CN111929440A - Method for detecting DNASE1L3 based on magnetic particle chemiluminescence immunoassay - Google Patents
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Abstract
The invention relates to the technical field of immunological detection, in particular to a method for detecting DNASE1L3 based on a magnetic particle chemiluminescence immunoassay. The method comprises the following steps: biotinylated anti-DNASE 1L3 antibody preparation: preparation of enzyme-labeled anti-DNASE 1L3 antibody: binding the biotinylated anti-DNASE 1L3 antibody to the anti-DNASE 1L3 antibody in the test sample; adding streptavidin coated magnetic particles, washing under the action of magnetic force after reaction, and removing unbound substances; adding enzyme-labeled anti-DNASE 1L3 antibody, reacting, washing under the action of magnetic force, and removing unbound substances; adding a buffer solution and a luminescent substrate solution for reaction, detecting the luminescent intensity on a luminometer, and calculating the concentration of DNASE1L3 in the sample. The method can quickly realize quantitative analysis of DNASE1L3, and the preparation method and application of the g-c3n4moo3 composite photocatalytic material with the mortise and tenon structure are high in detection sensitivity and strong in specificity.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for detecting DNASE1L3 based on a magnetic particle chemiluminescence immunoassay.
Background
DNASE1L3, DNASE 1-like protein 3, is an endonuclease in the DNASE superfamily, is mainly a secreted protein produced by dendritic cells and myeloid cells such as macrophages and entering blood, has DNA hydrolytic activity, can cleave single-stranded and double-stranded DNA, and can also cleave chromatin into nucleosome units and cleave nucleosome and liposome-coated DNA. Currently, studies have demonstrated that DNASE1L3 deficiency can lead to anti-DNA reactions and autoimmunity in humans and mice, and that blood DNASE1L3 is involved in circulating plasma DNA homeostasis by enhancing fragmentation and affecting end motif frequency. The reduction or lack of the DNASE1L3 level is related to the pathogenesis and clinical characteristics of Systemic Lupus Erythematosus (SLE) and the activity of the disease, and the DNASE1L3 enzyme can be used for treating autoimmune diseases such as systemic lupus erythematosus and the like. In addition, it is found that DNASE1L3 is also an independent prognostic factor for hepatocellular carcinoma. Therefore, the quantitative determination of DNASE1L3 in peripheral blood is not only beneficial to the clinical auxiliary diagnosis and curative effect evaluation of SLE, but also can evaluate the prognosis of hepatocellular carcinoma, and is an important biomarker.
At present, scholars at home and abroad mainly adopt an immunoblotting method and an ELISA method for research in order to detect DNASE1L3 in peripheral blood.
(1) Implementation scheme of Western Blot (WB)
The method comprises 6 main steps. The first step is as follows: loading, namely loading protein samples containing target proteins and molecular weight standards into SDS-polyacrylamide gel holes respectively; the second step is that: electrophoresis, under a certain current (voltage) condition, target protein and other proteins are separated due to molecular weight and charge difference; the third step: transferring, which transfers the bands separated in the gel onto a nitrocellulose membrane (NC); fourthly: adding primary antibody, adding the primary antibody to react with the target protein on the NC membrane, and washing redundant primary antibody and unreacted protein; fifth, the method comprises the following steps: adding a labeled secondary antibody, adding a second antibody capable of developing a color label, forming an antigen-antibody complex with the target protein and the primary antibody, and washing redundant primary antibody; sixth: and (3) developing or developing, adding a substrate to react with the developing label, and developing or developing the band of the target protein antigen-antibody complex. And finally, comparing the position of the sample with the position of the molecular weight standard band to determine whether the sample contains the target protein, and carrying out semi-quantification on the target protein according to the color depth of the color development band.
