LU500306B1

LU500306B1 - Method for detecting DNASE1L3 based on magnetic particle chemiluminescence immunoassay

Info

Publication number: LU500306B1
Application number: LU500306A
Authority: LU
Inventors: Yu Cheng; Jiabei Yang; Yuge Xi; Yongcan Guo
Original assignee: Affiliated Hospital Of Traditional Chinese Medicine Of Southwest Medical Univ
Priority date: 2020-07-29
Filing date: 2021-06-21
Publication date: 2021-12-21
Also published as: CN111929440A

Abstract

The invention relates to the technical field of immunological detection, in particular to a method for detecting DNASE1L3 based on magnetic particle chemiluminescence immunoassay. The method comprises the following steps: preparing biotinylated anti- DNASE1L3 antibody; preparing enzyme-labeled anti-DNASE1L3 antibody: combining the biotinylated anti-DNASE1L3 antibody with the anti-DNASE1L3 antibody in the sample to be detected; Adding streptavidin coated magnetic particles, washing under the action of magnetic force after reaction, and removing unbound substances; Adding enzyme-labeled anti-DNASE1L3 antibody, washing under the action of magnetic force after reaction, and removing unbound substances; Adding buffer solution and luminescent substrate solution for reaction, detecting luminescence intensity on luminometer, and calculating the concentration of DNASE1L3 in the sample. The method can quickly realize the quantitative analysis of DNASE1L3, and the preparation method and application of the tongue-and-groove structure gc3n4moo3 composite photocatalytic material have high detection sensitivity and strong specificity.

Description

DESCRIPTION Method for detecting DNASE1L3 based on magnetic particle chemiluminescence immunoassay

TECHNICAL FIELD The invention relates to the technical field of biological detection, in particular to a method for detecting DNASEI1L3 based on magnetic particle chemiluminescence Immunoassay.

BACKGROUND DNASEIL3, a nucleic acid endonuclease in the DNASE superfamily, is a secreted protein produced by myeloid cells such as dendritic cells and macrophages that enters the bloodstream and has DNA hydrolytic activity, cleaving single- and double-stranded DNA and also cleaving chromatin into nucleosomal units and cutting nucleosomal and liposomal-coated DNA. DNASEIL3 deficiency has been shown to cause anti-DNA responses and autoimmunity in humans and mice, and blood DNASE1L3 is involved in circulating plasma DNA homeostasis by enhancing fragmentation and affecting terminal motif frequency. DNASEIL3 enzyme may be used as a treatment for autoimmune diseases such as SLE. In addition, DNASE1L3 has been found to be an independent prognostic factor for hepatocellular liver cancer. Therefore, quantitative determination of DNASEIL3 in peripheral blood is an important biomarker not only for the clinical adjunct diagnosis and efficacy evaluation of SLE, but also for the assessment of the prognosis of hepatocellular hepatocellular carcinoma.

At present, in order to detect DNASEI1L3 in peripheral blood, western blotting and ELISA are mainly used by scholars at home and abroad for research.

There are six main steps. Step 1. Loading; loading protein samples containing target proteins and molecular weight standards into SDS- polyacrylamide gel pores respectively; Step 2: Electrophoresis, under a certain current (voltage) condition, the target protein and other protein are separated due to the difference of molecular weight and charge; Step 3: transfer, in which the separated bands in the gel are transferred to a nitrocellulose membrane (NC); Step 4, adding a primary antibody, adding a first antibody to react with the target protein on the NC membrane, and washing away redundant and primary antibody and unreacted protein; Step 5, adding a labeled secondary antibody, adding a second antibody capable of color development and labeling, forming an antigen- antibody complex with the target protein and the primary antibody, and washing away redundant and primary antibodies; And Step 6, coloration or development, namely adding a substrate to react with a developing label to color or develop the antigen-antibody complex strip containing the target protein. Finally, compared with the position of molecular weight standard band, it is determined whether the sample contains target protein or not, and the target protein is semi-quantitative according to the color depth of the color band.

