CN112067515B - Dynamic light scattering immune method for homogeneously detecting macromolecular antigen - Google Patents
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Abstract
The invention relates to the field of antigen detection, in particular to a homogeneous phase immunization method for detecting a macromolecular antigen, which is a method for constructing a dynamic light scattering homogeneous phase immunization detection macromolecular antigen by taking phage as a detection antibody, taking capture antibody-marked multi-dendritic colloidal gold as a dynamic light scattering signal enhancement probe, and taking the average hydration kinetic particle size change of colloidal gold solution before and after a sandwich structure as dynamic light scattering signal output. The method provided by the invention has the advantages of high detection sensitivity, strong stability of the colloidal probe, high antibody-antigen binding efficiency, fewer washing and separating steps, simpler operation and higher immunological reaction efficiency.
Description
Technical Field
The invention relates to the technical field of antigen detection, in particular to an antigen detection technology for dynamic light scattering homogeneous phase immunological analysis, and specifically relates to a homogeneous phase immunological method for detecting a macromolecular antigen.
Background
Immunological analysis methods are simple, rapid, sensitive detection techniques developed based on specific recognition and reversible binding reactions between antigens and antibodies. Because of the advantages of strong specificity, high sensitivity, simple operation, low cost, suitability for screening of large-scale samples on site and the like, the current immunological analysis method is widely applied to the analysis fields of clinical diagnosis (such as nucleic acid, biomarker protein and the like), environmental detection (such as bacteria, pesticides, veterinary drugs, environmental hormone, heavy metal pollutants and the like), food safety (such as food-borne mycotoxins, pathogenic bacteria, food additives and the like) and the like.
The traditional ELISA adsorption method has the advantages of low technical condition requirement, convenient carrying, simple and convenient operation, economy, long effective period, strong specificity, capability of realizing mass detection, easy commercialization and the like, and usually appears in the form of a kit, thus becoming the biological detection and analysis technology which is most widely applied and developed and mature. The double-antibody sandwich ELISA method plays an important role in detecting pathogenic microorganisms simply and rapidly. However, the conventional double-antibody sandwich elisa has three distinct disadvantages: 1. horseradish peroxidase or alkaline phosphatase is adopted to catalyze the color development of a chemical substrate as signal output, however, the molar extinction coefficient of the color development substrate is low, so that the sensitivity is low; 2. the need to prepare the enzyme-labeled antigen by a physical/chemical method and the relatively high affinity of the enzyme-labeled antigen or the competing antigen to the antibody results in the competing antigen being difficult to compete with the target analyte, resulting in lower sensitivity; 3. the traditional ELISA method needs to separate through repeated incubation and washing steps, and the steps are complicated; in addition, heterogeneous solid-liquid reaction patterns make the binding efficiency of antigen-antibody not high, resulting in reduced sensitivity. Therefore, the sensitivity of detection signals is improved or the affinity of competing antigens to antibodies is reduced, the operation steps are simplified, and the immunological binding efficiency is improved, so that the sensitivity of the traditional double-antibody sandwich ELISA method can be effectively improved.
In the traditional double-antibody sandwich enzyme-linked immunosorbent method, macromolecular antigens such as pathogenic microorganisms or biological macromolecular proteins depend on a pair of paired antibodies with good specificity for detection of the macromolecular antigens due to the structural characteristics of a plurality of antigenic determinants (a plurality of antibody binding sites) on the surfaces of the macromolecular antigens. Wherein the detection antibody is used for coating on an ELISA plate, and the detection antibody is used for coupling with carrier proteins (such as horseradish peroxidase, alkaline phosphatase and the like) to prepare a signal probe. Phage not only have the ability to detect antibodies binding to macromolecular antigens, but also have larger dimensions relative to detecting antibody-carrier protein complexes. The recognition site of phage binding to macromolecular antigen screened from the antibody library only exists on the surface of pVIII protein with the width of terminal position-6 nm, so that phage is used as a detection antibody of macromolecular antigen, not only specifically recognizes target antigen, but also can generate particle size signals, and further higher detection sensitivity is obtained. In addition, the use of phage as a detection antibody circumvents the limitations of traditional chemical or biosynthetic carrier protein-antibody conjugate analogs, such as complex and cumbersome procedures, time and effort consuming, and occasional large numbers.
In the homogeneous phase immunoassay, the detection of the target can be realized by simply mixing the signal output probe with the sample without additional washing and separation steps. The change in signal depends on the recognition of the signaling probe with the target by an immunological reaction. Compared with the enzyme-linked immunosorbent assay which is widely accepted, the homogeneous phase immunoassay has the characteristics of simple operation, convenience, economy, quick response and the like. Based on the above advantages, in combination with some more sensitive detection signals (such as plasma resonance, raman scattering spectrum, dynamic light scattering signals, fluorescent signals, chemiluminescent signals, electrochemical signals, pressure signals, plasma resonance signals, etc.), a homogeneous immunoassay method developed in recent years has become the most competitive analytical detection platform in the field of on-site timely detection. Among them, the homogeneous immunosensor based on dynamic light scattering is receiving more and more attention due to its ultra-high sensitivity and specificity. Currently, homogeneous immunosensors based on dynamic light scattering have been widely used to detect a variety of chemical and biological targets, including small molecule compounds, proteins, pathogenic microorganisms, and the like.
