CN115963252A - Method for coupling carboxyl microspheres with amino - Google Patents
Method for coupling carboxyl microspheres with amino Download PDFInfo
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- CN115963252A CN115963252A CN202310026467.2A CN202310026467A CN115963252A CN 115963252 A CN115963252 A CN 115963252A CN 202310026467 A CN202310026467 A CN 202310026467A CN 115963252 A CN115963252 A CN 115963252A
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- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims description 2
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Abstract
The invention discloses a method for coupling carboxyl microspheres with amino, wherein 4- (4, 6-dimethoxytriazine-2-yl) -4-methylmorpholine tetrafluoroborate or 4- (4, 6-dimethoxytriazine-2-yl) -4-methylmorpholine hexafluorophosphate is used as a cross-linking agent when the carboxyl is coupled with the amino. The invention is applied to the technical field of carboxyl microsphere coupling amino for the first time, and the microspheres coupled with amino are applied to the detection of proteins by a latex turbidimetric method, a chemiluminescence method and a flow fluorescence method.
Description
Technical Field
The invention relates to a method for coupling carboxyl microspheres with amino, belonging to the field of in-vitro diagnostic kits.
Background
In Vitro Diagnosis, i.e., IVD (In Vitro Diagnosis), refers to products and services for determining diseases or body functions by detecting human body samples (blood, body fluids, tissues, etc.) outside the human body to obtain clinical diagnostic information. The in vitro diagnostic industry and the laboratory medicine constitute an organic whole which is not only distinguished but also closely related to each other. The in vitro diagnosis industry is the tool and weapon of the inspection medicine, and the inspection medicine is the user and market of the in vitro diagnosis industry, and the common purpose of the two is to carry out the in vitro diagnosis. About 80% of clinical diagnostic information comes from in vitro diagnosis, and the cost thereof accounts for less than 20% of medical costs. In vitro diagnosis has become an increasingly important component in the prevention, diagnosis and treatment of human diseases.
The main methods for in vitro diagnosis include enzyme-linked immunosorbent assay, latex turbidimetry, chemiluminescence, flow-type fluorescence, molecular diagnosis and the like. The latex turbidimetry, chemiluminescence, and flow fluorescence methods involve the use of microsphere-labeled proteins. The microsphere labeled protein (antigen or antibody or avidin) method mainly includes adsorption method and covalent coupling method, and aims at coating the microsphere with the antibody or antigen corresponding to the matter to be detected and detecting the matter to be detected and the microsphere coated with the corresponding antigen and antibody to raise the sensitivity of the test. The microsphere surface commonly used in the covalent coupling method is coupled with a functional group-carboxyl. At present, there are two general methods for coupling carboxyl microspheres with amino groups: one is a one-step method using carbodiimide (EDC) as a cross-linking agent, and the other is a two-step method using carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as cross-linking agents.
The principle of the one-step method is as follows: EDC firstly reacts with carboxyl to form an intermediate product, the intermediate product reacts with amino, and then the intermediate product is removed, so that the coupling of the carboxyl and the amino is realized. The method has the defects that the EDC reagent is sensitive to water and is required to be prepared at present, an intermediate product is extremely unstable in an aqueous solution and is easy to hydrolyze, so that the coupling efficiency is low, the whole reaction is uncontrollable, and a large number of self-connection products of a plurality of proteins possibly exist in an end product besides a target product, so that the proteins are wasted, and the reaction efficiency is greatly reduced.
The principle of the two-step method is as follows: EDC reacts with carboxyl firstly, then reacts with NHS to form an intermediate product, the intermediate product reacts with amino, and then the intermediate product is removed, so that the coupling of carboxyl and amino is realized. The two-step process intermediate is slightly more stable in aqueous solution than the one-step process intermediate, but as the reaction proceeds, the formation of by-products inhibits the localization of carboxyl groups on the coupled protein, resulting in a decrease in coupling efficiency. Meanwhile, EDC is sensitive to pH value, the optimum pH value of activated carboxyl is 3.5-4.5, but the optimum pH value of coupling an intermediate product formed after the carboxyl is activated and amino is 4.5-7.5, and in the practical application process, the operation is complicated, and the reaction efficiency is influenced. In addition, EDC reacts with phosphate groups, so that a phosphate buffer cannot be used during the reaction in which EDC participates, but the phosphate buffer is a buffer that is commonly used in the art, easily prepared, and inexpensive. Therefore, both the one-step method and the two-step method in the prior art use EDC as a cross-linking agent, which causes reaction conditions to be limited, and the harsh operating conditions and the complicated operating steps further cause the high cost and the low coupling efficiency of the current carboxyl microsphere coupling amino method.
