CN114539401A

CN114539401A - Magnetic particle marking method and detection kit

Info

Publication number: CN114539401A
Application number: CN202210440696.4A
Authority: CN
Inventors: 柳静; 詹先发
Original assignee: Beijing Key Biotechnology Co ltd
Current assignee: Beijing Key Biotechnology Co ltd
Priority date: 2022-04-26
Filing date: 2022-04-26
Publication date: 2022-05-27
Anticipated expiration: 2042-04-26
Also published as: CN114539401B

Abstract

The invention relates to a magnetic particle marking method and a detection kit. The invention provides a monoclonal antibody aiming at serum amyloid A, the antibody has strong binding capacity with SAA and good specificity, an SAA immunochromatographic test strip prepared from the antibody to prepare magnetic particles has better lower detection limit and detection specificity, can be used for detecting specific SAA, and has good market application prospect.

Description

Magnetic particle marking method and detection kit

Technical Field

The invention relates to the field of biology, in particular to a magnetic particle marking method and a detection kit.

Background

Serum Amyloid A (SAA) is a positive acute phase response protein, a heterogeneous class of proteins in the apolipoprotein family, with a relative molecular weight of about 12000. SAA is an acute phase protein and binds to plasma High Density Lipoprotein (HDL).

SAA is a sensitive parameter that begins to rise after about 8 hours of inflammatory response and exceeds the upper limit of the reference range by a time earlier than CRP, whereas CRP differs from the upper limit of the reference range by about a factor of 10 in median values in normal humans. There is only a factor of 5 in SAA. Mild infections, e.g., many viral infections, are more common with elevated SAA than CRP. In infectious diseases, the absolute increase in SAA is higher than CRP, so SAA assays provide better discrimination, especially for "normal" versus minor acute phase reactions. SAA is typically elevated in about 2/3 cold patients, but CRP is also elevated in less than 1/2 patients. In the case of viral infections, elevated concentrations of SAA and CRP are seen in those infected with adenovirus. The reactive forms of SAA and CRP are parallel in the recovery phase of acute infection, which applies to both bacterial and viral infections. Lupus erythematosus and ulcerative colitis SAA did not rise. The metastatic stage of malignant tumors shows higher SAA elevation than the tumor-restricted organ stage. For transplant rejection, SAA detection is a rather sensitive indicator. In a study of one kidney transplant recipient, 97% of the rejection was examined as an increase in SAA. In the irreversible graft rejection test, the average concentration reaches 690 +/-29 mg/L, and the relevant level of reversible rejection attack cases is 271 +/-31 mg/L. Chronic elevation of SAA concentration in patients with rheumatoid arthritis, tuberculosis or leprosy is a prerequisite for the synthesis of AA-starch fibres, which are also used for the diagnosis of secondary amyloidosis. SAA also has certain auxiliary diagnosis effect on diseases such as transplant rejection, type 2 diabetes, chronic respiratory system diseases and the like.

Common detection methods for SAA include colloidal gold method, enzyme linked immunosorbent assay (ELISA), latex-enhanced immunoturbidimetry. The latex enhanced immunoturbidimetry is the most advanced, has higher sensitivity, wider linear range and relatively simple and convenient operation, and is widely adopted in clinic. Research shows that the analysis sensitivity of the latex enhanced immunoturbidimetry is 3.52 mg/L; the intra-batch variation coefficient is less than 8%, and the daytime is less than 10%; the anti-interference performance is strong, the hemoglobin is less than or equal to 4.0g/L, the bilirubin is less than or equal to 400 mu mol/L, the rheumatoid factor is less than or equal to 1621U/L, and the triglyceride is less than or equal to 10mmol/L, and the determination is not influenced. Latex-enhanced immunoturbidimetry captures SAA in a test sample using SAA antibodies adsorbed on latex microparticles of several tens to several hundreds of nanometers in diameter, thereby forming cross-links between the microparticles, changing the scattering degree or transmission absorbance (i.e., turbidity) of the solution.