Colloidal gold has been widely used for manufacturing various sensors due to its unique physicochemical and optical properties. Previous research reports indicate that the light scattering intensity of spherical colloidal gold is 2-3 orders of magnitude higher than that of polymer microspheres with the same particle size, so that strong light scattering signals can effectively shield interference signals generated by complex sample matrixes, and meanwhile, the colloidal gold has been widely used as an ideal signal probe as a signal enhancer in a dynamic light scattering sensor due to the advantage that the surface of the colloidal gold is easy to modify. Meanwhile, as the particle size of the colloidal gold increases, the light scattering cross section of the colloidal gold continuously increases; and when the particle size of the colloidal gold exceeds 80 nm, the scattering ability thereof increases drastically, so that the use of colloidal gold having a large particle size in a dynamic light scattering sensor can generate a stronger light scattering signal. In addition, our previous studies showed that the multi-dendritic colloidal gold (the morphology is similar to that of spherical surface with radial sharp branches or rough protrusions, etc., and can be divided into star, polygon, sea urchin, etc. due to the different shapes) has a stronger stability and a stronger light scattering ability than the spherical colloidal gold with the same particle size due to the enhanced three-dimensional surface of the surface tip-tip and core local electromagnetic fields. Therefore, the probe is more suitable for being used as a signal probe in a dynamic light scattering sensor. The excellent characteristics of the current multi-dendritic colloidal gold are attracting more and more attention, and the multi-dendritic colloidal gold is gradually applied to electrochemical technology, energy transfer technology, immunochromatography technology and other detection technologies. The invention provides a double-antibody sandwich dynamic light scattering homogeneous phase immunoassay method for detecting macromolecular antigens, which uses large-size phage to replace traditional complex preparation detection antibody formed by carrier protein coupled detection antibody, uses capture antibody coupled multi-dendritic colloidal gold, and is used as a signal output probe in a dynamic light scattering sensor.
Disclosure of Invention
Aiming at the technical defects of the prior art, the invention provides a detection method for a macromolecular antigen, which aims to solve the problems of low detection sensitivity, complex marking, low reaction efficiency of antigen-antibody reaction based on a semi-solid phase interface, unstable signal, false positive signal and the like of the traditional enzyme-linked immunosorbent assay for the macromolecular antigen in the prior art.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the method is aimed at the detection of the macromolecular antigen, and the carrier used for labeling the antibody in the method is multi-dendritic colloidal gold, and the multi-dendritic colloidal gold labeled with the antibody is simultaneously used as a signal probe of a dynamic light scattering sensor; the method, in which phages corresponding to the macromolecular antigens panned from the antibody library are used as competing antigens, outputs a dynamic light scattering signal as a signal. The method comprises the following steps:
(1) Labeling a macromolecular antigen specific monoclonal capture antibody on the surface of the multi-dendritic colloidal gold by using the multi-dendritic colloidal gold with a carboxyl chain modified on the surface as a carrier through an EDC one-step method to obtain capture antibody labeled multi-dendritic colloidal gold;
(2) Panning specific phage aiming at pathogenic microorganisms through an antibody library to obtain phage detection antibodies;
(3) Adding phage detection antibody and target object solution to be detected into the antibody-labeled multi-dendritic colloidal gold solution, reacting for 15-200min at 37 ℃, measuring the average hydration kinetic diameter of the solution on a Markov nanometer particle analyzer, and measuring the content of macromolecular antigen in a reaction sample to be detected by utilizing the change of the hydration kinetic diameter;
the macromolecular antigen is a protein marker or pathogenic microorganism having a plurality of antigenic determinants.
Further, the preparation method of the capture antibody labeled multi-dendritic colloidal gold in the step (1) comprises the following steps: synthesizing a multi-dendritic colloidal gold solution by using a colloidal gold seed mediated growth method, centrifuging the synthesized colloidal gold solution, replacing the solution with ultrapure water with the pH of 9.0-11.0, adding a sulfhydryl-carboxyl amphiphilic chain, and rotationally stirring at room temperature for 4-12h; centrifuging the mixed solution to remove redundant chains and obtain multi-dendritic colloidal gold with thiol carboxyl amphiphilic chains on the surface; adding the multi-dendritic colloidal gold with the surface sulfhydryl carboxyl amphiphilic chains into a buffer solution with the pH of 7.5PB (0.01 mol/L), adding a capture antibody, stirring at room temperature for reaction, adding EDC, stirring and supplementing twice, adding bovine serum albumin with the mass volume fraction of 10%, adding EDC, stirring at room temperature for 30 minutes, centrifuging, and separating the multi-dendritic colloidal gold coupled with the antibody to obtain the multi-dendritic colloidal gold marked by the capture antibody.
