CN113702632A - Immunochromatography detection card for rapidly detecting preeclampsia and preparation method and application thereof - Google Patents
Immunochromatography detection card for rapidly detecting preeclampsia and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an immunochromatographic detection card for rapidly detecting preeclampsia and a preparation method and application thereof, the detection card comprises a test strip, the test strip comprises a sample pad, a combination pad and a reaction membrane, the reaction membrane is provided with a first detection line, a second detection line and a quality control line, the first detection line is coated with a first anti-FKBPL monoclonal antibody, the second detection line is coated with a first anti-CD 44 monoclonal antibody or the first detection line is coated with a first anti-CD 44 monoclonal antibody, the second detection line is coated with a first anti-FKBPL monoclonal antibody, and the quality control line is coated with a first negative control antibody; the conjugate pad is coated with an upconversion luminescent nanoparticle-anti-CD 44 second monoclonal antibody, an upconversion luminescent nanoparticle-anti-FKBPL second monoclonal antibody, and an upconversion luminescent nanoparticle-second negative control antibody. The detection card can be used for simultaneously and quantitatively detecting the concentrations of CD44 and FKBPL, has high detection sensitivity and good stability, and the obtained concentration ratio of CD44 to FKBPL has important significance for the diagnosis of preeclampsia.
Description
Technical Field
The invention relates to the technical field of medical inspection, in particular to an immunochromatography detection card for rapidly detecting preeclampsia and a preparation method and application thereof.
Background
Preeclampsia (Preeclampsia) refers to the condition of elevated blood pressure, proteinuria, headache, dim eyesight, nausea, vomiting, epigastric discomfort and the like after 20 weeks of gestation, and belongs to pregnancy idiopathic hypertension. Eclampsia (Eclampsia) is a condition that progresses from preeclampsia to more severe, causing seizure episodes or coma that can lead to serious maternal-fetal complications that endanger the life of the mother. Currently, there is no effective diagnosis and treatment strategy for preeclampsia, and there is no other effective treatment method except for termination of pregnancy, and the existing treatment is to control the disease condition and strive to prolong the gestational period, so it is very important to diagnose preeclampsia in time.
There are two main types of preeclampsia: early pre-eclampsia diagnosed before 34 weeks of gestation and late pre-eclampsia diagnosed after 34 weeks. Most screening and monitoring strategies focus on early onset preeclampsia, accounting for 10-15% of all cases of preeclampsia, while late preeclampsia is largely overlooked.
Currently, the clinical diagnosis of preeclampsia needs to meet the following three criteria: (1) measuring blood pressure (systolic pressure/diastolic pressure is not less than 140/90 mmHg); (2) detecting albuminuria (more than or equal to 0.3g/24 h); (3) screening was performed by an experienced physician who speculated on patients with symptoms associated with preeclampsia. However, the sample collection process of this diagnostic method is cumbersome and time consuming. There is currently no qualitative or quantitative indicator of preeclampsia for patient screening. Therefore, there is a clinical urgent need for effective, rapid preeclampsia screening markers and related detection tools.
FKBPL is peptidyl prolyl cis-trans isomerase, belongs to immunoaffinity protein group, is a key protein for regulating angiogenesis, has strong anti-angiogenesis activity, and plays an important role in development, pathological angiogenesis and stem cell differentiation. FKBPL is dependent on binding to the cell surface receptor CD44 for disruption of angiogenesis. Studies have determined FKBPL to be a prognostic biomarker for preeclampsia. There was some difference in the concentrations of FKBPL and CD44 between healthy and pre-eclamptic pregnant women. Thus, CD44 and FKBPL were used as diagnostic and prognostic indicators for preeclampsia based on this study basis. At present, no relevant report of an preeclampsia immunochromatography detection card taking the concentration of CD44 and FKBPL in serum as detection indexes is found.
Disclosure of Invention
In order to solve the problems, the invention provides an immunochromatography detection card for rapidly detecting preeclampsia and a preparation method and application thereof. The invention takes CD44 and FKBPL as the biomarkers of preeclampsia, designs an immunochromatography detection card, can simultaneously carry out quantitative detection on the concentrations of CD44 and FKBPL in serum, and achieves the aims of quickly detecting and screening preeclampsia patients.
According to one aspect of the invention, an immunochromatographic detection card for rapidly detecting preeclampsia is provided, which comprises a test strip, wherein the test strip comprises a sample pad, a binding pad and a reaction membrane, the reaction membrane is provided with a first detection line, a second detection line and a quality control line, the first detection line is coated with a first anti-FKBPL monoclonal antibody, the second detection line is coated with a first anti-CD 44 monoclonal antibody or the first detection line is coated with a first anti-CD 44 monoclonal antibody, the second detection line is coated with a first anti-FKBPL monoclonal antibody, and the quality control line is coated with a first negative control antibody; the conjugate pad is coated with an upconversion luminescent nanoparticle-anti-CD 44 second monoclonal antibody, an upconversion luminescent nanoparticle-anti-FKBPL second monoclonal antibody, and an upconversion luminescent nanoparticle-second negative control antibody.
