CN111239403B - Beta 2 microglobulin latex enhanced immunoturbidimetry kit and application - Google Patents
Beta 2 microglobulin latex enhanced immunoturbidimetry kit and application Download PDFInfo
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Abstract
The invention relates to a beta 2 microglobulin latex enhanced immunoturbidimetry detection kit and a use method thereof. The kit provided by the invention comprises a beta 2 microglobulin R1 reagent, a beta 2 microglobulin R2 reagent and a beta 2 microglobulin calibrator, wherein the beta 2 microglobulin R1 reagent comprises an electrolyte, a coagulant, a stabilizer, a surfactant, a preservative and a buffer solution; the beta 2 microglobulin R2 reagent comprises anti-human beta 2 microglobulin polyclonal antibody coated methacrylic acid Glycidyl Methacrylate (GMA) latex, electrolyte, stabilizer, surfactant, preservative and buffer solution; the beta 2 microglobulin calibrator comprises a preservative, an electrolyte, a stabilizer, a beta 2 microglobulin antigen and a buffer solution. The kit has strong specificity, wide linear range, uniformity, stability, rapidness, simpleness and convenience, and can be used for various biochemical analyzers.
Description
Technical Field
The invention relates to a kit for determining the content of beta 2 microglobulin in human body fluid by using a latex enhanced immunoturbidimetry method, belonging to the field of medical immune in-vitro diagnosis.
Background
Beta 2 microglobulin (beta 2-microglobulin, beta 2-MG) is an endogenous low molecular weight serum protein, secreted by lymphocytes and most other nucleated cells, and plays an important role in immune response. The serum beta 2-MG can easily pass through a glomerular filtration membrane, 99.9 percent of the filtered beta 2-MG is reabsorbed and degraded by proximal tubular cells and does not flow back into the blood. The synthesis rate of normal human beta 2-MG and the amount released from cell membrane are very constant, so that the content of beta 2-MG is kept at a stable level. The clinical detection of the concentration of the beta 2-MG in blood or urine provides an early, reliable and sensitive index for clinical renal function determination, kidney transplantation survival, diabetic nephropathy, heavy metal cadmium, mercury poisoning and clinical diagnosis of certain malignant tumors. The clinical methods for measuring beta 2-MG include Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), time-resolved fluoroimmunoassay, immunotransmission turbidimetry, immunoscattering turbidimetry, latex-enhanced immunoturbidimetry, and the like. ELISA method (enzyme-linked immunosorbent assay): 0.8-2.4 mg/L.
There are many methods for measuring β 2-M in serum and urine reported so far, and some methods have low detection sensitivity, and can only be used for measuring the β 2-M concentration in serum samples, but cannot be used for measuring urine and other body fluid samples containing low β 2-M concentration. Experiments show that the latex immune scattering turbidimetry has the same accuracy, specificity and sensitivity as the RIA method, has no prozone phenomenon, is suitable for the detection of serum and urine, and is a convenient and practical automatic analysis method.
The safety range of the antigen excess is mainly related to the quality of the reagent, and the formula of the kit is important, and comprises various aspects of antibody quality (purity and titer), antibody concentration, a buffer system, molecular size and concentration selection of polyethylene glycol (PEG), a latex crosslinking technology and the like. If the analysis and measurement range of the formulated kit is wide enough, all clinically visible sample concentrations can be covered, and the hook effect cannot exist.
Disclosure of Invention
The present invention is provided to solve the above problems.
The kit of the invention adopts a latex enhanced immunoturbidimetry method to quantitatively detect the beta 2 microglobulin, and the principle is as follows: coating a specific anti-human beta 2 microglobulin antibody on the surface of methacrylic acid epoxy propyl ester (GMA) latex by using a chemical crosslinking method, carrying out antigen-antibody immunoreaction when beta 2 microglobulin antigen exists in a detected sample to form an antigen-antibody-GMA latex compound, wherein the formed compound can be connected with adjacent latex particles through oligonucleotide chain hybridization to enhance the binding force, and finally, the formed compound is crosslinked into a net shape to cause the obvious change of the turbidity of a reaction system, when the antibody in the reaction system is excessive, the change of the turbidity of the system after reaction is in direct proportion to the amount of the beta 2 microglobulin, and the quantitative analysis can be carried out according to a beta 2 microglobulin calibration curve.
