CN112067515A - Dynamic light scattering immunization method for homogeneous detection of macromolecular antigen - Google Patents
Dynamic light scattering immunization method for homogeneous detection of macromolecular antigen Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention relates to the field of antigen detection, in particular to a homogeneous phase immunoassay method for detecting a macromolecular antigen, which takes a bacteriophage as a detection antibody, captures multi-branch colloidal gold marked by the antibody as a dynamic light scattering signal enhancement probe, outputs the average hydration kinetic particle size change of colloidal gold solution before and after forming a sandwich structure as a dynamic light scattering signal, and constructs the method for detecting the macromolecular antigen by the dynamic light scattering homogeneous phase immunoassay. The method provided by the invention has the advantages of high detection sensitivity, strong stability of the colloidal probe, high antibody-antigen binding efficiency, few washing and separating steps, simpler operation and higher immunological reaction efficiency.
Description
Technical Field
The invention relates to the technical field of antigen detection, further relates to an antigen detection technology of dynamic light scattering homogeneous immunological analysis, and particularly relates to a homogeneous immunological method for detecting a macromolecular antigen.
Background
The immunological analysis method is a simple, rapid and sensitive detection technology developed based on the specific recognition and reversible binding reaction between antigen and antibody. Because immunoassay has the advantages of strong specificity, high sensitivity, simple operation, low cost, suitability for on-site large-scale sample screening and the like, the existing immunoassay 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 hormones, 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 enzyme-linked immunosorbent assay is often in the form of a kit and has become a biological detection and analysis technology which is most widely applied and developed due to the advantages of low technical condition requirement, convenient carrying, simple and economic operation, long validity period, strong specificity, capability of realizing large-scale detection, easy commercialization and the like. The double-antibody sandwich enzyme-linked immunosorbent assay plays an important role in the detection of pathogenic microorganisms due to simplicity and rapidness. However, the traditional double antibody sandwich enzyme-linked immunosorbent assay has three distinct disadvantages: firstly, horseradish peroxidase or alkaline phosphatase is adopted to catalyze a chemical substrate to develop color to be used as signal output, however, the developing substrate is low in molar extinction coefficient, and therefore sensitivity is low; secondly, enzyme-labeled antigen is prepared by a physical/chemical method, and the affinity of the enzyme-labeled antigen or competitive antigen and an antibody is relatively high, so that the competitive antigen is difficult to compete by a target analyte, and the sensitivity is low; thirdly, the traditional enzyme-linked immunosorbent assay needs to be separated through repeated incubation and washing steps, and the steps are complicated; in addition, the heterogeneous solid-liquid reaction mode makes the binding efficiency of antigen-antibody low, resulting in a decrease in sensitivity. Therefore, the sensitivity of the detection signal is improved or the affinity of the competitive antigen to the antibody is reduced, the operation steps are simplified, and the immunological binding efficiency is improved, so that the sensitivity of the traditional double-antibody sandwich enzyme-linked immunosorbent assay can be effectively improved.
In the traditional double-anti sandwich enzyme-linked immunosorbent assay, a macromolecular antigen such as a pathogenic microorganism or a biological macromolecular protein is required to depend on a pair of pairing antibodies with good specificity for detecting the macromolecular antigen due to the structural characteristics of a plurality of antigenic determinants (a plurality of antibody binding sites) on the surface of the macromolecular antigen. Wherein the detection antibody is used for coating on an enzyme label plate, and the detection antibody is used for coupling with carrier protein (such as horseradish peroxidase, alkaline phosphatase and the like) to prepare a signal probe. The phage not only has the ability to detect binding of the antibody to the macromolecular antigen, but also has a larger size relative to the detection antibody-carrier protein complex. The recognition site of the phage combined with the macromolecular antigen screened from the antibody library only exists on the surface of the pVIII protein with the width of end position-6 nm, so that the phage is used as the detection antibody of the macromolecular antigen, the target antigen is specifically recognized, particle size signals can be generated, and higher detection sensitivity is obtained. In addition, the phage is used as a detection antibody, so that the limitations of traditional chemical synthesis or biosynthesis of carrier protein-antibody coupling analogues, such as complex operation, time and labor waste, high contingency and the like, are avoided.
The homogeneous phase immunoassay method can realize the detection of the target object by simply mixing the signal output probe with the sample without additional washing and separation steps. The change in signal depends on the reaction of the signaling probe with the recognition of the target by an immunological reaction. Compared with the widely accepted enzyme-linked immunosorbent assay, the homogeneous immunoassay method has the characteristics of simple operation, convenience, economy, quick response and the like. Based on the above advantages, the homogeneous immunoassay developed by combining some more sensitive detection signals (such as plasmon resonance, raman scattering spectrum, dynamic light scattering signal, fluorescence signal, chemiluminescence signal, electrochemical signal, pressure signal, plasmon resonance signal, etc.) has become the most competitive analysis and detection platform in the field of on-site and in-time detection. Among them, homogeneous immunosensors based on dynamic light scattering have received increasing attention due to their ultra-high sensitivity and specificity. At present, homogeneous immunosensors based on dynamic light scattering have been widely used to detect various chemical and biological targets, including small molecule compounds, proteins, pathogenic microorganisms, and the like.