Preferably, the sample pad and the conjugate pad are both prepared from a glass cellulose membrane.
Preferably, the reaction membrane is prepared from a nitrocellulose membrane.
The detection card provided by the invention is provided with two detection lines for detecting CD44 and FKBPL respectively, can simultaneously and quantitatively detect the concentrations of CD44 and FKBPL in serum, and can obtain the concentration ratio of CD44 to FKBPL through calculation, so that the detection card can be used as a marker for predicting preeclampsia. The first anti-CD 44 monoclonal antibody and the second anti-CD 44 monoclonal antibody are anti-CD 44 monoclonal antibodies, and can be combined with CD 44; the first anti-FKBPL monoclonal antibody and the second anti-FKBPL monoclonal antibody are anti-FKBPL monoclonal antibodies, and can bind to FKBPL. Dripping a liquid sample to be detected on the sample pad, and carrying out chromatography on the sample to be detected from the sample pad to the combination pad; then the sample to be tested is chromatographed forwards to the binding pad, when the sample to be tested contains CD44 and FKBPL, CD44 and FKBPL can be respectively combined with the up-conversion luminescent nanoparticle-anti-CD 44 second monoclonal antibody and the up-conversion luminescent nanoparticle-anti-FKBPL second monoclonal antibody coated on the binding pad to form up-conversion luminescent nanoparticle-anti-CD 44 second monoclonal antibody-CD 44 and up-conversion luminescent nanoparticle-anti-FKBPL second monoclonal antibody-BPFKL; continuing the chromatography from the conjugate pad to the reaction membrane, when the up-converting luminescent nanoparticle-anti-FKBPL second monoclonal antibody-FKBPL and up-converting luminescent nanoparticle-anti-CD 44 second monoclonal antibody-CD 44 pass through two detection lines, they can be immobilized on the corresponding detection line coated with monoclonal antibody capable of binding to FKBPL, CD44 to form a double antibody sandwich structure of up-converting luminescent nanoparticle-anti-FKBPL second monoclonal antibody-FKBPL-anti-FKBPL first monoclonal antibody, up-converting luminescent nanoparticle-anti-CD 44 second monoclonal antibody-CD 44-CD 44 first monoclonal antibody. The action principle of the up-conversion luminescent nanoparticle-second negative control antibody coated on the bonding pad and the first negative control antibody coated on the quality control line is the same as that of the up-conversion luminescent nanoparticle-second negative control antibody coated on the bonding pad, and the up-conversion luminescent nanoparticle-second negative control antibody coated on the quality control line has the function of monitoring the correctness of the detection process and the failure of the immunochromatographic detection card.
The invention adopts an up-conversion luminescence technology, namely an immunochromatography technology taking up-conversion luminescence nano-particles formed by rare earth metal elements as a marker. Firstly, the up-conversion luminescent nano-particles adopted by the invention are nano-materials, have excellent signal amplification effect and can achieve good sensitivity and specificity. Secondly, the upconversion luminescent nanoparticles can emit visible light with different wavelengths under infrared excitation light, and the unique upconversion luminescent phenomenon eliminates the possibility that the spontaneous fluorescence of a biological sample to be detected causes interference on a detection result in the detection process by taking the upconversion luminescent nanoparticles as a marker. In addition, the upconversion luminescent nanoparticles can emit stronger emitted light under the irradiation of sufficient excitation light, and the detection sensitivity can be further improved. The characteristic of the up-conversion luminescence nano-particles enables the immunochromatography detection using the up-conversion luminescence nano-particles as the markers to be combined with an instrument, thereby realizing the high-sensitivity quantitative detection of the detected objects. Moreover, the upconversion luminescent nanoparticle has various characteristic spectrums (including an excitation spectrum and an emission spectrum), so that the immunochromatography detection based on the upconversion luminescent technology can perform multiple analyses with extremely high sensitivity, namely, the detection of multiple targets of a biological sample can be performed at one time.
Preferably, the particle size of the upconversion luminescent nanoparticles is between 25 and 200 nm.
Preferably, the particle size of the upconversion luminescent nanoparticle is 50 nm.
The fluorescent efficiency of the up-conversion luminescent nano-particles with the particle size smaller than 10nm is low, the nano-particles with the moderate particle size are selected for use in the scheme, the steric hindrance can be reduced, the combination of the nano-particles with specific antibodies is avoided, meanwhile, the fluorescent efficiency of the up-conversion luminescent nano-particles can be improved to a certain extent, and the detection sensitivity and stability are further improved. The concrete embodiment is as follows: the immunochromatography detection card can detect CD44 and FKBPL with the concentration of more than 0.1ng/mL, so that FKBPL with a low concentration level can be detected, false negatives are avoided to a certain extent, and the positive detection rate of the immunochromatography detection card on CD44 and FKBPL reaches 100%.