The invention is realized by the following technical scheme: wherein the reagent 1 is a diluent (containing a citric acid buffer solution with pH of 6.6 and 0.1mol/L and a 0.1% Proclin300 solution), the reagent 2 is a latex reagent (containing 0.3 μm diameter methacrylic acid epoxy propyl ester (GMA) latex (Shanghai Carboxyphenanthrene biological medicine science and technology Co., Ltd.) coated with a beta 2-microglobulin antibody and a 0.1% Proclin300 solution),
(1) the beta 2 microglobulin R1 reagent is a reagent for providing a proper reaction environment for a kit, keeping antigens in a natural conformation, and controlling the time and rate of reaction reaching an end point, and comprises an electrolyte, a coagulant, a stabilizer, a surfactant, a preservative, and a buffer.
(2) The beta 2 microglobulin R2 reagent is a uniform milky white liquid containing anti-human beta 2 microglobulin polyclonal antibody coated methacrylic acid epoxy propyl ester (GMA) latex, and comprises anti-human beta 2 microglobulin polyclonal antibody coated methacrylic acid epoxy propyl ester (GMA) latex, electrolyte, stabilizer, surfactant, preservative and buffer solution.
(3) A beta 2 microglobulin calibrator, used for comparison with samples for result calculation, purchased from cupadine bioengineering (beijing) ltd.
The kit for measuring the content of the beta 2 microglobulin in human body fluid (including serum and amniotic fluid) by using a latex immunoturbidimetry method comprises a R1 reagent, a R2 reagent and a beta 2 microglobulin calibrator, wherein each of the R1 reagent and the R2 reagent contains an electrolyte ammonium chloride, a coagulant PEG-800, a stabilizer propylene glycol alginate, a surfactant sucrose stearate monoester, a preservative Proclin300, a buffer pH6.6 citric acid buffer solution, the R2 reagent also contains methacrylic acid propylene oxide (GMA) latex coated by an anti-human beta 2 microglobulin antibody, and the R1 and the R2 reagents comprise the following components in percentage by mass: 2.5-3.0% of electrolyte, 3-5% of coagulant, 0.5-1% of stabilizer, 0.7-1.0% of surfactant, 0.05-0.1% of preservative and buffer solution with concentration range of 0.1 mol/L.
The diameter range of the methacrylic acid epoxy propyl ester (GMA) latex used by the invention is 300nm, and the concentration is 0.5-5 mg/mL. The methacrylic acid epoxy propyl ester (GMA) latex is methacrylic acid epoxy propyl ester (GMA) latex, and the surface of the methacrylic acid epoxy propyl ester (GMA) latex is modified with epoxy functional groups.
The method for coating the antibody on the surface of the methacrylic acid glycidyl ester (GMA) latex is a chemical crosslinking method, the method is carried out in a crosslinking buffer solution by a chemical crosslinking agent, latex with surface epoxy group modified and a polyclonal antibody of anti-human beta 2 microglobulin are adopted for reaction, and after the reaction is finished, residual site blocking is carried out by 4 oligonucleotides with 30bp length, so as to obtain the antibody latex conjugate. The amino end of the antibody is directly and covalently coupled through epoxy ring opening, and simultaneously, an electrically neutral hydrophilic hydroxyl group can be generated after epoxy group coupling amino ring opening, so that the nano particles of the covalently coupled antibody can still keep good water solubility.
Drawings
FIG. 1 dose-response curves for the three kits.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and embodiments. The following examples are only exemplary and are intended to illustrate the technical solutions of the present invention in further detail, and it should be understood by those skilled in the art that modifications or substitutions to the technical solutions without departing from the spirit and scope of the technical solutions of the present invention should be covered by the claims of the present invention.
Example 1:
1. preparation of beta 2 microglobulin R1 reagent
The reagent comprises the following components in percentage by mass: 2.5 to 3.0 percent of electrolyte, 3 to 5 percent of coagulant, 0.5 to 1 percent of stabilizer, 0.7 to 1.0 percent of surfactant, 0.05 to 0.1 percent of preservative and buffer solution with the concentration range of 0.1mol/L, namely the R1 reagent.
