CN114544946A - Fluorescence immunochromatographic test strip for simultaneously detecting two markers of bacterial infection - Google Patents

Fluorescence immunochromatographic test strip for simultaneously detecting two markers of bacterial infection Download PDF

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CN114544946A
CN114544946A CN202210074629.5A CN202210074629A CN114544946A CN 114544946 A CN114544946 A CN 114544946A CN 202210074629 A CN202210074629 A CN 202210074629A CN 114544946 A CN114544946 A CN 114544946A
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test strip
pad
antibody
solution
markers
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王丽
张阿玲
刘宏飞
程巧玲
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Jiangsu University
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Abstract

The invention belongs to the technical field of biomarker diagnosis, and particularly relates to a fluorescence immunochromatographic test strip for simultaneously detecting two markers of bacterial infection. The C-reactive protein and procalcitonin are used as biomarkers, and the biomarkers are detected by specific binding of specific antibodies by adopting the principle of an immune double-antibody sandwich method. According to the invention, the fluorescent quantum dots are respectively labeled with specific antibodies Ab of different markers by utilizing the fluorescent characteristics of the fluorescent quantum dots, then the specific antibodies Ab of the markers labeled with different specific sites are prepared into a solution according to corresponding concentrations and coated on a nitrocellulose membrane detection line, and the purpose of simultaneously detecting two biomarkers in one reaction is realized. The test strip prepared by the invention has the remarkable advantages of simple detection, simple and convenient operation, low cost, high automation level, short time and the like; simultaneously detects two bacterial infection markers of CRP and PCT, and has high sensitivity.

Description

Fluorescence immunochromatographic test strip for simultaneously detecting two markers of bacterial infection
Technical Field
The invention belongs to the technical field of biomarker diagnosis, and particularly relates to a fluorescence immunochromatographic test strip for simultaneously detecting two markers of bacterial infection.
Background
In recent years, with the development of science, the progress of science and technology and the medical needs of the masses, many technologies are gradually applied to clinical examination, such as fluorescence immunochromatography, biosensor technology, enzyme-linked immunosorbent assay, dry chemical assay technology, biochip technology, infrared and far infrared spectrophotometry technology and colloidal gold immunochromatography technology. The most widely applied technology is fluorescence immunochromatography and colloidal gold immunochromatography, which respectively focus on the research of quantitative detection technology and qualitative detection technology, and realize industrialization in a certain scale. Thus creating an industry that detects this instantly. Point-of-care testing (POCT), a sub-division of in vitro diagnostic devices (IVD), is a miniature mobile testing system that is located outside the laboratory, close to the test subject, and can report the results in time, facilitating clinical testing and bedside testing near the patient. The method is not necessarily performed by a clinical inspector, and is a new method for instantly analyzing in a sampling site and quickly obtaining an inspection result.
The fluorescence quantum dot immunization technology has the advantages of strong specificity, high sensitivity, high speed, no radioactive pollution, simple instrument and equipment and the like, and is widely applied to the fields of clinical diagnosis, life analysis, environmental science and the like. At present, a fluorescent quantum dot immunological technology is adopted to detect a biomarker reagent, but in the prior art, detection is carried out on a certain biomarker, although the method can obtain an accurate detection result, the detection is carried out by a single method, the detection cost is high, the operation is complex, the detection time is long, and the clinical application and popularization of various biomarkers are not facilitated.
Disclosure of Invention
In view of the above, the present invention provides a fluorescence immunochromatographic test strip for simultaneously detecting two markers of bacterial infection. The provided chromatographic test strip can simultaneously detect C-reactive protein and calcitonin. The method has the characteristics of simple operation, short reaction time, capability of detecting a plurality of biomarkers simultaneously and the like.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the invention provides a fluorescence immunochromatographic test strip for simultaneously detecting two markers, which comprises: a bottom plate 1, a sample pad 2, a combination pad 3, a coating film 4 and a water absorption pad 5; wherein, the bonding pad 3 comprises a specific antibody Ab1-1 solution of an inflammation marker CRP marked by fluorescent quantum dots and a specific antibody Ab2-1 solution of an inflammation marker PCT; the coating film is provided with two detection lines T1 and T2 and a quality control line.