Disclosure of Invention
In view of the above problems in the prior art, the present invention aims to obtain a novel method for coupling carboxyl microspheres with amino groups, which is low in cost and simple in operation.
In order to achieve one of the above objects, the technical scheme of the method for coupling carboxyl microspheres with amino groups adopted by the invention is as follows:
the invention uses 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine tetrafluoroborate or 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hexafluorophosphate as a cross-linking agent.
4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholinium tetrafluoroborate or 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholinium hexafluorophosphate is a reagent for activating carboxylic acid in solution or solid phase peptide synthesis, but the application of the reagent in the field of carboxyl microsphere labeled protein is not seen so far.
Preferably, the coupling method comprises the steps of:
a) Mixing carboxyl microspheres and protein to be coupled according to a mass ratio of 1:0.01-50, and mixing in coupling buffer solution; the mass volume ratio (w/v) of the carboxyl microspheres to the coupling buffer solution is 0.1-1%; adding 4- (4, 6-dimethoxytriazine-2-yl) -4-methylmorpholine tetrafluoroborate or 4- (4, 6-dimethoxytriazine-2-yl) -4-methylmorpholine hexafluorophosphate according to the molar ratio of the added salt to carboxyl contained in the microspheres being 1-10, and oscillating and uniformly mixing;
b) Centrifuging the uniformly mixed mixture obtained in the step a), then removing the supernatant, adding a Tris closed buffer solution containing 0.1-10% of BSA (bovine serum albumin), resuspending, then centrifuging, and removing the supernatant; repeating the steps for 1-2 times;
c) Adding 0.1% -10% of Tris blocking buffer solution containing BSA for resuspension, oscillating, centrifuging, removing supernatant, and adding 0.1% -10% of buffer solution containing BSA for resuspension.
Preferably, the coupling buffer comprises 2-morpholinoethanesulfonic acid, phosphate buffer, borate buffer, 3- (N-morpholino) propanesulfonic acid or N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid, the concentration is 10-200mM, and the pH value is 4.5-9.0.
Further, the pH value of the Tris buffer solution is 6.5-9.0.
More preferably, in the step a), the ratio of the carboxyl microspheres to the protein to be coupled is 1:0.02-10, wherein the mass-to-volume ratio (w/v) of the carboxyl microspheres to the coupling buffer solution is 0.2-0.5%; according to the molar ratio of 1:1-5 adding 4- (4, 6-dimethoxy triazine-2-yl) -4-methyl morpholine tetrafluoroborate or 4- (4, 6-dimethoxy triazine-2-yl) -4-methyl morpholine hexafluorophosphate.
More preferably, in the step a), the ratio of the carboxyl microspheres to the protein to be coupled is 1:0.05; according to the molar ratio of 1:1 adding 4- (4, 6-dimethoxy triazine-2-yl) -4-methyl morpholine tetrafluoroborate or 4- (4, 6-dimethoxy triazine-2-yl) -4-methyl morpholine hexafluorophosphate.
As a preferred embodiment, the shaking in step a) and step c) is carried out for a period of 1 to 24 hours.
As a preferred embodiment, the shaking time in step a) and step c) is preferably 2 to 4 hours.
When the time in the step a) is shorter than 4 hours, the coupling efficiency is poorer when the time is shorter; when the coupling time is more than 4 hours, the coupling time does not greatly affect the coupling efficiency, so 4 hours are generally selected. Sealing is better when the oscillation time is longer than 4 hours in the step c), but the background can be reduced to a certain extent, but the effect is very limited.
Preferably, the carboxyl microspheres are carboxyl latex microspheres or carboxyl magnetic microspheres, and the diameter of the microspheres is 50nm-9000nm.
The invention also discloses an application of the cross-linking agent, and the cross-linking agent is used in a method for carboxyl coupling of microspheres.
The use of the above cross-linking agent, namely 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholinium tetrafluoroborate or 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholinium hexafluorophosphate, in the coupling of carboxyl microspheres with amino groups.