Colloidal gold has been widely used to produce a variety of sensors due to its unique physicochemical and optical properties. Earlier research reports show 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 a strong light scattering signal can effectively shield an interference signal generated by a complex sample matrix, and meanwhile, the colloidal gold is widely applied as an ideal signal probe as a signal intensifier 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 is sharply increased, so that a stronger light scattering signal can be generated using the colloidal gold having a large particle size in the dynamic light scattering sensor. In addition, previous researches show that the multi-branch colloidal gold (the shape is similar to that of a spherical surface, radial sharp branches or rough bulges are distributed on the spherical surface, and the shape can be divided into a star shape, a polygonal shape, a sea-gall-shaped shape and the like due to different shapes) is endowed with stronger stability and stronger light scattering capacity than that of the spherical colloidal gold with the same particle size due to the three-dimensional surface enhanced by the local electromagnetic field from the tip to the tip of the surface and the core. Therefore, it is more suitable for use as a signal probe in a dynamic light scattering sensor. The excellent characteristics of the multi-branch colloidal gold make the multi-branch colloidal gold attract more and more attention at present, and the multi-branch 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 immunoassay method for detecting macromolecular antigens, which uses large-size phage to replace the traditional carrier protein coupled detection antibody to form a compound for preparing a detection antibody, uses a capture antibody coupled multi-dendritic colloidal gold as well as a signal output probe in a dynamic light scattering sensor, and is used for constructing the double-antibody sandwich dynamic light scattering homogeneous immunoassay method for detecting the macromolecular antigens.
Disclosure of Invention
The invention aims to provide a detection method for a macromolecular antigen aiming at the technical defects of the prior art, and aims to solve the problems that the traditional enzyme-linked immunosorbent assay for the macromolecular antigen in the prior art is low in detection sensitivity and tedious in labeling, and the antigen-antibody reaction is based on low semi-solid phase interface reaction efficiency, unstable in signal, false positive signal and the like.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a detection method for macromolecule antigen, said method belongs to the double-antibody sandwich immunoassay, said method is to the detection of the macromolecule antigen, the carrier used for marking antibody in said method is the colloidal gold of multiple branches, the colloidal gold of multiple branches marked with antibody is regarded as the signal probe of the dynamic light scattering sensor at the same time; the method is used for the competitive antigen of phage corresponding to macromolecular antigen panned from an antibody library, and the method takes dynamic light scattering signal as signal output. The method comprises the following steps:
(1) marking a macromolecular antigen specific monoclonal capture antibody on the surface of the multi-branch colloidal gold by using multi-branch colloidal gold with a surface modified carboxyl chain as a carrier by adopting an EDC one-step method to obtain the multi-branch colloidal gold marked by the capture antibody;
(2) panning out specific phage aiming at pathogenic microorganism through antibody library to obtain phage detection antibody;
(3) adding a phage detection antibody and a target solution to be detected into an antibody-labeled multi-branch colloidal gold solution, reacting at 37 ℃ for 15-200min, then determining the average hydration kinetic diameter of the solution on a Malvern nanometer particle size analyzer, and determining the content of macromolecular antigen in a sample to be detected by using the change of the hydration kinetic diameter;
the macromolecular antigen is a protein marker or a pathogenic microorganism with a plurality of antigenic determinants.
Further, the preparation method of the capture antibody labeled multi-branch colloidal gold in the step (1) comprises the following steps: synthesizing a multi-branch colloidal gold solution by using a colloidal gold seed mediated growth method, centrifuging the synthesized colloidal gold solution, replacing the centrifuged colloidal gold 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 4-12 hours; centrifuging the mixed solution, and removing redundant chains to obtain multi-branch colloidal gold with surface sulfhydryl and carboxyl amphiphilic chains; adding multi-branch colloidal gold with surface sulfhydryl carboxyl amphiphilic chains into a buffer solution with pH 7.5PB (0.01mol/L), adding a capture antibody, stirring for reaction at room temperature, adding EDC, stirring for two times, adding bovine serum albumin with the mass volume fraction of 10%, adding EDC, stirring at room temperature for 30 minutes, centrifuging to separate the multi-branch colloidal gold coupled with the antibody, and obtaining the multi-branch colloidal gold marked by the capture antibody.
