15
14.01
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are all within the protection scope of the technical solution of the present invention.