CN112098653A

CN112098653A - Method for rapidly detecting sildenafil in health-care product

Info

Publication number: CN112098653A
Application number: CN202010554891.0A
Authority: CN
Inventors: 谭贵良; 张世伟; 胡敏; 李向丽; 李雪雁; 李梅
Original assignee: Shenzhen Academy Of Metrology & Quality Inspection; University of Electronic Science and Technology of China Zhongshan Institute
Current assignee: Shenzhen Academy Of Metrology & Quality Inspection; University of Electronic Science and Technology of China Zhongshan Institute
Priority date: 2020-06-17
Filing date: 2020-06-17
Publication date: 2020-12-18

Abstract

The invention discloses a method for rapidly detecting sildenafil in a health-care product, which comprises the steps of screening sildenafil monoclonal antibody and preparing sildenafil antibody-fluorescent microsphere conjugate, and constructing a fluorescence detection test strip, wherein all the steps of combining and separating are completed in the affinity column, thereby avoiding the damage of affinity sites, enhancing the fluorescence signal of detection, the sildenafil detection method IC50 is 26.8ng/mL, the linear range is 1-81 ng/mL (y is-0.16 lnx +0.65(R2 is 0.97), so the method is suitable for rapid detection of illegally added sildenafil in health products, the whole detection process only needs 15min, and large-scale equipment is not needed, the method is simple and convenient to operate, high in sensitivity and reliable in result, and can be used for qualitative and semi-quantitative screening analysis of illegally added medicines of sildenafil in health care products.

Description

Method for rapidly detecting sildenafil in health-care product

[ technical field ] A method for producing a semiconductor device

The invention relates to a detection method of a health-care product, in particular to a method for rapidly detecting sildenafil in the health-care product.

[ background of the invention ]

Sildenafil with molecular formula C₂₂H₃₀N₆O₄S, a selective inhibitor of the cyclic guanosine monophosphate cGMP specific phosphodiesterase type 5 PDE5, is an oral drug for the treatment of male erectile dysfunction ED and is generally known under its commercial name Viagra. If the medicine is taken for a long time or in an overdose, serious damage can be caused to the cardiovascular system, the nervous system, the digestive system and the like, so that the addition of sildenafil in foods including health care products is forbidden in China. However, in order to pursue an ideal therapeutic effect and take effect in a short time, some illegal merchants illegally add the illegal drugs into the health care products for resisting fatigue and tonifying yang so as to achieve the aim of illegal profit, and undoubtedly bring huge potential safety hazards to consumers.

In the prior art, methods for detecting sildenafil are various, and mainly include liquid chromatography, a liquid chromatography-mass spectrometry combination method, ion mobility spectrometry, thin-layer chromatography, a capillary electrophoresis method, infrared spectrometry and the like. Although the methods can detect sildenafil and analogues thereof, most of the used instruments are expensive, the sample pretreatment is complex, the time consumption is long, the requirement on the professional technology of detection personnel is high, and the requirement on site rapid detection cannot be met. The ELISA method is a competitive enzyme-linked immune reaction, can shorten the detection time of sildenafil, has low requirements on instruments and professional technicians, but needs various matched reagents, has a complex operation process, can only realize qualitative detection generally and cannot achieve the purpose of quantitative detection.

The invention is developed and proposed aiming at the defects of the prior art.

[ summary of the invention ]

The invention aims to provide a method for rapidly detecting sildenafil in a health-care product. The sildenafil monoclonal antibody is obtained by screening through a cell fusion technology, the sildenafil antibody-fluorescent microsphere conjugate is prepared based on an antibody affinity site protection labeling method, and finally, the fluorescent detection test strip is successfully constructed. The method for protecting and marking the affinity sites based on the antibody is simple and stable to operate, all the steps of combining and separating are completed in the affinity column, the damage to the affinity sites can be effectively avoided, and the detected fluorescent signals are greatly enhanced. In addition, the fluorescence detection test strip consists of a sample pad, a combination pad, an NC membrane and absorbent paper. Coating antigen and goat anti-mouse polyclonal antibody are sprayed on an NC membrane to be respectively used as a sildenafil test strip detection line (T line) and a quality control line (C line), a sildenafil antibody coupling fluorescent microsphere is sprayed on a combination pad, finally, absorbent paper, the NC membrane, the combination pad and a pretreated sample pad are sequentially stuck on a PVC bottom plate, and the test strip with the width of 4mm is cut by an automatic slitter. The sildenafil detection method IC50 established by the invention is 26.8ng/mL, and the linear range is 1-81 ng/mL (y is-0.16 lnx +0.65 (R)²0.97)). The method is suitable for rapid detection of illegally added sildenafil in health products, the whole detection process only needs 15min, and large-scale equipment is not required. The test strip based on the fluorescent microsphere chromatography is simple and convenient to operate, high in sensitivity and reliable in result, and can be used for qualitative and semi-quantitative screening analysis of sildenafil in health care products.

The invention relates to a method for rapidly detecting sildenafil in a health-care product, which comprises the following specific steps:

A. synthesizing immunogen, namely respectively taking sildenafil immune hapten and sildenafil coated hapten, respectively coupling the hapten to carrier proteins BSA and OVA by adopting an active ester method to obtain immunogen AT-BSA and coated antigen ATA-OVA, and identifying whether the coupling of the hapten and the BSA and/or the OVA is successful or not by ultraviolet scanning;

B. screening sildenafil monoclonal antibody, immunizing an immunogen to test a BALB/C mouse, taking a spleen B cell of the mouse to fuse with a tumor cell, screening by using a monoclonal cell technology, implanting the spleen B cell into an abdominal cavity of the mouse to generate ascites, collecting the ascites, separating and purifying by using a Protein A column, and freeze-drying for long-term storage;