Implementation scheme of enzyme-linked immunosorbent assay (ELISA) The main steps are as follows: Step 1, the specific antibody is linked with the solid carrier to form the solid antibody. Washing to remove unbound antibodies and impurities; Step 2, combining the target protein in the sample with the solid-phase antibody to form a solid-phase antigen-antibody complex, and washing to remove other unbound substances; Step 3, the enzyme-labeled antibody is added to combine with the antigen on the solid immune complex and the enzyme-labeled antibody. Thoroughly washing unbound enzyme-labeled antibody; Step 4, adding substrate to develop color, detecting with microplate reader, and calculating the concentration of target protein by comparing with the absorbance of standard product.

At present, WB method and ELISA method are mainly used in scientific research, which have the disadvantages of tedious operation and long time-consuming, and their specificity, sensitivity and high-throughput processing ability need to be further improved.

References: 1 Sisirak V,Sally B,Clancy R Met al Digestion of Chromatin inApoptotic Cell Microparticles Prevents Autoimmunity[J].Cell,2016,166(1):1-14.

2.Zhao Q,Yang C,Wang J,L1 Y,Yang P.Serum level of DNasell3 in patients with dermatomyositis/polymyositis ,systemic lupus erythematosus and rheumatoid arthritis,and its association with disease activity.Clin ExpMed.2017; 17(4):459-465.

3 Wilber A,O'Connor TP Lu ML Karimi A Schneider MC .Dnasell3deficiency in lupus-prone MRL and NZB/W F1 mice.Clin Exp Immunol 2003; 134(1):46-52.

4. Jin Yuyang, Tu Yang, Song Rui, Chen Xiaoxiang, Zhang Yandong. Activity detection and clinical preliminary observation of DNase1L3. Modern Immunology. 2020; 40(01):22-7.

Wang S Ma Hi X Mo X Zhang H Yang L Deng Y,Yan Y,Yang G,Liu X,SunH .DNASF1L3 as an indicator of favorable survival in hepatocellular carcinomapatients following resection Aging(Albany NY).2020; 12(2):1171-85.

6 Roberts DE Kakar S Mehta N,Gill RM .A Point-based Histologic ScoringSystem for Hepatocellular Carcinoma Can Stratify Risk of Posttransplant TumorRecurrence. Am J Surg Pathol 2018; 42(7):855—865.

SUMMARY The main purpose of the invention is to provide a method for detecting DNASF1L3 based on magnetic particle chemiluminescence immunoassay, which can quickly realize quantitative analysis of DNASE1L3 with high detection sensitivity and strong specificity.

The invention adopts the following technical scheme: The method for detecting DNASEIL3 based on magnetic particle chemiluminescence immunoassay, comprising the following steps: Preparation of biotinylated anti-DNASE1L3 antibody; Preparation of enzyme-labeled anti-DNASE1L3 antibody; Combining the biotinylated anti-DNASE1L3 antibody with the anti-DNASE1L3 antibody in the sample to be detected to form a biotinylated anti-DNASE1L3 antibody antigen complex; Adding streptavidin to coat magnetic particles, washing under the action of magnetic force after reaction, and removing unbound substances; Adding an enzyme-labeled anti-DNASE1L3 antibody, washing under the action of magnetic force after reaction, and removing unbound substances to obtain a magnetic particle - DNASE1L3 antigen-antibody complex; Adding buffer solution and luminescent substrate solution to the magnetic particle -DNASEI1L3 antigen-antibody complex for reaction, detecting the luminescent intensity on a luminometer, detecting the standard substance by the same method, making a standard curve with the luminescent intensity of each concentration of the standard substance as the ordinate and the concentration as the abscissa, and calculating the concentration of DNASE1L3 in the sample.

