CN114409789B

CN114409789B - Colloidal gold particle marking method and detection kit

Info

Publication number: CN114409789B
Application number: CN202210320928.2A
Authority: CN
Inventors: 詹先发; 柳静
Original assignee: Beijing Key Biotechnology Co ltd
Current assignee: Beijing Key Biotechnology Co ltd
Priority date: 2022-03-30
Filing date: 2022-03-30
Publication date: 2022-06-07
Anticipated expiration: 2042-03-30
Also published as: CN114409789A

Abstract

The invention relates to a colloidal gold particle labeling method and a detection kit. The invention prepares and obtains the high-activity monoclonal antibody aiming at the CEA target, and develops a new colloidal gold particle marking method aiming at the antibody. The kit prepared by the monoclonal antibody has good detection specificity and good application prospect.

Description

Colloidal gold particle marking method and detection kit

Technical Field

The invention relates to the field of detection, and particularly relates to a colloidal gold particle labeling method and a detection kit.

Background

Carcinoembryonic antigen (CEA) is a polysaccharide-protein complex with a molecular weight of 180-200 kDa.A tumor-associated antigen was first extracted from colon adenomas and fetal intestines by academists in 1965. It is mainly present on rectal, colon cancer tissues and embryonic intestinal mucosa. This antigen is also known as carcinoembryonic antigen (EA) or Fetal Antigen (FA), because it is also present in gastrointestinal, liver and pancreatic tissues of 2-6 month embryos.

CEA is used as the most common tumor marker, is widely applied to diagnosis of various tumors, and has become one of important auxiliary indexes for clinical diagnosis. Serum CEA levels in healthy subjects are typically less than 5 ng/mL. Malignant tumors can cause the CEA expression level to be obviously increased, and when the CEA expression level is continuously increased by 5-10 times, the colon cancer is suggested to exist possibly. Medullary thyroid carcinoma, which is first indicated by elevated CEA levels, suggests that medullary thyroid carcinoma may be considered in identifying diseases with elevated CEA levels. CEA expression level is closely related to clinical staging of tumors. Research shows that the CEA positive rate of gastric cancer patients is increased along with the progressive increase of gastric cancer stages, and the CEA positive rates of gastric cancer patients in the IA, IB, II, IIIA, IIIB and IV stages are respectively 4.6%, 12.3%, 27.80%, 39.1%, 48.3% and 53.0%. CEA expression level of breast cancer patients is positively correlated with clinical stages of the disease, and is increased along with the increase of the clinical stages.

CEA can be used for the curative effect evaluation after the tumor operation, and the obvious reduction of the CEA level after the operation indicates that the operation effect is better. After effective colorectal cancer resection, the CEA level in peripheral blood generally recovers to a normal level within 4-6 weeks, and if the CEA level is not recovered to a normal level after 6 weeks, the early relapse of the tumor is suggested. In the treatment of tumors, radiotherapy and chemotherapy are as important as surgical treatment. During the course of radiotherapy and chemotherapy, the CEA level in serum should be detected regularly to monitor the curative effect of radiotherapy and chemotherapy. Generally, CEA levels are significantly reduced after radiation therapy.

The traditional method for determining CEA content is an immunoassay method, which is based on the specificity and sensitivity of an antibody-antigen. Three classical immunological techniques, namely immunofluorescence, radioimmunoassay and immunoenzyme, have appeared in succession since the last 40 s. The development of the immunization technology is promoted by the successive appearance of three classical immunization technologies. The immuno-technique advances the possibility of labeling antigen antibodies, the radioimmunoassay advances the sensitivity of immunoassays to a peak, and the immuno-enzyme technique exploits the field of immuno-techniques. The development of the I clinical detection technology is further promoted by the chemiluminescence immunoassay, the electrochemical immunoassay and the protein chip technology which are developed in recent years. At present, the methods for detecting the CEA concentration clinically mainly include enzyme-linked immunosorbent assay, fluorescence immunoassay, radioimmunoassay, electrochemiluminescence immunoassay, amperometric immunoassay, immuno-gold-labeling technology and protein chip technology.