C. preparing a sildenafil antibody-fluorescent microsphere conjugate, labeling fluorescent microspheres by a protection affinity site method, coupling amino sildenafil on agarose gel containing aldehyde group activation, reducing formed unstable carbon-oxygen double bonds by using sodium cyanoborohydride, mixing 2mL of 5mg/mL sildenafil antibody and 2mL of agarose gel subjected to coupling, incubating at room temperature for 10min, loading into a centrifugal column, centrifuging to remove unbound antibody, and washing the gel by 3 times by using 6mL of 0.01mol/L phosphate buffer PBS (phosphate buffer solution) with pH of 7.4; adding 0.2mg of fluorescent microspheres made of polystyrene into 2.7mL of PBS (0.01 mol/L), adding 1.0mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to activate, adding into a centrifugal column containing agarose gel, uniformly mixing by vortex, standing at room temperature for incubation for 1h, and centrifuging to remove liquid; eluting the agarose gel twice by using 4mL of 0.1mol/L glycine hydrochloric acid buffer solution with pH3.0 as eluent; immediately adjusting the pH of the eluent to 7.0 by using 0.1mol/L sodium hydroxide to obtain a marked sildenafil antibody-fluorescent microsphere conjugate;

D. constructing a fluorescent microsphere chromatography test strip, firstly soaking a sample pad in 20mmol/L phosphate buffer solution, and drying at 60 ℃ for 2 h; 2.5mg/mL coating antigen and 0.3mg/mL goat anti-mouse polyclonal antibody are sprayed on an NC membrane to be respectively used as a detection line and a quality control line of a sildenafil test strip, wherein the detection line is a T line, and the quality control line is a C line, and is dried in vacuum for 12h at 37 ℃, a sildenafil antibody coupling fluorescent microsphere is sprayed on a combination pad, and is dried in vacuum for 12h at room temperature, finally, absorbent paper, the NC membrane, the combination pad and a pretreated sample pad are sequentially stuck on a PVC base plate, and are cut into test strips with the width of 4mm by an automatic slitter, and the test strips are dried in vacuum at room temperature for standby use, so that the fluorescent microsphere chromatography test strip is obtained;

E. establishing a quantitative curve, and recording the T-line fluorescence signal as FIT, the C-line fluorescence signal as FIC and the FIT/FIC value as B under the negative condition₀The FIT/FIC of the sildenafil standard solution is denoted as B, in terms of B/B₀As ordinate, sildenafil standard solution mass concentrationThe logarithm of the degree is the abscissa, and a four-parameter fitting curve is established using Origin 8.0, as B/B₀And establishing a linear regression equation based on a least square method at a standard point of 0.2-0.8.

F. Evaluating the stability of the method, selecting a health-care product sample which is confirmed to be sildenafil negative by chromatography, wherein the sildenafil scalar is 0.5mg/kg, and continuously measuring the standard-added sample and the non-standard-added sample by using fluorescent test strips of the same batch and different batches for 6 d;

G. and (3) comparing and observing the samples, wherein the whole detection process only needs 15min, large-scale equipment is not needed, and the rapid quantitative detection of the content of sildenafil in the samples is realized by directly comparing and observing the samples to be detected and the inhibition condition of the fluorescent microsphere immunochromatographic strip of sildenafil in the standard series by naked eyes.

[ description of the drawings ]

The following detailed description of embodiments of the invention is provided in conjunction with the appended drawings, in which:

FIG. 1 is a UV scan of the hapten and antigen.

FIG. 2 is a graph showing the amino standard curve of BSA determined by TNBS method.

FIG. 3 is a TNBS amino standard curve diagram for OVA determination.

FIG. 4 is a graph of ELISA competition curves for monoclonal antibodies.

FIG. 5 is a standard curve diagram of fluorescence immunochromatography for labeling antibodies by different labeling methods (n-6).

FIG. 6 is a diagram of the inhibition of sildenafil fluorescent microsphere immunochromatographic strip by visual observation.

FIG. 7 is a graph comparing the effect of non-formulation fluorescent antibody dilutions on fluorescence signal attenuation.

[ detailed description ] embodiments

Embodiments of the present invention will be described in detail below with reference to fig. 1 to 7.

1.1 Experimental materials

1.1.1 Main instrumentation

1.1.2 Primary reagents

1.1.3 preparation of Main Medium and solution

1.1.3.1 RPMI-1640 basic culture solution

Dissolving in 1000mL of double distilled water, filtering with 0.22 μm filter membrane for sterilization, packaging, and storing at 4 deg.C.

1.1.3.2 RPMI-1640 complete culture solution

1.1.3.3 double antibody solution

Penicillin and streptomycin with the concentration of 100U/mL are used to prepare 100 times of stock solution, and the stock solution is frozen at the temperature of-20 ℃ for standby.

1.1.3.4 50％PEG

5g of PEG with the molecular weight of 4000 are taken, sterilized by autoclaving, and then 5mL of filter sterilized RPMI-1640 complete culture solution is added and stored in a refrigerator at 4 ℃ for later use.

1.1.3.5 cell cryopreservation solution

10mL of DMSO was added to 90mL of RPMI-1640 complete medium.

1.1.3.6 preparation of 100 × HT solutions

100 × H in HT solution: 1X 10-2mol/L, T: 1.6X 10-3 mol/L. Weighing H136.1mg and T38.8 mg, wherein H is Hypoxanthine Hypoxanthine, and T is thymine Thymidine, dropwise adding 0.1mol/L NaOH until dissolving, replenishing water to 90mL, fully dissolving in a water bath at 45-50 ℃, adjusting pH to be neutral by using 1mol/L HCl, replenishing water to 100mL, filtering and sterilizing through a filter membrane with the aperture of 0.22 mu m, subpackaging 2 mL/piece, and freezing and storing at-20 ℃.

1.1.3.7 preparation of 100 XHAT solution

100 × H in HAT solution: 1X 10-2mol/L, A: 4X 10-5mol/L, T: 1.6X 10-3 mol/L. Weighing 1.76mg of A, wherein A is Aminopterin, 136.1mg of H and 38.8mg of T, dropwise adding 0.1mol/L NaOH solution to dissolve the A, replenishing water to 90mL, adjusting pH to be neutral by using 1mol/L HCl, replenishing water to 100mL, finally, filtering and sterilizing through a filter membrane with the pore diameter of 0.22 mu m, subpackaging 2 mL/piece, and freezing and storing at-20 ℃.

1.1.3.8 preparation of various reagents for ELISA

(1) Coating buffer solution

The pH value of the coating buffer solution is 9.6, the concentration is 0.1mol/L carbonate buffer solution, and the specific configuration is as follows:

Na₂CO₃ 0.375g

NaHCO₃ 0.732g

distilled water was added to 250 mL.

(2) Washing buffer

The PBST of pH7.4 of the washing buffer solution is specifically configured as follows:

distilled water was added to 2000 mL.