Preferably, the upconversion luminescent nanoparticle is obtained by carboxyl modification. The upconversion luminescent nanoparticles often have hydrophobic groups, so that the upconversion luminescent nanoparticles are difficult to dissolve in water, the properties of the upconversion luminescent nanoparticles are greatly influenced by surface effects, and the fluorescence of the nanoparticles can be reduced by surface defects of the upconversion luminescent nanoparticles. The up-conversion luminescent nano-particles used in the scheme are obtained by carboxyl modification, so that the stability of the nano-particles can be improved, the luminescent performance of the nano-particles is more stable, and the accuracy of immunoassay is improved.
Preferably, the upconversion luminescent nanoparticle-anti-CD 44 second monoclonal antibody, the upconversion luminescent nanoparticle-anti-FKBPL second monoclonal antibody, and the upconversion luminescent nanoparticle-second negative control antibody are prepared by covalently crosslinking the upconversion luminescent nanoparticle with the anti-CD 44 second monoclonal antibody, the anti-FKBPL second monoclonal antibody, and the second negative control antibody, respectively. The upconversion luminescent nanoparticles are crosslinked with bioactive molecules in a covalent mode, so that the reliability and stability of the system are improved on the premise of ensuring the detection sensitivity.
Preferably, the preparation method of the upconversion luminescent nanoparticle-anti-CD 44 second monoclonal antibody comprises the following steps: preparing a suspension by using the up-conversion luminescent nano-particles and a PB buffer solution (phosphate buffer solution), and adding NHS (N-hydroxysuccinimide) and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) into the suspension in sequence for reaction; centrifuging to remove supernatant, adding PB buffer solution, and mixing; adding a second anti-CD 44 monoclonal antibody into the system for reaction, adding BSA (bovine serum albumin) for blocking after the reaction is finished, and performing centrifugal washing to obtain an upconversion luminescent nanoparticle-anti-CD 44 second monoclonal antibody;
the preparation method of the second monoclonal antibody of the upconversion luminescent nanoparticle-anti-FKBPL comprises the following steps: preparing a suspension by using the up-conversion luminescent nano particles and a PB buffer solution, and sequentially adding NHS and EDC into the suspension for reaction; centrifuging to remove supernatant, adding PB buffer solution, and mixing; adding a second anti-FKBPL monoclonal antibody into the system for reaction, adding BSA for sealing after the reaction is finished, and carrying out centrifugal washing to obtain the up-conversion luminescent nanoparticle-the second anti-FKBPL monoclonal antibody;
the preparation method of the up-conversion luminescent nanoparticle-second negative control antibody comprises the following steps: preparing a suspension by using the up-conversion luminescent nano particles and a PB buffer solution, and sequentially adding NHS and EDC into the suspension for reaction; centrifuging to remove supernatant, adding PB buffer solution, and mixing; and adding a second negative control antibody into the system for reaction, adding BSA (bovine serum albumin) for blocking after the reaction is finished, and performing centrifugal washing to obtain the upconversion luminescent nanoparticle-second negative control antibody.
Preferably, the mass ratio of the NHS to the upconversion luminescent nanoparticle is 1:2-5, and the mass ratio of the EDC to the upconversion luminescent nanoparticle is 1: 2-5.
Preferably, the mass ratio of the second anti-CD 44 monoclonal antibody, the second anti-FKBPL monoclonal antibody, the second negative control antibody and the upconversion luminescent nanoparticle is 1: 100-1000.
Preferably, the PB buffer has a pH of 7.2 and a concentration of 20 mM.
Preferably, the test strip further comprises a water absorption pad and an adhesive bottom plate, wherein the sample pad, the combination pad, the reaction membrane and the water absorption pad are partially overlapped in sequence and then adhered to the surface of the adhesive bottom plate.
Preferably, the test strip has a width of 3-5 mm.
Preferably, the test strip has a width of 3.5 mm.
Preferably, when the sample pad, the conjugate pad, the reaction membrane and the absorbent pad are partially overlapped in sequence, the distance between the overlapped parts is 1-2 mm.
Preferably, the sample pad, the combination pad, the reaction membrane and the absorbent pad are partially overlapped in sequence and then adhered to the surface of the adhesive base plate, wherein the sample pad is positioned above the combination pad at the overlapping part of the sample pad and the combination pad, the combination pad is positioned above the reaction membrane at the overlapping part of the combination pad and the reaction membrane, and the absorbent pad is positioned above the reaction membrane at the overlapping part of the reaction membrane and the absorbent pad.
According to the second aspect of the present invention, a method for simultaneously and quantitatively detecting CD44 and FKBPL concentrations is provided, which comprises: dropwise adding a sample to be detected on an immunochromatography detection card for rapidly detecting preeclampsia, irradiating the detection card by using a laser diode under the action of a hemispherical lens, and collecting signals by using a signal collector.
Preferably, the laser diode is a 790nm, 980nm or 1550nm laser diode.
