CN112379102A - Rhodium nanoparticle-ferritin polyclonal antibody compound and ferritin rapid detection test strip - Google Patents
Rhodium nanoparticle-ferritin polyclonal antibody compound and ferritin rapid detection test strip Download PDFInfo
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Abstract
本发明涉及免疫检测技术领域，尤其涉及一种铑纳米粒子‑铁蛋白多克隆抗体复合物和铁蛋白快速检测试纸条。本发明公开了一种铑纳米粒子‑铁蛋白多克隆抗体复合物，该复合物中铑纳米粒子具有类辣根过氧化物酶活性，可以代替胶体金在传统免疫试纸条的示踪作用。该复合物应用在试纸条中，与铁蛋白多克隆抗体组合，建立双抗夹心型免疫分析试纸条，该试纸条对铁蛋白特异性高，灵敏度好，检测限低，可快速准确的检测出血清中铁蛋白。
The invention relates to the technical field of immunodetection, in particular to a rhodium nanoparticle-ferritin polyclonal antibody complex and a ferritin rapid detection test strip. The invention discloses a rhodium nanoparticle-ferritin polyclonal antibody complex. The rhodium nanoparticle in the complex has horseradish peroxidase-like activity, which can replace the tracing effect of colloidal gold in traditional immunological test strips. The complex is used in test strips, combined with ferritin polyclonal antibodies to establish double-antibody sandwich type immunoassay test strips. The test strips have high specificity for ferritin, good sensitivity, low detection limit, and can be fast and accurate The detection of ferritin in serum.
Description
Technical Field
The invention relates to the technical field of immunodetection, in particular to a rhodium nanoparticle-ferritin polyclonal antibody compound and a ferritin rapid detection test strip.
Background
Ferritin is a metalloprotein ubiquitous in the body, having a nanoscale hydrated iron oxide core and a protein shell with a cage-like structure. Ferritin is widely found in liver and spleen tissues. Excessive iron deposition in the liver can lead to damage to hepatocytes. Typically, serum ferritin levels are maintained within a relatively stable range, whereas WHO dictates normal concentrations of ferritin in between 30-300ng/mL and 15-200ng/mL for men and women, respectively. Abnormal elevation of serum ferritin may reflect a variety of diseases such as hepatitis, cirrhosis, liver cancer, lung cancer, leukemia, etc. In addition, a reduction in serum ferritin can lead to anemia and malnutrition. Therefore, the detection of the ferritin in the serum has certain clinical guidance value. In the past decades, several methods for assaying ferritin have been established, such as electrochemical immunosensors, radioimmunoassays, and inductively coupled plasma mass spectrometry (ICP-MS). Although these methods are relatively sensitive and have excellent selectivity, these instruments are expensive, complicated to operate, require special expertise and highly trained operators.
The immunochromatography method can effectively improve the detection efficiency and accuracy of the content of the serum ferritin. At present, the materials for marking the immunochromatographic test strip are mainly colloidal gold nanoparticles, the colloidal gold is red, and the marker is relatively stable, so that the immunochromatographic test strip is widely applied to the immunochromatographic test strip. However, in practical application, it can be found that colloidal gold is easy to coagulate and discolor in a high-salt environment, so that a dead gold phenomenon is generated, and the detection effect is influenced.
Disclosure of Invention
The invention provides a rhodium nanoparticle-ferritin polyclonal antibody compound and a ferritin rapid detection test strip, and solves the problem that colloidal gold in the existing immunochromatographic test strip is easy to be coagulated and discolored in a high-salt environment, so that the detection effect is influenced.
The specific technical scheme is as follows:
the invention provides a rhodium nanoparticle-ferritin polyclonal antibody compound, which comprises the following components in percentage by weight: rhodium nanoparticles and ferritin polyclonal antibodies coupled to the rhodium nanoparticles.
The rhodium nanoparticles are stable, high-temperature resistant and pH resistant in a high-salt environment, the using efficiency of the immunochromatography method can be effectively improved, the rhodium nanoparticles used in the method have horseradish peroxidase-like activity, and a chromogenic signal can be improved through a catalytic reaction, so that the amplification of a detection signal is realized.