Preferably, the the preparation method of the biotinylated anti-DNASE1L3 antibody is as follow: diluting the mouse anti-human anti-DNASE1L3 antibody with buffer solution, dialyzing, adding N- hydroxysuccinimide biotin solution, magnetically stirring for 1-2h, adding ammonium chloride solution, continuously stirring for 10-20min, and dialyzing; Passing the dialyzed sample through a molecular sieve column to collect biotinylated anti-DNASE1L3 antibody; Biotinylated anti-DNASE1L3 antibody was added to PBS solution containing sodium azide and bovine serum albumin, and stored at 4°C away from light.

Preferably, the mouse anti-human anti-DNASEIL3 antibody is diluted to a concentration of 0.5-2mg/mL with boric acid buffer, and dialyzed with boric acid buffer.

Further, 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.

Optimally, the concentration of N- hydroxysuccinimide biotin solution is 0.5-

1.0mg/mL, and the solvent is dimethyl sulfoxide.

Preferably, the volume ratio of mouse anti-human anti-DNASEIL3 antibody solution to N- hydroxysuccinimide biotin solution is 1: 0.1-0.5; Preferably, the concentration of ammonium chloride solution is 0.5-1.0 m o I/L, and the addition amount of ammonium chloride solution is 8-10 %o of the volume of mouse anti-human anti-DNASE1L3 antibody solution.

Preferably, after adding ammonium chloride solution, dialysis is performed with PBS buffer; Preferably, the concentration of sodium azide is 0.1-0.5g/L, and the dosage of bovine serum albumin is 0.5-1. 0% w/w.

Furthermore, the preparation method of the enzyme-labeled anti-DNASF1L3 antibody is as follows:

Dissolving alkaline phosphatase in glutaraldehyde solution, standing overnight, passing through molecular sieve column, and eluting with physiological saline to obtain alkaline phosphatase solution; Dilute DNASF1L3 antibody with PBS buffer solution and add it into alkaline phosphatase solution with magnetic stirring; Adding lysinate solution, uniformly mixing, standing at room temperature for 3-5h, and dialyzing to obtain enzyme-labeled antibody solution; Activating magnetic particles with glutaraldehyde, stirring and mixing at room temperature for 3 hours, standing for 15min, pouring out supernatant, washing with PBS buffer solution, and suspending with PBS to prepare magnetic particle suspension with concentration of 50-100mg/mL; The concentration of PBS buffer used is 0.1M and the pH value of the solution is

7.4.

Further, the concentration of lysine solution used is 0.05-0.2m.

The biotinylated ant:-DNASE1L3 antibody reacts with the sample to be tested at 37 °C for 5 minutes; Preferably, that biotinylated anti-DNASE1L3 antibody antigen complex react with streptavidin coated magnetic particle at 37 °C for 5 minutes; Preferably, the enzyme-labeled anti-DNASEI1L3 antibody is added and reacted at 37°C for Smin.

The MES buff solution is added into the magnetic particle -DNASE1L3 antigen- antibody complex;

Preferably, the luminescent substrate solution is composed of 0.05mol/L trihydroxymethane, 0.5% sodium dodecyl sulfonate and 0 .4mg/mL dihydroacridine derivative, and the pH value of the solution is 9.0.

And a buff solution and a luminescent substrate solution are added to that magnetic particle -DNASE1L3 antigen-antibody complex and then react for 8 minutes at 37°C.

Compared with the prior art, the invention has the following advantages: 1) The detection method of the invention 1s rapid, and it takes about 25 minutes to detect a single sample, which is shorter than ELISA and WB methods; And the whole reaction is carried out in the same reaction tube, so that automatic operation is easy to realize.

2) The method has high detection precision, and the coefficient of variation (n = 10) CV% within the batch 1s 4.36%, and the coefficient of variation CV% between the batches is 8.94%.

3) The method disclosed by the invention has wide detection linear range and high detection sensitivity, and has good linearity in the range of 0.12-1276 ng/ml, and the lowest detection limit reaches 0.05ng/mL.

4) The detection accuracy of the method is high, and the recovery rate can reach over 96%.

5) The method of the invention has high specificity, and high concentration rheumatoid factor (RF), anti-streptococcus "O" and complement (C3, C4) do not interfere with the detection result of blood sample DNASE1L3.