(3) Sealing liquid

The confining liquid is prepared by dissolving 5g of skimmed milk powder in 100mL of distilled water.

(4) Diluent liquid

The diluent is prepared by adding 0.1g of bovine serum albumin BSA into 100mL of washing buffer solution, or preparing 5-10% of serum such as goat serum, rabbit serum and the like with the washing buffer solution for use.

(5) Color buffer

The specific configuration of the developing buffer is as follows:

Na₂HPO₄·12H₂O 14.2g

citric acid 9.33g

1000mL of deionized water was added.

(6) TMB substrate solution

The TMB substrate solution is prepared by dissolving 15mg of TMB in 10mL of DMF, and is frozen and stored by light-blocking subpackage.

(7) Stopping liquid

The stop solution is H with the concentration of 10 percent₂SO₄The water-soluble polymer is prepared by dropwise adding concentrated sulfuric acid with the concentration of 98% and the volume of 20mL into distilled water with the volume of 180 mL.

(8) Color developing liquid

The color developing solution is prepared in situ, namely 10mL of color developing buffer solution and 2 mu L of hydrogen peroxide solution with the concentration of 30 percent are taken, and then 150 mu L of substrate solution is added.

1.1.4 cell lines and laboratory animals

Rat myeloma cells SP2/0 Shenzhen City metrological quality testing research institute

Experimental animal center of Zhongshan medical university of clean-grade Balb/c pure female mice

Experimental animal center of Zhongshan medical university of common-grade female Kunming mouse

1.2 Experimental methods

1.2.1 Synthesis of hapten

Immunizing hapten: 1g of sildenafil, 1g of 4-bromobutyric acid and 0.1g of sodium hydroxide are added, the mixture is refluxed AT 80 ℃ for 24 hours in 100mL of anhydrous tetrahydrofuran, the solvent is distilled off under reduced pressure, 10mL of 0.01mol/L hydrochloric acid is added, the insoluble substances are filtered, and the mixture is recrystallized in ethanol to obtain an immunohapten, which is labeled AT-COOH.

Coating hapten: 1g of sildenafil, 0.5g of glyoxylic acid is added into the solution, the solution is stirred and dissolved in 50mL of methanol, the reaction is carried out at room temperature for 1h, insoluble substances are filtered, and the precipitate is washed by ethanol to obtain a coating hapten which is marked as ATA-COOH.

1.2.2 Synthesis of immunogens

1.2.2.1 coupling method of hapten to Carrier protein

0.1mmol of immune hapten and 0.1mmol of coating hapten are respectively dissolved in 2mL of DMF, 27.5mg of DCC and 14.4mg of NHS are added with stirring, magnetic stirring reaction is carried out at 4 ℃ overnight, supernatant fluid after centrifugation is taken as A fluid, and BSA or OVA 140mg is weighed and dissolved in 10mL of PBS with the concentration of 0.1mol/L, PH 8.0.0. Adding 1mL of DMF, stirring and dissolving to obtain solution B, gradually dripping the solution A into the solution B under magnetic stirring, and reacting at 4 ℃ for 12 h. Centrifuging, collecting supernatant, dialyzing with physiological saline AT 4 deg.C for 3d, and changing dialysate for 3 times per day to obtain whole antigen (artificial antigen, immunogen), i.e. immunogen AT-BSA and coating antigen ATA-OVA. The whole antigen was dispensed at a concentration of 1mg/mL into 0.5mL centrifuge tubes and frozen in a-20 ℃ freezer. Therefore, the whole antigen is prepared by the method, and the artificial antigen concentration is measured by a Bradford micro-scale method.

1.2.2.2 identification of Artificial antigens

And respectively measuring the ultraviolet absorption spectra of the immunogen, the coating antigen, the hapten, the BAS and the OVA at 200-400 nm to respectively identify whether the coupling of the hapten and the BSA and/or the OVA is successful.

1.2.32, 4, 6-trinitrobenzene sulfonic acid (TNBS) method for determining coupling ratio of conjugate

1.2.3.1 preparation of Standard Curve

Preparing protein into solutions with concentration of 0, 0.2, 0.4, 0.6, 0.8, 1.0mg/mL with distilled water, adding 0.5mL into 0.5mL Na with pH of 9.0 and concentration of 0.1mol/L₂CO₃、NaHCO₃0.5mL of 0.1% TNBS solution was added to the carbonate buffer solution, and the mixture was reacted at room temperature for 15min and then calorimetrically measured at a wavelength of 420 nm. And (4) making a standard curve according to the light absorption value and the protein concentration, wherein the slope of the curve is the light absorption value of the unit concentration of the standard protein.

1.2.3.2 sample determination procedures

Preparing immune antigen into solutions with the concentrations of 0, 0.2, 0.4, 0.6, 0.8 and 1.0mg/mL by using distilled water, and making a standard curve by the same operation as the standard curve in the other steps, wherein the slope of the curve is the light absorption value of unit concentration.

1.2.3.3 calculation of amino consumption Rate of antigen

The free amino groups of the carrier protein are mostly derived from lysine, wherein the number of lysines per molecule of BSA and OVA is 61 and 20, respectively. The coupling ratio of the hapten to the carrier protein in the artificial antigen can be obtained by multiplying the amino consumption rate of the artificial antigen by the number of the carrier protein amino groups.

1.2.4 animal immunization

Female balb/c mice were immunized with the whole antigen prepared with the immunizing hapten for 6 weeks, 3 per group, respectively. When the first immunization injection is carried out, 100 mu L of immune antigen with 100 mu g/mL and 100 mu L of equivalent Freund's complete adjuvant are fully emulsified, and the mixture is directly injected into an abdominal cavity. After two weeks, 100. mu.L of the same antigen was emulsified with 100. mu.L of incomplete adjuvant and injected in the same manner.

1.2.5 selection of optimal immunogens

1.2.5.1 measurement of potency

And (3) respectively taking blood from the tail of three immunized parallel mouse tails 10 days after the fourth immunization, and centrifuging at 10000rpm to obtain antiserum, wherein the higher the titer of the antibody in the serum is, the higher the effective antibody concentration is. The basic steps are as follows:

(1) coating: the homologous coaters were diluted to 1. mu.g/mL, 100. mu.L/well in coating buffer and left overnight at 4 ℃.