In the invention, the rabbit anti-human ferritin polyclonal antibody is obtained by immunizing animals with human ferritin, taking blood and centrifuging.
In the invention, the ferritin polyclonal antibody is a rabbit anti-human ferritin polyclonal antibody.
In the invention, the mass ratio of the rhodium nanoparticles to the ferritin polyclonal antibody is 800: (5-25), preferably 800: 10.
The invention also provides a preparation method of the rhodium nanoparticle-ferritin polyclonal antibody compound, which comprises the following steps:
step 1: reacting RhCl3Mixing the aqueous solution, hexadecyl trimethyl ammonium chloride and ascorbic acid, and reacting to obtain rhodium nanoparticles;
step 2: and mixing the aqueous solution of the rhodium nanoparticles, the aqueous alkali solution and the ferritin polyclonal antibody, reacting, and then adding a confining liquid for confining to obtain the rhodium nanoparticle-ferritin polyclonal antibody compound.
In step 1 of the present invention, RhCl3The dosage ratio of the hexadecyl trimethyl ammonium chloride to the ascorbic acid is (3-20) mg: (50-200) mg: (50-200) mL, preferably 10 mg: 10 mg: 1 mL;
the RhCl3The concentration of the aqueous solution is 0.3-5 mg/mL, preferably 2 mg/mL.
In step 2 of the invention, the alkali solution is preferably potassium carbonate solution, and the blocking solution is preferably bovine serum albumin;
the concentration of the alkali solution is 0.2M, the concentration of the ferritin polyclonal antibody is 1mg/mL, the concentration of the confining liquid is 0.1mg/mL, and the concentration of the aqueous solution of the rhodium nanoparticles is 1.5-1.6 mg/mL;
the volume ratio of the aqueous solution of the rhodium nanoparticles, the alkali solution, the ferritin polyclonal antibody and the blocking solution is (1000-4000): (0-8): (1-10): 100.
the invention also provides the application of the rhodium nanoparticle-ferritin polyclonal antibody compound or the rhodium nanoparticle-ferritin polyclonal antibody compound prepared by the preparation method in ferritin detection.
The invention also provides a ferritin rapid detection test strip, which comprises: a base plate;
the bottom plate is sequentially provided with a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper;
the combination pad is coated with the rhodium nanoparticle-ferritin polyclonal antibody compound or the rhodium nanoparticle-ferritin polyclonal antibody compound prepared by the preparation method, and the nitrocellulose membrane is sequentially scribed with a detection line of a ferritin monoclonal antibody and a quality control line of a goat anti-rabbit IgG antibody.
In the invention, the ferritin monoclonal antibody is a mouse anti-human ferritin monoclonal antibody.
In the invention, the concentration of the rhodium nanoparticle-ferritin polyclonal antibody compound is 5-30mg/mL, preferably 20mg/mL, and the spraying amount is 7.5-10.0 muL/cm, preferably 7.5 muL/cm or 8.5 muL/cm;
the concentration of the ferritin monoclonal antibody is 0.1-1.0mg/mL, preferably 0.2mg/mL, and the spraying amount is 1 muL/cm;
the concentration of the goat anti-rabbit IgG antibody is 0.1-1.0mg/mL, preferably 1.0mg/mL, and the spraying amount is 1 muL/cm.
In the invention, the distance between the quality control line and the detection line is 5 mm.
In the invention, the test strip further comprises: 3,3',5,5' -tetramethylbenzidine solution (TMB) and hydrogen peroxide solution. After the test of the serum to be detected is finished, preferably, the serum to be detected is dripped into the sample pad for 15min, the mixed solution is dripped into the detection line, the rhodium nanoparticles can catalyze TMB into oxidation type TMB, the oxidation type TMB is blue, the color of the rhodium nanoparticles is overlapped, the color development signal is improved, and the color development signal is used as an amplification signal for ferritin detection.
In the present invention, the amount of the mixed solution is 2 μ L, wherein the concentration of the TMB solution is 0.1M, the concentration of the hydrogen peroxide solution is 0.1M, and the molar ratio of TMB to hydrogen peroxide is 1: 1.
the invention also provides a method for rapidly detecting ferritin in serum, which comprises the following steps:
and (3) dropping serum to be detected on the sample pad in the ferritin rapid detection test strip, if the detection line and the quality control line are both colored, the ferritin in serum to be detected is detected, and if the detection line is not colored, and the quality control line is colored, the ferritin is not contained in the serum to be detected.