Further, the step (2) of panning the phage specific to the corresponding macromolecular antigen through the antibody library comprises the following operations: diluting the macromolecular antigen with PBS to a final concentration of 10 mug/mL, adding 100 mug/well into an enzyme-labeled well, and coating overnight at 4 ℃; after washing the plate, sealing; washing the plate, adding 100 mu L of phage library into the enzyme-labeled hole, reacting at 37 ℃, washing the plate after combination, washing out the unbound phage, adding acid for elution, taking out the phage eluent with specific combination, and neutralizing with a neutralizing solution until the pH value of the solution is neutral; then amplifying the obtained eluent to obtain a large amount of high-concentration specific phage; adding 25% glycerol, and storing in refrigerator at-20 deg.C.
Further, taking the multi-branch colloidal gold marked by the capture antibody obtained in the step (1), diluting with PBS (phosphate buffer solution) in a gradient manner, determining the average hydration kinetic diameter of the solution, and taking the lowest stable concentration of the average hydration kinetic diameter of the solution as the use concentration of the colloidal gold probe; at the concentration of the colloidal gold probe used, the amount of phage used as a detection antibody that does not produce a dynamic light scattering signal was measured.
Further, the amount of the capture antibody labeled on the multi-branched colloidal gold labeled with the capture antibody is determined by the maximum variation amount of the average hydrated particle size of the solution.
Furthermore, each buffer and the double solution were filtered through a 0.22 μm filter before use.
In the method, a signal output substrate is multi-branch colloidal gold. 1) When the concentration of the macromolecular antigen target in the solution is zero or extremely low, the phage serving as a detection antibody and the capture antibody on the surface of the multi-branch colloidal gold cannot be combined with the target antigen or cannot be combined with the target antigen, and a phage-antigen-multi-branch colloidal gold three-in-one sandwich structure cannot be formed, so that the average hydration kinetic diameter of the solution is slightly increased or not increased compared with a blank value (the multi-branch colloidal gold marked with the capture antibody); 2) along with the increase of the concentration of the macromolecular antigen in the solution, the bacteriophage used as a detection antibody and the capture antibody on the surface of the multi-branch colloidal gold are combined with a target antigen to form the bacteriophage-antigen-multi-branch 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 the blank value (the multi-branch colloidal gold marked with the capture antibody); 3) along with the change of the concentration of the macromolecular antigen, the increase of the average hydration kinetic diameter of the solution is linearly changed, 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 the macromolecular antigen in a sample to be detected in reaction, and a standard curve can be drawn by using a macromolecular antigen standard solution with known concentration and gradient distribution in actual operation by using the particle size of the solution as a vertical coordinate and using the concentration of the macromolecular antigen as a horizontal coordinate to calculate a linear equation. When the actual sample is detected, the particle size increase value of the sample is substituted into the standard curve, the concentration of the corresponding sample is read from the standard curve, and the actual concentration of the macromolecular antigen in the sample is obtained by multiplying the concentration by the corresponding dilution factor. The specific operation method can be arbitrarily selected according to the common technical knowledge in the technical field.
The method is suitable for quantitative detection of pathogenic microorganisms, such as pathogenic microorganisms with a plurality of antigenic determinants, such as microorganisms, cancer markers, environmentally harmful pathogenic microorganisms, and the like; the method is also suitable for quantitative detection of biological macromolecular proteins, such as disease protein biomarkers, environment-harmful macromolecular proteins and other biological macromolecular proteins with a plurality of antigenic determinants, and is particularly suitable for trace detection of target analytes. The sample is processed according to conventional processing method.
Compared with the prior art, the invention has the beneficial effects that:
the multi-branch 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 using a low-concentration multi-branch colloidal gold probe; on the other hand, the phage has the function of identifying specific antigens, and the identification unit is positioned at the end position of the pVIII protein, so that the false positive experiment result caused by crosslinking in the reaction process is avoided; meanwhile, the phage can generate larger particle size change than the conventional protein (usually about tens of nanometers) after being combined with a target antigen and a capture antibody labeled nano probe to form a sandwich structure by larger nano scale (width is 6 nanometers and length is 800 nanometers), so that the regulation range of the dynamic light scattering signal is widened; in addition, phage that are panned from the antibody library as detection antibodies have higher affinity with the 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 using amount of the antibody and increasing the particle size of the detection antibody; in addition, compared with the traditional double-antibody sandwich immunoassay method, the new method only needs to sample and mix and then test, 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 process of the present invention;
FIG. 2 is a standard curve for detecting Escherichia coli O157: H7 based on the dynamic light scattering homogeneous immunoassay of multi-branched colloidal gold;
FIG. 3 is a standard curve for norovirus detection based on the dynamic light scattering homogeneous immunoassay method using multi-dendrimer colloidal gold.