Compared with the prior art, the invention uses 4- (4, 6-dimethoxytriazine-2-yl) -4-methylmorpholine tetrafluoroborate or 4- (4, 6-dimethoxytriazine-2-yl) -4-methylmorpholine hexafluorophosphate as a cross-linking agent, so that the microspheres coupled with carboxyl on the surface and amino realize high-efficiency coupling, the whole process is simple and convenient to operate, the reaction conditions are mild and not harsh, the pH value of the buffer solution can be in the range of 4.5-9.0, the coupling efficiency is high, and the dosage of the reagent is small.
The invention provides a new application of the cross-linking agent, which is applied to the technical field of carboxyl microsphere coupling amino for the first time, the amino-coupled microspheres of the invention are applied to the detection of protein by a latex turbidimetric method, a chemiluminescence method and a flow fluorescence method, and the detection result shows that not only the main performance index meets the requirements, but also the reagent dosage is reduced, the cost is saved, the operation is simplified, and the cross-linking agent has better market application prospect and economic value. The method for coupling carboxyl microspheres with amino is simple and convenient to operate, can realize high-efficiency coupling of the carboxyl microspheres and the amino, and overcomes the defect of complicated operation of a one-step method and a two-step method in the prior art; the reaction condition is mild, phosphate buffer solution can be adopted, the requirement on the pH value of the phosphate buffer solution is wide, the high-efficiency coupling can be realized when the pH value of the phosphate buffer solution is 4.5-9.0, and the defects that the phosphate buffer solution cannot be used and the requirement on the pH value of the buffer solution is too narrow in the prior art are overcome.
Detailed Description
The method for coupling carboxyl microspheres with amino groups provided by the present invention is fully described in detail below with reference to the following examples. The following examples are illustrative only and are not to be construed as limiting the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; the reagents used in the examples are commercially available. The carboxyl latex microspheres used in the examples were obtained from JSR, the carboxyl magnetic microspheres from Merck, germany, and the magnetic separator from Beijing Bieaokang, and the crosslinkers described in the examples were all 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholinium tetrafluoroborate from Michelin. Biotin labeled TSH antibodies were purchased from roche.
EXAMPLE 1 labeling of CRP antibody (C-reactive protein) with carboxyl latex microspheres
Adding 1mL MES buffer (pH6.0), 5mg 145nm carboxyl latex microsphere (JSR company, carboxyl content is 0.104 meq/g) and 0.25mg CRP antibody into the reaction tube, and mixing; adding 144 mu g (0.104 mu mol) of cross-linking agent, and oscillating and reacting for 2 hours at room temperature; centrifuging at 18000rpm/min for 30 minutes, and discarding the supernatant; adding Tris blocking buffer solution (pH value is 6.5) containing 0.1% -10% of BSA for resuspension; centrifuging and discarding the supernatant; repeating the step of centrifuging and discarding the supernatant; adding Tris buffer (pH 6.5) containing 2% BSA for resuspension, and shaking at room temperature for 2 hr; centrifuging and discarding the supernatant; resuspend by addition of Tris buffer containing 2% BSA (pH 6.5).
The CRP calibrator linear range was determined using the reagents prepared above: 160mg/L, 40mg/L, 20mg/L, 5mg/L and 0mg/L of CRP calibrators with 5 different concentrations are taken for calibration. The measurement wavelength was 570nm, and the reagent R1 (10 mmol/L Tris buffer) in the CRP measurement kit was added to the detection tube, followed by 100ul reaction at 37 ℃ for 90 seconds. Respectively adding 2ul of calibrator with different concentrations into the detection tube, mixing uniformly, adding the carboxyl microsphere (R2) marked with the CRP antibody prepared according to the invention, measuring the absorbance values (A1 and A2) of the reaction at 50-80 seconds and 4-5 minutes, and calculating the difference value delta A = A2-A1 of the absorbance. And (3) taking the absorbance difference delta A of each calibration tube as a vertical coordinate and the corresponding concentration as a horizontal coordinate to prepare a concentration-absorbance difference calibration curve. Linear correlation analysis meterObviously, in the range of 0-160mg/L, the correlation coefficient R 2 =0.998, the linear correlation is good.
And (3) repeatability evaluation: the same batch of reagents R1 and R2 are adopted to perform 10 repeated measurement on a sample with a target value of 10mg/L, the CRP concentration is calculated by substituting the reagents into a calibration curve, the coefficient of variation CV (less than 6 percent) is calculated, and the measurement results are shown in Table 1.