(2) Enzyme-Linked Immunosorbent Assay (ELISA) implementation scheme
The method mainly comprises the following steps: in the first step, the specific antibody is coupled to a solid support to form a solid antibody. Washing to remove unbound antibodies and impurities; in the second step, the target protein in the sample is bound to the solid phase antibody to form a solid phase antigen-antibody complex. Washing to remove other unbound materials; and thirdly, adding an enzyme-labeled antibody, and combining the enzyme-labeled antibody with the antigen on the solid-phase immune complex. Washing the unbound enzyme-labeled antibody thoroughly; and fourthly, adding a substrate for color development, detecting by an enzyme-labeling instrument, and comparing with the absorbance of a standard substance to calculate the concentration of the target protein.
At present, the WB method and the ELISA method are mainly used for scientific research, have the defects of complex operation and long time consumption, and the specificity, the sensitivity and the high-flux processing capacity of the WB method and the ELISA method are required to be further improved.
Reference documents:
1.Sisirak V,Sally B,Clancy R M,et al.Digestion of Chromatin in Apoptotic Cell Microparticles Prevents Autoimmunity[J].Cell,2016,166(1):1-14.
2.Zhao Q,Yang C,Wang J,Li Y,Yang P.Serum level of DNase1l3 in patients with dermatomyositis/polymyositis,systemic lupus erythematosus and rheumatoid arthritis,and its association with disease activity.Clin Exp Med.2017；17(4):459–465.
3.Wilber A,O'Connor TP,Lu ML,Karimi A,Schneider MC.Dnase1l3 deficiency in lupus-prone MRL and NZB/W F1 mice.Clin Exp Immunol.2003；134(1):46–52.
4. the activity detection and clinical preliminary observation of Jinyuyan, Lingyang, Song Rui, Chengxiang, Zhangdong DNase1L3, modern immunology 2020; 40(01):22-7.
5.Wang S,Ma H,Li X,Mo X,Zhang H,Yang L,Deng Y,Yan Y,Yang G,Liu X,Sun H.DNASE1L3 as an indicator of favorable survival in hepatocellular carcinoma patients following resection.Aging(Albany NY).2020；12(2):1171-85.
6.Roberts DE,Kakar S,Mehta N,Gill RM.A Point-based Histologic Scoring System for Hepatocellular Carcinoma Can Stratify Risk of Posttransplant Tumor Recurrence.Am J Surg Pathol.2018；42(7):855–865.
Disclosure of Invention
The invention mainly aims to provide a method for detecting DNASE1L3 based on a magnetic particle chemiluminescence immunoassay, which can quickly realize quantitative analysis of DNASE1L3 and has high detection sensitivity and strong specificity.
The invention adopts the following technical scheme:
the invention provides a method for detecting DNASE1L3 based on a magnetic particle chemiluminescence immunoassay, which comprises the following steps:
biotinylated anti-DNASE 1L3 antibody preparation:
preparation of enzyme-labeled anti-DNASE 1L3 antibody:
combining the biotinylated anti-DNASE 1L3 antibody with DNASE1L3 in a sample to be detected to form a biotinylated anti-DNASE 1L3 antibody-antigen complex; adding streptavidin coated magnetic particles, washing under the action of magnetic force after reaction, and removing unbound substances; adding enzyme-labeled anti-DNASE 1L3 antibody, reacting, washing under the action of magnetic force, and removing unconjugated substance to obtain magnetic particle-DNASE 1L3 antigen-antibody complex; adding a buffer solution and a luminescent substrate solution into the magnetic particle-DNASE 1L3 antigen-antibody complex for reaction, detecting the luminous intensity on a luminometer, detecting a standard substance by the same method, preparing a standard curve by using the luminous intensity of each concentration of the standard substance as an ordinate and the concentration as an abscissa, and calculating the concentration of DNASE1L3 in a sample.
Preferably, the biotinylated anti-DNASE 1L3 antibody is prepared by: diluting the mouse anti-human DNASE1L3 antibody with buffer solution, dialyzing, adding N-hydroxysuccinimide biotin solution, magnetically stirring for 1-2h, adding ammonium chloride solution, stirring for 10-20min, and dialyzing; passing the dialyzed sample through a molecular sieve column, and collecting biotinylated anti-DNASE 1L3 antibody; the biotinylated anti-DNASE 1L3 antibody was added to PBS containing sodium azide and bovine serum albumin and stored at 4 ℃ in the dark.