FIG. 4 is a standard curve of a dynamic light scattering homogeneous immunoassay based on multi-branched colloidal gold for alpha-fetoprotein;
FIG. 5 is a standard curve of a dynamic light scattering homogeneous immunoassay based on multi-limbed colloidal gold for cardiac troponin I;
FIG. 6 is a standard curve of dynamic light scattering homogeneous immunoassay based on multi-branched colloidal gold for hepatitis B surface antigen;
FIG. 7 is a standard curve of a dynamic light scattering homogeneous immunoassay based on rampant colloidal gold for HIV p24 antigen;
FIG. 8 is a standard curve of a dynamic light scattering homogeneous immunoassay based on multi-branched colloidal gold for C-reactive protein.
Detailed Description
Hereinafter, the present invention will be described in further detail with reference to examples in order to make the present invention more clearly understood. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Preparation method of phosphate buffer solution (PBS, 0.05M, pH 7.4): NaCl 40g, Na2HPO4 13.5g,KH2PO41.0g of KCl and 1.0g of the mixture were dissolved in 1L of ultrapure water. Adjusting the pH value to 8.0-9.0 with 0.1M NaOH.
The murine IgG class monoclonal antibodies referred to in the examples: the anti-norovirus monoclonal antibody, the anti-Escherichia coli O157: H7 antibody, the anti-alpha fetoprotein monoclonal antibody, the anti-cardiac troponin I antibody, the anti-hepatitis B surface antigen monoclonal antibody, the anti-HIV p24 antigen monoclonal antibody and the anti-C reactive protein monoclonal antibody are purchased from the companies of Sigma, Abcam, Beijing thermopathic organisms and the like.
Example 1 application of detection of macromolecular antigen content
Preparation of 1 carboxyl modified multi-branch colloidal gold
1) Synthesizing multi-branch colloidal gold by a high-temperature one-step method; heating 100mL of ultrapure water system to 57 ℃, slowly stirring on a magnetic stirrer, turning off heat, and sequentially adding 2.5mL of 60nm seed gold and 1.5mL of L% HAuCl4The solution and 2.64mL of 1 percent trisodium citrate solution are added, the rotating speed is accelerated, 24mL of 30mmol/L hydroquinone solution is rapidly added at one time, the reaction is continued for L0 min, the solution is cooled to the room temperature, and the solution is stored for standby at 4 ℃.
The synthesis method of 60nm seed gold comprises the following steps: a) synthesizing 18-20nm colloidal gold by citric acid reduction method, adding 1mL of 1% chloroauric acid solution into 99mL of ultrapure water, slowly stirring at uniform speed, and heating to boil (about 20 mi)n large bubbles appear); b) 2.7mL of 1% trisodium citrate (Na) was added rapidly3C6H5O7) Rapidly stirring the solution for 10 min; stopping heating when the solution color becomes wine red and no longer changes, cooling to room temperature under stirring, and storing at 4 deg.C for use. c) Adding 1mL of the colloidal gold synthesized in the step b) into 100mL of ultrapure water solution, stirring vigorously, adding 0.8mL of 1% (mass volume fraction) chloroauric acid solution, quickly adding 0.2mL of 1% (w/v) trisodium citrate solution and 0.1mL of hydroquinone solution (30mM) as reducing agents of a reaction system after stirring uniformly, adding the two reducing agents at the same time every 10min, and circulating for 5 times. Stirring at room temperature for 30min, and storing at 4 deg.C.
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 4-12 h; 10mL of the synthesized multi-branched colloidal gold is taken, centrifuged at 3000rpm/min at 4 ℃ for 15min, the precipitate is redissolved by 1mL of ultrapure water solution with pH being more than 9, 20 mu g of amphiphilic thiol carboxyl chain is added, and the mixture is placed on a vertical mixer at room temperature to be stirred and reacted for 4 h.
3) Centrifuging the mixed solution in the step 2), rotating at 3000rmp/min for 15 minutes, removing excessive chains, re-dissolving the multi-branch colloidal gold with the surface connected with the amphiphilic chains in ultrapure water, and storing in a refrigerator at 4 ℃ for later use.
2 preparation of Multi-branched colloidal gold Probe labeled with Capture antibody
Adding 12 mu L of the multi-branch colloidal gold modified with the sulfhydryl carboxyl amphiphilic chain into 500 mu L of pH 7.5PB (0.01mol/L) buffer solution, adding 6-12 mu g of capture antibody corresponding to the macromolecular antigen, and stirring for reaction at room temperature for 30 minutes; the reaction was stirred further, after adding 0.25. mu.g of 1-ethyl- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the mixture was stirred at room temperature for 30 minutes, and two more times, 50. mu.L of bovine serum albumin with a mass volume fraction of 10% was 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-branched colloidal gold was separated by centrifugation, and the separated multi-branched colloidal gold was washed three times with ultrapure water. The washed multi-branch colloidal gold is re-dissolved in ultrapure water and stored at 4 ℃.