Further, step (2) panning the phage specific for the corresponding macromolecular antigen by means of the antibody repertoire, comprising the following operations: diluting macromolecular antigen with PBS to a final concentration of 10 μg/mL, adding 100 μl/well into the enzyme-labeled well, and coating at 4deg.C overnight; after washing the plate, sealing; washing the plate, adding 100 mu L of phage library into the enzyme-labeled well, reacting at 37 ℃, washing the plate after combining, washing unbound phage, adding acid for eluting, taking out specifically combined phage eluent, and neutralizing with a neutralizing solution until the pH value of the solution is neutral; amplifying the obtained eluent to obtain a large number of high-concentration specific phages; 25% glycerol was added and kept in a-20deg.C refrigerator for further use.
Further, taking the capture antibody-labeled multi-dendritic colloidal gold obtained in the step (1), carrying out gradient dilution by using PBS (phosphate buffer solution), measuring the average hydration kinetic diameter of the solution, and taking the lowest concentration with stable average hydration kinetic diameter of the solution as the use concentration of the colloidal gold probe; the amount of phage of the detection antibody that does not generate a dynamic light scattering signal was measured at the concentration at which the colloidal gold probe was used.
Further, the labeling amount of the capture antibody on the capture antibody-labeled multi-dendritic colloidal gold is determined by the maximum variation of the average hydration particle size of the solution.
Further, each buffer solution and the complex solution are used after being filtered by a 0.22 mu m filter membrane before being used.
In the method, the signal output substrate is multi-dendritic colloidal gold. 1) When the concentration of macromolecular antigen target in the solution is zero or even extremely low, the capture antibody on the surfaces of phage serving as a detection antibody and the multi-dendritic colloidal gold cannot be combined with the target antigen or cannot form a sandwich structure of three-in-one of phage-antigen-multi-dendritic colloidal gold, and the average hydration kinetic diameter of the solution is slightly increased or not increased compared with a blank value (the multi-dendritic colloidal gold marked with the capture antibody); 2) As the concentration of macromolecular antigen in the solution increases, the phage serving as a detection antibody and the capture antibody on the surface of the multi-dendritic colloidal gold are combined with the target antigen to form phage-antigen-multi-dendritic colloidal gold, the proportion of the three-in-one sandwich structure is gradually increased, and the average hydration kinetic diameter of the solution is increased more and more than a blank value (the multi-dendritic colloidal gold marked with the capture antibody); 3) Along with the change of the concentration of the macromolecular antigen, the average hydration kinetic diameter of the solution increases to generate linear change, and finally the quantitative and sensitive detection of the macromolecular antigen is realized.
The hydration kinetic diameter change is used for measuring the content of macromolecular antigens in a reaction sample to be detected, and in actual operation, macromolecular antigen standard solutions with known concentration and gradient distribution can be utilized, the solution particle size is taken as an ordinate, and the macromolecular antigen concentration is taken as an abscissa to draw a standard curve, so as to obtain a linear equation. When the actual sample detection is carried out, substituting the particle size increment value of the sample into a standard curve, reading the concentration of the corresponding sample from the standard curve, and multiplying the concentration by the corresponding dilution multiple to obtain the actual concentration of the macromolecular antigen in the sample. The specific method of operation may be arbitrarily selected according to common general knowledge in the art.
The method is suitable for quantitatively detecting pathogenic microorganisms, such as microorganisms, cancer markers, environmental harmful pathogenic microorganisms and the like, which have a plurality of antigenic determinants; the method is also suitable for quantitative detection of biological macromolecular proteins, such as biological macromolecular proteins with a plurality of antigenic determinants, such as disease protein biomarkers, environment-friendly macromolecular proteins and the like, and is particularly suitable for trace detection of target analytes. Sample treatment is carried out according to a conventional treatment method.
Compared with the prior art, the invention has the beneficial effects that:
the multi-dendritic colloidal gold has a larger light scattering cross section than spherical colloidal gold with the same particle size and concentration, so that the light scattering signal intensity of a dynamic light scattering analysis method can be greatly improved by a low-concentration multi-dendritic colloidal gold probe; on the other hand, the phage has the function of recognizing specific antigen, and the recognition unit is positioned at the end position of pVIII protein, so that false positive experimental results caused by crosslinking in the reaction process are avoided; meanwhile, the larger nanometer scale (the width is between 6 nanometers and the length is between 800 nanometers), so that the bacteriophage can generate larger particle size change than the conventional protein (usually about ten nanometers) after being combined with the target antigen and the nanometer probe marked by the capture antibody to form a sandwich structure, thereby widening the regulation and control range of dynamic light scattering signals; in addition, phage that are detection antibodies panned from the antibody repertoire have higher affinity for antigen. The detection sensitivity of the double-antibody sandwich immunoassay method can be improved by improving the light scattering intensity of the probe, reducing the dosage of the antibody and increasing the particle size of the detection antibody; in addition, compared with the traditional double-antibody sandwich immunoassay method, the novel method only needs to test after sample addition and mixing, and does not need repeated washing and separation processes, so that the operation is simpler and more convenient, the reagent is easier to store, and the detection sensitivity is higher.