With the development of scientific technology, especially molecular biology, antibody preparation technology has revolutionized the emergence of immunomagnetic particles. The magnetic particles coated with the monoclonal antibody can be specifically combined with a target substance containing a corresponding antigen to form a new complex. When passed through a magnetic field, the complex can be retained and separated from the other components, a process known as immunomagnetic separation. It is a new immunological technique which is a popular research in recent years at home and abroad and is based on immunology and permeates into various fields of physiology, pathology, pharmacology, microorganism, biochemistry, molecular genetics and the like. The immunomagnetic separation is simple and easy to implement, has high separation purity, retains the activity of a target substance, is efficient, rapid and low-toxic, and is widely applied to the fields of cell separation and purification, immunodetection, nucleic acid analysis and genetic engineering, targeting drug release carriers and the like.

Magnetic immunoassay technology (Magnetic immunoassay technology Tes, tMICT) marked by Magnetic particles is a Magnetic analysis detection technology developed and produced by combining modern physics and biotechnology and is first applied to the field of basic medicine. Unlike other labeling techniques, one significant advantage of this technique is that labeling with magnetic particles is not disturbed by colored impurities and can be used to directly measure colored samples such as blood, food, sewage, etc. The principle is that superparamagnetic nanometer particle is used as label, and high sensitivity magnetic detecting instrument is used to detect the local magnetic field effect produced by the magnetic particle combined on the immune complex, so as to obtain the quantitative result of the detected analyte. . It is based on the benefits of magnetic particles including magnetic microspheres that the development of magnetic particle-labeled SAA monoclonal antibodies for the detection of SAA is becoming urgent, and particularly the development of high affinity SAA monoclonal antibodies is of great importance.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides an improved monoclonal antibody aiming at SAA and application thereof.

In one aspect, the present invention provides a monoclonal antibody 2a4 directed against SAA, the heavy chain variable region sequence of which is set forth in SEQ ID NO:1, and the following components: EVQLEESATELARPGASVKLSCKASGYIFSCQDCCWIKQRPGQGLEWIGREKTKSEVKCKAFWEFGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGRMKRRGSWGLGTTLAVSS, the light chain variable region sequence is as shown in SEQ ID NO:2, as shown in the figure:

DIVITQRPALMAASPGEKVTITCREYFIAAHFGEFWYQQKSGISPKPWIYFCEWTEPGVPARFSGSGSGTSYSLTITSMEAEDAATYYCMAKQQHTGTFGAGTKLELK。

in another aspect of the present invention, the monoclonal antibody is preferably prepared by using ascites, and the method for preparing ascites is not particularly limited, and may be a method that is generally used in the art. The monoclonal antibody of the invention has specific reaction with SAA, which shows that the monoclonal antibody has good specificity; can be used as the antibody of the SAA detection test strip.

In some embodiments, an antibody or fragment thereof (e.g., an anti-SAA antibody provided herein) specifically binds to a target (SAA) with a binding constant of at least 10⁻⁹M or greater binding constant. In some embodiments, an antibody (e.g., a monoclonal antibody) or fragment thereof has an equilibrium constant (Kd) of 10nM or less, e.g., 9nM or less, 8.1nM or less, 8nM or less, 7nM or less, 6nM or less, 6.5nM or less, 6.3nM or less, 5nM or less, 4.3nM or less, 4nM or less, 3nM or less, 2nM or less, 5nM or less, 4nM or less, 3nM or less or 1.2nM or less. For example, an antibody or fragment thereof binds to a target such as GPC1 with a binding affinity of at least about 0.1X 106⁻⁸M, at least about 0.3X 10⁻⁸M, at least about 0.5X 10⁻⁸M, at least about 0.75X 10⁻⁸M, at least about 0X 10⁻⁸M, at least about 3X 10⁻⁸M is at least about 5X 10⁻ ⁸M, or at least about 2.0X 10⁻⁸M, at least about 2.5X 10⁻⁸At least about 3.0 x10⁻⁸At least about 3.5X 10⁻⁸At least about 4.0 x10⁻⁸At least about 4.5X 10⁻⁸At least about 5.0 x10⁻⁸M, at least about 1X 10⁻⁹M, at least about 3X 10⁻⁹M, at least about 5X 10⁻⁹M, at least about 2X10⁻⁹M, at least about 3X 10⁻⁹M, at least about 4X 10⁻⁹M, at least about 4.3X 10⁻⁹M, at least about 5X 10⁻ ⁹M, at least about 6X 10⁻⁹M, at least about 6.3X 10⁻⁹M, at least about 6.9X 10^0.9M, at least about 7X 10⁻⁹M, at leastAbout 8X 10⁻ ⁹M, at least about 8.1X 10⁻⁹M, or at least about 10X 10⁻⁹In certain embodiments, the specific binding agent that binds to the target has a dissociation constant (Kd) of<100nM， <10nM，<9nM，<8nM，<7nM，<6.9nM，<6.5nM，<6.3nM，<5nM，<4nM，<4.5nM，<3nM，<2nM，<1.5nM，<1nM，<0.1nM，<0.01nM or<0.001nM (e.g.,<10nM，<9nM，<8nM，<7nM，<6.9nM，<6.5nM，<6.3nM，<5nM，<4nM，<4.5nM，<3nM，<2nM，<1.5nM，<1nM，<0.1nM，<0.01nM or<0.001nM)。10⁻⁸M or less, e.g. 10⁻⁸M to 10⁻¹³M, e.g. 10⁻⁹M to 10⁻¹³In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of the antibody of interest and its antigen.

