CN111624347A - Application of gold nanorods in detecting serum circulating antigen by specific antibody of liver fluke - Google Patents

Abstract

The invention discloses a nanogold rod with a functionalized liver fluke specific antibody, which is prepared by adopting a seed-mediated growth method, then a sulfhydrylation liver fluke specific IgG antibody is coated on the nanogold rod, and the liver fluke specific IgG antibody functionalized nanogold antibody is screened by comparing LSPR (localized surface plasmon resonance) red peak displacement before and after coating. Also discloses a liver fluke specific antibody and application of the liver fluke specific antibody functionalized gold nanorod, and the liver fluke specific antibody is particularly applied to detection of serum circulating antigen. The invention realizes the advantages of detection sensitivity and early detection by using the nano gold rod biotechnology, has simple operation and low cost, and provides a new technical support for the early detection of the liver trematosis.

Description

Application of gold nanorods in detecting serum circulating antigen by specific antibody of liver fluke

Technical Field

The invention relates to the technical field of biological detection, in particular to application of a gold nanorod in detecting serum circulating antigen by using a specific antibody of liver fluke.

Background

The liver fluke, also known as Clonorchis sinensis, was first discovered in 1874 in the bile duct of a cadaver of Chinese, who answered by California, India. The testis is branched, so it is called the Clonorchis sinensis (Clonorchis sinensis). Adults are predominantly parasitic in the hepatobiliary ducts of humans and carnivorous mammals (e.g., cats and dogs), and occasionally also in the pancreatic ducts. The life history of liver flukes cannot be opened to the water environment. After the eggs in the excrement of the final host infected with the liver fluke enter water, the eggs are swallowed by the first intermediate host, mainly including the marshmallow, the hemiptera horn snail and the phaseolus coccineus. A large amount of cercaria are generated by incubation and differentiation in the snail body, and the mature cercaria invades fresh water fishes and shrimps (a second intermediate host) after escaping from the snail body and develops into cysticercosis. Humans or other animals such as cats, dogs, etc. may become infected when they feed freshwater fish and shrimp containing live cystates. Therefore, the key factor of the epidemic of the liver trematosis is the bad habit of people to eat fresh water fish and shrimp containing cysticercus either live or half live. Liver flukes are prevalent in asia, particularly in china, japan, korea and vietnam, where it is estimated that about 1500 million people are infected, mainly in the southeast and northeast regions of the provinces of guangdong, guangxi and heilongjiang. Sampling studies have shown that the prevalence of hepatic fluke disease in china has increased by 75% from 1990 to 2003. The modern social and economic development rapidly diversifies the diet and life style of people, and the number of people infected with liver fluke disease increases year by year.

As a food-borne zoonosis, the patients infected with liver fluke also have different clinical manifestations due to different infection degrees and infection durations. There are generally no obvious clinical symptoms in the early stages of infection, often resulting in missed diagnoses. In addition, liver trematosis is often misdiagnosed due to its non-specific clinical symptoms, such as fatigue, loss of appetite, nausea, abdominal pain, jaundice, and the like. Acute liver fluke disease can progress to chronic liver fluke disease if not correctly diagnosed and effectively treated. Except for common symptoms such as epigastric discomfort, dyspepsia, diarrhea, anorexia, jaundice, anemia, hepatomegaly (the left lobe is obvious), neurasthenia and the like, a few patients have splenomegaly, cirrhosis and ascites. The chronic infected patients can also be combined with cholecystitis, cholelithiasis, biliary colic, obstructive jaundice, peptic ulcer, liver cancer and primary cholangiocellular carcinoma. Almost 5000 patients with biliary tract cancer may be due to paragonimiasis infection, which occurs annually in east asia for the next decades. Therefore, liver flukes were classified as a class I carcinogenic factor causing cholangiocarcinoma by the world health organization international agency for research on cancer in 2009. Pediatric patients may cause developmental disorders. Therefore, early diagnosis of liver fluke disease is the key to timely treatment and recovery.

Currently, the conventional etiology detection is adopted, and the liver fluke ovum can be diagnosed only by finding the liver fluke ovum in the excrement of a patient or a carrier or in the duodenal fluid. Because patients are infected frequently in mild degrees, the egg discharging amount of liver flukes is not large, the number of eggs in excrement is generally small, and the individual number of the eggs of the liver flukes is small and is easily covered by excrement residues, so that the omission ratio is high. In addition, after the patient is infected with the liver fluke metacercaria, the patient can develop into adult worms and ovulate in about 30 days in the body, and the early diagnosis cannot be realized. The clinical routine immunology has poor early detection effect, low specificity, strong cross reaction with blood serum antibodies of other parasites such as schistosomes, epididymis, cocultivation and the like, and the curative effect of the medicine cannot be checked, because the antibodies exist in the blood serum for a long time even after effective treatment. After the host is infected with the liver fluke, the antigen is generated to induce the immune response of the host. The antigenic substance is continuously produced as long as living insect bodies exist. Although the antigen is combined with the specific antibody to form an antigen-antibody complex, trace CAg still exists in blood, and the method for detecting trace liver fluke circulating antigen in the serum by using a sensitive method has important values for early definite diagnosis and curative effect assessment.