(2) Washing: the well was decanted, 200 μ L of wash solution was added to each well, washed 3 times for 3min each time, and then the wash solution was spun off.

(3) And (3) sealing: add 250. mu.L of blocking solution to each well, block for 30min at 37 ℃ and wash the same as in step (2).

(4) Sample adding: except that the first row of wells was added with negative serum as a control, the other rows of wells were added with 100. mu.L of serum to be tested diluted at a rate of 1:1000 in each well, reacted at 37 ℃ for 1 hour, and washed in the same manner as in step (2).

(5) Adding an enzyme: mu.L of HRP-goat anti-mouse IgG was added to each well, diluted 15000-fold, reacted at 37 ℃ for 1 hour, and washed as in step (2).

(6) Color development: adding 100 μ L of color developing solution into each well, developing at 37 deg.C for 15min, and adding 50 μ L of 10% H into each well₂SO₄The reaction was terminated by using a stop solution.

(7) And (3) determination: each well A was assayed by an enzyme-linked immunosorbent assay_450nmThe absorption value of (2).

(8) And (4) judging the result: and (3) selecting the dilution of which the ELISA average light absorption value is near 1, and comparing the light absorption values based on the same dilution, wherein the higher the light absorption value is, the higher the titer is.

1.2.5.2 determination of optimal ELISA working conditions

The coating concentration and the serum dilution factor have a great influence on the sensitivity of the ELISA method. The invention determines the optimal serum dilution times and coating concentration by a maximum inhibition method, namely, the B/B of the same drug concentration under each condition is measured and calculated₀Values, where the left 8 × 8 well data is labeled B and the right 8 × 8 well is labeled B₀，B/B₀The smallest value is the best inhibition effect, the most sensitive reaction is, and the condition is the best working condition of ELISA. The ELISA plate arrangement is shown in table 1.

TABLE 1 maximum inhibition ELISA loading schematic

The specific operation steps are as follows:

(1) coating: the coated antigen was diluted in coating buffer at a doubling ratio of 4. mu.g/mL as shown in Table 1, and then stored at 100. mu.L/well at 4 ℃ overnight.

(2) Washing: the well was decanted, 200 μ L of wash solution was added to each well, washed 3 times, and then the wash solution was spun off.

(3) And (3) sealing: add 250. mu.L of blocking solution to each well, block for 30min at 37 ℃ and then wash.

(4) Sample adding: as shown in Table 3, 50. mu.L of 500ng/mL 2NPAOZ standard and 50. mu.L serum dilutions were added to the left 8X 8 wells. The right 8X 8 wells were filled with 50. mu.L PBST and 50. mu.L serum dilutions. Incubate 1h at 37 ℃ and wash.

(5) Adding an enzyme: mu.L of HRP-goat anti-mouse IgG diluted 15000 times was added to each well and incubated at 37 ℃ for 1 h. Washing the above step (2).

(6) Color development: each timeAdding 100 μ L of color developing solution into each well, developing at 37 deg.C for 15min, and adding 50 μ L of 10% H into each well₂SO₄The reaction was terminated by using a stop solution.

(7) And (3) determination: each well A was assayed by an enzyme-linked immunosorbent assay_450nmThe absorption value of (2). The left 8 × 8 well data is labeled B. The right 8X 8 wells are marked B₀. Selection of B₀>1, corresponding to B/B₀The serum dilution and coating concentration with the smallest ratio are the best ELISA working conditions.

The optimal ELISA working parameters of different antibodies and coatinggen are determined according to the method.

1.2.5.3 measurement of antisera sensitivity

In order to compare the sensitivity of antiserum obtained from different mice to target drugs, an indirect competitive ELISA method is adopted, 10 mu g/mL, 5 mu g/mL, 2 mu g/mL, 0.5 mu g/mL, 0.2 mu g/mL, 0.05 mu g/mL, 0.02 mu g/mL and 0.005 mu g/mL of sildenafil standard solution are respectively added into antiserum diluted according to 40000 times, a diluent is also added into negative serum as a control, pre-incubation is carried out in advance, then the antiserum is added onto an enzyme-labeled OD plate for reaction, the value is measured, and the IC value is calculated by using a four-parameter fitting module of Originlab₅₀The value is obtained. The basic steps are as follows:

(1) coating: the coating antigen was diluted to the optimum coating concentration with the coating buffer, 100. mu.L/well was added to the coated microplate, and left overnight at 4 ℃.

(4) Sample adding: add 50. mu.L of antibody (antiserum) at optimal concentration and 50. mu.L of sildenafil standard solution at a certain concentration, incubate for 1h at 37 ℃ and wash.

(5) Adding an enzyme: mu.L of HRP-goat anti-mouse IgG was diluted 15000-fold per well and incubated at 37 ℃ for 1 h. Washing is carried out in the same step (2).

(6) Color development: adding 100 μ L of color developing solution into each well, developing at 37 deg.C for 15min, and adding 50 μ L of stop solution (concentrated solution) into each wellH with a degree of 10%₂SO₄Solution) to terminate the reaction.

(8) And (3) calculating: IC calculation Using the four parameter fitting Module of Originlab 7.5₅₀Values, the lowest mouse was used for cell fusion.

1.2.6 preparation of monoclonal antibodies

1.2.6.1 preparation of feeder cells

Kunming mice were sacrificed by pulling their necks 1d before or on the day of cell fusion, soaked in 70% alcohol, and disinfected on the body surface. Fixing Kunming mouse on wax plate with pin, cutting abdomen on super clean bench, picking up peritoneum with small forceps, injecting 5mL RPMI-1640 complete culture solution, gently rubbing abdomen with hand, transferring the in vivo liquid into 75mL HAT complete culture solution with sterile suction tube, repeating for 2-3 times, wherein 75mL HAT complete culture solution is 75mL RPMI-1640 complete culture solution added with 0.75mL100 XHAT solution. Then mixing with a pipette, spreading in 24-well plate with 0.5mL per well, and placing in CO at 37 deg.C₂An incubator.

1.2.6.2 preparation of splenocytes

The method comprises the following steps of bleeding the orbit of a mouse, collecting serum, pulling the neck to kill, soaking and disinfecting the body surface with 70% alcohol, taking out the spleen aseptically, putting the spleen into RPMI-1640 basic culture solution, carefully removing fascia and fat, shearing, putting the spleen into a 100-mesh stainless steel sieve, carrying out aseptic grinding, releasing single spleen cells, sucking liquid containing the spleen cells, putting the liquid into a 50mL aseptic centrifuge tube, and centrifuging.