3 preparation of phage for detection of antibodies
The method adopts an antibody library to carry out panning on phage with corresponding macromolecular antigen specificity as a detection antibody, and specifically comprises the following operations:
1) diluting AFP antigen with PBS to final concentration of 10 μ g/mL (the coating concentration of the second and third rounds of panning antigen is 5 μ g/mL and 2.5 μ g/mL respectively), adding into enzyme-labeled well at 100 μ L/well, and coating overnight at 4 deg.C; (ii) a
2) Discarding the coating solution, washing with PBS 3 times, adding 300 μ L of 3% BSA-PBS (OVA-PBS and BSA-PBS for the second and third rounds respectively) blocking solution into each well, and blocking at 37 deg.C for 2 hr;
3) PBS wash 6 times, add 100 u L phage library, phage number is about 6.2X 1011cfu (the input amount of phage library in the second round and the third round is 8.4X 10 respectively11cfu、1.1×1012cfu), incubation for 1.5h at 37 ℃ (the binding time of the second round and the third round is 1 h);
4) the unbound phage were aspirated and washed 8 times with PBST (12 and 15 washes for the second and third rounds, respectively), and 8 times with PBS (12 and 15 washes for the second and third rounds, respectively);
5) adding 100 mu L of Gly-HCl eluent, incubating for 6-8 min at 37 ℃, and eluting the specifically combined phage; transferring the eluate to a sterile centrifuge tube, and rapidly neutralizing with 12 μ L Tris-HCl neutralization buffer;
6) adding the eluent obtained in the step 5) into 20mL of Escherichia coli ER2738 culture solution in the early logarithmic growth stage for amplification culture at 37 ℃ for 4.5 hours;
7) taking 10 mu L for gradient dilution, determining titer, calculating elutriation recovery rate, mixing the rest eluates, amplifying and purifying for next round of affinity elutriation;
8) amplification of the post-panning library:
a) mixing the elutriation eluate with 3mL of E.coli ER2738 culture in early logarithmic growth stage, shaking and culturing at 37 ℃ and 220r/min for 45min, transferring to 20mL of 2 XYT-A liquid culture medium, shaking and culturing at 37 ℃ and 220r/min for 2h, and performing cell: phase 1: adding M13K07 phage at the ratio of 20, standing for 15min at 37 ℃, and performing shaking culture at 220r/min for 30-45 min;
b) the culture obtained in the step a) is subpackaged in a centrifuge tube, the temperature is 4 ℃, 3500r/min and 10min, the cell sediment is resuspended in 25mL of 2 XYT-AK liquid culture medium, and the shaking culture is carried out at 30 ℃ and 250r/min overnight:
c) centrifuging the overnight culture at 4 ℃ at 12000r/min for 15min, transferring the supernatant to a new centrifuge tube, adding 1/5 volumes of PEG-NaCl, mixing uniformly, and standing at 4 ℃ for more than 1 h;
d) removing supernatant at 12000r/min at 4 ℃ for 15min, suspending the precipitate in 1mL PBS, adding 1/5 volume of PEG/NaCl, mixing uniformly, and standing at 4 ℃ for more than 1 h;
e)12000r/min, 5min, removing supernatant, suspending the precipitate in 200 μ L PBS to obtain amplification product, and determining titer for next round of panning or analysis.
9) And (3) rescuing the phage:
a) selecting a plate with the titer of the eluate from the third round of panning (the number of colonies is 30-200), randomly selecting 48 single clones by using a sterilized toothpick, inoculating the single clones into 1mL of 2 XYT-GA, and carrying out shake culture at 37 ℃ and 220r/min for 12 hours;
b) inoculating to 2 XYT-GA at 37 deg.C and 220r/min, and culturing to logarithmic growth stage;
c) according to the cell: phase 1: adding M13K07 phage at a ratio of 20, standing at 37 deg.C for 15min, and shake culturing at 220r/min for 30-45 min
d) Centrifuging at 4 deg.C and 3500r/min for 10min, resuspending the precipitate with equal volume of 2 XYT-AK, and culturing at 30 deg.C under vigorous shaking overnight;
e) centrifuging at 12000rpm for 10min the next day, discarding supernatant, adding 25% glycerol solution into the precipitate for redissolving, and storing at-20 deg.C for use.
4 detecting the content of macromolecular antigen
When the novel dynamic light scattering homogeneous immunoassay method is used for detecting the content of macromolecular antigens, the method is implemented by the following steps: sample pretreatment, detection by the detection method and result analysis.
1) Sample pretreatment: diluting the purchased pathogenic microorganism standard substance to a corresponding concentration gradient, wherein the concentration is determined according to a required actual detection limit; the diluted antigen sample is placed in a refrigerator at 4 ℃ for standby. The purification method of the target analyte in different samples to be detected is specifically completed by referring to national standards.