Drawings
FIG. 1 is a schematic diagram of the method of the present invention;
FIG. 2 is a standard curve for detecting Escherichia coli O157:H27 by a multi-branched colloid Jin Dongtai light scattering homogeneous phase immunoassay method;
FIG. 3 is a standard curve for detection of norovirus based on a multi-branched colloid Jin Dongtai light scattering homogeneous immunoassay.
FIG. 4 is a standard curve of a multi-dendritic colloidal gold-based dynamic light scattering homogeneous immunoassay for alpha fetoprotein;
FIG. 5 is a standard curve of a dynamic light scattering homogeneous immunoassay based on a multi-dendritic colloidal gold for cardiac troponin I;
FIG. 6 is a standard curve of a multi-dendritic colloidal gold-based dynamic light scattering homogeneous immunoassay for hepatitis B surface antigen;
FIG. 7 is a standard curve of a dynamic light scattering homogeneous immunoassay based on a multi-dendritic colloidal gold for HIV p24 antigen;
FIG. 8 is a standard curve of a dynamic light scattering homogeneous immunoassay based on a multi-branched colloidal gold for C-reactive protein.
Detailed Description
The present invention will be described in further detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The method for preparing the phosphate buffer (PBS, 0.05M,pH 7.4) comprises the following steps: naCl 40g, na 2 HPO 4 13.5g,KH 2 PO 4 1.0g of KCl 1.0g was dissolved in 1L of ultrapure water. The pH value is adjusted to 8.0-9.0 by 0.1M NaOH.
The murine IgG class monoclonal antibodies referred to in the examples: anti-norovirus monoclonal antibodies, anti-E.coli O157: H7 antibodies, anti-alpha fetoprotein monoclonal antibodies, anti-cardiac troponin I antibodies, anti-hepatitis B surface antigen monoclonal antibodies, anti-HIV p24 antigen monoclonal antibodies, and anti-C reactive protein monoclonal antibodies, purchased from Sigma, abcam, beijing heat scenery organisms, and the like.
Example 1 application for detecting macromolecular antigen content
Preparation of 1-carboxyl-modified multi-dendritic colloidal gold
1) Synthesizing multi-dendritic colloidal gold by a high-temperature one-step method; heating 100mL of ultrapure water system to 57 ℃, slowly stirring on a magnetic stirrer, closing heat, and sequentially adding 2.5mL of 60nm seed gold and 1.5mL of%HAuCl 4 The solution and 2.64mL of 1% trisodium citrate solution are added with 24mL of 30mmol/L hydroquinone solution at one time rapidly, the reaction is continued for 0min, the solution is cooled to room temperature, and the solution is preserved at 4 ℃ for standby.
The synthesis method of 60nm seed gold comprises the following steps: a) Synthesizing 18-20nm colloidal gold by a citric acid reduction method, adding 1mL 1% chloroauric acid solution into 99mL ultrapure water, and heating to boiling (large bubbles appear in about 20 min) under the condition of slow and uniform stirring; b) 2.7mL of 1% trisodium citrate (Na 3 C 6 H 5 O 7 ) The solution is rapidly stirred for 10min; stopping heating when the color of the solution changes to wine red and no change occurs, cooling to room temperature under stirring, and preserving at 4deg.C for use. c) 1mL of the colloidal gold synthesized in b) is added into 100mL of ultrapure water solution, the mixture is vigorously stirred, then 0.8mL of 1% (mass volume fraction) chloroauric acid solution is added, after uniform stirring, 0.2mL of 1% (w/v) trisodium citrate solution and 0.1mL of hydroquinone solution (30 mM) are rapidly added as reducing agents of a reaction system, two reducing agents are added at intervals of 10min, and the cycle is 5 times. Stirring at room temperature for 30min, and storing at 4deg.C for use.
2) Centrifuging the synthesized colloidal gold solution, replacing the solution with ultrapure water with the pH of 9.0-11.0, adding a sulfhydryl-carboxyl amphiphilic chain, and rotationally stirring at room temperature for 4-12h; 10mL of the synthesized multi-dendritic colloidal gold is taken, the mixture is centrifuged at 3000rpm/min for 15min at 4 ℃,1mL of ultrapure water solution with pH more than 9 is used for re-dissolving the precipitate, 20 mug of amphiphilic mercapto carboxyl chain is added, and the mixture is placed on a vertical mixer at room temperature for stirring reaction for 4h.
3) Centrifuging the mixed solution in the step 2), removing redundant chains at the rotating speed of 3000rmp/min for 15 minutes, redissolving the multi-dendritic colloidal gold with the surface connected with the amphiphilic chains in ultrapure water, and storing in a refrigerator at the temperature of 4 ℃ for later use.