Preferably, the laser diode is a 980nm laser diode, which is near infrared excitation light.
Preferably, the signal collected by the signal collector is a 540nm green light signal. When the immunochromatography detection card is used for detecting CD44 and FKBPL, green light (540nm) visible to the naked eye can be emitted under the irradiation of a 980nm laser diode, so that the detection result is convenient to observe.
Preferably, the energy density of the laser diode under the action of the hemispherical lens is more than 0.001mW/cm3。
The immunochromatographic detection card for rapidly detecting preeclampsia provided by the invention can screen whether a pregnant woman has preeclampsia: if the concentration ratio of CD44 to FKBPL in the serum of the subject is obviously higher than the ratio of the CD44 to FKBPL in the serum of the subject at 20 weeks of pregnancy and is also obviously higher than the concentration ratio of CD44 to FKBPL in the serum of a normal pregnant woman, determining that the risk of preeclampsia of the subject is high; otherwise, the risk of preeclampsia is small.
The invention has the following beneficial effects:
1. the invention provides an immunoassay chromatography card adopting new detection indexes (namely CD44 and FKBPL) for the detection of preeclampsia.
2. The invention is based on the up-conversion luminescence immunochromatography technology, utilizes the unique up-conversion luminescence characteristic of the up-conversion luminescence nano-particles, takes the up-conversion luminescence nano-particles as markers, has the advantages of no background interference in detection, high sensitivity, stable luminescence performance and difficult quenching, and can simultaneously and quantitatively detect the concentrations of CD44 and FKBPL in serum by combining with a matched instrument.
3. When the immunochromatography detection card is used for detecting the concentrations of CD44 and FKBPL, the lowest detection concentration is 0.1ng/mL, and the sensitivity is high.
4. The immunoassay chromatographic card is simple to use and operate, the time for detecting samples is short, and the detection speed is greatly improved compared with the detection method generally used clinically.
5. The immunochromatography detection card has the advantages of simple structure, easy processing and manufacturing, low production cost and convenient industrial production.
CD44 and FKBPL may be novel pre-eclampsia markers and the ratio of the concentration of CD44 to FKBPL may be of great importance for the diagnosis and assessment of the risk of pre-eclampsia in the early and late stages of pregnancy.
Drawings
FIG. 1 is a schematic structural diagram of a single-well immunochromatographic assay card for rapid detection of preeclampsia in accordance with the present invention.
The reference signs are: 1 sample pad, 2 combination pads, 3 first detection lines, 4 second detection lines, 5 quality control lines, 6 reaction membranes, 7 water absorption pads and 8 sticky bottom plates.
FIG. 2 is a schematic structural diagram of a double-well immunochromatographic assay card for rapid detection of preeclampsia in accordance with the present invention.
The reference signs are: c quality control line, T detection line and S sample adding hole.
Detailed Description
Technical features in the technical solutions provided by the present invention are further clearly and completely described below with reference to specific embodiments, and it is obvious that the described embodiments are only a part of 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.
Example 1
A single-hole immunochromatographic assay for rapidly detecting preeclampsia is prepared by the following steps:
(1) preparation of Up-converting luminescent nanoparticles (UCNPs) -antibodies
Adding UCNPs with the surface subjected to carboxylation modification into a PB buffer solution with the pH of 7.2 and the concentration of 20mM to prepare a suspension with the concentration of 1 mg/mL; adding 50mg/mL NHS (NHS: UCNPs mass ratio of 1:4) and 50mg/mL EDC (EDC: UCNPs mass ratio of 1:4) in sequence, and reacting at 4-30 ℃ for 30 min; centrifuging to remove redundant NHS and EDC, then adding an equivalent amount of 0.02mol/L PB buffer solution, and uniformly dispersing the suspension; adding a second anti-CD 44 monoclonal antibody, reacting for 2 hours at 4-30 ℃, wherein the mass ratio of the added second anti-CD 44 monoclonal antibody to UCNPs is 1: 100; after the reaction is finished, adding BSA with the final concentration of 1% for blocking for 1.5 h; after the reaction is finished, centrifugally washing for 1 time at 4 ℃ to obtain an upconversion luminescent nanoparticle-anti-CD 44 second monoclonal antibody, storing in 0.02M PB buffer solution, and placing at 2-8 ℃ for later use;