Further preferably, the mouse anti-human DNASE1L3 antibody is diluted with a boric acid buffer solution to a concentration of 0.5-2mg/mL and dialyzed against the boric acid buffer solution;
further preferably, the concentration of the boric acid buffer solution is 0.5-1.0mmol/L, and the pH value of the solution is 8.0-8.6.
Further preferably, the concentration of the N-hydroxysuccinimide biotin solution is 0.5-1.0mg/mL, and the solvent is dimethyl sulfoxide;
further preferably, the volume ratio of the mouse anti-human DNASE1L3 antibody solution to the N-hydroxysuccinimide biotin solution is 1: 0.1-0.5;
further preferably, the concentration of the ammonium chloride solution is 0.5-1.0mol/L, and the addition amount of the ammonium chloride solution is 8-10 per mill of the volume of the mouse anti-human DNASE1L3 antibody solution;
further preferably, the ammonium chloride solution is added and then dialyzed with a PBS buffer solution;
further preferably, the sodium azide concentration is 0.1-0.5g/L and the bovine serum albumin is added in an amount of 0.5-1.0% w/w.
Preferably, the enzyme-labeled anti-DNASE 1L3 antibody is prepared by the method comprising:
dissolving alkaline phosphatase in glutaraldehyde solution, standing overnight, passing through a molecular sieve column, and eluting with normal saline to obtain alkaline phosphatase solution; diluting the DNASE1L3 antibody with PBS buffer solution, and adding the diluted antibody into alkaline phosphatase solution under magnetic stirring; adding lysine solution, mixing, standing at room temperature for 3-5h, and dialyzing to obtain enzyme-labeled antibody solution;
activating magnetic particles by glutaraldehyde, stirring at room temperature, uniformly mixing for 3 hours, standing for 15min, pouring out supernatant, washing by PBS buffer solution, and suspending by PBS to prepare magnetic particle suspension with the concentration of 50-100 mg/mL;
further preferably, the PBS buffer is used at a concentration of 0.1M and the solution pH is 7.4.
Further preferably, the lysine solution is used in a concentration of 0.05-0.2M.
Preferably, the biotinylated anti-DNASE 1L3 antibody is reacted with the test sample at 37 ℃ for 5 min;
preferably, the biotinylated anti-DNASE 1L3 antibody antigen complex is reacted with streptavidin-coated magnetic microparticles at 37 ℃ for 5 min;
preferably, the reaction is carried out at 37 ℃ for 5min after addition of the enzyme-labeled anti-DNASE 1L3 antibody.
Preferably, MES buffer is added to the magnetic particle-DNASE 1L3 antigen-antibody complex;
preferably, the luminogenic substrate solution is: consists of 0.05mol/L trihydroxymethane, 0.5 percent sodium dodecyl sulfate and 0.4mg/mL dihydroacridine derivative, and the pH value of the solution is 9.0.
Preferably, the magnetic particle-DNASE 1L3 antigen-antibody complex is added with a buffer solution and a luminescent substrate solution and then reacted for 8min at 37 ℃.
Compared with the prior art, the invention has the following advantages:
1) the detection method is rapid, takes about 25 minutes for detecting a single sample, and is shorter than ELISA and WB methods; and the operation is easy, the whole reaction is carried out in the same reaction tube, and the automatic operation is easy to realize.
2) The method has high detection precision, the CV percent of the intra-batch variation coefficient (n is 10) is 4.36 percent, and the CV percent of the inter-batch variation coefficient is 8.94 percent.
3) The method disclosed by the invention has the advantages of wide detection linear range, high detection sensitivity, good linearity within the range of 0.12-1276 ng/mL, and the lowest detection limit of 0.05 ng/mL.
4) The method has high detection accuracy and the recovery rate can reach more than 96%.