The recent developed nanogold biotechnology has attracted more and more attention to people with unique physical, chemical and biological characteristics, and the rapid development of biomacromolecules containing a nanogold system in the aspects of biomedical detection, diagnosis, treatment and the like is promoted. The nanogold can mark biomolecules in a large range, and the activity of the marked biomolecules is hardly changed, so that the nanogold marking technology becomes a stable, high-efficiency, high-sensitivity and high-flux immune marking technology, and the advantages of the nanogold marking technology are embodied in the four immune marking technologies. However, how to utilize the nano-gold biotechnology to solve the problems that the early stage of the liver fluke cannot be accurately detected due to low content, and the method for detecting the liver fluke in the prior art has the defects of low sensitivity, long time and the like becomes an urgent need for scientific research personnel.

Disclosure of Invention

The invention aims to provide application of a liver fluke specificity IgG antibody functionalized nano gold rod in detecting circulating antigens in all final hosts of liver flukes, wherein the optimal concentration of liver fluke specificity IgG antibodies marked on the surface of the nano gold rod is screened out by constructing the nano gold rod and combining the nano gold rod with high-purity liver fluke specificity IgG antibodies, so that the liver fluke specificity IgG antibody functionalized nano gold rod is obtained and applied to detecting serum circulating antigens, and the detection effect of the functionalized nano gold rod on early stage or low-degree infection serum circulating antigens of liver flukes is realized.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a nanogold rod with a functionalized specific antibody against liver flukes, which is prepared by adopting a seed-mediated growth method, then a sulfhydrylation specific IgG antibody against liver flukes is coated on the nanogold rod, and the functionalized nanogold antibody against the specific IgG antibody against liver flukes is screened by comparing the LSPR red peak displacement before and after coating.

Preferably, the method for preparing the gold nanorod by using the seed-mediated growth method comprises the following steps:

step 1, preparing gold crystal seed liquid: mixing and stirring CTAB, chloroauric acid solution and pure water uniformly, adding precooled sodium borohydride solution into the mixed solution, continuously stirring until the mixed solution is dark brown, then shaking for 3-5min, and placing at 28 ℃ for incubation for 2 h;

step 2, preparation of growth solution: sequentially adding CTAB, pure water, chloroauric acid solution, silver nitrate solution and reducing agent into a container, uniformly mixing until the color becomes colorless, and storing at 27-30 ℃;

and 3, mixing the gold seed crystal liquid prepared in the step 1 and the growth solution prepared in the step 2 according to a volume ratio of 1: 230-240, standing and growing at 27 ℃, and obtaining the nano gold rod solution after 12 h.

Preferably, in the step 1, CTAB, chloroauric acid solution and pure water are mixed according to the volume ratio of (1-2): (0.1-0.5): 1, mixing;

in the step 2, CTAB, pure water, chloroauric acid solution, silver nitrate solution and reducing agent are mixed according to the volume ratio of (1-2.5): 1: (0.1-1): (0.01-0.03): (0.01-0.03).

Preferably, the preparation of the gold nanorod by the seed-mediated growth method further comprises a purification process, specifically: centrifuging the obtained nano-gold rod solution at 8000-.

Preferably, the specific antibody of the liver fluke is obtained by constructing a rat model infected by the cysticercus cellulosae of the liver fluke, collecting serum of the liver fluke with different infection degrees and different infection times, and purifying the serum by a chromatographic column.

The invention also provides a kit for detecting serum circulating antigen, which comprises the liver fluke specific antibody functionalized gold nanorod and the liver fluke specific antibody.

The invention also provides application of the liver fluke specific antibody functionalized gold nanorod, which is applied to detection of serum circulating antigen by the liver fluke specific antibody.

Preferably, the detection of the serum circulating antigen by the liver fluke specific antibody is realized by labeling the specific IgG antibody on the surface of a gold nanorod and then combining the serum circulating antigen;

wherein, the optimal concentration of the specific IgG antibody marked on the surface of the gold nanorod is 20 mug/mL.