1.2.6.3 cell fusion

Adding myeloma cells and the prepared spleen cells into a same 50mL centrifuge tube according to the ratio of 5:1, adding 20mL of RPMI-1640 basic culture solution which is warm-bathed at 37 ℃, uniformly mixing, and centrifuging at the rotating speed of 1500rpm for 6 min. Removing supernatant, and flicking the bottom of the centrifuge tube with finger to mix the precipitate into paste. Taking 1mL of PEG preheated at 37 ℃ by a pipette, dripping into a centrifuge tube, standing for 1min, dripping 10mL of RPMI-1640 complete culture solution into a 37 ℃ water bath within 2min, centrifuging at the centrifugal speed of 1000rpm for 6min, and removingAdding 75mL HAT culture solution into supernatant, mixing, subpackaging the mixed suspension into 24-well plate with feeder cells, each well is 0.5mL, and culturing at 37 deg.C under CO₂Incubate at 5% concentration in an incubator saturated with humidity.

1.2.6.4 hybridoma cell screening and identification

And 6-9 days after cell fusion in the above steps, half amount of HT culture solution is used for replacing the culture solution for 1 time, and the complete culture solution is used according to proliferation conditions after 12-14 days. The number of wells and total number of cells in which the hybridoma cells grew were counted when the cells adhered to the wells of the wells 1/3. Taking supernatant, and adopting indirect ELISA to select positive hybridoma with high titer and indirect competitive ELISA to select drug inhibition.

1.2.6.5 Positive cell subcloning and establishment

And (3) screening positive hybridoma cells by adopting an indirect ELISA method and an indirect competition ELISA method, wherein the hole which shows positive and has competition inhibition reaction is a hole for producing the sildenafil antibody, and can be used for further subcloning.

And (3) eluting cells in the positive holes under the aseptic condition, transferring the cells to a 96-hole culture plate previously plated with feeder cells by using an elbow pipette, cloning each original hole into 8 holes, taking supernatant after the cells grow to 1/2-1/3 hole bottoms in a full adherent manner, and carrying out indirect ELISA detection. And (3) taking the subclones with strong positive, repeating the steps for 2-5 times, when the antibody positive rate in the cloned supernatant of 8 holes is 100%, picking single-cell clones, transferring the clones detected to be all positive to a 24-hole cell culture plate or a 25mL cell culture bottle for expanded culture, establishing strains, subpackaging and freezing.

1.2.6.6 preparation of ascites

(1) One week earlier, 0.5mL of pristanane was injected into the abdominal cavity of Balb/c mice.

(2) And (3) taking the frozen cell strain, recovering, culturing and propagating in a large scale, collecting the cells, washing twice by using an incomplete culture medium, suspending by using 10mL of the incomplete culture medium, and counting.

(3) The cells were injected intraperitoneally into the abdomen of mice, 1mL each, containing 3.1X 10⁷And after 10-15 days, aseptically collecting ascites by using a No. 16 syringe when the abdomen of the mouse is obviously enlarged.

(4) Centrifuging at 2000rpm for 10min, removing upper layer fat, lower layer fibrin and cells, collecting middle layer, measuring titer by ELISA method, and packaging the rest at-70 deg.C.

1.2.6.7 separation and purification of antibody by caprylic acid-ammonium sulfate precipitation method

Approximately 3mL of ascites fluid was taken and 2 volumes of 0.06mol/L, pH 4.5.5 sodium acetate buffer were added. Adding 33 μ g/mL ascites, i.e. caprylic acid, into the sample slowly and dropwise with stirring, continuing to stir for 30min after the adding, centrifuging at 10000rpm at 4 ℃ for 30min, and removing albumin and other non-IgG protein precipitates. The supernatant was filtered through a 0.45 μm microporous membrane and mixed with 1/10 volumes of 10 x PBS with the following formulation parameters: 80g NaCl, 2g KCl, 11.5g Na₂HPO₄、2g KH₂PO₄0.5845g EDTA, 1000mL distilled water, pH 7.4.

Then the pH value is adjusted to 7.4 by using 1mol/L NaOH solution. The supernatant was cooled to 4 ℃ and 0.277g/mL of ammonium sulfate was added to bring the final saturation to 45%. Stirring for 30min, centrifuging at 4 deg.C at 10000rpm for 30min, and discarding the supernatant. Dissolving the precipitate with a small amount of PBS solution, dialyzing with 50-100 times volume of PBS overnight, and changing the solution for 3 times. The dialyzed solution was appropriately concentrated with PEG-6000 and stored at 4 ℃ for further use.

1.2.7 Effect of the coating antigen Structure on ELISA sensitivity

The invention uses indirect non-competitive ELISA method to determine the titer of antiserum, and determines the optimal working concentration of coating antigen and antiserum by chessboard analysis method. The indirect non-competitive ELISA procedure was as follows:

1.2.7.1 Effect of different coaters on antibody affinity

The antibody affinity curve reflects the affinity of the antibody for the coating antigen. The basic steps of the affinity curve are as follows:

(1) coating: the coating source was diluted to 1. mu.g/mL, 100. mu.L/well with coating buffer and left overnight at 4 ℃.

(4) Sample adding: except that the first row of wells was added with negative serum as a control, the remaining rows of wells were added with 100. mu.L of the test antibody diluted at a rate of 1:1000 in each well, reacted at 37 ℃ for 1 hour, and washed as in step (2).

(5) Adding an enzyme: mu.L of HRP-goat anti-mouse IgG diluted at 15000 was added to each well, reacted at 37 ℃ for 1 hour, and washed as in step (2).

(6) Color development: adding 100 μ L of developing solution into each well, developing at 37 deg.C for 15min, and adding 50 μ L of 10% H into each well₂SO₄The reaction was terminated by using a stop solution.

(8) And (4) judging the result: and (4) drawing an affinity curve by taking the dilution factor as an abscissa and the OD value as an ordinate.

1.2.7.2 determination of optimal ELISA working conditions

The optimal antibody dilution factor and optimal coating concentration were determined as described in 1.2.7.1.