TABLE 1 repeatability evaluation results sheet
Inter-batch difference evaluation: three batches of reagent R2 were tested on samples with a target value of 10mg/L, 3 times for each batch of reagent, with a batch-to-batch difference of less than 15%. The results are shown in Table 2. The results show that when the carboxyl microspheres marked with the CRP antibody prepared in the embodiment are applied to the latex turbidimetry for detecting CRP, the detection result is accurate, the repeatability is good, and the effect of applying the carboxyl latex microspheres and the CRP antibody to the latex turbidimetry for detecting CRP after coupling is achieved by adopting a one-step method or a two-step method. Compared with the prior art, the method for coupling the carboxyl latex microspheres and the amino groups has the advantages of simple and convenient operation, controllable process, mild and non-harsh reaction conditions and greatly reduced operation difficulty and complexity.
TABLE 2 evaluation results of inter-batch Difference
Inter-batch difference = (max-min)/total mean value 100%
Example 2 labeling of streptavidin with carboxyl magnetic microspheres
Adding 1mL HEPES buffer solution (pH8.5), 1mg carboxyl magnetic microsphere (Merck, carboxyl content is 0.0156 meq/g) and 50ug streptavidin into a reaction tube, and mixing; adding 44 mu g (0.156 mu mol) of cross-linking agent to react overnight at room temperature with shaking; magnetic separation and supernatant removal; resuspending by adding Tris buffer containing 2% BSA (pH 9.0); magnetic separation, removing supernatant; repeating the steps of centrifuging, discarding the supernatant and resuspending; adding Tris buffer (pH 9.0) containing 2% BSA for resuspension, and shaking at room temperature for 2 hr; magnetic separation and supernatant removal; tris buffer (pH 9.0) containing 2% BSA was added for resuspension.
The linear range of the TSH standard is detected by using the magnetic beads (M1) marked by the method and the commercial magnetic beads (M2) by using a double antibody sandwich method, and the test steps are as follows:
respectively adding 50 mu L of biotin-labeled TSH antibody, 50 mu L of alkaline phosphatase-labeled TSH antibody and 30 mu L of human serum sample or TSH standard substance into the reaction tube, and uniformly mixing; reacting at 37 ℃ for 20 minutes; adding 300 μ L of lotion, and washing for 5 times; adding 50 mu L of the streptavidin marked magnetic particles prepared in the embodiment; reacting for 15 minutes at 37 ℃; adding 300 μ L of lotion, and washing for 5 times; adding 200 mu L of chemiluminescence substrate solution; the chemiluminescence detector detects the RLU value of the luminescence intensity. The concentrations of the TSH standards and the corresponding luminescence values are given in the following table:
TABLE 3TSH Standard Linear Range test results
And (3) making a linear regression graph on the concentration of the TSH standard substance and the RLU value of the luminous intensity to obtain a correlation coefficient between the concentration of the TSH standard substance and a corresponding luminous value, wherein the correlation coefficient R2 of M1 is =0.9983, and the correlation coefficient R2 of M2 is =0.9995, so that the TSH standard substance and the luminous intensity have good linear relation and no difference.
The cost of the commercial magnetic beads M2 is compared with that of the labeled magnetic beads M1 of the present invention in Table 4. As can be seen from Table 4, M2 (Thermo company) has a selling price of about 160 RMB per mg, and the selling price of the same product of other companies is also basically about 160 RMB. The carboxyl magnetic beads used in the invention are purchased from Merck company in Germany, streptavidin is purchased from Roche company, and a cross-linking agent is purchased from Thermo company, the M1 obtained by the method of the invention is about 10.51 yuan per milligram compared with RMB, the cost of each milligram is reduced by about 149.49 yuan compared with M2, and the cost reduction reaches 93.4%.
TABLE 4 cost per mg comparison of M1 and M2 (Yuan)
| Composition of | Monovalent (mg) | M1(mg) | M2(mg) |
| Carboxyl magnetic bead | 5 | 5 | —— |
| Streptavidin | 110 | 5.5 | —— |
| Crosslinking agent | 0.05 | 0.01 | —— |
| Streptavidin labeled magnetic beads | —— | 10.51 | 160 |
Example 3 Effect of comparative hydrochloride and tetrafluoroborate salts on microsphere coupling Effect
The same microsphere is activated and coupled with the same antibody by using different salts, and the conditions are completely the same except for the different salts. And incubation was performed with PE-labeled secondary antibody, and after incubation, fluorescence signal was detected using flow cytometry. Commercially available 3 sets of microspheres were selected for detection. The comparative test results are shown in table 5 below.