The invention discloses the following technical effects:

the invention firstly prepares a specific and specific nano gold rod, and successfully prepares a functionalized nano gold rod by using the nano gold rod to mark the specific IgG antibody of the liver fluke to detect serum circulating antigens with different infection degrees, different infection times and different dilution degrees. The thiolated antibody is marked on the surface of the gold nanorod through a stable Au-S covalent bond, when circulating antigen in serum is combined with a specific antibody on the gold nanorod, the inherent frequency of the gold nanorod is changed, the ultraviolet spectrophotometer scans a map to find that a longitudinal plasma resonance absorption peak of the gold nanorod generates red shift, and weak antigen-antibody reaction signals are amplified into optical signals which are easy to detect and observe through the optical phenomenon to observe the change rule of the circulating antigen in a host body, so that the early and sensitive detection of the circulating antigen of the rat infected with the liver fluke is realized. Specifically, a rat model infected by the fasciola hepatica metacercaria is successfully established, and the blood serum of the fasciola hepatica with different infection degrees and different infection times is collected and purified to obtain a high-purity specific IgG antibody of the fasciola hepatica; and a high-purity liver fluke specificity IgG antibody is marked by a gold nanorod, and the fact that the maximum red shift of the surface longitudinal plasma resonance absorption peak is 20nm and the peak shape is normal compared with the peak before marking is found when the marking concentration is 20 mug/mL through scanning of an ultraviolet spectrophotometer, namely the optimal coating concentration is determined to be 20 mug/mL.

In addition, experiments verify that the liver fluke specific IgG antibody functionalized gold nanorods can detect positive results in light, medium and severe infected rat models at day 3, and can identify serum circulating antigen with the lowest infection amount of 50 cysticercosis. Whereas conventional ELISE detects positivity at 17d for mild infections and on day 10 for moderate to severe infections. As can be seen, the positive detection time of the functionalized gold nanorods is 14d and 7d earlier than that of ELISE. The maximum dilution of positive serum circulating antigen detected by the functionalized gold nanorods is 1:400, and the maximum dilution detected by an ELISE detection method is 1: 100. Therefore, the application of the gold nanorod disclosed by the invention in the detection of serum circulating antigen by the specific antibody of the liver fluke realizes the advantages of detection sensitivity and early stage by applying the gold nanorod biotechnology, is simple to operate and low in cost, and provides a new technical support for the early detection of the liver fluke disease.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the cysticercosis separated after the digestion of the pseudorasbora parva of the present invention;

FIG. 2 shows the results of determining the infectivity of the liver fluke metacercaria of the invention to SD rats; a: separating the intrahepatic bile duct system into adults; b: imago;

FIG. 3 is an SDS-PAGE electrophoresis of the fractions of the original serum and antibody purification according to the invention; 1: raw serum; 2. 3: purifying the antibody; 4, discharging liquid; 5: washing the impurity liquid; mr: protein relative molecular mass standard;

FIG. 4 is a spectrum scanning curve of the antibody functionalized gold nanorods with different concentrations;

FIG. 5 is a histogram of the displacement of the red peaks before and after the gold nanorods are labeled with antibodies of different concentrations;

FIG. 6 is a graph of a standard protein determined by the BCA method of the present invention;

FIG. 7 is an SDS-PAGE electropherogram of each phase of the serum of the invention; 1: 3d serum; 2: serum 10 d; 3: 17d serum; 4: serum 31 d; mr: protein relative molecular mass standard;

FIG. 8 shows the variation of serum antibodies of rat liver fluke ELISE in different time periods of mild infection;

FIG. 9 shows the change of serum antibodies of rat liver fluke ELISE at different time of moderate infection;

FIG. 10 shows the variation of the serum antibodies of the rat liver fluke ELISE in different time periods of severe infection;

FIG. 11 shows the variation of antibody value of positive serum in different dilutions detected by the rat liver fluke ELISE of the present invention.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

1. Preparation of liver fluke antiserum and specific IgG antibody

1.1 establishment of hepatic fascioliasis model of SD rat

1.1.1 Collection and isolation of liver fluke metacercaria

Generally, 1-2 muscles on the back of a fish are taken (a part of fish skin is picked up by scissors, a piece of fish meat with the size of soybean is cut from the skin without the fish skin), the cut fish meat is placed on a prepared glass slide, a cover glass is lightly pressed, the fish meat is thinned to be semitransparent for convenient observation, and cysticercosis is detected under a dissecting mirror. Scraping observed positive fish meat from glass slide, preheating to about 40 deg.C with prepared digestive juice (digestive juice preparation: adding 5ml hydrochloric acid into 1000ml normal saline, adding 7.0g pepsin 1:3000, adding into the above mixture, and mixing thoroughly), wherein the ratio of fish meat to artificial digestive juice is 1:10 (adding 10ml digestive juice into one gram of fish meat), and digesting in constant temperature shaking table at 37 deg.C overnight for about 10 hr. The digested fish meat was filtered through a 60 mesh screen into a large conical measuring cup. After 40 minutes of first precipitating the filtrate, the supernatant is decanted, the precipitate is left and filled with water, after another 30 minutes, the supernatant is decanted again, water is added and the precipitate is left to stand, this is repeated, usually 4 to 5 times with water change, until the water becomes clear. Transferring the rest precipitate into small conical measuring cup, standing for precipitation, collecting precipitate, and checking for cyst, wherein appropriate amount of normal saline can be added if the precipitate is turbid (at least five to eight ml of precipitate is left).