Furthermore, the mass concentration of the antibody Ab1-1 solution and the mass concentration of the antibody Ab2-1 solution are both 20-60 mug/mL.
The mass ratio of the labeled antibody Ab1-1 solution to the labeled antibody Ab2-1 solution is 1: 3.
preferably, the antibody Ab1-1 solution and the antibody Ab2-1 solution both use phosphate buffer with pH7.4 and 20mM as a solvent, and further comprise the following content components: 20-60 mu g/mL of fluorescent quantum dot-labeled C-reactive protein specific antibody Ab1-1, glycine with the mass concentration of 0.375%, sucrose 10%, potassium carbonate 0.2%, BSA 1% and sucrose 10%.
The two detection lines are respectively coated with a C-reactive protein specific antibody Ab1-2 coupled with quantum dots and a procalcitonin specific antibody Ab2-2 coupled with quantum dots; the quantum dots are self-made, and the emission wavelength of the quantum dots is 610 nm.
The concentrations of the Ab1-2 and the Ab2-2 are both 1 mg/mL; the volume ratio of the labeled Ab1-1 solution to the labeled Ab2-1 solution was 1: 3.
preferably, Ab1-2 and Ab2-2 coated on the coating film are coated at a streaking speed of 0.7 μ L/cm.
Furthermore, the water absorption pad 5 is arranged at one end of the bottom plate 1, the sample pad 2 is arranged at the other end of the bottom plate, the coating film 4 and the combination pad 3 are arranged between the sample pad 2 and the water absorption pad 5, and the water absorption pad 5 is erected at one end of the coating film 4; the combination pad 3 is arranged at the other end of the coating film 4, and the sample pad 2 is arranged on the combination pad 3; the sample pad 2 chromatographs in the direction of the conjugate pad 3 and the coating film 4.
Further, the invention also provides a kit, which comprises the fluorescent immunochromatographic test strip for simultaneously detecting the two markers. The preparation method of the test strip comprises the following steps: spraying gold on a specific antibody Ab1-1 solution of CRP marked by fluorescent quantum dots and a specific antibody Ab2-1 solution of PCT in a gold spraying mode of a gold spraying film scratching instrument, drying to obtain a binding pad, combining the binding pad with a sample pad, a bottom plate, a coated film, absorbent paper and a card shell, adding a sample with detection into a sample adding hole, waiting for 15min, reading by a dry fluorescence immunoassay analyzer to detect fluorescence intensity, and substituting the fluorescence intensity into a regression equation of each marker to obtain the concentration of each biomarker in the sample.
The invention also provides application of the fluorescence immunochromatographic test strip for simultaneously detecting the two markers in simultaneously detecting two markers of bacterial infection, namely CRP and PCT.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a fluorescence immunochromatographic test strip for simultaneously detecting two markers. The test strip takes C-reactive protein (hsCRP) and Procalcitonin (PCT) as biomarkers, and adopts the principle of an immune double-antibody sandwich method to detect the biomarkers by specific binding of specific antibodies. According to the invention, the fluorescent quantum dots are respectively labeled with specific antibodies Ab of different markers by utilizing the fluorescent characteristics of the fluorescent quantum dots, then the specific antibodies Ab of the markers labeled with different specific sites are prepared into a solution according to corresponding concentrations and are coated on a nitrocellulose membrane detection line (T line), and the purpose of simultaneously detecting two biomarkers in one reaction is realized. The test strip prepared by the invention can effectively distinguish the fluorescence intensity of different biomarkers, and the existence of different biomarkers can not influence the detection results of other types of biomarkers, and has no cross reaction. The method has the remarkable advantages of simple detection, simple and convenient operation, low cost, high automation level, short time and the like; the two bacterial infection markers of CRP and PCT are detected simultaneously, and the sensitivity is high; the test strip has no special requirements on a detection sample, and does not need to be subjected to complex sample treatment. The invention has wide application prospect in the field of instant examination by exploring and optimizing the marking process and the formula of the sample pad.