The immuno-gold labeling (ICG) technique uses colloidal gold as a label, utilizes specific antigen-antibody reaction, and carries out positioning, qualitative and quantitative researches on antigen or antibody substances under a microscope. The research shows that the same specimen is simultaneously subjected to CEA detection by adopting a radioimmunoassay, an enzyme-linked immunosorbent assay and a gold-labeling assay, and the result shows that the gold-labeling assay is a simple, convenient and feasible preliminary screening method. At present, the gold-labeled technology is mainly developed towards high sensitivity, diversified detection, quantitative or semi-quantitative detection. However, there is room for further improvement in the detection of CEA, and a method of labeling colloidal gold particles for CEA detection has yet to be further improved, and particularly, studies on more preferable antibodies against CEA have yet to be further improved.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides an improved colloidal gold particle labeling method, a monoclonal antibody aiming at CEA, a labeling method and a kit for truly developing the antibody.

In one aspect, the present invention provides a monoclonal antibody 2A03 against CEA, the light chain variable region of which is (SEQ ID NO: 1)

DIVITQRPALMAASPGEKVTITCRPFSTKEFEVMKWYQQKSGISPKPWIYNLQNAADGVPARFSGSGSGTSYSLTITSMEAEDAATYYCTCHHMAPCYFGAGTKLELK

Heavy chain variable region (SEQ ID NO: 2)

EVQLEESATELARPGASVKLSCKASGYIFSFTFWAWIKQRPGQGLEWIGAHLHCRCNINLFIYLNGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGKYQCHAHWGLGTTLAVSS。

Wherein, the invention also provides a recombinant DNA expression vector containing the polynucleotide sequence or the combination of the polynucleotide sequence and the amino acid sequence; the recombinant DNA expression vector further comprises the amino acid sequences encoding the heavy chain variable region, the heavy chain constant region, the light chain variable region and the light chain constant region of the anti-2A 03 antibody in the DNA sequence.

The invention also provides a host cell transfected with the recombinant DNA expression vector, wherein the host cell comprises prokaryotic cells, yeast, insect or mammalian cells.

Among them, the prokaryotic cell is preferably Escherichia coli.

Wherein the mammalian cell is preferably a HEK293E cell, a CHO cell or a NS0 cell.

Further, the monoclonal antibody of the present invention can also be produced by ascites in mice.

Further, the invention also provides a method for labeling the colloidal gold particles, which specifically comprises the following steps: washing various glassware for experiments with distilled water for many times, washing with double distilled water for many times, and drying; and the vessel is put into concentrated sulfuric acid solution to be soaked for 24 hours, and ultrapure water is soaked and washed for many times, and then is dried, so that pollution of dust and the like is avoided. Sterilizing by autoclaving, washing with deionized water for several times, and drying at high temperature. Preparing colloidal gold:one 250mL triangular flask is taken, double distilled water and 1% gold chloride solution are added, and the mixture is filtered by a 0.22 mu m filter membrane and heated to boil. Filtering sodium citrate with 0.22 μm filter membrane, adding into the above solution, rapidly mixing, boiling for 10min, cooling to room temperature, and supplementing with double distilled water to original volume K₂CO₃Adjusting the pH value to 6.5, preparing a wine red colloidal gold solution, filtering the solution by a filter membrane of 0.22 mu m, scanning the colloidal gold solution in the range of visible light (400-600nm) by using an ultraviolet-visible spectrophotometer to obtain the colloidal gold solution with the maximum absorption wavelength of a visible light region of 521nm and smaller absorption peak width, which indicates that the colloidal gold solution has uniform particle size and better quality. Adding colloidal gold into 2A03 monoclonal antibody solution according to the proportion of 1/10(V/V), reacting at room temperature, adding polyethylene glycol, centrifuging, and sucking the supernatant. Washed with PBS containing Bovine Serum Albumin (BSA). Finally, the precipitate is resuspended in 1% BSA solution corresponding to the original volume of 1/5, and the monoclonal antibody labeled with the colloidal gold particles is prepared after preservation at 4 ℃. By adopting the technical scheme, if the particle size of the colloidal gold is too large, the moving speed of the colloidal gold on the detection pad can be influenced, and if the particle size of the colloidal gold is too small, the color development depth of each detection line can be influenced. Both of them can influence the reading of the detection result by the detection personnel, and reduce the detection accuracy of the test strip. The colloidal gold prepared by the application can further accelerate the detection speed of the test strip and ensure the detection accuracy of the test strip on the basis of ensuring the color development of each detection line to be clear.