BRIEF DESCRIPTION OF THE FIGURES The accompanying drawings of the specification, which form a part of the invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention, and do not constitute an improper limitation of the invention.

Fig. 1 is a schematic diagram of a method for detecting DNASEIL3 based on magnetic particle chemiluminescence immunoassay according to a specific embodiment of the present invention.

Fig. 2 is a DNASF1L3 standard graph according to a specific embodiment of the present invention; Fig. 3 is a fitting curve of the relationship between measured concentration and theoretical concentration.

DESCRIPTION OF THE INVENTION It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

It should be noted that the terms used here are only for describing specific embodiments, and are not intended to limit exemplary embodiments according to the present invention. As used herein, the singular form is also intended to include the plural form unless the context clearly indicates otherwise, and it should also be understood that when the terms "comprising" and/or "including" are used in this specification, they indicate the presence of features, steps, operations and/or combinations thereof.

Example 1

Preparation of biotinylated anti-DNASE1L3 antibody: The mouse anti-human anti- DNASE1L3 antibody (purchased) was diluted to 1mg/mL with 0 .5mmol/L boric acid buffer (pH = 8. 6), and then dialyzed three times in dialysis bag (MW CO 8000) with 0 .5mmol/L boric acid buffer (pH = 8.6). Dissolve N- hydroxysuccinimide biotin (NHSB, purchased) with dimethyl sulfoxide (DMSO) at a ratio of 1mL:1mg, with a final concentration of 1 mg/ml; Add 120uL of the above NHSB solution to ImL antibody solution, magnetically stir for 2 hours at room temperature, add 9.6u 1 of 1mol/l ammonium chloride and stir for 10 minutes; Dialysis with PBS buffer at 4°C for 3 times to remove free biotin, Sample that dialyzed sample on a molecular sieve column (G25), elute slowly with PBS, and collecting biotinylated anti-DNASE1L3 antibody; At last, biotinylated antibody was added into sodium azide (final concentration 0.5g/L) and 1. 0% (w/w) bovine serum albumin PBS solution. Store the binding product at 4°C and keep it away from light.

Preparation of alkaline phosphatase (ALP)-labeled anti-DNASEIL3 antibody: Dissolve 10mg of ALP in glutaraldehyde solution (1.5%), let it stand at room temperature overnight, then pass through G25 column chromatography, and elute with 0.9% normal saline; The antibody DNASF1L3 was weighed, diluted with SmL PBS buffer (pH=7.4,

0.1M), and added to the chromatographic enzyme solution with magnetic stirring. Add

0.2M with lysine 0.2mL, mix well, leave it at room temperature for 3 hours, and put it on standby after dialysis.

Example 2

Step 1: Add SOuL sample into 100uL biotinylated anti-DNASEI1L3 antibody reaction tube prepared by the method of Example 1, and react at 37°C for 5 minutes to form biotinylated anti-DNASE1L3 antibody antigen complex; Step 2: Add 60uL 1mg/mL streptavidin coated magnetic particles (purchased, with a particle size of 31 m) into the above reaction tube, react at 37°C for 5 minutes, and fix the anti-DNASE1L3 antibody antigen complex on the magnetic particles through the specific binding reaction between streptavidin and biotin; Step 3: Absorb the liquid part of the reaction tube under the action of magnetic force, then wash it with Tris buffer solution, remove the liquid under the action of magnetic force, and repeat the operation for 5 times; Step 4: Add 100uL of ALP labeled anti-DNASEIL3 antibody reagent into the reaction tube, react at 37°C for 5 minutes, magnetically separate and wash with Tris buffer. Repeat the operation 5 times.

Step 5: Add SOuL MES buffer solution and 100uL substrate solution into the above reaction tube, mix and stir, react at 37°C for 8 minutes, and detect the luminous intensity on the luminometer, and detect the standard substance by the same method. Take the luminous intensity of each concentration of the standard substance as ordinate and the concentration as abscissa, make a standard curve, and calculate the concentration of DNASEI1LS3 in the sample.