Drawings
FIG. 1 is a schematic diagram of a test strip architecture;
FIG. 2 is a schematic diagram of a test strip card;
FIG. 3 is a diagram showing the judgment of the test result of the test paper;
FIG. 4 is a CRP standard curve;
fig. 5 is a PCT standard curve.
In the figure: 1. the device comprises a base plate, 2, a sample pad, 3, a combination pad, 4, a coating film, 5, absorbent paper, 6, a detection line, 7, a quality control line, 8, a sample adding hole, 9, a window, 10 and a clamping shell.
Detailed Description
The invention discloses a fluorescence immunochromatographic test strip for simultaneously detecting CRP and PCT and a preparation method thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the fluorescence immunochromatographic strip for simultaneously detecting CRP and PCT described herein has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the present invention may be implemented and applied by modifying or appropriately modifying and combining the fluorescence immunochromatographic strip for simultaneously detecting CRP and PCT described herein without departing from the spirit, scope and spirit of the present invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Reagent: the fluorescent quantum dots are self-made by a subject group; the code of the C-reactive protein specific antibody Ab1-1 is Kinry hsCRP-12D4 #; the cat # of the C-reactive protein specific antibody Ab1-2 is Kinry CRP-2R4 #; procalcitonin specific antibody Ab2-1 has the code number Holms PCT-1020E #; the product number of the procalcitonin-specific antibody Ab2-2 is PCT-1020A #.
Example 1
The fluorescence immunochromatographic test strip for simultaneously detecting two markers of bacterial infection as shown in fig. 1 comprises a base plate 1, a sample pad 2, a binding pad 3, a coating film 4 and a water absorption pad 5. The water absorption pad 5 is arranged at one end of the bottom plate 1, the sample pad 2 is arranged at the other end of the bottom plate, the coating film 4 and the combination pad 3 are arranged between the sample pad 2 and the water absorption pad 5, and the water absorption pad 5 is erected at one end of the coating film 4; the combination pad 3 is arranged at the other end of the coating film 4, and the sample pad 2 is arranged on the combination pad 3; the sample liquid for detection is chromatographed through the sample pad 2 in the direction of the conjugate pad 3 and the coating film 4; the binding pad 3 comprises inflammation markers CRP marked by fluorescent quantum dots, antibodies Ab1-1 and Ab2-1 of PCT, the mass concentrations of the antibodies Ab1-1 and Ab2-1 are both 20-60 mug/mL, and the prepared test strip is arranged in a detection card to facilitate further detection of a blood sample.
The coated membrane 4 is respectively provided with two detection lines 6 of T1 and T2 and a quality control line 7, a CPR antibody Ab1-2 is coated at the position of T1, a PCT antibody Ab2-2 is coated at the position of T2, the concentrations are 0.5-1.5 mg/mL, and the membrane scratching amount is 0.7 muL/cm; the quality control line 7 is coated with a goat anti-mouse IgG polyclonal antibody, the concentration is 1-2 mg/mL, and the membrane scratching amount is 0.7 muL/cm. The interval between the T2 detection line and the T1 detection line at the detection line 6 is 3.5mm, and the interval between the T1 detection line and the quality control line 7 is 3.5 mm. The sample pad 2 and the combination pad 3 are made of a glass cellulose membrane, the coating membrane 4 is made of a nitrocellulose membrane (NC membrane), the bottom plate 1 is made of a PVC rubber plate, and the water absorption pad 5 is made of water absorption paper.