In the invention, the dosage of the serum to be detected is 80-150 mu L, preferably 100 mu L
In the invention, the principle of the ferritin rapid detection test strip is as follows:
after the serum to be detected is dripped into the test strip sample pad, the sample solution diffuses to the other end due to the capillary action of the nitrocellulose membrane carrier. During the movement, corresponding antigen-antibody reaction occurs, and the reaction is shown by the color of the rhodium nano-particles. If the sample solution to be detected contains ferritin, the ferritin firstly reacts with the rhodium nanoparticle-ferritin polyclonal antibody on the binding pad. When the rhodium nanoparticles diffuse to the detection line along with the sample solution, the ferritin monoclonal antibody is specifically combined with ferritin again to display a detection trace, and the goat anti-rabbit IgG antibody can be combined with the antibody again to color the quality control line, preferably, a mixed solution of TMB and hydrogen peroxide is dripped on the detection line to serve as an amplification signal of the ferritin on the detection line; on the contrary, when no ferritin exists in serum to be detected, the rhodium nanoparticle-ferritin polyclonal antibody and the ferritin monoclonal antibody do not have antigen-antibody reaction, the detection line does not develop color, and the goat anti-rabbit IgG antibody in the quality control area can be combined with the antibody and only the quality control line develops color; if the quality control line on the cellulose membrane and the detection line do not develop color, the test strip is invalid.
The invention adopts a double-resistance sandwich type immunoassay method which has high specificity and good sensitivity.
According to the technical scheme, the invention has the following advantages:
the invention provides a rhodium nanoparticle-ferritin polyclonal antibody compound, wherein the rhodium nanoparticle in the compound has horseradish peroxidase-like activity and can replace the tracing effect of colloidal gold on the traditional immune test strip. The compound is applied to a test strip and combined with a ferritin polyclonal antibody, a double-antibody sandwich type immunoassay test strip is proposed, the test strip has high specificity to ferritin, good sensitivity and low detection limit (0.29ng/mL), and ferritin in serum can be rapidly and accurately detected.
Drawings
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 only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without inventive exercise.
FIG. 1 is a TEM image of rhodium nanoparticles prepared in example 1 of the present invention;
FIG. 2 is a diagram showing the results of pH value optimization in the rhodium nanoparticle-ferritin polyclonal antibody complex in example 2 of the present invention;
FIG. 3 is a schematic diagram showing the results of exploring and optimizing the usage amount of rhodium nanoparticles in example 2 of the present invention;
FIG. 4 is a schematic diagram showing the results of the optimization of the amount of ferritin polyclonal antibody used in example 2 of the present invention;
FIG. 5 is a graph showing the ferritin standard substance detected by the test strip prepared in example 3 of the present invention and the corresponding standard curve;
fig. 6 is a working schematic diagram of the test strip prepared in embodiment 3 of the present invention.
Detailed Description
In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it should be apparent that the embodiments described below are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the examples of the present invention, ferritin monoclonal antibodies (mouse anti-human ferritin monoclonal antibodies) were obtained from Wuhanyun clone science and technology GmbH, and human ferritin standards were obtained from Fitzgerald, England.
Example 1 preparation of rhodium nanoparticles
The synthesis of rhodium nanoparticles was as follows: 10mg RhCl3Dissolved in 4mL of water, 100mg of CTAC was added, followed by 1mL of 0.1M ascorbic acid, which was freshly prepared, and after stirring uniformly, the mixture was placed in an oven at 80 ℃ and reacted for 16 hours. After the reaction is finished, cooling at room temperature, 10000rpm, centrifuging for 30 minutes, taking out the precipitate, redissolving with 20mL of water, and storing the solution at normal temperature, wherein the solution is black.
The morphology of the rhodium nanoparticles prepared in this example was characterized. As shown in FIG. 1, the successful synthesis of rhodium nanoparticles with an average particle size of 48nm can be observed by a scanning electron microscope.