Precision test: the quality control product with the concentration of 75.0ng/mL was prepared with DNASEIL3, and its intra-batch precision was obtained by repeated detection for 10 times; The quality control product was tested twice a day to get the average value, and it was tested continuously for 10 days to calculate the inter-batch precision of this method. It can be seen from Table 1 that the detection precision of this method 1s high, and the coefficient of variation (n = 10) CV% within the batch 1s 4. 36%, and the coefficient of variation CV% between batches 1s 8. 94%.

Table 1 Intra-batch and inter-batch precision of the method (n = 10) as LEE EEE IE RE assay _Interassay [1 [2 [3 [4 |5 Je 7 [8 |9 [10 | Linear experiment: Collect one sample of mixed serum with high and low concentrations, which are labeled S1 and S5 respectively. S2 is 3 parts of S1 mixed with 1 part of S5; S3 is 2 parts S1 mixed with 2 parts S5; S4 is 1 part S1 mixed with 3 parts SS; Except that the concentration of DNASEIL3 in S1 and SS is measured three times (no outliers) by this method, the theoretical concentration of S2 ~ S4 is calculated according to the formula C = (C1 X V1+C5 X V5)/(V1+V5), where C1 and CS refer to the concentration of S1 and S5 respectively, and V1 and V5 refer to the volume of S1 and S5 respectively. Five samples were randomly arranged, and each concentration was determined twice, and the results were recorded and analyzed.

As shown in Fig. 2 and Fig. 3, the linear range of the standard curve obtained by the method of the present invention is wide, and the linearity is good in the range of 0.97 ~ 1276 ng/mL.

Specificity test: the potential cross-reactants rheumatoid factor (RF), anti- streptococcus "O" (ASO), complement C3 and complement C4 were added to serum samples with DNASE1L3 concentration of 130ng/mL, and the changes of DNASEIL3 concentration before and after adding potential interferents were detected to judge the cross-reactivity of this method (analysis specificity).

It can be seen from Table 2 that the method of the present invention has strong specificity, and the detection result is not affected by high concentration of rheumatoid factor (RF), anti-streptococcal bacteria "O" and complement (C3, C4). Table 2 Potential cross reactant Additive concentration | Additive concentration Rheumatoid factor 400IU/mL Anspocosens 0200010 Complement complement CS Accuracy verification test Two clinical blood samples were taken with DNASEIL3 concentrations of

30.68ng/mL and 101.31ng/mL respectively, and DNASE1L3 recombinant standards of 20ng/mL and 40ng/mL were added to them, and three parallel tests were performed for each concentration to calculate the recovery rate. The results are shown in Table 3. Table 3 Recovery rate of method Sample Addition Estimated Average Recovery Average concentrationng/mL | concentrationng/mL | valueng/mL valueng/mL rate (%) value (%)

30.68 20 48.72 96.1

30.68 40 66.22 93.7

101.31 20 113.07 93.2

101.31 40 132.46 93.7

It can be seen from Table 3 that the recovery rate of this method is between 93. 2% and 96. 1%.

To sum up, the method is easy to operate, fast 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 embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principles of the present invention shall be equivalent substitutions and are included in the scope of protection of the present invention.

Claims

1. The method for detecting DNASE1L3 based on magnetic particle chemiluminescence immunoassay, comprising the following steps: preparation of biotinylated anti-DNASE1L3 antibody; preparation of enzyme-labeled anti-DNASE1L3 antibody; combining the biotinylated anti-DNASE1L3 antibody with the anti-DNASE1L3 antibody in the sample to be detected to form a biotinylated anti-DNASEI1L3 antibody antigen complex; adding streptavidin to coat magnetic particles, washing under the action of magnetic force after reaction, and removing unbound substances; adding an enzyme- labeled anti-DNASE1L3 antibody, washing under the action of magnetic force after reaction, and removing unbound substances to obtain a magnetic particle -DNASE1L3 antigen-antibody complex; adding buffer solution and luminescent substrate solution to the magnetic particle -DNASE1L3 antigen-antibody complex for reaction, detecting the luminescent intensity on a luminometer, detecting the standard substance by the same method, making a standard curve with the luminescent intensity of each concentration of the standard substance as the ordinate and the concentration as the abscissa, and calculating the concentration of DNASE1L3 in the sample.