FIG. 2 is a schematic diagram of a test strip card; as shown in fig. 2, a sample hole 8 and a window 9 are provided on the detection card, the sample hole 8 corresponds to the sample pad 2 of the quantum dot immunochromatographic test strip, and the window 9 corresponds to the detection line 6 and the quality control line 7.
The invention adopts quantum dot immunochromatography, combines chromatography technology with quantum dot marking technology, and quantitatively detects two inflammation markers in a human blood sample: CRP, PCT; the self-made quantum dots are respectively coupled with two mouse anti-human CRP antibodies with high specificity, PCT antibodies Ab1-1 and Ab2-1 to form Ab-Q, and gold is pre-sprayed on a glass cellulose membrane of a binding pad; antibodies (Ab 1-2/Ab 2-2) containing different epitopes of two antigens (CRP, PCT) coated on the nitrocellulose membrane at T1, T2 respectively at detection line 6 on the nitrocellulose membrane (NC membrane) on the upper side of the loading well 8; the NC membrane is coated with goat anti-mouse IgG polyclonal antibody at the position of a quality control line 7. When a sample contains one or more antigens (Ag) to be detected, the Ag is combined with Ab-1 marked by quantum dots to form an antigen-antibody complex Ag-Ab-1-Q, and the complex moves forwards along a test strip due to chromatography and reacts with pre-coated Ab-2 when passing through the area of a detection line 6 to form an immune sandwich complex Ab-2-Ag-1-Ab-Q and show a fluorescent strip; samples that did not bind to Ab-2 continued to move forward, bound to goat anti-mouse IgG via the region of quality control line 7 to form a complex IgG-Ab-1-Q and visualized as a fluorescent band. If the sample does not contain the antigen Ag to be detected, only the quality control line 7 shows a fluorescence strip; when a sample is detected, the detection card is placed on a clean and flat table top, then 100 muL of human blood sample is vertically and slowly dripped into a sample adding hole 8 of the detection card, after waiting for 15 minutes, a dry type fluorescence immunoassay analyzer is used for reading to detect fluorescence intensity, and the result is judged according to the fluorescence conditions of the detection line 6 and the quality control line 7. And (3) bringing the fluorescence intensity into a regression equation of each marker to obtain the concentration of each biomarker in the sample. FIG. 3 is a diagram showing the judgment of the test result of the test paper; as shown in fig. 3, when both the control line and both the detection lines had a color band, a double positive was indicated; when the quality control line has a strip, only one strip exists in the detection line, and the result is single positive; the quality control line has a strip, and the detection line has no strip, which indicates that the result is negative; other cases indicated that the test strip was not effective.
Example 2
In this embodiment, the preparation of the inflammation markers CRP antibody and PCT antibody containing quantum dot markers comprises the following specific steps:
the quantum dot labeled antibody is coupled by using 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) activation coupling method. Diluting 10 muL of Quantum Dot (QD) solution to 60 muL with 20mM PB (pH7.4), shaking the added EDC for two minutes, adding NHS, carrying out vortex for 30min in a dark place for activation, then adding inflammation marker antibodies Ab1-1 and Ab2-1 respectively, and marking for 3h under the conditions of 37 ℃ and 265rmp in a shaking table after vortex for 15min at a low speed. After 3h, blocking was performed by adding Bovine Serum Albumin (BSA) for 1 h. After the blocking is finished, centrifuging for 20min at the rotation speed of 18500 r at the temperature of 4 ℃, discarding the supernatant, recovering the volume of the PCT to 200 mu L by using a recovery solution (0.375% of glycine, 1% of BSA, 0.2% of potassium carbonate and 10% of sucrose), and recovering the volume of the CRP to 100 mu L, namely the prepared quantum dot labeled CRP antibody and quantum dot labeled PCT antibody.
Example 3
In this example, a conjugate pad, a coating film, and a sample pad were prepared.