preparing an upconversion luminescent nanoparticle-anti-FKBPL second monoclonal antibody and an upconversion luminescent nanoparticle-second negative control antibody according to the above preparation method;
(2) preparation of sample pad
Selecting a glass cellulose membrane as a sample pad material, cutting the sample pad material into strips with the specification of 1.5cm x 30cm, soaking the strips in PB buffer solution with the pH value of 7.2, the concentration of 0.02M and the content of 0.1% wt Tween-20 for 10min, and drying the strips at 37 ℃ after soaking to obtain a sample pad 1;
(3) preparation of the bonding pad
Selecting a glass cellulose membrane as a binding pad material, cutting the glass cellulose membrane into strips with the specification of 1cm x 30cm, soaking the strips in a binding pad treatment solution for 10min, wherein the components of the binding pad treatment solution comprise 0.1% BSA, 2% sucrose and 0.1% Tween-20, drying the strips at 37 ℃, mixing the up-conversion luminescent nanoparticles-anti-CD 44 second monoclonal antibody, the up-conversion luminescent nanoparticles-anti-FKBPL second monoclonal antibody and the up-conversion luminescent nanoparticles-second negative control antibody prepared in the step (1) according to the proportion of 2:2:1 by using a metal spraying instrument, uniformly spraying the mixture on the glass cellulose membrane, and drying the mixture at 37 ℃ to obtain a binding pad 2;
(4) preparation of the reaction film
Cutting a nitrocellulose membrane (NC membrane) into strips with the specification of 2.5cm by 30cm, pasting the obtained strips in the middle of an adhesive bottom plate 8, respectively scribing a first anti-FKBPL monoclonal antibody, a first anti-CD 44 monoclonal antibody and a first negative control antibody on different positions of the NC membrane by using a membrane scribing instrument to serve as a first detection line 3, a second detection line 4 and a quality control line 5, and drying at 37 ℃ to obtain a reaction membrane 6;
(5) assembled immunochromatography test strip
And (3) respectively sticking the sample pad 1, the combination pad 2 and the water absorption pad 7 to two sides of the reaction membrane 6 prepared in the step (4), ensuring that the pad intervals of all components are 1.5mm, alternately laminating, cutting into test strips with the width of 3.5mm, and installing the test strips in a customized card shell to obtain the single-hole immunochromatography detection card for rapidly detecting preeclampsia as shown in figure 1.
Example 2
A double-hole immunochromatographic assay card for rapidly detecting preeclampsia comprises a test strip, wherein the test strip comprises a first test strip and a second test strip.
The preparation method of the first test strip specifically comprises the following steps:
(1) preparation of Up-converting luminescent nanoparticles (UCNPs) -antibodies
Adding UCNPs with the surface subjected to carboxylation modification into a PB buffer solution with the pH of 7.2 and the concentration of 20mM to prepare a suspension with the concentration of 1 mg/mL; adding 50mg/mL NHS (NHS: UCNPs mass ratio of 1:4) and 50mg/mL EDC (EDC: UCNPs mass ratio of 1:4) in sequence, and reacting at 4-30 ℃ for 30 min; centrifuging to remove redundant NHS and EDC, then adding an equivalent amount of 0.02mol/L PB buffer solution, and uniformly dispersing the suspension; adding a second anti-CD 44 monoclonal antibody, reacting for 2 hours at 4-30 ℃, wherein the mass ratio of the added second anti-CD 44 monoclonal antibody to UCNPs is 1: 100; after the reaction is finished, adding BSA with the final concentration of 1% for blocking for 1.5 h; after the reaction is finished, centrifugally washing for 1 time at 4 ℃ to obtain an upconversion luminescent nanoparticle-anti-CD 44 second monoclonal antibody, storing in 0.02M PB buffer solution, and placing at 2-8 ℃ for later use;
preparing an upconversion luminescent nanoparticle-second negative control antibody according to the above preparation method;
(2) preparation of sample pad
Selecting a glass cellulose membrane as a sample pad material, cutting the sample pad material into strips with the specification of 1.5cm x 30cm, soaking the strips in PB buffer solution with the pH value of 7.2, the concentration of 0.02M and the content of 0.1% wt Tween-20 for 10min, and drying the strips at 37 ℃ after soaking to obtain a sample pad;
(3) preparation of the bonding pad
Selecting a glass cellulose membrane as a binding pad material, cutting the glass cellulose membrane into strips with the specification of 1cm x 30cm, soaking the strips for 10min by using a binding pad treatment solution, wherein the components of the binding pad treatment solution comprise 0.1% BSA, 2% sucrose and 0.1% Tween-20, then drying the strips at 37 ℃, mixing the up-conversion luminescent nanoparticles-anti-CD 44 second monoclonal antibody and the up-conversion luminescent nanoparticles-second negative control antibody prepared in the step (1) according to the ratio of 2:1 by using a gold spraying instrument, uniformly spraying the mixture on the glass cellulose membrane, and drying the mixture at 37 ℃ to obtain a binding pad;