Preparation of 2 capture antibody labeled multi-dendritic colloidal gold probe
Adding 12 mu L of the multi-dendritic colloidal gold modified with the sulfhydryl carboxyl amphiphilic chain into 500 mu L of buffer solution with pH of 7.5PB (0.01 mol/L), adding 6-12 mu g of capture antibody corresponding to macromolecular antigen, and stirring and reacting for 30 minutes at room temperature; the reaction was continued with stirring, and after 0.25. Mu.g of 1-ethyl- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was added, the mixture was stirred at room temperature for 30 minutes, and two additional portions of 50. Mu.L of bovine serum albumin having a mass/volume fraction of 10% were added, and 0.25. Mu.g of 1-ethyl- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was added. After stirring at room temperature for 30 minutes, the antibody-coupled multi-dendritic colloidal gold was separated by centrifugation, and the separated multi-dendritic colloidal gold was washed three times with ultrapure water. The washed multi-dendritic colloidal gold is redissolved in ultrapure water and stored at 4 ℃.
3 preparation of detection antibody phage
Panning phage specific to the corresponding macromolecular antigen by using an antibody library as a detection antibody, wherein the method specifically comprises the following steps of:
1) Diluting AFP antigen with PBS to a final concentration of 10 μg/mL (second round and third round of panning antigen coating concentration are 5 μg/mL and 2.5 μg/mL respectively), adding 100 μl/well into enzyme-labeled wells, and coating at 4deg.C overnight; the method comprises the steps of carrying out a first treatment on the surface of the
2) The coating solution is discarded, washed 3 times by PBS, 300 mu L of 3% BSA-PBS blocking solution (OVA-PBS and BSA-PBS are respectively used for the second round and the third round) is added into each hole, and the mixture is blocked for 2 hours at 37 ℃;
3) PBS was washed 6 times, 100. Mu.L phage library was added, and the number of phages was about 6.2X10 11 cfu (phage library input of the second round and third round was 8.4X10, respectively) 11 cfu、1.1×10 12 cfu), incubation at 37 ℃ for 1.5h (binding time of the second round and the third round is 1 h);
4) Unbound phage were aspirated and washed 8 times with PBST (12 and 15 times for the second and third rounds, respectively) and 8 times with PBS (12 and 15 times for the second and third rounds, respectively);
5) Adding 100 mu L Gly-HCl eluent, incubating for 6-8 min at 37 ℃, and eluting specifically bound phage; transferring the eluate to a sterile centrifuge tube, and rapidly neutralizing with 12 mu LTris-HCl neutralization buffer;
6) Adding the eluent obtained in the step 5) into 20mL of escherichia coli Escherichia coli ER2738 culture solution in the early logarithmic growth phase for amplification culture at 37 ℃ for 4.5 hours;
7) Taking 10 mu L to carry out gradient dilution, measuring titer, calculating elutriation recovery rate, mixing other eluents, amplifying and purifying for next round of affinity elutriation;
8) Amplification of library after panning:
a) Mixing elutriation eluate with 3mL of E.coli ER2738 culture at logarithmic growth phase, shake culturing at 37deg.C at 220r/min for 45min, transferring to 20mL of 2 XYT-A liquid culture medium, shake culturing at 37deg.C at 220r/min for 2h, and mixing with the culture medium according to cell: phage=1: 20, adding M13K07 phage according to the proportion, standing for 15min at 37 ℃, and culturing for 30-45 min at 220r/min in a shaking way;
b) Sub-packaging the culture obtained in the step a) into a centrifuge tube, culturing at 4 ℃ at 3500r/min for 10min, re-suspending the cell sediment with 25mL of 2 XYT-AK liquid culture medium at 30 ℃ at 250r/min for overnight:
c) Centrifuging overnight culture at 4deg.C for 15min at 12000r/min, transferring supernatant to a new centrifuge tube, adding 1/5 volume of PEG-NaCl, mixing, and standing at 4deg.C for more than 1 hr;
d) Removing supernatant at 4deg.C for 12000r/min and 15min, re-suspending the precipitate in 1mL PBS, adding 1/5 volume of PEG/NaCl, mixing, and standing at 4deg.C for more than 1 hr;
e) 12000r/min,5min, removing supernatant, suspending the precipitate in 200 μl PBS to obtain amplified product, and determining titer for next round of panning or analysis.
9) Rescue of phage:
a) Randomly picking 48 monoclonals from a third round of elutriation eluate titer plate (the colony number is 30-200), inoculating the monoclonals into 1mL of 2 XYT-GA, and culturing at 37 ℃ under 220r/min in a shaking way for 12h;
b) Inoculating to 2 XYT-GA according to 1% inoculum size, culturing at 37deg.C and 220r/min to logarithmic growth prophase;
c) According to the cell: phage=1: 20, adding M13K07 phage according to the proportion, standing for 15min at 37 ℃, and shaking and culturing for 30-45 min at 220r/min
d) Centrifuging at 4deg.C at 3500r/min for 10min, re-suspending the precipitate with equal volume of 2 XYT-AK, and shaking vigorously at 30deg.C for overnight;
e) The next day was centrifuged at 12000rpm for 10min, the supernatant was discarded, and the precipitate was reconstituted with 25% glycerol solution and stored at-20℃for further use.
4 detecting the content of macromolecular antigen
When the novel dynamic light scattering homogeneous phase immunity detection method is used for detecting the content of macromolecular antigens, the method is implemented through the following steps: sample pretreatment, detection and analysis result by the detection method.