2. Preparation of antibody-coated latex
The method comprises the steps of selecting a rabbit anti-human beta 2 microglobulin antibody and methacrylic acid epoxy propyl ester (GMA) latex for coupling, and sealing the active site of the latex by adopting a nucleic acid fragment with 30 basic groups after coupling.
The purchased surface-epoxidized propylene methacrylate latex particles are added into a corresponding antibody dilution solution (10 mu g/mL, 0.5-1.0% (w) trehalose, PBS solution) for resisting beta 2 microglobulin, the dosage of the surface-epoxidized latex particles is 0.3mg in each milliliter of the antibody dilution solution, and the latex particles are placed at 4 ℃ for 12 hours to obtain the latex particle conjugate of the covalent coupling antibody. The anti-beta 2 microglobulin antibody can be added in combination with a monoclonal antibody and a polyclonal antibody or independently. Weighing 0.5 mg of GMA microspheres and a proper amount of anti-beta 2 microglobulin antibody, adding PBS into a conical flask filled with a certain amount of GMA microspheres in batches to prepare 1 mu g/mL of anti-beta 2 microglobulin monoclonal antibody solution, placing the conical flask in a shaking table at 4 ℃, carrying out oscillation reaction for a plurality of hours at 25 ℃. After centrifugal separation, the solid particles are washed for 4 times by PBS solution, and free antibodies are removed, thus obtaining the anti-beta 2 microglobulin antibody coupling microspheres. In the reaction process, determining the mass fraction of the residual anti-beta 2 microglobulin monoclonal antibody in the reaction liquid by an enzyme-linked immunosorbent assay, and concluding that the microspheres fully complete the coupling reaction with the antibody when the mass fraction of the antibody is unchanged; after the reaction is finished, the composite microspheres combined with the anti-beta 2 microglobulin antibody are subjected to oscillation and sealing for 24 hours at 25 ℃ by using 1mol/L of 4 equimolar oligonucleotide solutions with 30 repeated bases, so that the specific adsorption of the antibody on the surfaces of the microspheres and the antigen in a sample in the next reaction is enhanced.
The preparation of the R2 reagent comprises the following three steps: antibody cross-linking, latex washing, latex suspension, antibody cross-linking: see example 2
Latex cleaning: centrifuging to remove the supernatant to remove the redundant antibodies, cross-linking agents, cross-linking reaction byproducts and the like, and obtaining the bottom sediment which is the coated latex. Washed 3 times with 0.1mol/L citrate buffer pH 6.6.
Suspending latex: adding 20mL of R1 into the precipitate, performing ultrasonic treatment for 4min, and mixing uniformly to obtain a uniform milky latex suspension, namely the R2 reagent.
Wherein R1 contains electrolyte ammonium chloride, coagulant PEG-800, stabilizer propylene glycol alginate, surfactant sucrose stearate monoester, preservative Proclin300, buffer pH6.6 citric acid buffer, and ensures that the methacrylic acid epoxy propyl ester (GMA) latex is in uniform and stable suspension state.
Comparative example 1: antibodies were coupled using commercially available carboxylated polystyrene microspheres and the general NHS/EDC activation method. Such as the beta 2 microglobulin latex enhanced turbidimetry kit purchased from beijing lidman bio-chemical ltd.
Comparative example 2: step 2 in example 1 used glycine solution as blocking reagent.
Experimental example 1 detection of excessive amounts of 3 antigens in kit by latex agglutination method
Diluting the high-value samples of the items with physiological saline in different proportions to form samples with different number of concentration gradients, wherein the beta 2-microglobulin has 27 concentration gradients, and the theoretical sample concentration range is 1.1-226.3 mg/L. The three kits described in the above examples were used to measure the standard and serum samples, respectively, using a two-reagent endpoint method with a dominant wavelength of 540 nm, a minor wavelength of 700 nm, a temperature of 37 ℃, a ratio of serum to reagent 1 to reagent 2 of 3 μ l to 270 μ l to 30 μ l, starting reagents added after 3 min, a reaction time of 8min, and calculating the linear range.