Example 2 preparation of rhodium nanoparticle-ferritin polyclonal antibody complexes
Preparation of ferritin polyclonal antibody:
1. animal immunization
2 New Zealand white rabbits (female) of 1.5-2kg are selected. The immunizing antigen (human ferritin) was dissolved in PBS buffer and mixed with Freund's adjuvant in equal proportion. Continuously oscillating the mixture on a vortex instrument, fully emulsifying and mixing, dropping the mixture into deionized water after 10-15min if the solution becomes paste, observing whether the liquid drops diffuse in the water, and gently oscillating the water surface, if so, continuing to emulsify, otherwise, proving that the emulsification is successful. The emulsified immunizing antigen was then immunized against New Zealand white rabbits. The experimental protocol is shown in table 1:
TABLE 1 New Zealand white rabbit immunization protocol
2. Serum preparation
After the immunization for 63 days, the rabbit anti-human ferritin polyclonal antibody is obtained by taking blood from the ear marginal vein, and the collected rabbit blood is placed at 4 ℃ overnight, so that the blood clot can be completely contracted. Centrifuging at 4 deg.C and 1000rpm for 10min, collecting supernatant, and storing at-20 deg.C for use as ferritin polyclonal antibody.
Adding K to rhodium nanoparticle solution2CO3And then adding a ferritin polyclonal antibody, reacting for 1 hour at room temperature, adding a bovine serum albumin solution for sealing the unbound sites, reacting for one hour, centrifuging and taking a precipitate to obtain the rhodium nanoparticle-ferritin polyclonal antibody compound. Dissolving a rhodium nanoparticle-ferritin polyclonal antibody compound in a redissolution, wherein the formula of the redissolution is as follows: PBS buffer containing 5% sucrose, 2% fucose, 1% PEG20000, 1% BSA and 0.25% Tween-20.
1. Exploring the pH of the coupled System
Adding 0, 2, 4, 6 and 8 mu L of 0.2M K into 1mL rhodium nanoparticle aqueous solution with the mass ratio of 1:1 and the concentration of 1.5mg/mL2CO3To change the pH of the solution, 2. mu.L of 1mg/mL anti-ferritin polyclonal antibody was then added,after reaction at room temperature for one hour, 100. mu.L of 0.1mg/mL bovine serum albumin solution was added to block unbound sites, and after reaction for one hour, the pellet was centrifuged. Dissolving the conjugate in a redissolution, wherein the formula of the redissolution is as follows: PBS buffer containing 5% sucrose, 2% fucose, 1% PEG20000, 1% BSA and 0.25% Tween-20. The conjugate was then sprayed onto a conjugate pad and observed for color development at different pH.
As shown in FIG. 2, the quality control line and the detection line have no obvious change with the increase of the pH value of the rhodium nanoparticle aqueous solution, and 0 μ L of 0.2M K is preferably added for balancing the visible signal output and the economic cost2CO3An aqueous rhodium nanoparticle solution.
2. The dosage of rhodium nano-particles is explored
Diluting 1mL of rhodium nanoparticle aqueous solution with concentration of 1.6mg/mL according to volume ratio of 1:1, 1:2, 1:4, 1:8 and 1:16, respectively adding 2 μ L of 0.2M K2CO3To change the pH of the solution, 2. mu.L of 1mg/mL anti-ferritin polyclonal antibody was added, the reaction was allowed to proceed for one hour at room temperature, and then 100. mu.L of 0.1mg/mL bovine serum albumin solution was added to block unbound sites. After one hour of reaction, the precipitate was centrifuged. Dissolving the conjugate in a redissolution, wherein the formula of the redissolution is as follows: PBS buffer containing 5% sucrose, 2% fucose, 1% PEG20000, 1% BSA and 0.25% Tween-20. The conjugate was then sprayed onto a conjugate pad and the color development of different concentrations of rhodium nanoparticle solution was observed.
As shown in fig. 3, the intensity of the quality control line and the detection line gradually decreased as the amount of rhodium nanoparticles decreased. To balance the visible signal output and economic cost considerations, a 1:2 dilution ratio of rhodium nanoparticles.