Sucking more than a proper amount of the settled precipitate by a small suction tube, dripping the precipitate on a glass slide, carrying out operation examination under a low power microscope, separating and counting the coenuruses of the liver fluke from other coenuruses by a thin dissecting needle (paying attention to distinguish the coenuruses of the liver fluke from the coenuruses of the east secondary testicles), sucking out the counted coenuruses of the liver fluke by the small suction tube, and putting the coenuruses of the liver fluke into a small test tube containing normal saline for gastric lavage (figure 1).

1.1.2 Final host infection

The SD rats to be infected were divided into three groups of 20 rats each. Rats were given gavage, i.e. mild, moderate and severe infections, according to the infection amount of 50, 100 and 200.

1.2 serum sample Collection at different infection times and different degrees of infection

According to the dynamic changes of the circulating antigen content and the antibody level of the liver fluke metacercaria entering the rat body, the rat is anesthetized by injecting the prepared chloral hydrate into the abdominal cavity after 3d, 10d, 17d, 31d, 38d, 45d and 56d respectively after infection, and the experiment is carried out smoothly. Blood samples were collected at the angular venous plexus of the rat eye using a blood collection needle and left for several hours at 4 ℃ for centrifugation, and the centrifuged serum was stored at-80 ℃.

1.3 infectivity of liver fluke metacercaria on SD rats

After 60 days of infection of the rats with cysticercosis, each rat was sacrificed after blood was taken from the abdominal aorta, and the rats were dissected and the hepatobiliary system was removed. A large amount of mature liver fluke adults can be seen by flushing the hepatobiliary duct with normal saline, and a small amount of adults can be separated from the liver. The success rate of infection was demonstrated to be 100% (fig. 2).

1.4 purification and purity identification of serum-specific IgG antibodies

1.4.1 column purification of specific IgG antibodies

1mL of thawed serum was diluted to 5mL with conjugate Buffer to ensure the appropriate ionic strength and pH before application to the column. The diluted sample was filtered through a 0.22um filter to prevent impurities from clogging the column, ensuring antibody purification efficiency.

The filtered sample was added to well-balanced rProntein G Beads 4FF, 50mL of the wash Buffer was withdrawn and slowly added to the column to wash the non-specifically adsorbed protein, and the wash containing the hetero-protein was collected. 25ml of elution Buffer was used, and the eluate was collected, i.e., purified antibody IgG. The eluted fractions were immediately neutralized using an eluted fraction volume 1/10 of neutralization solution.

The packing was equilibrated with 15mL of binding Buffer and 25mL of deionized water in this order, and finally equilibrated with 25mL of 20% ethanol, and stored at 4 ℃ to prevent contamination of the packing by bacteria.

1.4.2 purity characterization of SDS-PAGE antibody IgG

Respectively using polyacrylamide gel electrophoresis to collect the effluent part, the impurity washing part, the elution component and the original serum sample in the purification process, and detecting and analyzing the purification effect of the chromatographic column by observing an electrophoresis band. (FIG. 3)

Example 2

1. Preparation of liver fluke specificity IgG antibody functionalized gold nanorod

1.1 preparation and purification of Nanogold rods

1.1.1 preparation of gold seed solution

Firstly, preparing gold nanoparticle seeds with small particle size. Mixed with 1.88mL of cetyltrimethylammonium bromide solution (CTAB0.2mmol/L) and chloroauric acid solution (HAuCl)₄2mmol/L) 625. mu.L, 1.37mL pure water and stirred well. The freshly prepared sodium borohydride (NaBH 40.01M) in the 4 ℃ freezer was removed and 450. mu.L of this mixture was added, stirring was accelerated for several minutes until the solution became dark-red, shaking was continued for 5 minutes and incubation was carried out in a 28 ℃ incubator for 2 hours.

1.1.2 growth solution preparation

Several 50mL centrifuge tubes were prepared according to the experimental requirements, and 11.875mL of CTAB (0.2M) which had been prepared and completely dissolved in advance was added to each centrifuge tube, and 7.71mL of pure water and HAuCl were sequentially aspirated₄(2M)5mL，AgNO₃150 μ L (10mM) and 160 μ L ascorbic acid (AA 100mM) were added to the tubes and mixed well, and the color of the growth solution in each tube turned colorless. The prepared growth medium is placed in an environment of about 27 ℃ to prevent CTAB from being separated out at low temperature.

1.1.3 polymerization of Nanogold rods

In order to induce the growth of the seeds, 108 mu L of gold crystal seed liquid is absorbed by a micro liquid transfer gun and mixed and added into 25mL of growth solution, the growth solution is placed in a thermostat at 27 ℃ for standing and growing, and after 12 hours, the nano gold rod solution can be obtained, and an ultraviolet spectrophotometer scans and analyzes a spectrogram.