In some embodiments, the VH domain of the monoclonal antibody has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, identical to 198% or greater than 99% of SEQ ID NO; and/or the amino acid sequence of the VL domain of the monoclonal antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID No. 2. In certain non-limiting embodiments, the sequence of the VH domain of the monoclonal antibody comprises the composition of SEQ ID NO. 1, and/or the sequence of the VL domain of the monoclonal antibody comprises the composition of SEQ ID NO. 2.

Further, the antibody may be conjugated to a detectable label; for example, a detectable label detectable by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (e.g., Computed Tomography (CT), Computed Axial Tomography (CAT) scan, Magnetic Resonance Imaging (MRI), magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optics and laparoscopy). Specific, non-limiting examples of detectable labels include fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). For example, useful detectable labels include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers are also useful, such as luciferase, Green Fluorescent Protein (GFP) and Yellow Fluorescent Protein (YFP). The antibody or antigen-binding fragment may also be conjugated to an enzyme that can be used for detection, such as horseradish peroxidase, -galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When the antibody or antigen-binding fragment is conjugated to a detectable enzyme, it may be detected by the addition of additional reagents for the enzyme to produce a reaction product that can be recognized. For example, when the reagent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that is visually detectable. The antibody or antigen binding fragment may also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin may itself be conjugated to an enzyme or fluorescent label.

Further, the invention provides application of the SAA monoclonal antibody in preparation of a test strip for detecting SAA.

The invention also provides a test strip for rapidly detecting SAA, which comprises a detection line and a quality control line;

the preparation method of the detection line comprises the steps of spraying the monoclonal antibody on a nitrocellulose membrane;

the preparation method of the quality control line comprises the step of spraying the goat anti-mouse IgG antibody on the nitrocellulose membrane.

The structure of the test strip is not particularly limited, and preferably includes a test strip structure conventional in the art.

The invention also provides magnetic particle detection test paper for rapidly detecting the SAA, which comprises the following components that are sequentially stuck on a back plate from left to right: sample pad, magnetic particle pad, nitrocellulose membrane and absorbent paper.

Advantageous effects

The invention provides a monoclonal antibody aiming at serum amyloid A, the antibody has strong binding capacity with SAA and good specificity, an SAA immunochromatographic test strip prepared from the antibody to prepare magnetic particles has better lower detection limit and detection specificity, can be used for detecting specific SAA, and has good market application prospect.

Drawings

FIG. 1 is a graph showing the results of the surface charge conditions of magnetic particles before and after coupling