(4) preparation of the reaction film
Cutting a nitrocellulose membrane (NC membrane) into strips with the specification of 2.5cm by 30cm, sticking the obtained strips in the middle of a viscous bottom plate, respectively scribing a first monoclonal antibody resisting CD44 and a first negative control antibody on different positions of the NC membrane by using a scribing instrument as a detection line (T) and a quality control line (C), and drying at 37 ℃ to obtain a reaction membrane;
(5) assembled immunochromatography test strip
Respectively sticking a sample pad, a combination pad and a water absorption pad on two sides of the reaction membrane prepared in the step (4), ensuring that the distance between the component pads is 1.5mm, and alternately stacking, and then cutting into test strips with the width of 3.5mm to obtain first test strips;
the preparation method of the second test strip specifically comprises the following steps:
(1) preparation of Up-converting luminescent nanoparticles (UCNPs) -antibodies
Adding UCNPs with the surface subjected to carboxylation modification into a PB buffer solution with the pH of 7.2 and the concentration of 20mM to prepare a suspension with the concentration of 1 mg/mL; adding 50mg/mL NHS (NHS: UCNPs mass ratio of 1:4) and 50mg/mL EDC (EDC: UCNPs mass ratio of 1:4) in sequence, and reacting at 4-30 ℃ for 30 min; centrifuging to remove redundant NHS and EDC, then adding an equivalent amount of 0.02mol/L PB buffer solution, and uniformly dispersing the suspension; adding a second anti-FKBPL monoclonal antibody into the mixture, and reacting for 2 hours at 4-30 ℃, wherein the mass ratio of the added second anti-FKBPL monoclonal antibody to UCNPs is 1: 100; after the reaction is finished, adding BSA with the final concentration of 1% for blocking for 1.5 h; after the reaction is finished, centrifugally washing for 1 time at 4 ℃ to obtain an up-conversion luminescent nanoparticle-anti-FKBPL second monoclonal antibody, storing in 0.02M PB buffer solution, and placing at 2-8 ℃ for later use;
preparing an upconversion luminescent nanoparticle-second negative control antibody according to the above preparation method;
(2) preparation of sample pad
Selecting a glass cellulose membrane as a sample pad material, cutting the sample pad material into strips with the specification of 1.5cm x 30cm, soaking the strips in PB buffer solution with the pH value of 7.2, the concentration of 0.02M and the content of 0.1% wt Tween-20 for 10min, and drying the strips at 37 ℃ after soaking to obtain a sample pad;
(3) preparation of the bonding pad
Selecting a glass cellulose membrane as a binding pad material, cutting the glass cellulose membrane into strips with the specification of 1cm x 30cm, soaking the strips in a binding pad treatment solution for 10min, wherein the components of the binding pad treatment solution comprise 0.1% BSA, 2% sucrose and 0.1% Tween-20, then drying the strips at 37 ℃, mixing the up-conversion luminescent nanoparticles-anti-FKBPL second monoclonal antibody and the up-conversion luminescent nanoparticles-second negative control antibody prepared in the step (1) according to the ratio of 2:1 by using a gold spraying instrument, uniformly spraying the mixture on the glass cellulose membrane, and drying the mixture at 37 ℃ to obtain a sample pad;
(4) preparation of the reaction film
Cutting a nitrocellulose membrane (NC membrane) into strips with the specification of 2.5cm by 30cm, pasting the obtained strips in the middle of an adhesive bottom plate, respectively scribing a first monoclonal antibody resisting FKBPL and a first negative control antibody on different positions of the NC membrane by using a scribing instrument to serve as a detection line (T) and a quality control line (C), and drying at 37 ℃ to obtain a reaction membrane;
(5) assembled immunochromatography test strip
Respectively sticking a sample pad, a combination pad and a water absorption pad on two sides of the reaction membrane prepared in the step (4), ensuring that the distance between the component pads is 1.5mm, and alternately stacking, and then cutting into test strips with the width of 3.5mm to obtain second test strips;
the first test strip and the second test strip obtained above are mounted in customized CD44 and FKBPL card shells to obtain the double-hole immunochromatographic detection card for rapidly detecting preeclampsia as shown in fig. 2, wherein the card shell of the double-hole immunochromatographic detection card contains two sample application holes S, and both are located above the sample pad.