3. Explore the dosage of the ferritin polyclonal antibody
Rhodium nanoparticle aqueous solution (1.6mg/mL) was added to each of the solutions at a dilution ratio of 1: 21 mL, and each of the solutions was 2. mu. L0.2M K2CO3To change the pH of the solution, 1, 5, 10, 15, 20, 25. mu.L of 1mg/mL ferritin polyclonal antibody was added, reacted at room temperature for one hour, and then 100. mu.L of 0.1mg/mL bovine serum albumin solution was added for blocking unbound sites.After one hour of reaction, the precipitate was centrifuged. Dissolving the conjugate in a redissolution, wherein the formula of the redissolution is as follows: PBS buffer containing 5% sucrose, 2% fucose, 1% PEG20000, 1% BSA and 0.25% Tween-20. The conjugate was then sprayed onto a conjugate pad and the development of different concentrations of the anti-ferritin polyclonal antibody was observed.
As shown in FIG. 4, in the range of 1-10. mu.L of 1mg/mL ferritin polyclonal antibody, the color intensity of the detection line increased and the color intensity of the quality control line decreased with the increase of the content of the ferritin polyclonal antibody. In a typical double antibody sandwich, the more antibodies, the better the exposure and recognition of Fab and the better the recognition of antigen or target, while the control line recognizes mainly the Fc of the antibody. By adding the anti-ferritin polyclonal antibody in the range of 10-15. mu.L, the intensity of the detection line and the quality control line were almost the same, while the color intensity of the T line and the C line decreased at 20. mu.L. In view of economic cost and sensitivity, 10. mu.L is preferred as the optimum amount of the anti-ferritin polyclonal antibody.
Example 3 preparation of test strips
And the ferritin monoclonal antibody and the goat anti-rabbit antibody which are drawn on the NC membrane in a sample loading amount of 1 mu L/cm are respectively used as a detection line and a quality control line, and the rhodium nanoparticle-anti-ferritin polyclonal antibody compound is sprayed on the combination pad. The NC film is pasted on the bottom plate, the water absorption pad (one end close to the quality control line) is pasted above the bottom plate, and the combination pad and the sample pad are pasted below the bottom plate. Wherein, the interval between matter accuse line and detection line is 5mm, stacks each other with 1 mm's distance between the pad and the pad, pad and the bottom plate. Drying at 37 ℃ to obtain the test strip.
1. Optimization of the streaking concentration of antibodies on a detection line
0.1-1.0mg/mL ferritin monoclonal antibody and 1.0mg/mL goat anti-rabbit antibody are respectively drawn on an NC membrane by a sample loading amount of 1 muL/cm as a detection line and a quality control line, and simultaneously, a rhodium nanoparticle-ferritin polyclonal antibody compound of 20mg/mL is jet-printed on a binding pad by a sample loading amount of 7.5 muL/cm. The NC film is pasted on the bottom plate, the water absorption pad (one end close to the quality control line) is pasted on the bottom plate, and the combination pad and the sample pad are pasted below the bottom plate. Wherein, the interval between matter accuse line and detection line is 5mm, stacks each other with 1 mm's distance between the pad and the pad, pad and the bottom plate. After drying at 37 ℃, 100. mu.L of 2. mu.g/mL ferritin was added to observe color development.
The results showed that the detection line was darker as the concentration of ferritin monoclonal antibody loaded on the detection line increased at 0.1-0.2mg/mL, but there was no significant difference in the detection line at a gradient of 0.2-1mg/mL, at which time ferritin-binding antibody on the detection line had already been saturated, so the streaking concentration of the detection line with 0.2mg/mL antibody was preferred.
2. Streaking concentration optimization of antibodies on quality control line
0.2mg/mL of anti-ferritin monoclonal antibody and 0.1-1.0mg/mL of goat anti-rabbit antibody are respectively drawn on an NC membrane by a sample loading amount of 1 mu L/cm as a detection line and a quality control line, and meanwhile, a rhodium nanoparticle-anti-ferritin polyclonal antibody compound is jet-printed on a binding pad by a sample loading amount of 7.5 mu L/cm. The NC film is pasted on the bottom plate, the water absorption pad (one end close to the quality control line) is pasted on the bottom plate, and the combination pad and the sample pad are pasted below the bottom plate. Wherein, the interval between matter accuse line and detection line is 5mm, stacks each other with 1 mm's distance between the pad and the pad, pad and the bottom plate. After drying at 37 ℃, 100. mu.L of 2. mu.g/mL ferritin was added to observe color development.