2) The detection method of the invention is used for detecting the content of macromolecular antigens with a plurality of antigenic determinants, such as microorganisms, cancer markers, environment harmful macromolecular proteins/pathogenic microorganisms and the like.
3) And (6) analyzing the result.
The corresponding average hydration kinetic diameters of the solutions were tested on a malvern nanometer particle size analyzer using the standards of different concentrations prepared above.
And drawing a standard curve by taking the particle size of the solution as a vertical coordinate and the concentration of the macromolecular antigen as a horizontal coordinate to obtain a linear equation. When the actual sample is detected, the particle size increase value of the sample is substituted into the standard curve, the concentration of the corresponding sample is read from the standard curve, and the actual concentration of the macromolecular antigen in the sample is obtained by multiplying the concentration by the corresponding dilution factor.
When the concentration of pathogenic microorganism is detected, the standard substance can be selected from 0CFU/mL, 100CFU/mL, 10 CFU/mL1CFU/mL、102CFU/mL、103CFU/mL、104CFU/mL、105CFU/mL concentration gradient.
For the detection of the concentration of the protein marker, the standard substance may be selected from concentration gradients 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 test substance of food-borne pathogenic bacterium Escherichia coli O157H 7
mu.L of Escherichia coli O157: H7 Capture antibody labeled Multi-branched colloidal gold (0.02pM), 150. mu.L of Escherichia coli O157: H7 solution of different concentrations, 150. mu.L of bacteriophage specifically binding to Escherichia coli O157: H7 (titer 2.1X 109) Mixing, incubating at 37 ℃ for 100 minutes, and taking out the average hydration kinetic diameter change of the solution measured at 25 ℃ by a Malvern nanometer particle size analyzer. Calculating the average value and substituting the average value into a standard curve to obtain a sample to be detectedConcentration of Escherichia coli O157: H7. The specific experimental results are as follows: the linear standard curve is y-0.9466 ln (x) +129, R20.9864, see fig. 2. The minimum detection limit of this method is defined as the antigen concentration required for the mean hydrated particle size at 20 first standards (mean hydrated particle size of solution at 0 standard) plus 3 times the standard deviation (3 times the standard deviation of three parallel samples of the first standard sample), the desired antigen concentration. The lowest detection line was calculated from the standard curve to be 2.5 CFU/mL.
The method is not limited to E.coli O157: the detection of H7 can also be used for the detection of other food-borne pathogenic bacteria, such as Listeria monocytogenes, Bacillus cereus, Salmonella, Enterobacter flexus, Shigella, Enterobacter sakazakii, Vibrio parahaemolyticus, Staphylococcus aureus, etc.
EXAMPLE 3 food-borne Virus, norovirus, was used as test substance
100 μ L of multi-branch colloidal gold labeled with norovirus capture antibody (0.0125pM), 150 μ L of standard norovirus at different concentrations, and 150 μ L of detection antibody phage corresponding to norovirus (titer 10)9) Mixing, incubating at 37 ℃ for 100 minutes, and taking out the average hydration kinetic diameter change of the solution measured at 25 ℃ by a Malvern nanometer particle size analyzer. And calculating the average value and substituting the average value into a standard curve to obtain the concentration of the norovirus in the sample to be detected. The specific experimental results are as follows: the linear standard curve is y-0.9648 ln (x) +131.02, R20.9793, see fig. 3. The minimum detection limit of this method is defined as the antigen concentration required for the mean hydrated particle size at 20 first standards (mean hydrated particle size of solution at 0 standard) plus 3 times the standard deviation (3 times the standard deviation of three parallel samples of the first standard sample), the desired antigen concentration. The lowest detection line calculated from the standard curve was 3.2 PFU/mL.
The method is not limited to the detection of norovirus, and 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
mu.L of alpha-fetoprotein capture antibody-labeled ramose colloidal gold (0).0125pM) and 150. mu.L of standard substance of alpha-fetoprotein with different concentrations, and detection antibody phage corresponding to 150. mu.L of alpha-fetoprotein (titer is 10)9) Mixing, incubating at 37 ℃ for 100 minutes, and taking out the average hydration kinetic diameter change of the solution measured at 25 ℃ by a Malvern nanometer particle size analyzer. And calculating the average value and substituting the average value into a standard curve to obtain the concentration of the alpha-fetoprotein in the sample to be detected. The specific experimental results are as follows: the linear standard curve is y-0.9534 ln (x) +132.1, R20.9762, see fig. 4. The minimum detection limit of this method is defined as the antigen concentration required for the mean hydrated particle size at 20 first standards (mean hydrated particle size of solution at 0 standard) plus 3 times the standard deviation (3 times the standard deviation of three parallel samples of the first standard sample), the desired antigen concentration. The lowest detection line calculated from the standard curve was 0.18 pg/mL.