1.1.4 purification of Nanogold rods

To remove excess CTAB in solution and perform shape and aspect ratio separation, approximately 25mL of the nanogold rod suspension synthesized in each centrifuge tube was centrifuged at 8500r/min for 20-40min using a SIGMA centrifuge, and the supernatant was carefully discarded with a micropipette without disturbing the bottom of the nanogold rod particles. Then, 1.5ml of ultrapure water was added to each tube to resuspend the solid precipitate, and centrifuged again at 13000r/min for 10 min. 1.5mL of 0.5mM CTAB solution was added to the resulting precipitate from each centrifuge tube to obtain concentrated gold nanorod samples for antibody coating. The temperature is adjusted to about 27 ℃ in the whole centrifugation process by attention to prevent CTAB from separating out and influencing the purification effect.

1.2 Nanogold rod-labeled liver fluke specific IgG antibody

1.2.1 thiolation of antibodies at various concentrations

1.2.1.1 the concentration of IgG purified antibody from example 1 was determined using the BCA protein quantification kit and diluted to different concentrations of 10. mu.g/mL, 20. mu.g/mL, 30. mu.g/mL, 40. mu.g/mL.

1.2.1.2 the prepared PBS buffer solution containing EDTA is respectively filled in 2mL centrifuge tubes, 0.8mL is filled in each centrifuge tube, 0.2mL of the antibodies with different concentrations are respectively and sequentially added, and the mixture is fully and evenly mixed. Then, 5. mu.L of Traut's reagent (5mg/mL) was pipetted into the above mixture and left at 27 ℃ for 1 hour.

1.2.1.3 repeating the desalting treatment of the antibody mixture solution after sulfhydrylation for multiple times to remove the hybrid antibody which is not sulfhydrylated until the required antibody is collected, thereby improving the experimental efficiency.

1.2.2 different thiolated antibodies functionalized gold nanorods

mu.L of the thiolated antibody mixture (100. mu.L) of the above antibody mixtures (10. mu.g/mL, 20. mu.g/mL, 30. mu.g/mL, and 40. mu.g/mL) at different concentrations was added to 2mL of the nanogold rod solution, 1000. mu.L of 150mM PEG6000 was slowly added thereto, and the mixture was left at 28 ℃ for 2 hours.

1.2.3 ultraviolet spectrophotometer scanning antibody mark absorption wave contrast screening

And scanning by an ultraviolet spectrophotometer, comparing the longitudinal plasma resonance absorption peak displacement change conditions of the front and back surfaces of the antibody-coated nano gold rod with different concentrations, and determining the optimal labeling concentration of the antibody according to the red peak displacement.

As shown in FIGS. 4 and 5, the eluted antibodies were diluted to different concentrations of 10. mu.g/mL, 20. mu.g/mL, 30. mu.g/mL and 40. mu.g/mL, and the optimized gold nanorods were labeled with the above-mentioned concentrations of the eluted antibodies, and then scanned by an ultraviolet spectrophotometer, and it was found that the LSPR on the surface of the gold nanorods before and after labeling exhibited different degrees of red peak shifts of 10nm, 20nm, 15nm and 4nm, respectively. When the labeling concentration was 20. mu.g/mL, the red peak shift was 20nm at the maximum. That is, the optimal labeling concentration for the purified antibody is 20. mu.g/mL.

Example 3

1. Screening and identification of circulating antigen-associated components

1.1SDS-PAGE analysis of circulating antigen-associated fractions

1.1.1SDS-PAGE purification of excreted secretory antigens

1/4 volumes of SDS-PAGE protein loading buffer (5X) were added to the serum samples on days 3, 10, 17, and 31, respectively, and mixed well. The mixture was heated in a boiling water bath for 5 to 10 minutes, after which time the mixture was centrifuged briefly and collected at the bottom of the tube. The loading amount and volume of the sample were determined from the protein concentration measured by the BCA protein quantification kit (fig. 6 and table 1). And after electrophoresis treatment, dyeing and decoloring are carried out in sequence to obtain clearer protein strips and completely remove dyeing backgrounds, and then photographing and recording can be carried out.

Finally, the proteins in the gel were eluted and recovered using a PAGE gel minirecovery kit.

TABLE 1 protein concentration Table

1.2 antigenic identification of excretory secretory purified antigens

1.2.1 preparation of functionalized gold nanorods for excreting, secreting and purifying antigens by liver flukes

1.2.1.1 preparation and purification of Nanogold rods (same procedure as in example 21.1)

1.2.1.2 thiolation of secretory purified antigens with different periods of excretion

To 0.4mL of EDTA-containing PBS buffer, 0.1mL of the purified antigen excreted and secreted at different times was added, and the mixture was thoroughly mixed. Then, 5. mu.L of Traut's reagent (5mg/mL) was pipetted into the above mixture and left for 1 hour. And (3) respectively repeating the sulfhydrylation of the excretory secretory antigen mixed solution for a plurality of times of desalting treatment by using a gravity desalting column to remove the excretory secretory antigen foreign proteins which are not sulfhydrylated until the required excretory secretory antigens are collected, so that the experimental efficiency is improved.