1) Sample pretreatment: diluting the purchased pathogenic microorganism standard substance to a corresponding concentration gradient, wherein the concentration is determined according to the required actual detection limit; the diluted antigen sample is put into a refrigerator with the temperature of 4 ℃ for standby. The purification methods of target analytes in different samples to be detected are specifically finished by referring to national standards.
2) The detection method of the present invention is used to detect the contents of macromolecular antigens having a plurality of antigenic determinants, such as microorganisms, cancer markers, and environmentally harmful macromolecular proteins/pathogenic microorganisms.
3) And analyzing the result.
The average hydration kinetic diameters corresponding to the solutions were measured on a malvern nanoparticle analyzer using the standards prepared at different concentrations as described above.
And drawing a standard curve by taking the grain size of the solution as an ordinate and the concentration of the macromolecular antigen as an abscissa, and solving a linear equation. When the actual sample detection is carried out, substituting the particle size increment value of the sample into a standard curve, reading the concentration of the corresponding sample from the standard curve, and multiplying the concentration by the corresponding dilution multiple to obtain the actual concentration of the macromolecular antigen in the sample.
For detection of the concentration of pathogenic microorganisms, the standard may be selected from, for example, 0CFU/mL, 100CFU/mL, 10 1 CFU/mL、10 2 CFU/mL、10 3 CFU/mL、10 4 CFU/mL、10 5 Concentration gradient of CFU/mL.
For detection of protein marker concentration, the standard may be selected from, for example, a concentration gradient of 0pg/mL, 0.19pg/mL, 0.39pg/mL, 0.78pg/mL, 1.56pg/mL, 3.12pg/mL, 6.25pg/mL, 12.5pg/mL, 25pg/mL, 50pg/mL, 100pg/mL, and 1000 pg/mL.
Example 2 food-borne pathogenic bacterium, E.coli O157: H7 as specimen
100. Mu.L of E.coli O157:H7 capture antibody-labeled multi-dendritic colloidal gold (0.02 pM) was specifically bound to 150. Mu.L of E.coli O157:H7 solution, 150. Mu.L of E.coli O157:H7 phage (titer 2.1X10) 9 ) The mixture was incubated at 37℃for 100 minutes, and the change in average hydration kinetic diameter of the solution was measured at 25℃using a Markov nanosize analyzer. And obtaining the concentration of the escherichia coli O157:H7 in the sample to be detected by substituting the average value into a standard curve. The specific experimental results are as follows: the linear standard curve is y= 0.9466ln (x) +129, r 2 = 0.9864, see fig. 2. The minimum limit of detection for this method is defined as the average hydrated particle size at 20 first standards (average hydrated particle size of solution at 0 standard) plus 3 standard deviations (standard deviation of three parallel samples of 3 times the first standard sample) of the desired antigen concentration. The lowest detection line was calculated to be 2.5CFU/mL from the standard curve.
The method is not limited to E.coli O157: h7 detection can also be used for detecting other food-borne pathogenic bacteria, such as Listeria monocytogenes, bacillus cereus, salmonella, enterobacter campylobacter, shigella, enterobacter sakazakii, vibrio parahaemolyticus, and Staphylococcus aureus.
Example 3 food-borne Virus-norovirus as test substance
100. Mu.L of norovirus capture antibody labeled multi-dendritic colloidal gold (0.0125 pM) was used to capture antibodies against different concentrations of norovirus standard 150. Mu.L, detection antibody phage corresponding to norovirus 150. Mu.L (titer 10) 9 ) The mixture was incubated at 37℃for 100 minutes, and the change in average hydration kinetic diameter of the solution was measured at 25℃using a Markov nanosize analyzer. And obtaining the concentration of the norovirus in the sample to be detected by substituting the average value into a standard curve. The specific experimental results are as follows: the linear standard curve is y= 0.9648ln (x) +131.02, r 2 = 0.9793, see fig. 3. The minimum limit of detection for this method is defined as the average hydrated particle size at 20 first standards (average hydrated particle size of solution at 0 standard) plus 3 standard deviations (standard deviation of three parallel samples of 3 times the first standard sample) of the desired antigen concentration. The lowest detection line was calculated to be 3.2PFU/mL from the standard curve.
The method is not limited to the detection of norovirus, but can also be used for the detection of other food-borne viruses, such as avian influenza virus, foot-and-mouth disease virus, rotavirus, adenovirus and the like.
Example 4 cancer marker protein-alpha fetoprotein as test substance
100. Mu.L of alpha-fetoprotein capturing antibody labeled multi-dendritic colloidal gold (0.0125 pM) and 150. Mu.L of alpha-fetoprotein standard with different concentrations, 150. Mu.L of detection antibody phage (titer 10) 9 ) The mixture was incubated at 37℃for 100 minutes, and the change in average hydration kinetic diameter of the solution was measured at 25℃using a Markov nanosize analyzer. And obtaining the concentration of alpha fetoprotein in the sample to be detected by substituting the average value into a standard curve. The specific experimental results are as follows: the linear standard curve is y= 0.9534ln (x) +132.1, r 2 = 0.9762, see fig. 4. The minimum limit of detection of this method is defined as the average hydrated particle size at 20 first standards (average hydrated particle size of solution at 0 standard) plus 3 standard deviations (3 times the first standard sample three parallel samples)Standard deviation), the desired antigen concentration. The lowest detection line was calculated to be 0.18pg/mL by this standard curve.