1.2.8 preparation of sildenafil antibody-fluorescent microsphere conjugate

The fluorescent microspheres are marked by a protection affinity site method. AminoLink Plus Immobilisation Kit was used to couple amino sildenafil to aldehyde-containing activated agarose gels, and the unstable carbon-oxygen double bond formed was reduced with sodium cyanoborohydride, the specific procedure was described in the Kit instructions. 2mL of sildenafil antibody at 5mg/mL and 2mL of the above-mentioned agarose gel after coupling were mixed, incubated at room temperature for 10min, loaded on a spin column, centrifuged to remove unbound antibody, and the gel was washed 3 times with 6mL of 0.01mol/L phosphate buffered saline PBS pH 7.4. Adding 0.2mg of fluorescent microspheres made of polystyrene into 2.7mL of PBS (0.01 mol/L), adding 1.0mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide for activation, adding the activated microspheres into a centrifugal column containing agarose gel, uniformly mixing the activated microspheres by vortex, standing the mixture at room temperature for 1 hour, and centrifuging the mixture to remove liquid. The agarose gel was rinsed twice with 4mL of 0.1mol/L glycine-HCl buffer, pH3.0, as eluent. Immediately adjusting the pH of the eluent to 7.0 by using 0.1mol/L sodium hydroxide to obtain the marked sildenafil antibody-fluorescent microsphere conjugate. Meanwhile, the fluorescent microspheres are marked by the material feeding ratio of 1:10, 1:30 and 1:100 in the edge separation process. The coupled antibody is diluted by the following fluorescent antibody diluents with different formulas respectively for preparing test strips:

formula 1: pH7.4, 0.01mol/L PBS, containing 2% fructose, 1% PEG 40000, 5% sucrose, 1% BSA, 0.4% Tween-20.

And (2) formula: pH7.4, 0.01mol/L PBS, 2% fructose, 1% PEG 40000, 5% sucrose, 1% BSA, 0.4% Tween-20, 0.1% NaN₃。

And (3) formula: pH7.4, 0.01mol/L PBS, 5% trehalose, 1% PEG 40000, 1% BSA, 0.4% Tween-20, 0.1% NaN₃。

And (4) formula: pH7.4, 0.01mol/L PBS, 5% trehalose, 1% PEG 40000, 1% PVP K-40, 1% BSA, 0.4% Tween-20, 0.1% NaN₃。

And (5) formula: pH7.4, 0.01mol/L PBS, containing 5% trehalose, 0.2% arginine, 1% PEG 40000, 1% PVP K-40, 1% BSA, 0.4% Tween-20, 0.1% NaN₃。

1.2.9 preparation of fluorescent test paper strip

The sample pad was first soaked in 20mmol/L phosphate buffer, pH7.4 for 20mmol/L phosphate buffer, 1.0% BSA, 0.25% Tween-20, 1% PVP K-40, 0.5% PEG 40000 and 0.1% NaN₃. Drying at 60 deg.C for 2 h; 2.5mg/mL of coating antigen and 0.3mg/mL of goat anti-mouse polyclonal antibody are sprayed on an NC membrane to be respectively used as a detection line and a quality control line of the sildenafil test strip, wherein the detection line is a T line, and the quality control line is a C line, and the samples are dried for 12 hours in vacuum at 37 ℃. The combined pad is sprayed with sildenafil antibody coupled fluorescent microspheres and dried for 12h in vacuum at room temperature. And finally, sequentially adhering the absorbent paper, the NC membrane, the combination pad and the pretreated sample pad on a PVC (polyvinyl chloride) bottom plate, cutting the test paper into test strips with the width of 4mm by using an automatic strip cutting machine, and performing vacuum drying at room temperature for later use.

1.2.10 establishment of quantitative Curve

In the case of negative conditions, the fluorescence signal of T-line was designated as FIT, that of C-line as FIC, and the FIT/FIC value as B₀FIT @ of standard solutions of sildenafilFIC is marked as B, and B/B is₀On the ordinate, the logarithm of the mass concentration of sildenafil standard solution is plotted on the abscissa, and a four-parameter fitting curve is established using Origin 8.0. With B/B₀And establishing a linear regression equation based on a least square method at a standard point of 0.2-0.8.

1.2.11 method stability evaluation

And selecting a health product sample which is confirmed to be sildenafil negative by chromatography, wherein the scalar of sildenafil is 0.5 mg/kg. The labeled and unlabeled samples were assayed using the same and different batches of fluorescent test strips in serial 6 d.

1.3 results and analysis

1.3.1 Artificial antigen identification

As shown in FIG. 1, by UV scanning and comparing with BSA of equal concentration, the maximum absorption peaks of immunogen and coatingen appear at 281nm and 272nm, respectively, while the maximum absorption peaks of immunohapten and coatingen appear at 288 nm. And BSA and OVA do not absorb after 305nm, while the absorption of immunogen and coating antigen is weakened after 320nm, which indicates that the two artificial antigens of sildenafil immunogen and coating antigen are successfully synthesized.

1.3.2 Artificial antigen coupling ratio calculation

BSA, OVA were made into solutions of 0, 0.1, 0.2, 0.5, 0.8, 1.0mg/mL in distilled water, and 0.5mL was added to 0.5mL of pH10.0 carbonate buffer (0.1mol/L Na)₂CO₃、NaHCO₃) To this solution, 0.5mL of 0.1% TNBS solution was added, and the mixture was reacted at room temperature for 15min and then subjected to colorimetry at a wavelength of 420 nm. And (3) making a standard curve according to the light absorption value and the protein concentration, wherein the slope of the curve is the light absorption value of the unit concentration of the standard protein as shown in figures 2 and 3. The coupling ratio of the artificial antigen was calculated according to step 1.2.3.3 and the results are shown in Table 2. The results of the TNBS assay showed that the artificial antigen conjugation ratios in AT-BSA and ATA-OVA were 13:1 and 10:1, respectively, and that the calculated conjugation ratios indicated that the amino groups on the protein had been attached, thus confirming that the antigen synthesis was successful.

TABLE 2 Artificial antigen coupling ratio

Immunogens	Absorbance per unit concentration	Coupling ratio	Coating source	Absorbance per unit concentration	Coupling ratio
						AT-BSA	0.36	13:1	ATA-OVA	0.20	10:1
BSA	0.36		OVA	0.28

1.3.3 selection of optimal immunogens

1.3.3.1 determination of potency

The absorbance values of ELISA were compared at 40000-fold dilution of antiserum, and the most potent antiserum of the three groups was selected as shown in Table 3 for the next sensitivity measurement.