1.2.1.3 purified antigen excreted and secreted by gold nanorod-labeled sulfhydrylation at different periods

Respectively sucking 200 mu L of the purified excretory secretion antigen subjected to thiolation in different periods, adding the purified excretory secretion antigen into 4mL of nano gold rod solution for reaction for 3min, slowly adding PEG 60002 mL, and mixing and reacting at 28 ℃ for 2 h. And the LSPR on the surface of the gold nanorod is subjected to red shift before and after the ultraviolet spectrophotometer scans and marks, which indicates that the marking is successful, and the gold nanorod can be used for detection.

1.2.2 liver fluke excretion and secretion purified antigen functionalized gold nanorods for detecting positive serum antibody of liver fluke

Adding 40 μ L of positive serum antibody into 2mL of the functional gold nanorods for the liver fluke excretion and secretion antigen at different periods, and reacting for 10min at about 28 ℃.

As shown in FIG. 7, the 3d, 10d, 17d, and 31d bands of the original serum electrophoresis all have the excretory secretion antigen band with a molecular weight of 26kD and 28 kD.

As shown in Table 2, it can be seen that the Nanogold-labeled 3 rd, 10 th, 17 th and 31 th excreted and secreted purified antigens detect that the positive serum antibodies of the liver fluke all have red peak shift changes, i.e., the positive antiserum of the liver fluke can be identified, so that the excreted and secreted purified antigens at each stage have immunoreactivity, and the serum circulating antigen can be used as a serum diagnosis antigen of the liver fluke disease.

TABLE 2 displacement chart of liver fluke serum antibody peak displacement by excretion, secretion and purification of antigen functional nano gold rod in each period

Example 4

Liver fluke specificity IgG antibody functionalized gold nanorod detection serum circulating antigen

1.1 detection of circulating antigens in rat serum at different infection times and different infection degrees

Adding 40 μ L of serum circulating antigen of light, medium and severe infected liver fluke metacercaria on days 3, 10, 17 and 31 into 2mL liver fluke specific IgG antibody functionalized gold nanorod solution, and reacting at 28 deg.C for 10 min.

1.2 detection of circulating antigens in Positive rat serum at different dilutions

Adding (1:5-1:800) diluted positive rat serum circulating antigen 40 μ L into 2mL liver fluke specific IgG antibody functionalized gold nanorod solution, and reacting at 28 deg.C for 10 min.

1.3 ultraviolet spectrophotometer scanning spectrum evaluation functionalized nano gold rod detection serum circulating antigen

And comparing the displacement change of the longitudinal plasma resonance absorption peak of the functionalized nano gold rod before and after scanning detection by an ultraviolet spectrophotometer, and analyzing the detection result.

As shown in Table 3, it can be seen that the maximum red peak shift of 17.5nm occurred at the 10 th d of the mildly infected liver fluke, and that there were corresponding red peak shifts at the 3 rd, 10d, and 31 d. The result shows that the liver fluke specific antibody IgG functionalized gold nanorods can detect serum circulating antigens of 3d, 10d, 17d and 31d of mildly infected liver flukes, and the circulating antigen content in the serum is higher when the liver flukes are infected at 10 d.

TABLE 3 functional nano gold bar chart for detecting mild infection different time serum circulating antigen red peak displacement

As shown in Table 4, the gold nanorods functionalized by the liver fluke-specific IgG antibody can detect the serum circulating antigens of 3d, 10d, 17d and 31d of moderate infection. After infection at 10d, the host's immune system is activated and circulating antigens in the serum are more neutralized by serum specific antibodies, thus, there is less red shift upon infection at 17 d. Along with the extension of modeling time and the development and maturity of more polypide, the egg discharge amount and excretion secretion are increased, so that the amount of circulating antigen in serum is gradually increased, the more circulating antigen combined with the antibody marked by the nano gold rod, and the red peak displacement is in an increasing trend.

TABLE 4 functional nano gold bar table for detecting circulating antigen red peak displacement of serum at different time of moderate infection

As shown in Table 5, the maximum red peak shift was 34.5nm on day 31 of the severe infection, and 12nm and 32.5nm on day 31 of the mild and moderate infections, respectively. Indicating that the amount of circulating antigen in the serum is larger with the increase of the infection amount, i.e. the amount of circulating antigen in the serum is positively correlated with the insect charge.