Comparative example 1
An immunochromatography detection card for qualitatively detecting CD44 and FKBPL is prepared by the following steps:
(1) preparation of sample pad
Selecting a glass cellulose membrane as a sample pad material, cutting the sample pad material into strips with the specification of 1.5cm x 30cm, soaking the strips in PB buffer solution with the pH value of 7.2, the concentration of 0.02M and the content of 0.1% wt Tween-20 for 10min, and drying the strips at 37 ℃ after soaking to obtain a sample pad;
(2) preparation of gold label pad
Dissolving chloroauric acid in deionized water to form a chloroauric acid solution with the concentration of 0.2g/100mL, heating the chloroauric acid solution until the solution boils, adding a trisodium citrate solution with the concentration of 1g/100mL into the boiled chloroauric acid solution under stirring, wherein the volume ratio of the trisodium citrate solution to the chloroauric acid solution is 1.4:50, stopping heating when the mixed solution is transparent wine red (about 6 min), naturally cooling the mixed solution to room temperature, and then using K to cool the mixed solution naturally2CO3Adjusting the pH value of the solution to 7.5 to obtain a colloidal gold solution (the average particle size of the colloidal gold particles is 40 nm);
secondly, preparing a CD44 second monoclonal antibody solution with the concentration of 1mg/mL by using a phosphate buffer solution with the pH value of 7.2 and the concentration of 0.02mol/L as a solvent and using a second anti-CD 44 monoclonal antibody as a solute, mixing 10 mu L of the second monoclonal antibody solution with 1mL of the colloidal gold solution prepared in the step I, wrapping the centrifuge tube with tinfoil paper after uniform mixing, and marking the centrifuge for 30min by a shaking table; then 20. mu.L of bovine serum albumin solution with a concentration of 5g/100mL is added and mixed well, and the labeling is continued in the shaker for 20 min. After the labeling is finished, centrifuging for 30min at 11000rpm/min to obtain a separation product, namely a second gold-labeled anti-CD 44 monoclonal antibody, and dissolving the second gold-labeled anti-CD 44 monoclonal antibody in 50 mu L of a gold-labeled complex solution to obtain a second gold-labeled anti-CD 44 monoclonal antibody solution with the concentration of 0.2mg/mL (stored at 4 ℃ for later use); the gold-labeled complex solution is prepared by dissolving trihydroxymethyl aminomethane, bovine serum albumin and sodium azide in deionized water, wherein the concentration of the trihydroxymethyl aminomethane is 0.02mol/L, the concentration of the bovine serum albumin is 1g/100mL, and the concentration of the sodium azide is 0.2g/100 mL;
the preparation method of the gold-labeled second anti-FKBPL monoclonal antibody solution and the gold-labeled second negative control antibody solution is the same as that of the gold-labeled second anti-CD 44 monoclonal antibody solution;
③ willCutting the glass cellulose membrane for making gold label pad into 1cm × 30cm, placing on the running platform of gold spraying label machine, setting spraying amount to be 4 μ L/cm2Operating a gold spraying label machine to fill a mixed solution containing a gold-labeled anti-CD 44 second monoclonal antibody solution, a gold-labeled anti-FKBPL second monoclonal antibody solution and a gold-labeled second negative control antibody in a mass ratio of 2:2:1, completing gold spraying operation, and drying the glass cellulose membrane at 37 ℃ for 12 hours by using an electrothermal constant-temperature drying oven to obtain the gold-labeled pad.
(3) Preparation of the reaction film
Preparing a second detection line solution with the concentration of the first anti-CD 44 monoclonal antibody of 2mg/mL by using phosphate buffer solution with the pH value of 7.2 and the concentration of 0.02mol/L as a solvent and using the first anti-CD 44 monoclonal antibody as a solute; preparing a first detection line solution with the concentration of the first anti-FKBPL monoclonal antibody of 2mg/mL by using a phosphate buffer solution with the pH of 7.2 and the concentration of 0.02mol/L as a solvent and using the first anti-FKBPL monoclonal antibody as a solute; preparing a quality control line solution with the concentration of the first negative control antibody of 2mg/mL by using phosphate buffer solution with the pH value of 7.2 and the concentration of 0.02mol/L as a solvent and the first negative control antibody as a solute;
cutting a nitrocellulose membrane (NC membrane) into strips with the specification of 2.5cm x 30cm, sticking the obtained strips in the middle of a viscous bottom plate, respectively scribing a first detection line solution, a second detection line solution and a quality control line solution on a first detection line, a second detection line and a quality control line of the NC membrane by using a scribing instrument, and drying at 37 ℃ to obtain a reaction membrane;
(4) assembled immunochromatography test strip
And (3) respectively sticking the sample pad, the combination pad and the water absorption pad on two sides of the reaction membrane prepared in the step (3), ensuring that the distance between the component pads is 1.5mm, staggering and laminating, cutting into test strips with the width of 3.5mm, and installing the test strips in a customized card shell to obtain the immunochromatography detection card for qualitatively detecting CD44 and FKBPL.
Test example
1. Experimental construction mode
(1) Subject and sample
Subject: 30 normal pregnant women and 30 preeclamptic pregnant women are selected, the preeclamptic pregnant women are diagnosed as preeclamptic patients by gynecologic main and conception physicians with clinical experience of more than 15 years through relevant symptoms, and the reliability is high.
Sample preparation: when the pregnancy period is 15 weeks and 20 weeks, the normal pregnant woman and the preeclamptic pregnant woman respectively take 3-4mL of venous blood, stand at room temperature for 30min, centrifuge at the rotation speed of 2500rpm/min for 5min, transfer the serum to a new EP tube, subpackage and store at-80 ℃.
(2) Detection of CD44 and FKBPL
The immunochromatographic test cards of examples 1-2 and comparative example 1 were used to perform CD44 and FKBPL tests, respectively, on the serum samples of the pregnant women. The immunochromatography detection card of example 1-2 can quantitatively detect the concentrations of CD44 and FKBPL and calculate the concentration ratio of CD 44/FKBPL; whereas the immunochromatographic test card of comparative example 1 can perform qualitative test only for CD44 and FKBPL. Previous studies show that the ratio of CD44/FKBPL concentration increases gradually from 15 weeks to 20 weeks during pregnancy of pregnant women, and when the ratio of CD44/FKBPL concentration is significantly higher than the ratio of CD44/FKBPL concentration of normal pregnant women during 20 weeks of pregnancy, the pregnant women are judged to have preeclampsia.