1.3.3.2 determination of antiserum sensitivity

As can be seen from Table 4, when the immunizing antigen was used at an immunizing dose of 2mg, the sensitivity of the mouse serum was the highest, and therefore the mouse was selected for cell fusion.

TABLE 3 comparison of antiserum titers from different mice

Immunization dose (mg)	Mouse A	Mouse B	Mouse C
					1	1.62	1.44	1.60
2	1.88	0.87	1.53
				3	2.14	0.99	1.88
4	1.67	0.89	1.84
				5	1.22	1.11	1.65
6	1.35	1.43	0.81
				6	1.66	1.55	1.42
7	1.10	1.54	1.92
				8	0.98	1.43	1.53

TABLE 4 different antibody IC₅₀Value of

Immunization dose (mg)	IC₅₀(ng/mL)
		1	321
2	122
		3	359
4	543
		5	877
6	987
		6	843
7	788
		8	932

1.3.4 selection of monoclonal antibodies

Two monoclonal antibodies, 4C3 and 6F2, were screened by cell fusion techniques, i.e. the sensitivity was determined by ELISA in step 1.2.7.2. As shown in FIG. 4, the IC50 values for the two monoclonal antibodies were 2.1ng/mL and 15ng/mL, respectively. Therefore, 4C3 is used as an optimal antibody for the establishment of a subsequent immunochromatography method.

1.3.5 comparison of labeling methods

The fluorescent microsphere marking of the antibody is the key point for establishing the fluorescence immunochromatographic method and is also the experimental difficulty. The loss of the activity of the antibody can be caused by the over-high feeding ratio of the fluorescent microspheres and the antibody, and the reduction of the detection sensitivity can be caused by the over-low influence on the fluorescent signal intensity, so that the feeding ratio can not be correctly selected in the prior art, the workload is very large, the result is unstable, and the reagent batch-to-batch ratio is highThe difference is large. The invention relates to a method for marking affinity sites of a protective antibody, which is established by the method, the antibody is subjected to affinity on agarose gel of an immobilized antigen to protect the active sites of the antibody, and then excessive activated fluorescent microspheres are added to mark the fluorescent microspheres on the antibody as much as possible, so that the fluorescent signals of the antibody are greatly increased on the basis of not influencing the activity of the antibody, and the fluorescent signals of the labeled antibody prepared in each batch are uniform because the charge ratio of the fluorescent microspheres and the antibody is excessive. As shown in FIG. 5, the effect of the antibody active site protection labeling method established in the present invention and the conventional labeling method on the detection sensitivity is compared. The traditional method examines 3 feed ratios, wherein 1:30 optimum sensitivity, IC thereof₅₀The concentration was 136 ng/mL. And the method IC used in the present invention₅₀The sensitivity is improved to about 8 times of the original sensitivity when the concentration is 26.8 ng/mL. The affinity column after the labeling and elution by the method can be repeatedly used for 4-5 times, and the excessively added fluorescent microspheres can be continuously used after being centrifugally recovered, so that the labeling cost is greatly reduced. The method also performs enzyme labeling and biotin labeling of the antibody, and achieves good effects.

1.3.6 detection method parameters

As shown in fig. 6, the immunochromatographic method established based on the fluorescent microsphere labeled antibody of the protective marker detects the effect of the test strips of sildenafil with different mass concentrations at the excitation wavelength of 980 nm. According to the standard curve interpretation established by the scanner on the standard strip, the linear range is 1-81 ng/mL (R)²> 0.98) and the regression equation is-0.16 lnx +0.65 (R)²0.97). In addition, as shown in table 5, it shows that the method has cross-reaction with each kind of drug, indicating that it is a broad-spectrum screening method for that kind of drug. Table 6 shows that the method has no non-specific identification on other common illegal drugs in the health care product.

TABLE 5 Cross-reactivity with each nafil class

TABLE 6 Cross-reactivity with the commonly illegally added drugs of each health product

1.3.7 Effect of fluorescent antibody Diluent formulations on the Effect of reagent expiration date

The fluorescence immunochromatography is more and more used in the field of in vitro diagnosis because the sensitivity is greatly improved compared with the traditional colloidal gold immunochromatography method. However, the food sample matrix is more complex than the clinical sample, and the direct labeled fluorescein is easily interfered by the matrix to cause the quenching of the signal. According to the fluorescent microsphere, a large amount of fluorescent materials are wrapped in the polymer material, so that higher fluorescence intensity is shown, the interference of a matrix on a fluorescence signal can be effectively prevented, and the optical stability of the fluorescent materials is improved. Aiming at the problem that the particle diameter of the colloidal gold particles is 10-100 times that of the colloidal gold particles, the large-diameter particles are easy to gather and adsorb on a bonding pad after being placed for a long time, and effective chromatography cannot be achieved. If the antibody marked by the fluorescent microspheres is additionally added into the enzyme-labeled hole and is mixed uniformly manually in use, the use is inconvenient. In the invention, the fluorescent antibody is sprayed on the combination pad and is arranged at the front end of the sample pad, and the influence of 5 formulations of fluorescent antibody diluent on the effective period of the reagent is examined as shown in figure 7. Formula 1 is a formula of fluorescent antibody diluent which is relatively universal at present, however, after the fluorescent antibody diluent is sprayed on a combination pad, a fluorescent signal is greatly attenuated, and after a preservative is added in formula 2 and a trehalose is added in formula 3 to protect an antibody, although the result is improved, the commercial requirement cannot be met. After observing the test paper, the fluorescent microspheres form a precipitate which cannot be chromatographed on the binding pad. 1% of PVP K-40 is added into the formula 4, so that the dispersity of the fluorescent microspheres is improved, and the fluorescent signal after 6 months is greatly improved. The arginine protection fluorescence signal of 0.2 percent is added into the formula 5, so that the fluorescence attenuation of the compound in 6 months is less than 30 percent, and the commercialization condition is basically met. Therefore, formulation 5 was chosen as the optimal fluorescent antibody diluent.