The method is not limited to the detection of alpha-fetoprotein, and can also 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
mu.L of multi-branched colloidal gold labeled with capture antibody for cardiac troponin I (0.02pM), 150. mu.L of cardiac troponin I solution with different concentrations, and 150. mu.L of phage specifically binding to cardiac troponin I (titer of 2.1X 10)9) Mixing, incubating at 37 ℃ for 100 minutes, and taking out the average hydration kinetic diameter change of the solution measured at 25 ℃ by a Malvern nanometer particle size analyzer. And calculating the average value and substituting the average value into a standard curve to obtain the concentration of the cardiac troponin I in the sample to be detected. The specific experimental results are as follows: the linear standard curve is that y is 0.9862ln (x) +127.7, R20.9813, see fig. 5. The minimum detection limit of this method is defined as the antigen concentration required for the mean hydrated particle size at 20 first standards (mean hydrated particle size of solution at 0 standard) plus 3 times the standard deviation (3 times the standard deviation of three parallel samples of the first standard sample), the desired antigen concentration. The lowest detection line calculated from the standard curve was 0.31 pg/mL.
The method is not limited to the detection of cardiac troponin I, and can also be used for the detection of other cardiovascular disease marker proteins, such as Creatine Kinase (CK), creatine kinase isozyme (CK-MB), myoglobin (Mb/Myo), B Natriuretic Peptide (BNP), N-terminal pre-B natriuretic peptide (NT-proBNP), and the like.
Example 6 hepatitis B surface antigen as a marker protein for liver diseases
100 mu L of multi-branch colloidal gold (0.02pM) marked by the capture antibody of the hepatitis B surface antigen, 150 mu L of hepatitis B surface antigen solution with different concentrations and 150 mu L of bacteriophage (the titer is 2.1 multiplied by 10) specifically combined with the hepatitis B surface antigen9) Mixing, incubating at 37 ℃ for 100 minutes, and taking out the average hydration kinetic diameter change of the solution measured at 25 ℃ by a Malvern nanometer particle size analyzer. The average value is calculated and then substituted into a standard curve to obtain the concentration of the hepatitis B surface antigen in the sample to be detected. The specific experimental results are as follows: the linear standard curve is that y is 0.8954ln (x) +128.5, R20.9768, see fig. 6. The minimum detection limit of this method is defined as the antigen concentration required for the mean hydrated particle size at 20 first standards (mean hydrated particle size of solution at 0 standard) plus 3 times the standard deviation (3 times the standard deviation of three parallel samples of the first standard sample), the desired antigen concentration. The lowest detection line calculated from the standard curve was 0.29 pg/mL.
The method is not limited to the detection of hepatitis B surface antigen, and can also be used for the detection of liver disease marker protein, such as hepatitis B surface antibody, core antibody, e antigen, e antibody, hepatitis C virus core antigen, hepatitis E virus antibody and the like.
Example 7 AIDS marker protein-HIVp 24 antigen as test substance
100 u LHIVp24 antigen capture antibody labeled multi-branch colloidal gold (0.02pM) and 150 u L HIV p24 antigen solution of different concentrations, 150 u LHIV p24 antigen specific binding phage (titer 2.1X 10)9) Mixing, incubating at 37 ℃ for 100 minutes, and taking out the average hydration kinetic diameter change of the solution measured at 25 ℃ by a Malvern nanometer particle size analyzer. And calculating the average value and substituting the average value into a standard curve to obtain the concentration of the HIVp24 antigen in the sample to be detected. The specific experimental results are as follows: the linear standard curve is y-0.9142 ln (x) +130.4, R20.9824, see fig. 7. Minimum detection of the methodThe assay limit was defined as the antigen concentration required for the mean hydrated particle size +3 times the standard deviation (3 times the standard deviation of three replicates of the first standard) for the 20 first standards (0 standard time mean hydrated particle size of the solution). The lowest detection line calculated from the standard curve was 0.42 pg/mL.
The method is not limited to detection of HIV p24 antigen, and can also be used for detection of other AIDS marker proteins, such as HIV 1/2 antibody, HIV gp120 antigen, etc.