Further, the invention also provides a detection kit for preparing the colloidal gold labeled antibody, the kit contains a test strip, and the preparation method of the test strip comprises the following steps: using a sample applicator to apply a 0.5-5mg/L concentration 2A03 monoclonal antibody solution and a rabbit anti-mouse IgG solution in a straight line to A, B of an NC membrane in parallel, wherein the distance between A and B is 3 mm; solidifying the colloidal gold labeled antibody: mixing colloidal gold labeled anti-CEA monoclonal antibody solution diluted by PBS containing BSA in equal volume, adding onto the conjugate pad, and drying; sticking and assembling the reagent strip: and assembling according to a conventional mode, firstly adhering the NC film coated with the antibody to a plastic bottom plate with single-sided adhesive, and then sequentially adhering the colloidal gold conjugate pad, the sample pad and the water absorption pad.

Furthermore, each pad is arranged in a box body, a sample adding hole is formed in the position, corresponding to the sample pad, of the box body, and an observation window and position marks of the detection line and the quality control line are arranged in the position, corresponding to the detection line and the quality control line, of the box body.

Specifically, the NC membrane may also be a cellulose acetate membrane or a mixed membrane; the sample pad may be glass or polyester or a blend of fibers; the absorbent pad is made of an absorbent material, such as absorbent paper.

Furthermore, the width of the NC film is controlled to be 20-30 mm; the width of the water absorption pad can be 20-40 mm, and the width of the colloidal gold conjugate pad is 5-10 mm; the width of the sample pad is 10-40 mm.

Furthermore, the working concentration of the monoclonal antibody in the detection line is 0.1-2.0 mg/mL, and the spraying amount is 0.5-10 muL/cm; the working concentration of the quality control line is 0.1-2.0 mg/mL, and the spraying amount is 0.5-10 mu L/cm; working concentration of colloidal gold labeled probe monoclonal antibody (in protein concentration): 1-100 μ g/mL, and the spraying amount is 1-100 μ L/cm.

Furthermore, the back plate material of the test strip back is various, and can be a plastic plate, such as a polyvinyl chloride (PVC) plate.

Further, the immunochromatographic test strip with the back plate is cut into pieces with the width of 3-5 mm, and the pieces are put into a box body of a detection card.

The sample detected by the kit of the invention can be CEA protein or human whole blood or serum.

When the sample is whole blood or serum, the detection method is to dilute 50 mu L of sample whole blood or 25 mu L of serum/plasma by using 50 mu L of PBS buffer solution, add the sample into the prepared sample adding hole of the test strip for detection, and observe the detection result after 10 min.

Carcinoembryonic antigen (CEA) is a broad-spectrum tumor marker, the content of serum in normal human is extremely low and less than 5ng/ml, and the CEA level is increased and is commonly found in colorectal cancer, gastric cancer, lung cancer, breast cancer, medullary thyroid cancer and the like. Therefore, when the kit is used for detecting CEA expressed at high concentration, the kit can be used for judging the occurrence of cancer.

Advantageous effects

The invention aims at the CEA target to prepare and obtain the high-activity monoclonal antibody, and develops a new colloidal gold particle marking method aiming at the antibody. The kit prepared by the monoclonal antibody has good detection specificity and good application prospect.

Drawings

FIG. 1 shows the result of recombinant CEA purification

FIG. 2 shows a structure of a test strip

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the present disclosure and fall within the scope of the appended claims.