The detection parameters of each item are set on a BS-2000 biochemical module of the SAL8000 biochemical immune all-in-one machine according to the kit specification of each manufacturer, the instrument is calibrated by using respective matched calibrators, the indoor quality control is carried out on quality control products with 2 concentration levels, samples with different concentration gradients of the item are tested under the condition of ensuring the indoor quality control, and each sample is repeatedly detected for 2 times. And (3) drawing a dose-effect curve by taking the mean value of 2 detection results of each sample as a vertical coordinate and the theoretical concentration as a horizontal coordinate, and determining the back band limit (a concentration point at the right side of the dose-effect curve and equal to the absorbance signal at the upper limit of the analysis and measurement range), namely the upper limit of the safety range (the concentration range from the upper limit of the analysis and measurement range to the back band limit) of detection when the antigen is excessive.
When the kit is used for measuring beta 2-microglobulin, the hook effect still does not appear around 226mg/L, and the upper limits of the safety ranges of the comparative example 1 and the comparative example 2 are respectively around 70mg/L and 128 mg/L;
experimental example 2 comparative experiment 50 serum samples were respectively measured by latex agglutination turbidimetry and radioimmunoassay on a fully-automatic biochemical analyzer, and the difference between serum β 2-microglobulin y = 0.527x + 11.02, R = 0.981, t = 0.845, and P > 0.05 was insignificant.
Experimental example 3 precision experiment 15 serum samples were repeatedly measured 5 times per day and 5 d times between batches in a batch, and the results showed good reproducibility, with a CV value of 2.5% in the serum batch and 3.68% between batches.
Experimental example 4 recovery experiment the serum specimen with the concentration of 1.34 mg/L was divided into 5 specimens with the same volume, and standard solutions (9.30, 4.75, 2.38, 1.19, 0.60 mg/L) with different concentrations were added to carry out the recovery experiment, and the recovery rate was 97% to 103%, and the average was 99.3%.
Claims (3)
1. A β 2 microglobulin assay kit for increasing the linear range, the kit comprising: r1 reagent, R2 reagent and beta 2 microglobulin calibrator, characterized in that: the R1 reagent and the R2 reagent both contain electrolyte ammonium chloride, and the mass percent is 2.5-3.0%; coagulant PEG-800 with the mass percent of 3-5%; the stabilizer is 3 to 5 percent of propylene glycol alginate by mass percent; 0.7-1.0% of sucrose stearate monoester serving as a surfactant; a preservative Proclin300 with the mass percent of 0.05-0.1%, a pH6.6 citric acid buffer solution with the concentration of 0.1mol/L, and an R2 reagent also comprises anti-human beta 2 microglobulin antibody coated Glycidyl Methacrylate (GMA) latex, wherein the preparation method of the glycidyl methacrylate latex comprises the following steps: latex with surface epoxy group modified and an anti-human beta 2 microglobulin antibody are adopted for reaction, residual site blocking is carried out through 4 oligonucleotides with 30bp length after the reaction is finished, and the obtained conjugate is subjected to heavy suspension according to a certain proportion by using the R1 reagent.
2. The linear range enhanced β 2 microglobulin assay kit of claim 1, wherein: the anti-human beta 2 microglobulin antibody is a monoclonal antibody or a polyclonal antibody or a mixture of the monoclonal antibody and the polyclonal antibody.
3. The beta 2 microglobulin assay kit for increasing the linear range of any of claims 1 to 2, wherein: the beta 2 microglobulin determination kit for improving the linear range is used for determining the content of beta 2 microglobulin in human serum.
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| WO2004092733A1 (en) * | 2003-04-14 | 2004-10-28 | Caliper Life Sciences, Inc. | Reduction of migration shift assay interference |
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| EP2006685A1 (en) * | 2006-03-31 | 2008-12-24 | Nissui Pharmaceutical Co., Ltd. | Immune agglutination reaction reagent kit and method of assaying antigen |
| CN103123319A (en) * | 2012-12-20 | 2013-05-29 | 武汉生之源生物科技有限公司 | Heart-type fatty acid binding protein content detection kit and preparation method thereof |
| CN105393119A (en) * | 2013-07-30 | 2016-03-09 | 生物辐射实验室股份有限公司 | Multiplex blocker beads for immunoassays |
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