5) The method has high specificity, and the high concentration Rheumatoid Factor (RF), streptococcus resisting O and complement (C3, C4) do not interfere the detection result of the blood sample DNASE1L 3.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description, serve to explain the invention and not to limit the invention;
FIG. 1 is a schematic diagram of a method for detecting DNASE1L3 based on magnetic particle chemiluminescence immunoassay according to an embodiment of the invention;
FIG. 2 is a DNASE1L3 standard curve diagram according to an embodiment of the invention;
FIG. 3 is a graph of the relationship between the measured concentration and the theoretical concentration.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
Example 1
Biotinylated anti-DNASE 1L3 antibody preparation: mouse anti-human DNASE1L3 antibody (purchased) was diluted to 1mg/mL with 0.5mmol/L boric acid buffer (pH 8.6) and then dialyzed in dialysis bag (MW CO 8000) with 0.5mmol/L boric acid buffer (pH 8.6) for 3 times; dissolving N-hydroxysuccinimide biotin (NHSB, purchased) in dimethyl sulfoxide (DMSO) at a ratio of 1mL:1mg to a final concentration of 1 mg/mL; adding 120 mu L of the NHSB solution into 1mL of antibody solution, magnetically stirring at room temperature for 2 hours, adding 9.6 mu L of 1mol/L ammonium chloride, and stirring for 10 minutes; dialyzing with PBS buffer at 4 deg.C for 3 times to remove free biotin; loading the dialyzed sample on a molecular sieve column (G25), slowly eluting with PBS, and collecting biotinylated anti-DNASE 1L3 antibody; finally, biotinylated antibody was added to sodium azide (final concentration 0.5g/L) and 1.0% (w/w) bovine serum albumin PBS. The bound product was kept at 4 ℃ in the dark.
Alkaline phosphatase (ALP) -labeled anti-DNASE 1L3 antibody preparation: weighing 10mg of alkaline phosphatase, dissolving the alkaline phosphatase in a glutaraldehyde solution (1.5%), standing the solution at room temperature overnight, performing G25 chromatographic column chromatography, and eluting the solution by 0.9% physiological saline; DNASE1L3 antibody was weighed, diluted with 5mL of PBS buffer (pH 7.4,0.1M), and added to the enzyme solution for chromatography under magnetic stirring; adding 0.2mL of 0.2M lysine, mixing uniformly, standing at room temperature for 3 hours, and dialyzing for later use.
Example 2
Step 1: adding 50 μ L of the sample to 100 μ L of the biotinylated anti-DNASE 1L3 antibody reaction tube prepared by the method of example 1, and reacting at 37 ℃ for 5 minutes to form a biotinylated anti-DNASE 1L3 antibody-antigen complex;
step 2, adding 60 mu L of streptavidin coated magnetic particles (with the particle size of 3 mu m, purchased) with the concentration of 1mg/mL into the reaction tube, placing the reaction tube at 37 ℃ for 5 minutes for reaction, and fixing the anti-DNASE 1L3 antibody antigen complex on the magnetic particles through the specific binding reaction of the streptavidin and the biotin;
and step 3: sucking the liquid part of the reaction tube under the action of magnetic force; washing with Tris buffer solution, removing liquid under magnetic action, and repeating the operation for 5 times;
and 4, step 4: mu.L of ALP-labeled anti-DNASE 1L3 antibody reagent was added to the reaction tube, and the mixture was reacted at 37 ℃ for 5 minutes, magnetically separated, and washed with Tris buffer. The operation was repeated 5 times.
And 5: adding 50 mu L of MES buffer solution and 100 mu L of substrate solution into the reaction tube, uniformly mixing and stirring, placing at 37 ℃ for reacting for 8 minutes, detecting the luminous intensity on a luminometer, detecting a standard product by the same method, taking the luminous intensity of each concentration of the standard product as the ordinate and the concentration as the abscissa, preparing a standard curve, and calculating the concentration of DNASE1L3 in the sample.