TABLE 5 comparison of the effect of hydrochloride and tetrafluoroborate on coupling of microspheres
Since the fluorescence signal is proportional to the coupling efficiency of the microspheres. As can be seen from table 5, the prevalence of the coupling efficiency improvement was verified by three types of microspheres (three types of microspheres in the table are carboxyl microspheres purchased from three manufacturers: microsphere 1 is new longitudinal, microsphere 2 is degree, and microsphere 3 is bangs.), and it can be seen that the tetrafluoroborate coupling efficiency was improved by 60.2% compared to the hydrochloride coupled microspheres.
Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.
Claims (8)
1. A method for coupling carboxyl microspheres with amino is characterized in that 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholinium tetrafluoroborate or 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholinium hexafluorophosphate is used as a crosslinking agent when the carboxyl microspheres are coupled with the amino.
2. The method for coupling carboxyl microspheres with amino groups according to claim 1, wherein the coupling method comprises the following steps:
a) And (2) mixing the carboxyl microspheres with the protein to be coupled according to the mass ratio of 1:0.01-50, and mixing in coupling buffer solution; the mass volume ratio (w/v) of the carboxyl microspheres to the coupling buffer solution is 0.1-1%; adding 4- (4, 6-dimethoxytriazine-2-yl) -4-methylmorpholine tetrafluoroborate or 4- (4, 6-dimethoxytriazine-2-yl) -4-methylmorpholine hexafluorophosphate according to the molar ratio of the 4- (4, 6-dimethoxytriazine-2-yl) -4-methylmorpholine hexafluorophosphate to carboxyl contained in the microspheres, and uniformly mixing by oscillation;
b) Centrifuging the uniformly mixed mixture obtained in the step a), removing the supernatant, adding a Tris blocking buffer solution containing 0.1-10% of BSA (bovine serum albumin) for resuspension, centrifuging again, and removing the supernatant; repeating the steps for 1-2 times;
c) Adding Tris blocking buffer solution with 0.1-10% of BSA for resuspension, oscillating, centrifuging, discarding the supernatant, and adding buffer solution with 0.1-10% of BSA for resuspension.
3. The method for coupling carboxyl microspheres with amino groups, according to claim 2, wherein the coupling buffer comprises 2-morpholinoethanesulfonic acid, phosphate buffer, borate buffer, 3- (N-morpholino) propanesulfonic acid or N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid, at a concentration of 10-200mM and a pH of 4.5-9.0.
4. The method for coupling carboxyl microspheres with amino groups according to claim 2, wherein in the step a), the mass ratio of the carboxyl microspheres to the protein to be coupled is 1:0.02-10, wherein the mass-to-volume ratio (w/v) of the carboxyl microspheres to the coupling buffer solution is 0.2-0.5%; according to the molar ratio of 1:1-5 adding 4- (4, 6-dimethoxy triazine-2-yl) -4-methyl morpholine tetrafluoroborate or 4- (4, 6-dimethoxy triazine-2-yl) -4-methyl morpholine hexafluorophosphate.
5. The method for coupling carboxyl microspheres with amino groups according to claim 2, wherein in the step a), the mass ratio of the carboxyl microspheres to the protein to be coupled is 1:0.05; according to the molar ratio of 1:1 adding 4- (4, 6-dimethoxy triazine-2-yl) -4-methyl morpholine tetrafluoroborate or 4- (4, 6-dimethoxy triazine-2-yl) -4-methyl morpholine hexafluorophosphate.
6. The method for coupling carboxyl microspheres with amino groups according to any one of claims 1 to 5, wherein the carboxyl microspheres are carboxyl latex microspheres or carboxyl magnetic microspheres, and the diameter of the microspheres is 50nm to 9000nm.
7. Use of a cross-linking agent according to any of claims 1 to 5 in a method of carboxyl coupling of microspheres.
8. Use of a cross-linking agent according to claims 1 to 5, wherein the cross-linking agent comprises 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholinium tetrafluoroborate or 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholinium hexafluorophosphate.
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