EXAMPLE 1 CEA protein expression

The primer sequences designed for CEA gene PCR are as follows:

and (5) end: 5' -GCGGCCGCGCCACCATGGAGTCTCCCTCGGCCCCT-3' (base shown below is NotI cleavage site), 3-terminal: 5' -GGAATTCCTATATCAGAGCAACCCCAACCAG-3' (base underlined is EcoRI cleavage site).

Extracting CEA gene fragment from human blood DNA by a PCR method, carrying out low melting point agarose gel electrophoresis on a specific PCR product, recovering and purifying, carrying out double digestion by NotI and EcoRI, and connecting an amplification product to NotI and EcoRI sites of a Pichia pastoris secretory expression vector pPIC 9-his. The SalI enzyme-linearized pPIC9k-CEA expression plasmid was electroporated into the GS115 strain and plated in MD. Inoculating single colony to YPD plate containing G418 concentration (mg/ml) of 1 and 2, culturing at 30 deg.C for 2d, dissolving colony in 20 μ l sterile water, heating at 95 deg.C for 10min, performing colony PCR at 2 μ l, and selecting colony identified as recombinant plasmid inserted into yeast genome for induced expression.

Resistant clones were picked and inoculated in BMGY (100mmol/ml K)₃PO₄1.34% YNB,0.00004% biotin, 1% glycerol) at 30 deg.C until D₆₀₀At 5 hours, the cells were harvested by centrifugation and suspended in 1/10 volumes of BMMY (100mmol/ml K)₃PO₄1.34% YNB,0.00004% biotin, 1% glycerol, 0.5% methanol) culture medium, and supplementing 0.5% methanol every day for induction expression. The culture supernatant was purified by Ni column, and SDS-PAGE was performed to verify that the CEA was purified to a relatively pure concentration of 1mg/mL as shown in FIG. 1. The blank had no protein band.

EXAMPLE 2 preparation of CEA monoclonal antibody

BALB/c mice were used as immunized mice, and 100. mu.g of BTN3A3-mFc protein was injected subcutaneously in the back and neck of the mice at the time of first immunization. Day 21 post prime, 2 nd immunization was performed, with 100 μ g CEA protein injected subcutaneously in the back of the neck of each mouse. On day 14 after the 2 nd immunization, 3 rd immunization was performed, and 100. mu.g of CEA protein was intraperitoneally injected into each mouse, and blood was collected from orbital venous plexus of mice. On day 14 after the 3 rd immunization, the 4 th immunization was performed, and the mice were injected with 50 μ g of CEA protein in the vicinity of the spleen and the abdominal cavity, respectively. Blood is collected through orbital venous plexus after 7 days of 4 th immunization, and the blood serum titer is detected by ELISA together with the three-immune serum. Mice were injected with 100 μ g of CEA protein diluted in physiological saline 3-4 days before fusion. Spleen cells of the immunized mice were fused with Sp2/0 cells according to a conventional method. Screening by an indirect ELISA method and cloning by a limiting dilution method.

Cell fusion, screening and cloning are carried out to obtain 5 hybridoma cell strains capable of stably secreting CEA monoclonal antibody, wherein 3 clone numbers with the best effect are respectively 2A03, 4D05 and 7E 08. Respectively preparing mouse ascites from the hybridoma cell strains according to a conventional method, and purifying the ascites to obtain the monoclonal antibody.

The CEA monoclonal antibody was detected using a mouse Ig class/subclass/subtype detection kit, and the results are shown in Table 1.

TABLE 1 CEA monoclonal antibody subtype detection

Clone number	Subclass of heavy chain	Light chain subclass
			2A03	IgM	κ
4D05	IgM	κ
			7E08	IgM	κ

As can be seen from the results in Table 1, the three monoclonal antibodies 2A03, 4D05, and 7E08 were all IgM in the heavy chain subclasses and kappa in the light chains.