Example 8 inflammation marker protein-C reactive protein
mu.L of multi-branched colloidal gold (0.02pM) labeled with capture antibody for reactive protein 100. mu.L, 150. mu.L of C reactive protein solution with different concentrations, and 150. mu.L of phage specifically binding to reactive protein 150. mu.L (titer 2.1X 10)9) Mixing, incubating at 37 ℃ for 100 minutes, and taking out the average hydration kinetic diameter change of the solution measured at 25 ℃ by a Malvern nanometer particle size analyzer. And calculating the average value and substituting the average value into a standard curve to obtain the concentration of the C-reactive protein in the sample to be detected. The specific experimental results are as follows: the linear standard curve is y-0.8796 ln (x) +128.9, R20.9913, see fig. 8. The minimum detection limit of this method is defined as the antigen concentration required for the mean hydrated particle size at 20 first standards (mean hydrated particle size of solution at 0 standard) plus 3 times the standard deviation (3 times the standard deviation of three parallel samples of the first standard sample), the desired antigen concentration. The lowest detection line calculated from the standard curve was 0.39 pg/mL.
The method is not limited to the detection of C-reactive protein, and can also be used for the detection of other inflammation marker proteins, such as hypersensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), tumor necrosis factor-alpha, Procalcitonin (PCT) human serum amyloid A (SAA1), human trypsinogen 2(PRSS2), Lipopolysaccharide Binding Protein (LBP), and the like.
Claims (5)
1. A homogeneous phase immune method for detecting macromolecular antigen is characterized in that specific phage is used as a detection antibody and multi-branch colloidal gold labeled by a matched capture antibody is used as a dynamic light scattering signal enhancement probe, average hydration kinetic particle size change of a solution before and after forming a sandwich structure is used as dynamic light scattering signal output, and the content of the macromolecular antigen in a sample to be detected is measured and reacted by utilizing the hydration kinetic diameter change, and the homogeneous phase immune method comprises the following steps:
(1) marking a macromolecular antigen specific monoclonal capture antibody on the surface of the multi-branch colloidal gold by using multi-branch colloidal gold with a surface modified carboxyl chain as a carrier by adopting an EDC one-step method to obtain the multi-branch colloidal gold marked by the capture antibody;
(2) panning out specific phage aiming at pathogenic microorganism through antibody library to obtain phage detection antibody;
(3) adding a phage detection antibody and a target solution to be detected into an antibody-labeled multi-branch colloidal gold solution, reacting at 37 ℃ for 15-200min, then determining the average hydration kinetic diameter of the solution on a Malvern nanometer particle size analyzer, and determining the content of macromolecular antigen in a sample to be detected by using the change of the hydration kinetic diameter;
the macromolecular antigen is a protein marker or a pathogenic microorganism with a plurality of antigenic determinants.
2. The homogeneous immunoassay method for detecting a macromolecular antigen according to claim 1, wherein the capture antibody-labeled rampant colloidal gold in step (1) is prepared by: synthesizing a multi-branch colloidal gold solution by using a colloidal gold seed mediated growth method, centrifuging the synthesized colloidal gold solution, replacing the centrifuged colloidal gold 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 4-12 hours; centrifuging the mixed solution, and removing redundant chains to obtain multi-branch colloidal gold with surface sulfhydryl and carboxyl amphiphilic chains; adding multi-branch colloidal gold with surface sulfhydryl carboxyl amphiphilic chains into a buffer solution with pH 7.5PB (0.01mol/L), adding a capture antibody, stirring for reaction at room temperature, adding EDC, stirring for two times, adding bovine serum albumin with the mass volume fraction of 10%, adding EDC, stirring at room temperature for 30 minutes, centrifuging to separate the multi-branch colloidal gold coupled with the antibody, and obtaining the multi-branch colloidal gold marked by the capture antibody.
3. The homogeneous immunological method for detecting macromolecular antigens according to claim 1, wherein the step (2) of panning the phage specific to the corresponding macromolecular antigen through an antibody library comprises the following operations: diluting the macromolecular antigen with PBS to a final concentration of 10 mug/mL, adding 100 mug/well into an enzyme-labeled well, and coating overnight at 4 ℃; after washing the plate, sealing; washing the plate, adding 100 mu L of phage library into the enzyme-labeled hole, reacting at 37 ℃, washing the plate after combination, washing out the unbound phage, adding acid for elution, taking out the phage eluent with specific combination, and neutralizing with a neutralizing solution until the pH value of the solution is neutral; then amplifying the obtained eluent to obtain a large amount of high-concentration specific phage; adding 25% glycerol, and storing in refrigerator at-20 deg.C.
4. The homogeneous immunological method for detecting macromolecular antigens according to claim 1, wherein the average hydration kinetic diameter of the solution is determined after the capture antibody-labeled multi-branched colloidal gold obtained in step (1) is taken and diluted with PBS gradient, and the lowest concentration at which the average hydration kinetic diameter of the solution is stable is taken as the use concentration of the colloidal gold probe; at the concentration of the colloidal gold probe used, the amount of phage used as a detection antibody that does not produce a dynamic light scattering signal was measured.
5. The homogeneous immunological method of claim 4, wherein the amount of the capture antibody labeled on the capture antibody-labeled rampant colloidal gold is determined by the maximum variation of the average hydrated particle size of the solution.
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