TABLE 5 functionalized nano gold rod table for detecting the peak-to-red displacement of circulating serum antigen at different time of severe infection

As shown in Table 6, it can be seen that the maximum longitudinal surface plasmon resonance red peak shift is 41nm when the serum dilution ratio is 1:5 by scanning with an ultraviolet spectrophotometer when the liver fluke specific antibody functionalized gold nanorods detect serum circulating antigens diluted by times with different dilutions (1:5-1: 800). When the dilution was 1:600, the red-shift was 1nm, and the amount of circulating antigen in the serum was considered to be very small, possibly due to errors, and negligible. Therefore, the maximum dilution factor of the serum circulating antigen detected by the functionalized gold nanorods is 1: 400. The red peak shift gradually decreased with increasing dilution, indicating that the red peak shift gradually decreased with less binding of specific antibody on the gold nanorods as the serum circulating antigen content decreased. When the dilution ratio is 1:800, the aspect ratio of the functionalized gold nanorods is continuously reduced, the surface longitudinal plasma resonance absorption peak is blue-shifted from 796.5nm to 781nm, and the property of circulating antigen in serum is converted to negative.

TABLE 6 results of serum circulating antigen detection of different dilutions by nano-gold labeling technique

Comparative example

The detection of the serum antibody by the rat liver fluke ELISE detection kit specifically comprises the following steps:

1. the coated plates in the ELISE kit were removed from the 4 ℃ environment and allowed to equilibrate at room temperature for 15 to 30 minutes. Preparation of a washing solution: 20mL of 30 Xconcentrated washing solution was poured into a 1000mL large flask, and distilled water was made up to 600 mL.

2. Numbering: all samples were numbered in order of microwell, each plate should have two wells for negative control, 2 wells for positive control and 1 well for blank control.

3, sample treatment and sample application: the serum was removed in a-80 ℃ freezer, allowed to stand for a period of time in a-20 ℃ freezer, and thawed in a 4 ℃ freezer for approximately 1 hour. Sample adding: 50mL of negative control and positive control samples were added to the negative and positive control wells, respectively. Then, 40 μ L of the sample diluent and 10 μ L of the sample to be measured are sequentially added to the sample well to be measured. And (3) in the sample adding process, the sample is added to the bottom of the hole of the enzyme label plate without contacting the hole wall as much as possible, and the sample is mixed uniformly by slight shaking.

4, incubation: after sealing the sample addition plate with a sealing plate membrane, the plate was incubated at 37 ℃ for 30 minutes.

5, washing: the sealing membrane was gently removed, the liquid removed and air dried, and each well was filled with wash solution and held for 30 seconds, repeated five times and patted dry.

6, adding enzyme reaction: in addition to the blank, a drop of enzyme conjugate was used to cover each of the other wells and the sealing membrane was replaced. And (4) reacting for 30 minutes at 37 ℃ in a dark environment, removing liquid in the holes after the reaction is finished, and washing the plate in the same way as the step (5).

7, color development reaction: adding the color developing agent A, B into each well in turn, shaking slightly until mixing well, and placing in a dark environment at 37 deg.C for 15min to develop color.

And 8, terminating: to each well was added in turn 50. mu.L of stop solution, and the reaction was terminated (in this case, the blue color changed to yellow).

9, measurement: the absorbance (OD value) of each well was measured sequentially at a wavelength of zero and 450nm in a blank air conditioner. The measurement should be performed within 15 minutes after the addition of the stop solution.

10 analysis of results

Testing accuracy: a control group (positive avg is more than or equal to 1.00, negative avg is less than or equal to 0.10);

critical value (CUT OFF) estimate: cutoff value negative avg + 0.15;

negative judgment: the sample OD value is less than the critical value (CUT OFF), and the sample is negative to the liver fluke antibody IgG (LF-IgG);

and (4) positive judgment: the sample with the OD value larger than or equal to the critical value (CUT OFF) is positive to the liver fluke antibody IgG (LF-IgG).

As shown in Table 7 and FIG. 8, it can be seen that the antibody detection results were negative in the 3d and 10d cases of the mild infections with the Fasciola hepatica metacercaria. That is, the IgG antibody in rat serum started to rise around 17d (2 weeks) after the initial infection, and the IgG antibody in serum reached its peak and was maintained at a high level around 56d (8 weeks), and the IgG antibody was maintained in vivo for a long time.

TABLE 7 rat liver fluke ELISE detection of serum antibody OD values at different time of mild infection

As shown in Table 8 and FIG. 9, it was found that antibody positivity was detected at 10d in the case of moderate infection with coenurosis of liver fluke, the detection time was one week earlier than that in the case of mild infection, and the IgG antibody concentration was continuously increased. It is possible that as the worm develops, more and more new antigens are continuously produced, which stimulates the rat body to continuously produce new antibodies, and when the antibody titer reaches the peak value, the antibody level is always maintained at the peak value.