The specific preparation method of the bonding pad comprises the following steps: the mixture was uniformly coated on a glass cellulose membrane with a conjugate pad treatment solution (20 mM pH7.4 PB, 0.2% Tween, 0.2% Casein, 0.5% S17), and the membrane was placed in an oven at 37 ℃ and dried overnight. And (3) carrying out gold spraying on the solution of the fluorescent quantum dot-labeled C-reactive protein specific antibody Ab1-1 in a gold spraying mode of a gold spraying film scratching instrument, carrying out gold spraying on the solution of the fluorescent quantum dot-labeled procalcitonin specific antibody Ab2-1 in the gold spraying mode of the gold spraying film scratching instrument, and drying at 37 ℃ for 30min to obtain the binding pad containing the fluorescent quantum dot-primary antibody compound.
The preparation method of the coating film comprises the following steps: adjusting the antigen CRP and an antibody of different epitopes of PCT, namely a secondary antibody (Ab 1-2/Ab 2-2) to be 0.5-1.5 mg/mL by using 20mM PB (pH7.2-7.4), and respectively scribing at T1 and T2 of a detection line 6 of the NC membrane by 0.7 mu L/cm; adjusting the goat anti-mouse IgG to be 1-2 mg/mL by using 20mMPBS (pH 7.2-7.4), and marking the 1 muL/cm on a quality control line 7 of an NC membrane; the interval between the T2 detection line and the T1 detection line at the detection line 6 is 3.5mm, and the interval between the T1 detection line and the quality control line 7 is 3.5 mm. After the streaking was completed, the plate was placed in an oven for air drying at 37 ℃ overnight.
The preparation method of the sample pad comprises the following steps: the sample pad treatment solution (20 mM pH7.4 PB, 0.2% Tween, 0.2% Casein, 0.5% S17, 0.06mg/mLHRB-5, 0.03 mg/mLHRB-7) was uniformly coated on a glass cellulose film, placed in an oven at 37 ℃ and dried overnight.
Example 4
The method for treating the coupling solution containing two inflammation marker (CRP/PCT) antibodies (Ab) marked by quantum dots comprises the following specific steps:
the solution (CRPAb 1-1) of the fluorescent quantum dot labeled C-reactive protein primary antibody is coated on a nitrocellulose membrane by a gold scratching process: firstly, drawing a piece of confining liquid at a position 8mm below a T2 line on a nitrocellulose membrane under the condition of 1.5 muL/cm, after drying in an oven for 10min, drawing gold at the same position at the speed of 0.5 muL/cm, and drying at 37 ℃ for 30 min. The solution (PCTAb 2-1) of the fluorescent quantum dot-labeled procalcitonin primary antibody is sprayed on a bonding pad by a gold spraying process at the speed of 4 mu L/cm, and is placed in an oven to be dried by air blowing at 37 ℃ for 0.5 hour.
Example 5: drawing of standard curve
(1) CRP Standard Curve preparation
The CRP calibrators were subjected to test calibration at different concentrations and the calibration data are shown in table 1 and fig. 4.
TABLE 1 CRP calibrator calibration data
Concentration (ng/mL) 0.098 0.195 0.391 0.781 1.563 3.125 6.250
Fluorescence value 650 680 702.5 731 750 806.5 924
Concentration (ng/mL) 12.500 25.000 50.000 100.000 170.000 200.000 340.000
Fluorescence value 1943.5 3248 5602 10721.5 16022 17042 26546.5
The results show that the CRP scaling equation is y = 2E-07x2 + 0.0085x - 4.737，R2＝0.9986。
(2) Preparation of PCT Standard Curve
Test calibrations were performed with different concentrations of PCT calibrators, and the calibration data are shown in table 2 and fig. 5.