Detailed Description

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

EXAMPLE 1 preparation of monoclonal antibodies

Experimental animals BALB/C (SPF) female 10 mice, 6-8 weeks old, weight 18-22 g. Serum amyloid A (cat # SX01146, Shanghai river bend Daizhi Ltd.) was used as the immunizing antigen. Antigen 80. mu.g/mouse was first immunized. 40 μ g/mouse was used as a booster. Antigen emulsification: preparing 0.01M pH7.2PBS buffer solution, and requiring sterility; if the tube bottom has granular precipitate before emulsification, the emulsification operation is carried out after ultrasonic pulverization, and two 5ml syringes are used for respectively sucking the same amount of PBS buffer solution containing antigen and Freund's adjuvant. The disposable converter is connected with the injector, and the push-pull operation is repeated for 30-60 min. The complete emulsification standard is that the emulsion is not dispersed in water after being dropped into the water, namely the emulsification is successful. Immune site: the antibody preparation process can adopt multiple injections at the same part, and also can adopt a multi-part combined method for immunization, but subcutaneous injection is recommended for the first immunization, and intraperitoneal injection can be adopted for boosting the immunization. First immunization: the total amount injected per mouse was 0.4ml (80. mu.g antigen/mouse). And (3) boosting immunity: the total amount of 0.2ml (40. mu.g antigen/mouse) per mouse was injected, and the antibody peak in vivo was observed approximately 10 to 15 days after the first immunization of the mice, and the subsequent booster immunization was selected to be performed within 2 weeks of the first immunization. ELISA for detecting the serum titer of the mice: preheating the tail of the mouse in water at 37-40 deg.C for about 5min, subtracting the tip part of about 1cm, and squeezing blood along the tail of the mouse by hand into an EP tube. The blood was left at room temperature for 1h and then at 4 ℃ overnight. The next day, the ELISA was used to detect the titer of mouse sera, and 7 of these sera showed that the titer of polyclonal antibodies was over 1:10000 by indirect ELISA. The best 3 mice were selected and re-immunized 3 days before the fusion experiment at a dose of 100. mu.g/mouse. Mice with acceptable titer were selected by indirect ELISA.

NS-1 was selected as the hybrid myeloma cell and was in the logarithmic growth phase prior to the fusion procedure to satisfy the conditions suitable for fusion. On the day of fusion, NS-1 cells were collected, centrifuged at 1000rpm for 5min, and the supernatant was discarded. Adding pure culture medium, washing once, centrifuging under the above conditions, discarding supernatant, and resuspending in a certain amount of pure culture medium for fusion operation.

Mice were sacrificed and disinfected in 75% alcohol. The abdominal skin and peritoneum were cut open and the spleen was removed. Connective tissue and fat were washed in 10ml1640 dishes with medium alone. The spleen was gently squeezed by the inner core of the syringe and blown into a single cell suspension. Filtered through a copper mesh, 1000rpm, centrifuged for 5 min. The medium alone was washed 1-2 times and resuspended in 10ml1640 medium alone.

Mouse NS-1 cells: splenocytes =1:5, gently mixed at this ratio, centrifuged at 1000rpm for 5 min. The entire supernatant from the tube was discarded (ensuring the concentration of PEG 4000) and placed in a 37 ℃ water bath, where 1ml of 50% PEG4000 was added to a 50ml centrifuge 1 tube and stirred slowly for 1 minute. 1ml of 371640 ℃ medium was added within 1 min. 10ml of 1640 medium was added within 2min, and centrifuged at 800rpm for 5min, and the supernatant was discarded. Resuspending the cells in HAT medium, plating the cells in 96-well culture plates, adding feeder cells, CO₂Culturing in an incubator. The preparation method of the feeder cells comprises the following steps: mice were sacrificed and disinfected, abdominal skin was cut open, and peritoneum was exposed. Lifting mouse peritoneum with forceps, cutting a small opening, sucking a certain amount of 1640 simple culture medium, sucking several times in mouse abdominal cavity, placing into centrifuge tube, and centrifugingAnd counting. 96-well culture plates require 2x10 per well⁴The individual feeder cells were cultured in an incubator at 37 ℃.

5 days after the fusion, half-liquid HAT medium was changed. HAT medium was replaced with HT at day 8. Thereafter, the cells were cultured in 1640 complete medium. When the hybridoma cells grew to above the dish bottom 1/8, the positive wells were determined by detecting cell supernatant titers by indirect ELISA. Preparing hybridoma cells with qualified potency into cell suspension, diluting according to a certain proportion after cell counting until each ml contains 10 hybridoma cells, laying 96-hole culture plate, culturing at 37 deg.C with CO₂Culturing in an incubator; individual hybridoma cell culture wells were selected and labeled. ELISA detects the titer of the antibody of the cell supernatant to obtain a positive hole, 7 hybridoma cell strains capable of stably expressing the monoclonal antibody are finally obtained by screening through 2 times of subclone screening, and 2A4 and 3C5 with the best positive effect are selected for producing ascites.