And (3) precision test: preparing a quality control product with the concentration of 75.0ng/mL by using DNASE1L3, and repeatedly detecting for 10 times to obtain the batch precision; the quality control product is used for detecting twice a day to obtain an average value, the detection is continuously carried out for 10 days, and the batch precision of the method is calculated. As can be seen from table 1, the method of the present invention has high detection precision, with a coefficient of variation (n ═ 10) CV% of 4.36% in batch and a coefficient of variation CV% of 8.94% between batches.
Table 1 method precision batch to batch (n ═ 10)
Linear experiments: 1 part of each of the high and low concentration pooled serum samples was collected and designated as S1 and S5. S2 is 3 parts S1 mixed with 1 part S5; s3 is 2 parts S1 mixed with 2 parts S5; s4 is 1 part S1 mixed with 3 parts S5; the concentration of DNASE1L3 in S1 and S5 is divided by taking the mean value of the method after 3 times of detection (without outlier) as the concentration index value, and the theoretical concentrations of S2-S4 are calculated according to the formula C (C1 × V1+ C5 × V5)/(V1+ V5), wherein C1 and C5 refer to the concentrations of S1 and S5, and V1 and V5 refer to the volumes of S1 and S5, respectively. 5 samples were randomly arranged and the assay was repeated 2 times for each concentration and the results were recorded for analysis.
As shown in FIG. 2 and FIG. 3, the standard curve obtained by the method of the present invention has a wide linear range, and has good linearity in the range of 0.97-1276 ng/mL.
And (3) specificity test: the potential cross-reactants Rheumatoid Factor (RF), anti-streptococcal "O" (ASO), complement C3 and complement C4 were added to serum samples with DNASE1L3 concentration of 130ng/mL, and the change in DNASE1L3 concentration before and after the addition of the potential interferents was examined to determine the cross-reactivity (assay specificity) of the method.
As can be seen from Table 2, the method of the present invention has high specificity, and the detection results are not affected by high concentrations of Rheumatoid Factor (RF), Streptococcus mutans "O" and complement (C3, C4).
TABLE 2
| Potential cross-reactants | Concentration of additive | Cross reactivity (%) |
| Rheumatoid factor | 400IU/mL | -0.03 |
| Anti-streptococci 'O' | 2000IU/mL | 0.01 |
| Complement C3 | 4.5g/L | 0.02 |
| Complement C4 | 2.0g/L | 0.01 |
Accuracy verification test
Two clinical blood samples were taken and DNASE1L3 concentration was 30.68ng/mL and 101.31ng/mL, respectively, to which DNASE1L3 recombinant protein standard was added at 20ng/mL and 40ng/mL, respectively, and each concentration was tested in parallel for 3, and recovery was calculated. The results are shown in Table 3.
TABLE 3 recovery of the process
As can be seen from Table 3, the recovery rate of the process of the present invention is between 93.2% and 96.1%.
In conclusion, the method is easy to operate, high in detection speed, high in precision, strong in specificity and high in sensitivity.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for detecting DNASE1L3 based on magnetic particle chemiluminescence immunoassay, the method comprising:
biotinylated anti-DNASE 1L3 antibody preparation:
preparation of enzyme-labeled anti-DNASE 1L3 antibody:
combining the biotinylated anti-DNASE 1L3 antibody with the anti-DNASE 1L3 antibody in the sample to be tested to form a biotinylated anti-DNASE 1L3 antibody-antigen complex; adding streptavidin coated magnetic particles, washing under the action of magnetic force after reaction, and removing unbound substances; adding enzyme-labeled anti-DNASE 1L3 antibody, reacting, washing under the action of magnetic force, and removing unconjugated substance to obtain magnetic particle-DNASE 1L3 antigen-antibody complex; adding a buffer solution and a luminescent substrate solution into the magnetic particle-DNASE 1L3 antigen-antibody complex for reaction, detecting the luminous intensity on a luminometer, detecting a standard substance by the same method, preparing a standard curve by using the luminous intensity of each concentration of the standard substance as an ordinate and the concentration as an abscissa, and calculating the concentration of DNASE1L3 in a sample.