TABLE 8 rat liver fluke ELISE detection of moderate infection serum antibody OD values at different times

As shown in Table 9 and FIG. 10, it can be seen that the OD value of the serum antibody of the rat infected with the coenurosis of liver fluke is larger than that of the light and medium infected persons, i.e., the serum antibody titer of the rat may be related to the infection degree. Moreover, the difference is not very obvious in the early infection stage, and the difference of the OD values of serum antibodies with different infection degrees is obvious around 38 days after infection, but the difference is not obvious as the time is prolonged.

TABLE 9 rat liver fluke ELISE detection of OD values of rat serum at different times of severe infection

As shown in Table 10 and FIG. 11, the positive sera from rats infected with liver flukes were diluted at different ratios, and it was found that the OD values of the serum antibodies gradually decreased as the degree of dilution increased (FIG. 11). The maximum dilution at which positive serum antibodies could be detected by ELISE was 1:100 (Table 10).

TABLE 10 rat liver fluke ELISE detection of different dilutions of positive serum antibody OD values

As can be seen by comparing tables 3, 4 and 5 with tables 7, 8 and 9, the functionalized gold nanorods all have red peak shift changes at the 3 rd infection, the moderate infection and the severe infection, namely all positive results can be detected. While the ELISE method did not detect positivity on the third day of light, medium and severe infections. Mild infection is positive from 17d of infection, 14d later than functionalized nano gold rods, and the time of positive of serum antibodies of medium and severe infection is 10d after infection, and is positive 7d later than functionalized nano gold rods. The comparison result shows that the nano gold rod has the capability of early identification and can realize early detection. The functionalized gold nanorods not only can be used for early detection, but also have higher sensitivity, and by comparing table 6 with table 10, positive serum circulating antigen with the maximum dilution of 1:400 can be detected by the functionalized gold nanorods, and the maximum dilution factor of 1:100 can be detected by ELISE.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (8)

1. The nano gold rod with the functionalized liver fluke specific antibody is characterized in that a seed-mediated growth method is adopted to prepare the nano gold rod, sulfhydrylation liver fluke specific IgG antibody is coated on the nano gold rod, and the liver fluke specific IgG antibody functionalized nano antibody is screened by comparing LSPR (localized surface plasmon resonance) red peak displacement before and after coating.

2. The liver fluke-specific antibody functionalized gold nanorods according to claim 1, wherein the seed-mediated growth method produces gold nanorods comprising the steps of:

step 1, preparing gold crystal seed liquid: mixing and stirring CTAB, chloroauric acid solution and pure water uniformly, adding precooled sodium borohydride solution into the mixed solution, continuously stirring until the mixed solution is dark brown, then shaking for 3-5min, and placing at 28 ℃ for incubation for 2 h;

step 2, preparation of growth solution: sequentially adding CTAB, pure water, chloroauric acid solution, silver nitrate solution and reducing agent into a container, uniformly mixing until the color becomes colorless, and storing at 27-30 ℃;

and 3, mixing the gold seed crystal liquid prepared in the step 1 and the growth solution prepared in the step 2 according to a volume ratio of 1: 230-240, standing and growing at 27 ℃, and obtaining the nano gold rod solution after 12 h.

3. The NanoAu rod functionalized with a liver fluke-specific antibody according to claim 2, wherein in the step 1, CTAB, chloroauric acid solution and pure water are mixed in a volume ratio of (1-2): (0.1-0.5): 1, mixing;

in the step 2, CTAB, pure water, chloroauric acid solution, silver nitrate solution and reducing agent are mixed according to the volume ratio of (1-2.5): 1: (0.1-1): (0.01-0.03): (0.01-0.03).

4. The liver fluke-specific antibody functionalized gold nanorods according to claim 2, wherein the seed-mediated growth method for preparing gold nanorods further comprises a purification process, specifically: centrifuging the obtained nano-gold rod solution at 8000-.

5. The NanoAu rod functionalized with the liver fluke-specific antibody according to claim 1, wherein the liver fluke-specific antibody is obtained by constructing a rat model infected with liver fluke cysticercosis, collecting liver fluke serum with different infection degrees and different infection times, and purifying by a chromatographic column.

6. A kit for detecting circulating serum antigens, comprising the liver fluke-specific antibody functionalized gold nanorods according to claims 1 to 5 and the liver fluke-specific antibody.

7. Use of nanogold rods functionalized with antibody specific to liver fluke according to any one of claims 1 to 5 in the detection of circulating serum antigen by said antibody specific to liver fluke.

8. The use of gold nanorods functionalized with liver fluke-specific antibodies according to claim 7, wherein the detection of serum circulating antigens by liver fluke-specific antibodies is achieved by labeling specific IgG antibodies on the surface of the gold nanorods and then combining with serum circulating antigens;

wherein, the optimal concentration of the specific IgG antibody marked on the surface of the gold nanorod is 20 mug/mL.

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