SPF-grade BALB/C mice were 3 each, and were first inoculated with Freund's incomplete adjuvant 0.3ml each on the abdomen. After 10 days, the logarithmic growth phase hybridoma cells 2A4 and 3C5 were collected and prepared to a concentration of 10⁶Cell suspension per ml. Hybridoma cells were injected intraperitoneally at a rate of 0.3 ml/cell. Observing the growth condition of the ascites. If the abdomen is enlarged, ascites of the mouse can be extracted, and the ascites corresponding to each hybridoma can be directly detected by ELISA after being mixed, and the results are shown in Table 1.

TABLE 1 ELISA test results

Dilution factor	2A4 ascites	3C5 ascites
			1:2000	1.532	1.418
1:5000	0.923	0.902
			1:10000	0.574	0.539
1:20000	0.264	0.233
			1:40000	0.132	0.109

As can be seen from the results in Table 1, the ascites effect of each hybridoma reached 1:20000, which is a good effect. The remaining ascites was centrifuged at 2000rpm for 5 min. And taking the supernatant, purifying to obtain the 2A4 monoclonal antibody and the 3C5 monoclonal antibody, and respectively subpackaging and storing at-80 ℃.

EXAMPLE 22 characterization of the A4 monoclonal antibody

The affinity constant for the monoclonal antibody was measured using a biosensor IAsysPlus manufactured by AffinitySensors corporation. The sample cell was pretreated with carboxymethyl dextran (CMD), different concentrations of SAA protein were added to the cell, and after 5min the free carboxyl groups were blocked with ethanolamine for 3 min. Then, free and non-specifically bound protein molecules are washed away with 1mol/L formic acid. And (4) placing the sample pool coated with the SAA into a biosensor, and balancing for 10 min. Baseline 50. mu.l of 0.01mol/L PBS at pH7.2 was added and a stable baseline was established after 5 min. Association (asso-association) PBS was aspirated, 45. mu.l PBS and 5. mu.l monoclonal antibody were added, and the monoclonal antibody solution was aspirated when the monoclonal antibody was saturated. Dissociation (dissociation) 50. mu.l PBS was added and the monoclonal antibody binding and dissociation were equilibrated. Regeneration (regeneration) 20mmol/LHCl 50. mu.l was allowed to act for 2min to completely elute bound mAb. Return to baseline PBS was changed and returned to baseline again, i.e. the next 1 cycle was started. Diluting each monoclonal antibody to 5 different concentrations by PBS, sequentially and respectively measuring the binding rate and the dissociation rate of each concentration monoclonal antibody and the envelope antigen, and calculating the affinity constant of each monoclonal antibody through special software FASTpit attached randomly. The results are shown in Table 2.

TABLE 2 affinity constants of antibodies

Name of antibody	Affinity constant (nM)
		2A4 monoclonal antibody	0.89±0.02

As can be seen from the results in Table 2, the 2A4 monoclonal antibody has better affinity and binding ability.

The subclass of the monoclonal antibody was identified using a mouse antibody subclass identification kit. ELISA plates were coated with antigen, 50 ul/well, and left overnight at 4 ℃. The supernatant was discarded, 200 ul/well of blocking solution was added, and the mixture was incubated at 37 ℃ for 1 hour. The washing solution was washed 3 times, and 2A4 mAb was added to 9 different wells, 50 ul/well, and wells were repeated. Positive controls were added to well 10 and incubated at 37 ℃ for l hours. The liquid was removed and the wash washed 3 times. Normal rabbit serum was added to well 1, specific antibodies against immunoglobulin class or subclass of rabbit were added to wells 2-9, respectively, positive control wells were added with rabbit anti-mouse IgGl antibody, 50 ul/well, and incubated at 37 ℃ for 1 hour. The liquid was removed and the wash washed 3 times. HRP-goat anti-rabbit lgG antibody was added at 50 ul/well and incubated at 37 ℃ for l h. The liquid was removed and the wash washed 3 times. TMB color developing solution is added, 50 ul/hole. Developing at room temperature for 5min, adding 100ul stop solution to terminate the reaction, measuring OD value at 450nm, recording and judging the result. The results showed that the 2a4 monoclonal antibody was an IgM subtype and the light chain was a kappa chain.