The results show that at 0.1-1.0mg/mL, the color of the quality control line is darker as the concentration of goat anti-rabbit antibody loaded on the quality control line is increased, and at the same time, no significant difference in the color of the quality control line can be observed at the concentration of 0.2-1.0 mg/mL. Therefore, 0.2mg/mL of antibody is preferred as the streaked concentration of the control line.
3. Metal spray dosage optimization
Respectively drawing 0.2mg/mL ferritin monoclonal antibody and 0.1mg/mL goat anti-rabbit antibody on an NC membrane by using a sample loading amount of 1 mu L/cm as a detection line and a quality control line, and simultaneously spraying and printing a rhodium nanoparticle-anti-ferritin polyclonal antibody compound on a binding pad by using a sample loading amount of 7.5-10.0 mu L/cm. The NC film is pasted on the bottom plate, the water absorption pad (one end close to the quality control line) is pasted on the bottom plate, and the combination pad and the sample pad are pasted below the bottom plate. Wherein, the interval between matter accuse line and detection line is 5mm, stacks each other with 1 mm's distance between the pad and the pad, pad and the bottom plate. After drying at 37 ℃, ferritin was added at 2. mu.g/mL to observe color development.
The results showed that the color of the detection line was darker with the increase of the sprayed amount at 7.5 to 8.5. mu.L/cm, but there was no significant difference in the detection line at the gradient of 8.5 to 10.0. mu.L/cm, and it was found that the rhodium nanoparticle-ferritin polyclonal antibody complex could form a visually recognizable signal with the antibody on the detection line at the sample loading amount of 8.5. mu.L/cm, and therefore, the sample loading amount of 8.5. mu.L/cm was preferable.
4. Testing of the Standard sample
And respectively drawing 0.2mg/mL ferritin monoclonal antibody and 0.2mg/mL goat anti-rabbit antibody on an NC membrane by using a sample loading amount of 1 mu L/cm as a detection line and a quality control line, and simultaneously spraying and printing a rhodium nanoparticle-anti-ferritin polyclonal antibody compound on a binding pad by using a sample loading amount of 8.5 mu L/cm. The NC film is pasted on the bottom plate, the water absorption pad (one end close to the quality control line) is pasted on the bottom plate, and the combination pad and the sample pad are pasted below the bottom plate. Wherein, the interval between matter accuse line and detection line is 5mm, stacks each other with 1 mm's distance between the pad and the pad, pad and the bottom plate. After drying at 37 ℃, adding 100 mu L of human ferritin standard solutions (0, 1, 10, 100 and 500ng/mL) with different concentrations, observing by naked eyes after 15 minutes, and shooting images by a smart phone for gray scale calculation. And establishing a four-parameter fitting curve by taking the protein content as an abscissa and the gray value as an ordinate.
At ferritin concentrations above 50ng/mL, saturation of the developed intensity was observed based on the rhodium nanoparticle immunochromatographic strip, the lowest visually detectable amount was 1ng/mL, the difference in developed intensity was plotted as the Y-axis and ferritin concentration was plotted as the X-axis, and fitted using a four-parameter logistic equation defined below, Y ═ (a-D)/[1+ (X/C) B]+ D where a is the response at high asymptote, B is the slope factor, C is the concentration corresponding to 50% specific binding (IC50), D is the response at low asymptote, X is the calibration concentration. The four-parameter logarithmic equation is-27459.93/[ 1+ (x/4265.75) ^0.58]+26699.75，R2＝0.999。
After 15 minutes, 2 μ L of a mixed solution of 0.1M TMB solution and 0.1M hydrogen peroxide solution (molar ratio of 1: 1) was dropped on the detection line, and an image was photographed with a smartphone to perform gray scale calculation. And establishing a four-parameter fitting curve by taking the protein content as an abscissa and the gray value as an ordinate.