Detection of monoclonal antibody specificity by Western blotting: respectively preparing a Western blot sample from lysate of escherichia coli cells and human breast cancer cells and CEA and BSA proteins prepared in example 1, carrying out electrophoresis and membrane conversion, sequentially incubating 3 monoclonal antibodies to be detected, reacting overnight at 4 ℃, incubating goat anti-mouse IgG secondary antibody marked by HRP for 40min at room temperature, and developing. The results show that the three monoclonal antibodies 2A03, 4D05 and 7E08 can form a single band in human epithelial cell lysate and CEA prepared in example 1, which indicates that CEA can be specifically detected, and the three monoclonal antibodies have no band in Escherichia coli and BSA, which indicates that the monoclonal antibodies have better specificity.

The affinity of the whole antibody was determined by a capture method. Goat anti-mouse IgG was coupled to the CM5 chip surface and three mabs were diluted separately to ensure that approximately 200RU of antibody was captured by goat anti-human IgG. CEA was applied in a series of concentration gradients (200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5625nM, 0.78125nM) across the stationary phase surface and the affinity of the antibody was determined. As a result, the three antibodies were found to have high affinity (see Table 2).

TABLE 2 CEA monoclonal antibody affinity identification

Clone number	Affinity constants KD (M)
		2A03	7.52E-10
4D05	4.81E-10
		7E08	5.64E-10

As can be seen from the results in Table 2, the affinity of the three monoclonal antibodies 2A03, 4D05 and 7E08 is better, especially 4D05, and the affinity reaches 4.81E-10.

EXAMPLE 32A 03 identification of monoclonal antibody sequences

2A03 hybridoma cell line is expanded and cultured, cells in logarithmic growth phase are collected, and the whole set of reagents for cloning the variable region gene of the Novagen murine antibody are used, and the heavy chain variable region gene and the light chain variable region gene of the antibody are cloned and sequenced according to the instructions. The specific method route is as follows: total RNA was isolated from the collected hybridoma cell lines using StraightA's TMmRNAI (TM) primers, followed by synthesis of the first cDNA strand using first StrandcDNASynthesis kit and Ig-3' constantregionprimer, PCR amplification using Ig-5' primers and NovaTaqDNApolymerase using the first cDNA strand as a template, cloning of the resulting PCR amplification product into a cloning vector using VectorCloning kit, screening, isolation of DNA and gene sequencing. The sequences of the variable regions of the light chain and the heavy chain of the antibody were obtained by analysis as follows:

light chain variable region (SEQ ID NO: 1)

Heavy chain variable region (SEQ ID NO: 2)

Example 4 preparation of test paper for colloidal gold labeled 2A03 monoclonal antibody

Washing various glassware for experiments with distilled water for many times, washing with double distilled water for many times, and drying; and the vessel is put into concentrated sulfuric acid solution to be soaked for 24 hours, and ultrapure water is soaked and washed for many times, and then is dried, so that pollution of dust and the like is avoided. Sterilizing by autoclaving, washing with deionized water for several times, and drying at high temperature.

Preparing colloidal gold: one 250mL triangular flask was taken, and 99mL double distilled water and 1mL 1% gold chloride solution were added, filtered through a 0.22 μm filter membrane, and boiled for 3 min. Filtering 2mL of 1% sodium citrate with 0.22 μm filter membrane, adding into the above solution, rapidly mixing, boiling for 10min, changing the color of the solution in the conical flask from light yellow to purple to wine red during the stirring process, cooling to room temperature, and supplementing with double distilled water to original volume, 0.1 mol/mL K₂CO₃Adjusting pH to 6.5, preparing into wine red colloidal gold solution, filtering with 0.22 μm filter membrane, scanning with ultraviolet-visible spectrophotometer in visible light (400-600nm) range to obtain colloidal gold with maximum absorption wavelength of visible light region521nm and smaller absorption peak width, which shows that the colloidal gold solution has uniform particle size and better quality.

Adding 0.3mg/mL 2A03 monoclonal antibody solution into the colloidal gold according to the proportion of 1/10(V/V), reacting for 15min at room temperature, adding 1% polyethylene glycol, centrifuging for 30min at 8000r/min, and sucking the supernatant. Washed 2 times with 0.02mol/L PBS containing 1% Bovine Serum Albumin (BSA). Finally the pellet was resuspended in 1% BSA solution corresponding to the original volume of 1/5 and stored at 4 ℃.