And extracting total RNA of the hybridoma cells by using the kit, and performing reverse transcription to synthesize cDNA. Designing primers, amplifying heavy chains and light chains, sequencing, analyzing sequencing results by software, and obtaining a monoclonal light and heavy chain sequence through sequence identification, wherein the heavy chain variable region sequence is shown as SEQ ID NO:1, and the following components: EVQLEESATELARPGASVKLSCKASGYIFSCQDCCWIKQRPGQGLEWIGREKTKSEVKCKAFWEFGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGRMKRRGSWGLGTTLAVSS, the light chain variable region sequence is as shown in SEQ ID NO:2, as shown in the figure:

example 32A 4 specific identification of monoclonal antibodies

The specificity identification adopts a Western blot method: performing SDS-PAGE on SAA, BSA, AFP and beta globulin, transferring the gel protein to a nitrocellulose membrane after electrification, sequentially reacting with 2A4 and HRP-goat anti-mouse IgG, developing with DAB, performing cross reactivity detection, and adopting indirect ELISA.

Western blot identification results show that the 2A4 monoclonal antibody can recognize an SAA band with the Mr of about 12KD, which indicates that the SAA band is combined with the target SAA protein; and has no cross reaction with BSA, AFP, beta globulin and the like. The 2A4 monoclonal antibody has no binding reaction with other proteins and has better specificity through indirect ELISA method determination.

Example 4 preparation of magnetic microparticle SAA immunochromatographic test strip

Firstly, preparing SAA paramagnetic particles:

magnetic particles were purchased from sienna millennium biotechnology limited, cat #: 82023, 150nm in diameter.

1) Sucking 200 μ L (2mg) of magnetic microparticles, rinsing 3 times with 500 μ L of 10mmol/L MES buffer, pH5.5, washing buffer containing 0.05% Tween-20, adding 5mg/mL freshly prepared EDC and NHS, respectively, rotary activating at 37 deg.C for 4h, and washing twice with MES buffer to remove unreacted activating agent after activation.

2) Washing the activated magnetic particles twice with 0.005M of a pH9.0 BS coupling buffer (0.95 g of sodium tetraborate powder is weighed and dissolved in 50mL of pure water, 8mL is mixed with 2mL of 0.05M boric acid buffer, the pH is adjusted to 9.0 with 0.1M NaOH, then diluted by 40 times, the pH is adjusted to 9.0 with 0.1M NaOH, and the magnetic particles are washed twice with 500. mu.L of the coupling buffer; adding 475 mul of coupling buffer solution to resuspend the washed magnetic particles, adding 25 mul of 2A4 monoclonal antibody of 150 mug, contacting the activated hydroxyl on the surface of the magnetic particles with the amino of the antibody, reacting for 3 hours at 37 ℃, and coupling the antibody on the surface of the magnetic particles to obtain the immune magnetic particles.

3) Magnetic separation adsorbing magnetic particles after removing supernatant, adding 1mL coupling buffer containing blocking agent (0.25 g skimmed milk powder added 10mL 0.005M BS coupling buffer), blocking overnight at 37 deg.C; 4) washing the blocked magnetic particles with coupling buffer 4 times, storing in 500. mu.L appropriate storage buffer (0.1 g BSA and 5mg Tween-20 added to 10mL 0.005M BS coupling buffer, and adding 0.01% NaN₃And storing in a refrigerator at 4 deg.C for use. The content of the coupled magnetic particle antibodies is analyzed by a BCA protein quantitative detection kit, and the result shows that the amount of the coupled antibodies on the surface of each milligram of magnetic particles is 38 mug, so that the coupling amount is better.

The measurement of the ZETA potential is sometimes referred to as a "charge" measurement and is used to evaluate the electrostatic force stabilization effect of the dispersion. And (3) measuring the potentials of the magnetic particle stock solution and the coupled immune magnetic particles ZETA by using a high-performance nano particle size analyzer at the temperature of 25 ℃. The results are shown in FIG. 1.

FIG. 1 shows the surface charge of magnetic particles before and after coupling. The carboxyl groups on the surface of the magnetic particles enable the magnetic particles to have negative charges (-7.92 +/-0.91) mV, after the antibody is coupled, the carboxyl groups are combined with the amino groups of the antibody, the number of the carboxyl groups is reduced, and the surface charge amount of the immunized magnetic particles is reduced (-2.71 +/-0.51) mV. Since the reduction of the surface charge amount is not favorable for the stability of the magnetic particles, the total amount of the negative charges on the surface of the magnetic particles is increased, and the pH of the immune magnetic particle preservation solution is set to 9.0, so that the stability of the immune magnetic particles is improved.