As shown in fig. 5, the rhodium nanoparticles can catalyze TMB to oxidized TMB, and the solution turns blue, demonstrating that the rhodium nanoparticles have enzyme-like activity. The blue color of the oxidized TMB was superimposed with the color of the rhodium nanoparticles, thereby amplifying the detection signal of ferritin. In addition, ferritin was fitted with a linear relationship between ferritin concentration and color intensity after signal enhancement at 0.01-5ng/mL with the equations of y 711.41x +2367.90 and R2 0.973, while the linear fit equation for the pre-catalytic immunochromatographic strip at 0.1-10ng/mL was y 2173.298x +2459.02 and R2 0.917. The sensitivity before and after catalysis is improved by 1.3 times through comparison.
The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A rhodium nanoparticle-ferritin polyclonal antibody complex comprising: rhodium nanoparticles and ferritin polyclonal antibodies coupled to the rhodium nanoparticles.
2. The rhodium nanoparticle-ferritin polyclonal antibody complex according to claim 1, wherein the mass ratio of rhodium nanoparticle to ferritin polyclonal antibody is 800: (5-25).
3. A preparation method of a rhodium nanoparticle-ferritin polyclonal antibody compound is characterized by comprising the following steps:
step 1: will be provided withRhCl3Mixing the aqueous solution, hexadecyl trimethyl ammonium chloride and ascorbic acid, and reacting to obtain rhodium nanoparticles;
step 2: and mixing the aqueous solution of the rhodium nanoparticles, an alkali solution and the anti-ferritin polyclonal antibody, reacting, and then adding a confining liquid for confining to obtain the rhodium nanoparticle-ferritin polyclonal antibody compound.
4. The method according to claim 3, wherein the concentration of the aqueous rhodium nanoparticle solution is 1.5 to 1.6mg/mL, the concentration of the alkali solution is 0.2M, the concentration of the ferritin polyclonal antibody is 1mg/mL, the concentration of the blocking solution is 0.1mg/mL, and the concentration of the aqueous rhodium nanoparticle solution is 1.5 to 1.6 mg/mL;
the volume ratio of the aqueous solution of the rhodium nanoparticles, the alkali solution, the ferritin polyclonal antibody and the blocking solution is (1000-4000): (0-8): (1-10): 100.
5. use of the rhodium nanoparticle-ferritin polyclonal antibody complex according to claim 1 or 2 or the rhodium nanoparticle-ferritin polyclonal antibody complex prepared by the preparation method according to claim 3 or 4 in ferritin detection.
6. A ferritin rapid detection test strip is characterized by comprising: a base plate;
the bottom plate is sequentially provided with a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper;
the combination pad is coated with the rhodium nanoparticle-ferritin polyclonal antibody compound of claim 1 or 2 or the rhodium nanoparticle-ferritin polyclonal antibody compound prepared by the preparation method of claim 3 or 4, and the detection line of the ferritin monoclonal antibody and the quality control line of the goat anti-rabbit IgG antibody are scribed on the nitrocellulose membrane in sequence.
7. The ferritin rapid detection test strip according to claim 6 wherein the concentration of rhodium nanoparticle-ferritin polyclonal antibody complex is 5-30mg/mL and the spray dose is 7.5-10.0 μ L/cm;
the concentration of the ferritin monoclonal antibody is 0.1-1.0mg/mL, and the spraying amount is 1 muL/cm;
the concentration of the goat anti-rabbit IgG antibody is 0.1-1.0mg/mL, and the spraying amount is 1 muL/cm.
8. The ferritin rapid detection test strip of claim 6 further comprising: a mixed solution of a 3,3',5,5' -tetramethylbenzidine solution and a hydrogen peroxide solution.
9. A method for rapidly detecting ferritin in serum is characterized by comprising the following steps:
dropping the serum to be detected on the sample pad in the test strip for rapid detection of ferritin according to claim 6, wherein if the detection line and the quality control line both develop color, the serum to be detected contains ferritin, and if the detection line does not develop color and the quality control line develops color, the serum to be detected does not contain ferritin.
10. The method for rapidly detecting ferritin in serum according to claim 9 wherein the amount of serum to be detected is 80-150 μ L.
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