1.3.8 method stability evaluation

The stability is an important index of the fluorescence immunochromatographic test strip. According to the invention, the test strips of the same batch and different batches prepared on the same day of the experiment are used for testing the same sample, negative samples are not detected, the result of testing the added samples is shown in table 7, and the adding amount is 0.5 mg/kg. The RSD of the test strip 6d of the same lot was 11% and the RSD of the different lots was 16%. The recovery rate is between 60 and 100 percent.

TABLE 7 Intra-lot and inter-lot differences of sildenafil fluorescence test paper

4.4 conclusion

The sildenafil-resistant fluorescent microsphere prepared by the antibody affinity site protection labeling method has the advantages of good affinity, large fluorescence intensity, small difference among batches and the like. And a fluorescent antibody diluent formula is provided, so that the fluorescence attenuation of the fluorescent labeled antibody integrally assembled on the test card for 6 months is less than 30%, and the technology can be commercially applied in the form of the test card. By matching with a simple one-step extraction pretreatment method, the whole detection process of the newly-built method only needs 15min, and large-scale equipment is not needed, so that the defects of the field monitoring technology of sildenafil are overcome, and the rapid quantitative detection is realized.

Claims

1. A method for rapidly detecting sildenafil in a health product is characterized by comprising the following steps:

C. preparing a sildenafil antibody-fluorescent microsphere conjugate, labeling fluorescent microspheres by adopting a protection affinity site method, coupling amino sildenafil on agarose gel containing aldehyde group activation, reducing formed unstable carbon-oxygen double bonds by using sodium cyanoborohydride, mixing the sildenafil antibody and the agarose gel after coupling, incubating at room temperature, loading into a centrifugal column, centrifuging to remove unbound antibody, and washing the gel for multiple times by using phosphate buffer solution PBS; adding fluorescent microspheres made of polystyrene into phosphate buffer solution PBS, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide for activation, then adding the activated fluorescent microspheres into a centrifugal column containing agarose gel, uniformly mixing the fluorescent microspheres by vortex, standing the mixture at room temperature for incubation, and centrifuging the mixture to remove liquid; eluting the agarose gel by using a glycine-hydrochloric acid buffer solution as an eluent; immediately adjusting the pH of the eluent to 7.0 by using sodium hydroxide to obtain a labeled sildenafil antibody-fluorescent microsphere conjugate;

D. constructing a fluorescent microsphere chromatography test strip, firstly soaking a sample pad in a phosphate buffer solution, and drying at 55-70 ℃; coating a coating antigen and goat anti-mouse polyclonal antibody on an NC membrane to be respectively used as a sildenafil test strip detection line and a quality control line, wherein the detection line is a T line, the quality control line is a C line, vacuum drying is carried out at 30-40 ℃, a sildenafil antibody coupling fluorescent microsphere is sprayed on a combination pad, vacuum drying is carried out at room temperature, finally, absorbent paper, the NC membrane, the combination pad and a pretreated sample pad are sequentially stuck on a PVC bottom plate, cut into test strips by an automatic slitter, and vacuum drying is carried out at room temperature for standby application, thus obtaining the fluorescent microsphere chromatography test strip;

E. establishing a quantitative curve, and recording the T-line fluorescence signal as FIT, the C-line fluorescence signal as FIC and the FIT/FIC value as B under the negative condition₀The FIT/FIC of the sildenafil standard solution is denoted as B, in terms of B/B₀Using the logarithm of the mass concentration of the sildenafil standard solution as the ordinate and the logarithm of the mass concentration of the sildenafil standard solution as the abscissa, and using Origin 8.0 to establish a four-parameter fitting curve, and using the B/B₀And establishing a linear regression equation based on a least square method at a standard point of 0.2-0.8.

F. Evaluating the stability of the method, selecting a health-care product sample which is confirmed to be sildenafil negative by chromatography, wherein the sildenafil scalar is 0.3-1mg/kg, and measuring the standard-added sample and the non-standard-added sample by using fluorescent test strips of the same batch and different batches continuously for multiple days;

G. and (4) comparing and observing the samples, namely, directly comparing and observing the inhibition conditions of the fluorescent microsphere immunochromatographic strip of the sildenafil in the sample to be detected and the standard series by naked eyes.

2. The method for rapidly detecting sildenafil in a health product according to claim 1, wherein in the step C, 2mL of 5mg/mL sildenafil antibody and 2mL of the agarose gel after coupling are mixed, incubated at room temperature for 10min, loaded on a spin column, centrifuged to remove unbound antibody, and the gel is washed 3 times with 6mL of 0.01mol/L Phosphate Buffered Saline (PBS) with pH 7.4; adding 0.2mg of fluorescent microspheres into 2.7mL of PBS (0.01 mol/L), adding 1.0mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide for activation, adding the activated microspheres into a centrifugal column containing agarose gel, uniformly mixing the microspheres by vortex, incubating the mixture at room temperature for 1 hour, and centrifuging the mixture to remove liquid; eluting the agarose gel twice by using 4mL of 0.1mol/L glycine hydrochloric acid buffer solution with pH of 3.0 as eluent; immediately adjusting the pH of the eluent to 7.0 by using 0.1mol/L sodium hydroxide to obtain the marked sildenafil antibody-fluorescent microsphere conjugate.

3. The method for rapidly detecting sildenafil in a health product according to claim 1, wherein in the step D, the sample pad is soaked in 20mmol/L phosphate buffer solution and dried at 60 ℃ for 2 h; 2.5mg/mL coating antigen and 0.3mg/mL goat anti-mouse polyclonal antibody are sprayed on an NC membrane to be respectively used as a detection line and a quality control line of a sildenafil test strip, wherein the detection line is a T line, and the quality control line is a C line, and is dried in vacuum for 12h at 37 ℃, a sildenafil antibody coupling fluorescent microsphere is sprayed on a combination pad, and is dried in vacuum for 12h at room temperature, finally, absorbent paper, the NC membrane, the combination pad and a pretreated sample pad are sequentially stuck on a PVC base plate, and are cut into test strips with the width of 4mm by an automatic slitter, and are dried in vacuum at room temperature for later use, so that the fluorescent microsphere chromatography test strip is obtained.

4. The method of claim 1, wherein in step F, the sildenafil addition amount is 0.5mg/kg, and the samples with or without added standard are measured by using fluorescence test strips of the same batch and different batches for 6d continuously.