Using a sample injector to parallelly point the optimized 2A03 monoclonal antibody solution with the concentration of 1.5mg/ml and the rabbit anti-mouse IgG solution to A, B positions on an NC membrane in a straight line manner, wherein the distance between A and B is 3 mm; solidifying the colloidal gold labeled antibody: adding a colloidal gold labeled anti-CEA monoclonal antibody (Cat. Z211N113, Beijing Kagaokadai Biotech Co., Ltd.) solution diluted with 0.02mol/L PBS containing 1% BSA at a ratio of 30uL/cm onto the conjugate pad, and drying for use; sticking and assembling the reagent strip: assembling according to a conventional mode, firstly adhering the NC film coated with the antibody on a plastic bottom plate with single-sided glue, then sequentially adhering the colloidal gold conjugate pad, the sample pad and the water absorption pad, and respectively overlapping 2mm between every two pads for adhering, as shown in figure 2.

The CEA standard solution with different concentrations is used for measurement according to the conventional measurement steps. When the CEA standard solution is 3ng/ml, the determination line of the area A begins to develop color, and is obviously different from a negative result; the concentration is measured for 5 times, the color of the measuring line is developed every time, and the color development depth is basically consistent, so that the sensitivity of the test strip for detecting CEA by the double-antibody sandwich method is 3 ng/ml.

Example 5 specificity detection of test strips of colloidal gold labeled 2A03 monoclonal antibody

The test paper strip is specifically prepared into standard solutions of CEA, prostate specific antigen PSA, tumor marker antigen CA125, Ferritin (Ferritin) and human serum albumin, and the concentrations are 0, 10, 50, 100 and 200ng/mL respectively. And (4) detecting by using a test strip, and repeating each concentration for 3 times to judge the specificity of the test strip. The results are shown in Table 3.

TABLE 3 specificity test results of the test strips of colloidal gold labeled 2A03 monoclonal antibody

As can be seen from the results in Table 3, when the test strip is used to detect CEA, prostate specific antigen PSA, tumor marker antigen CA125, Ferritin (Ferritin), human serum albumin, etc., it is found that the test strip has no cross reaction with other proteins, but only has a specific reaction with CEA.

It is obvious to those skilled in the art that the present invention is not limited to the above embodiments, and it is within the scope of the present invention to adopt various insubstantial modifications of the method concept and technical scheme of the present invention, or to directly apply the concept and technical scheme of the present invention to other occasions without modification.

Sequence listing

<110> Beijing Kongzheng Zhongzhong Biotechnology Co., Ltd

<120> colloidal gold particle labeling method and detection kit

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Asp Ile Val Ile Thr Gln Arg Pro Ala Leu Met Ala Ala Ser Pro Gly

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Claims

1. A monoclonal antibody 2A03 directed against CEA, the light chain variable region sequence of the antibody is as set forth in SEQ ID NO: 1 is shown in the specification; the heavy chain variable region sequence is shown as SEQ ID NO: 2, respectively.

2. A method for labeling an antibody with colloidal gold particles, comprising: the colloidal gold particle labeling method comprises adding 0.3mg/mL monoclonal antibody 2A03 solution according to 1/10V/V, reacting at room temperature for 15min, adding 1% polyethylene glycol, centrifuging at 8000r/min for 30min, and removing supernatant; washing with 0.02mol/L PBS containing 1% bovine serum albumin for 2 times; finally, the precipitate was resuspended in 1% BSA solution corresponding to the original volume of 1/5, and the monoclonal antibody labeled with colloidal gold particles was prepared.

3. A CEA detection kit, characterized in that the kit contains a test strip, and the test strip is marked with the monoclonal antibody marked by the colloidal gold particles in claim 2.

4. Use of a colloidal gold particle-labeled monoclonal antibody as described in claim 2 for the preparation of a kit for CEA detection.

5. Use according to claim 4, characterized in that the sample detected by the kit is serum, blood or CEA protein.