The method is not limited to the detection of alpha fetoprotein, and can be used for the detection of other cancer marker proteins, prostate specific antigen, carcinoembryonic antigen and the like.
Example 5 cardiovascular disease marker protein-cardiac troponin I as test substance
100. Mu.L of cardiac troponin I capture antibody labeled multi-dendritic colloidal gold (0.02 pM) was specifically bound to 150. Mu.L of cardiac troponin I solution at different concentrations, 150. Mu.L of phage (titer 2.1X10) 9 ) The mixture was incubated at 37℃for 100 minutes, and the change in average hydration kinetic diameter of the solution was measured at 25℃using a Markov nanosize analyzer. And obtaining the concentration of the cardiac troponin I in the sample to be detected by substituting the average value into a standard curve. The specific experimental results are as follows: the linear standard curve is y= 0.9862ln (x) +127.7, r 2 = 0.9813, see fig. 5. The minimum limit of detection for this method is defined as the average hydrated particle size at 20 first standards (average hydrated particle size of solution at 0 standard) plus 3 standard deviations (standard deviation of three parallel samples of 3 times the first standard sample) of the desired antigen concentration. The lowest detection line was calculated to be 0.31pg/mL by this standard curve.
The method is not limited to the detection of cardiac troponin I, but can also be used for the detection of other cardiovascular disease marker proteins, such as Creatine Kinase (CK), creatine kinase isozymes (CK-MB), myoglobin (Mb/Myo), B Natriuretic Peptide (BNP), N-terminal pre-B natriuretic peptide (NT-proBNP), and the like.
Example 6 liver disease marker protein-hepatitis B surface antigen as test substance
100 mu L of hepatitis B surface antigen capture antibody labeled multi-dendritic colloidal gold (0.02 pM) specifically bound to 150 mu L of hepatitis B surface antigen solution of different concentrations and 150 mu L of hepatitis B surface antigen phage (titer 2.1X10) 9 ) The mixture was incubated at 37℃for 100 minutes, and the change in average hydration kinetic diameter of the solution was measured at 25℃using a Markov nanosize analyzer. By calculating the average value and substituting it into the standardAnd obtaining the concentration of hepatitis B surface antigen in the sample to be detected by a curve. The specific experimental results are as follows: the linear standard curve is y=0.8954 ln (x) +128.5, r 2 = 0.9768, see fig. 6. The minimum limit of detection for this method is defined as the average hydrated particle size at 20 first standards (average hydrated particle size of solution at 0 standard) plus 3 standard deviations (standard deviation of three parallel samples of 3 times the first standard sample) of the desired antigen concentration. The lowest detection line was calculated to be 0.29pg/mL by this standard curve.
The method is not limited to the detection of hepatitis B surface antigen, but can also be used for the detection of liver disease marker proteins, such as hepatitis B surface antibody, core antibody, e antigen, e antibody, hepatitis C virus core antigen, hepatitis E virus antibody, etc.
Example 7 AIDS marker protein-HIVp 24 antigen as test object
100 mu LHIVp24 antigen capture antibody labelled multi-dendritic colloidal gold (0.02 pM) with 150 mu L HIV p24 antigen solution at different concentrations, phage with 150 mu LHIV p24 antigen specific binding (titer 2.1X10) 9 ) The mixture was incubated at 37℃for 100 minutes, and the change in average hydration kinetic diameter of the solution was measured at 25℃using a Markov nanosize analyzer. And obtaining the concentration of the HIVp24 antigen in the sample to be detected by substituting the calculated average value into a standard curve. The specific experimental results are as follows: the linear standard curve is y= 0.9142ln (x) +130.4, r 2 = 0.9824, see fig. 7. The minimum limit of detection for this method is defined as the average hydrated particle size at 20 first standards (average hydrated particle size of solution at 0 standard) plus 3 standard deviations (standard deviation of three parallel samples of 3 times the first standard sample) of the desired antigen concentration. The lowest detection line was calculated to be 0.42pg/mL by this standard curve.
The method is not limited to detection of HIV p24 antigen, but can also be used for detection of other HIV marker proteins, such as HIV 1/2 antibody, HIV gp120 antigen, etc.