2. The method accord to claim 1, wherein the preparation method of the biotinylated anti- DNASEIL3 antibody is as follow: diluting the mouse anti-human anti-DNASE1L3 antibody with buffer solution, dialyzing, adding N- hydroxysuccinimide biotin solution, magnetically stirring for 1-2h, adding ammonium chloride solution, continuously stirring for 10-20min, and dialyzing; passing the dialyzed sample through a molecular sieve column to collect biotinylated anti-DNASE1L3 antibody; biotinylated anti-DNASE1L3 antibody was added to PBS solution containing sodium azide and bovine serum albumin, and stored at 4°C away from light.

3. The method according to claim 2, wherein the mouse anti-human anti-DNASF1L3 antibody is diluted to a concentration of 0.5-2mg/mL with boric acid buffer, and dialyzed with 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 according to claim 2, wherein the concentration of N- hydroxysuccinimide biotin solution is 0.5-1.0mg/mL, and the solvent is dimethyl sulfoxide; preferably, the volume ratio of mouse anti-human anti-DNASE1L3 antibody solution to N- hydroxysuccinimide biotin solution is 1: 0.1-0.5; preferably, the concentration of ammonium chloride solution is 0.5-1.0 m o I/L, and the addition amount of ammonium chloride solution is 8-10 %o of the volume of mouse anti- human anti-DNASE1L3 antibody solution; preferably, after adding ammonium chloride solution, dialysis is performed with PBS buffer; preferably, the concentration of sodium azide is 0.1-0.5g/L, and the dosage of bovine serum albumin is 0.5-1. 0% w/w.

5. The method according to claim 1, wherein the preparation method of the enzyme- labeled anti-DNASE1L3 antibody is as follows: dissolving alkaline phosphatase in glutaraldehyde solution, standing overnight, passing through molecular sieve column, and eluting with physiological saline to obtain alkaline phosphatase solution; dilute DNASE1L3 antibody with PBS buffer solution and add it into alkaline phosphatase solution with magnetic stirring; adding lysinate solution, uniformly mixing, standing at room temperature for 3-5h, and dialyzing to obtain enzyme-labeled antibody solution; activating magnetic particles with glutaraldehyde, stirring and mixing at room temperature for 3 hours, standing for 15min, pouring out supernatant, washing with PBS buffer solution, and suspending with PBS to prepare magnetic particle suspension with concentration of 50-100mg/mL; the magnetic particle suspension and the enzyme-labeled antibody solution were stirred at 4°C overnight, then stood for 10-15min under the magnetic field, sucked off the supernatant, sealed with 0.5% bovine serum albumin and 1% skim milk in PBS buffer at room temperature for 3-4h, and finally washed with PBS and prepared into working fluid.

6. The method according to claim 5, wherein the concentration of PBS buffer used is

0.1M and the pH value of the solution is 7.4.

7. The method according to claim 5, wherein the concentration of lysine solution used is

0.05-0.2m.

8. The method accord to claim 1, wherein the biotinylated anti-DNASE1L3 antibody reacts with the sample to be tested at 37 °C for 5 minutes; preferably, that biotinylated anti-DNASEI1L3 antibody antigen complex react with streptavidin coated magnetic particle at 37 °C for 5 minutes; preferably, the enzyme-labeled anti-DNASEI1L3 antibody is added and reacted at 37°C for Smin.

9. The method accord to claim 1, wherein the MES buff solution is added into the magnetic particle -DNASE1L3 antigen-antibody complex;

10. The method accord to claim 1, wherein a buff solution and a luminescent substrate solution are added to that magnetic particle -DNASF1L3 antigen-antibody complex and then react for 8 minutes at 37°C.