TABLE 2 PCT calibrator data
Concentration (ng/mL) 0.024 0.049 0.098 0.195 0.391 0.781
Fluorescence value 100 233 229.5 358.5 729 1465.5
Concentration (ng/mL) 1.563 3.125 6.250 12.500 25.000 50.000
Fluorescence value 3137 7101 8918.5 15731.5 23292 29004
The results show that the PCT scaling equation is y = 2E-07x2 + 0.0085x - 4.737，R2＝0.9986。
Therefore, the detection kit provided by the invention has good linearity, which indicates that the detection kit has the characteristics of accurate and reliable detection results, and meanwhile, the kit can be used for simultaneously detecting two biomarkers, and has the characteristics of simple and convenient operation and good result accuracy.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. The fluorescent immunochromatographic test strip for simultaneously detecting two markers is characterized by comprising the following components in parts by weight: the device comprises a bottom plate (1), a sample pad (2), a combination pad (3), a coating film (4) and a water absorption pad (5); wherein, the bonding pad (3) contains a specific antibody Ab1-1 solution of an inflammation marker CRP marked by fluorescent quantum dots and a specific antibody Ab2-1 solution of an inflammation marker PCT; the coating film is provided with two detection lines T1 and T2 and a quality control line.
2. The test strip according to claim 1, wherein the mass concentrations of the labeled antibody Ab1-1 solution and the labeled antibody Ab2-1 solution are both 20-60 μ g/mL; the mass ratio of the antibody Ab1-1 solution to the antibody Ab2-1 solution is 1: 3.
3. the test strip of claim 1, wherein the antibody Ab1-1 solution and the antibody Ab2-1 solution each use a phosphate buffer solution with a ph of 7.4 and a concentration of 20mM as a solvent, and further comprise the following components: 60 mu g/mL fluorescent quantum dot-labeled C-reactive protein specific antibody Ab1-1, glycine with the mass concentration of 0.375%, sucrose 10%, potassium carbonate 0.2%, BSA 1% and sucrose 10%.
4. The test strip of claim 1, wherein the two detection lines are coated with a quantum dot-conjugated C-reactive protein specific antibody Ab1-2 and a quantum dot-conjugated procalcitonin specific antibody Ab2-2, respectively.
5. The test strip of claim 4, wherein the quantum dots are self-made quantum dots, and the emission wavelength of the quantum dots is 610 nm.
6. The test strip of claim 1, wherein each of the Ab1-2 and Ab2-2 is at a concentration of 1 mg/mL; the volume ratio of Ab1-2 to Ab2-2 is 1: 1.
7. the test strip of claim 1, wherein the Ab1-2 and Ab2-2 coated on the coating film are coated at a streaking speed of 0.7 μ L/cm.
8. The test strip of claim 1, wherein the absorbent pad (5) is arranged at one end of the base plate (1), the sample pad (2) is arranged at the other end of the base plate, the envelope (4) and the combination pad (3) are arranged between the sample pad (2) and the absorbent pad (5), and the absorbent pad (5) is arranged at one end of the envelope (4); the combination pad (3) is arranged at the other end of the coating film (4), and the sample pad (2) is arranged on the combination pad (3); the sample pad (2) chromatographs in the direction of the binding pad (3) and the coating film (4).
9. A kit comprising the test strip of any one of claims 1 to 8.
10. The use of the fluorescence immunochromatographic test strip for simultaneous detection of two markers according to claim 1 for simultaneous detection of inflammation markers CRP and PCT.
CN202210074629.5A 2022-01-21 2022-01-21 Fluorescence immunochromatographic test strip for simultaneously detecting two markers of bacterial infection Pending CN114544946A (en)

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CN202210074629.5A CN114544946A (en) 2022-01-21 2022-01-21 Fluorescence immunochromatographic test strip for simultaneously detecting two markers of bacterial infection

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CN202210074629.5A CN114544946A (en) 2022-01-21 2022-01-21 Fluorescence immunochromatographic test strip for simultaneously detecting two markers of bacterial infection

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CN114544946A true CN114544946A (en) 2022-05-27

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