2. The method of claim 1, wherein the biotinylated anti-DNASE 1L3 antibody is prepared by: diluting the mouse anti-human DNASE1L3 antibody with buffer solution, dialyzing, adding N-hydroxysuccinimide biotin solution, magnetically stirring for 1-2h, adding ammonium chloride solution, stirring for 10-20min, and dialyzing; passing the dialyzed sample through a molecular sieve column, and collecting biotinylated anti-DNASE 1L3 antibody; the biotinylated anti-DNASE 1L3 antibody was added to PBS solution containing sodium azide and bovine serum albumin, and stored at 4 ℃ in the dark.
3. The method according to claim 2, wherein the murine anti-human anti-DNASE 1L3 antibody is diluted with a boric acid buffer to a concentration of 0.5 to 2mg/mL and dialyzed against the boric acid buffer;
preferably, the concentration of the boric acid buffer solution is 0.5-1.0mmol/L, and the pH value of the solution is 8.0-8.6.
4. The method of claim 2, wherein the concentration of the N-hydroxysuccinimide biotin solution is 0.5-1.0mg/mL, and the solvent is dimethyl sulfoxide;
preferably, the volume ratio of the mouse anti-human DNASE1L3 antibody solution to the N-hydroxysuccinimide biotin solution is 1: 0.1-0.5;
preferably, the concentration of the ammonium chloride solution is 0.5-1.0mol/L, and the addition amount of the ammonium chloride solution is 8-10 per mill of the volume of the mouse anti-human DNASE1L3 antibody solution;
preferably, ammonium chloride solution is added and then dialyzed with PBS buffer;
preferably, the sodium azide concentration is 0.1-0.5g/L and the bovine serum albumin is added in an amount of 0.5-1.0% w/w.
5. The method of claim 1, wherein the enzyme-labeled anti-DNASE 1L3 antibody is prepared by:
dissolving alkaline phosphatase in glutaraldehyde solution, standing overnight, passing through a molecular sieve column, and eluting with normal saline to obtain alkaline phosphatase solution; diluting the DNASE1L3 antibody with PBS buffer solution, and adding the diluted antibody into alkaline phosphatase solution under magnetic stirring; adding lysine solution, mixing, standing at room temperature for 3-5h, and dialyzing to obtain enzyme-labeled antibody solution;
activating magnetic particles by glutaraldehyde, stirring at room temperature, uniformly mixing for 3 hours, standing for 15min, pouring out supernatant, washing by PBS buffer solution, and suspending by PBS to prepare magnetic particle suspension with the concentration of 50-100 mg/mL;
stirring the magnetic particle suspension and the enzyme-labeled antibody solution at 4 ℃ overnight, standing for 10-15min under a magnetic field, removing a supernatant, sealing with 0.5% bovine serum albumin and 1% skim milk in PBS buffer solution at room temperature for 3-4h, and finally washing with PBS to prepare a working solution.
6. The method of claim 5, wherein the PBS buffer is used at a concentration of 0.1M and the solution has a pH of 7.4.
7. The method of claim 5, wherein the lysine solution is used at a concentration of 0.05 to 0.2M.
8. The method of claim 1, wherein the biotinylated anti-DNASE 1L3 antibody is reacted with the test sample at 37 ℃ for 5 min;
preferably, the biotinylated anti-DNASE 1L3 antibody antigen complex is reacted with streptavidin-coated magnetic microparticles at 37 ℃ for 5 min;
preferably, the reaction is carried out at 37 ℃ for 5min after addition of the enzyme-labeled anti-DNASE 1L3 antibody.
9. The method of claim 1, wherein MES buffer is added to the magnetic particle-DNASE 1L3 antigen-antibody complex;
preferably, the luminescent substrate solution: consists of 0.05mol/L trihydroxymethane, 0.5 percent sodium dodecyl sulfate and 0.4mg/mL dihydroacridine derivative, and the pH value of the solution is 9.0.
10. The method of claim 1, wherein the reaction is carried out at 37 ℃ for 8min after adding the buffer and the luminescent substrate solution to the magnetic particle-DNASE 1L3 antigen-antibody complex.
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