Secondly, preparation of the test paper strip

1) Sample pad pretreatment: the glass cellulose membrane cut to an appropriate size was completely immersed in a sample pad treatment solution (0.01 mol/L PBS, pH7.4, 0.5% BSA, 0.5% TritonX-100) for 4 hours, and then naturally dried at room temperature.

2) Preparation of magnetic particle bonding pad: and taking a proper amount of prepared monoclonal antibody-labeled magnetic particles, sucking the proper amount of magnetic particles by using a pipette gun, spotting the magnetic particles on the bonding pad, and ventilating and drying at room temperature for 5 hours.

3) Preparing an NC film: commercially available SAA monoclonal antibody (Shanghai Yu Bo Biotech Co., Ltd., product No. MAA885Hu 22) and commercially available goat anti-mouse secondary antibody diluted to a concentration of 1mg/mL were sprayed onto the corresponding positions of the NC membrane by a film dispenser at a rate of 1. mu.L/cm, respectively, to obtain a detection T-line and a quality control C-line.

4) Assembling the immunochromatography test strip: and sequentially sticking the prepared sample pad, the bonding pad, the NC membrane and the water absorption pad on the PVC back plate, overlapping the adjacent pad parts, cutting the assembled pad parts into test strips with the width of 3mm by using a test strip cutting machine, and putting the test strips into a sealing bag containing a drying agent for storage at 4 ℃.

Test sensitivity test of the test strip:

and (3) determining the detection sensitivity: the SAA standard antigen is diluted in human normal serum with the concentration range of 0-1000 ng/mL. The constructed test strips are used for detecting 0, 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL and 1000ng/mL serum samples respectively, and a magnetic intensity detector (MAR) is used for analyzing the relation between T/C and sample concentration after 15 minutes. As the sensitivity of the test strip quantitative detection is the lowest concentration deviating from the test strip background signal, the quantitative detection sensitivity of the SAA detection calculated by adding 3 times of standard deviation to the T/C average value of the blank sample is 0.18 ng/mL.

And (3) specific identification: the prepared SAA, CEA, BSA, AFP and HCG standard solutions are dropwise added onto a test strip, the detection result is observed after 15 minutes, the result shows that only the standard solution containing the SAA shows a positive result, and the rest are negative, as shown in Table 3, the detection system has higher specificity.

TABLE 3 test strip Cross-reactivity

Detection of antigens	The result of the detection
		SAA	+
CEA	-
		BSA	-
AFP	-
		HCG	-

+ indicates positive and-negative.

It is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of components set forth in the following description and/or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

While the invention has been described and illustrated in detail as being sufficient to enable those skilled in the art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein represent preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications thereof and other uses will occur to those skilled in the art. Such modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

Sequence listing

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Gly Arg Glu Lys Thr Lys Ser Glu Val Lys Cys Lys Ala Phe Trp Glu

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Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Met Ala Lys Gln Gln His Thr

85 90 95

Gly Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

Claims

1. Monoclonal antibody 2a4 directed against SAA, characterized in that its heavy chain variable region sequence is as set forth in SEQ ID NO:1 is shown in the specification; the light chain variable region sequence is shown as SEQ ID NO:2, respectively.

2. Use of the monoclonal antibody 2a4 of claim 1 in the preparation of a magnetic microparticle-labeled SAA detection kit.

3. The use according to claim 2, wherein the kit comprises a test strip labeled with magnetic particles.

4. The use of claim 3, wherein the test strip comprises a detection line and a quality control line.

5. The use of claim 4, wherein the magnetic particle detection test paper comprises a sample pad, a magnetic particle pad, a nitrocellulose membrane and absorbent paper, which are sequentially adhered to a backing plate.

6. The use of claim 5, wherein the immunochromatographic strip is assembled by the steps of: and sequentially sticking the prepared sample pad, the bonding pad, the NC membrane and the water absorption pad on the PVC back plate, overlapping the adjacent pad parts, cutting the assembled pad parts into test strips with the width of 3mm by using a test strip cutting machine, and putting the test strips into a sealing bag containing a drying agent for storage at 4 ℃.