Example 8 inflammatory marker protein-C-reactive protein as test substance
100 mu LC-reactive protein capture antibody-labeled multi-dendritic colloidal gold (0.02 pM) and150. Mu.L of C-reactive protein solution of different concentrations, phage to which 150. Mu.L of LC-reactive protein specifically binds (titer 2.1X10) 9 ) The mixture was incubated at 37℃for 100 minutes, and the change in average hydration kinetic diameter of the solution was measured at 25℃using a Markov nanosize analyzer. And obtaining the concentration of the C-reactive protein in the sample to be detected by substituting the calculated average value into a standard curve. The specific experimental results are as follows: the linear standard curve is y= 0.8796ln (x) +128.9, r 2 = 0.9913, see fig. 8. The minimum limit of detection for this method is defined as the average hydrated particle size at 20 first standards (average hydrated particle size of solution at 0 standard) plus 3 standard deviations (standard deviation of three parallel samples of 3 times the first standard sample) of the desired antigen concentration. The lowest detection line was calculated to be 0.39pg/mL from this standard curve.
The method is not limited to detection of C-reactive protein, but can be used for detection of other inflammatory marker proteins, such as hypersensitive C-reactive protein (hs-CRP), interleukin-6 (IL-6), tumor necrosis factor-alpha, procalcitonin (PCT) human serum amyloid A (SAA 1), human trypsinogen 2 (PRSS 2), lipopolysaccharide Binding Protein (LBP), etc.
Claims (3)
1. A homogeneous immunization method for detecting macromolecular antigens is characterized in that a specific phage is used as a detection antibody and a capture antibody labeled multi-dendritic colloidal gold matched with the detection antibody is used as a dynamic light scattering signal enhancement probe, so that the average hydration kinetic particle size change of a solution before and after a sandwich structure is formed and is used as dynamic light scattering signal output, and the hydration kinetic particle size change is used for measuring the content of the macromolecular antigens in a reaction sample to be detected, and the method comprises the following steps:
(1) Labeling a macromolecular antigen specific monoclonal capture antibody on the surface of the multi-dendritic colloidal gold by using the multi-dendritic colloidal gold with a carboxyl chain modified on the surface as a carrier through an EDC one-step method to obtain capture antibody labeled multi-dendritic colloidal gold;
(2) Panning specific phage aiming at pathogenic microorganisms through an antibody library to obtain phage detection antibodies; the specific phage has a recognition unit of a specific antigen, and the recognition unit is positioned at the pIII protein terminal position;
(3) Adding phage detection antibody and target object solution to be detected into the antibody-labeled multi-dendritic colloidal gold solution, reacting for 15-200min at 37 ℃, measuring the average hydration kinetic diameter of the solution on a Markov nanometer particle analyzer, and measuring the content of macromolecular antigen in a reaction sample to be detected by utilizing the change of the hydration kinetic diameter;
the macromolecular antigen is a protein marker or pathogenic microorganism with a plurality of antigenic determinants;
taking the capture antibody marked multi-dendritic colloidal gold obtained in the step (1), carrying out gradient dilution by using PBS, measuring the average hydration kinetic diameter of the solution, and taking the lowest concentration with stable average hydration kinetic diameter of the solution as the use concentration of the colloidal gold probe; measuring the amount of the detection antibody phage which does not generate a dynamic light scattering signal under the use concentration of the colloidal gold probe;
the labeling amount of the capture antibody on the capture antibody labeled multi-dendritic colloidal gold is determined by the maximum variation of the average hydration particle size of the solution.
2. The homogeneous immunization method for detecting macromolecular antigens according to claim 1, wherein said preparation method of capture antibody-labeled multi-dendritic colloidal gold in step (1) comprises the steps of: synthesizing a multi-dendritic colloidal gold solution by using a colloidal gold seed mediated growth method, centrifuging the synthesized colloidal gold solution, replacing the solution with ultrapure water with the pH of 9.0-11.0, adding a sulfhydryl-carboxyl amphiphilic chain, and rotationally stirring at room temperature for 4-12h; centrifuging the mixed solution to remove redundant chains and obtain multi-dendritic colloidal gold with thiol carboxyl amphiphilic chains on the surface; adding the multi-dendritic colloidal gold with the surface sulfhydryl carboxyl amphiphilic chains into PB buffer solution with the pH of 7.5 and the concentration of 0.01mol/L, adding a capture antibody, stirring at room temperature for reaction, adding EDC, stirring and supplementing twice, adding bovine serum albumin with the mass volume fraction of 10%, adding EDC, stirring at room temperature for 30 minutes, centrifuging, and separating the multi-dendritic colloidal gold coupled with the antibody to obtain the multi-dendritic colloidal gold marked by the capture antibody.
3. A homogeneous immunization method for detecting a macromolecular antigen according to claim 1, wherein step (2) panning for phage specific for the corresponding macromolecular antigen by a pool of antibodies comprises the following operations: diluting macromolecular antigen with PBS to a final concentration of 10 μg/mL, adding 100 μl/well into the enzyme-labeled well, and coating at 4deg.C overnight; after washing the plate, sealing; washing the plate, adding 100 mu L of phage library into the enzyme-labeled well, reacting at 37 ℃, washing the plate after combining, washing unbound phage, adding acid for eluting, taking out specifically combined phage eluent, and neutralizing with a neutralizing solution until the pH value of the solution is neutral; amplifying the obtained eluent to obtain a large number of high-concentration specific phages; 25% glycerol was added and kept in a-20deg.C refrigerator for further use.
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