(3) Sensitivity testing
After quantification of CD44 and FKBPL concentrations in the serum samples of preeclamptic pregnancies at 15 and 20 weeks of pregnancy, the remaining serum samples were accurately diluted to give serum samples with CD44 concentrations of 0.05ng/mL, 0.1ng/mL, 0.5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 60ng/mL, 80ng/mL, 100ng/mL, and 120ng/mL, and FKBPL concentrations of 0.05ng/mL, 0.10ng/mL, 0.20ng/mL, 0.30ng/mL, 0.40ng/mL, 0.50ng/mL, 0.60ng/mL, 0.70ng/mL, 0.80ng/mL, 0.90ng/mL, and 1.00 ng/mL.
The serum samples obtained by the dilution are respectively tested for multiple times by using the immunochromatographic test cards of examples 1-2 and comparative example 1, and the experimental results are averaged.
(4) Method for using immunoassay chromatography card and detection principle
Use of the immunochromatographic test card of example 1-2 and the test principle
Dropping a serum sample to be detected on a sample pad or a sample adding hole, carrying out forward chromatography on the sample, moving the sample to a binding pad, wherein CD44 and FKBPL are respectively combined with an up-conversion luminescent nanoparticle-anti-CD 44 second monoclonal antibody, an up-conversion luminescent nanoparticle particle-anti-FKBPL second monoclonal antibody on the binding pad to form an up-conversion luminescent nanoparticle-anti-CD 44 second monoclonal antibody-CD 44, an up-conversion luminescent nanoparticle particle-anti-FKBPL second monoclonal antibody-BPFKL, continuing chromatography on a reaction membrane to form an up-conversion luminescent nanoparticle-anti-CD 44 second monoclonal antibody-CD 44-anti-CD 44 first monoclonal antibody, an up-conversion luminescent nanoparticle-anti-FKBPL second monoclonal antibody-FKBPL first monoclonal antibody (double antibody sandwich structure) on two corresponding detection lines, and is fixed on the corresponding detection line.
The detection card is irradiated by a 980nm laser diode (near infrared exciting light) under the action of a hemispherical lens, green light (540nm) visible to the naked eye can be emitted, and signals are collected by a signal collector. If the green light signal can be collected on the quality control line, the green light signal cannot be collected on the detection line, which indicates that the serum sample does not contain CD44 and FKBPL; green signals can be collected on the quality control line and the detection line, and the serum sample contains CD44 and FKBPL; when no green light signal is detected on the quality control line, the immunochromatography detection card is invalid. And specific concentrations of CD44 and FKBPL are obtained through signal acquisition results, and the concentration ratio of CD44 to FKBPL is calculated.
② use method and detection principle of immunochromatography detection card of comparative example 1
And dropwise adding the serum sample to be detected on a sample pad, carrying out forward chromatography on the sample, moving the sample to a binding pad, wherein CD44 and FKBPL are respectively bound with a second gold-labeled anti-CD 44 monoclonal antibody and a second gold-labeled anti-FKBPL monoclonal antibody on the binding pad to form a second gold-labeled anti-CD 44 monoclonal antibody-CD 44 and a second gold-labeled anti-FKBPL monoclonal antibody-FKBPL, continuously carrying out chromatography on a reaction membrane, forming double-antibody sandwich structures of the first gold-labeled anti-CD 44 monoclonal antibody-CD 44-anti-CD 44 monoclonal antibody and the second gold-labeled anti-BPFKL monoclonal antibody-FKBPL-anti-FKBPL monoclonal antibody on two corresponding detection lines, and fixing the double-antibody sandwich structures on the corresponding detection lines.
Observing the reaction result within 10-30min, and determining the result to be invalid after 30 min. Only red bands appeared on the quality control line, no red bands appeared on the detection line, indicating that no CD44 and FKBPL were contained in the serum sample; red strips appear on the quality control line and the detection line, and the strip color of the detection line is lighter than the color of the quality control line or the strip color is uniform in shade, which indicates that the serum sample contains CD44 and FKBPL; when no colored band appears on the quality control line, the immunochromatography detection card is invalid.
2. Results of the experiment
(1) Detection results of normal pregnant woman and preeclamptic pregnant woman
TABLE 1 detection results of Normal pregnant women and preeclamptic pregnant women
Note: + indicates that CD44 or FKBPL can be detected and the detection line is red;
-indicates that CD44 or FKBPL could not be detected and the detection line did not develop color.
Table 1 shows the average value of the experimental results, and the immunochromatographic test card provided in examples 1-2 of the present invention can be used to test serum samples of normal pregnant women and preeclamptic pregnant women at 15 weeks and 20 weeks, and can quantitatively detect the concentrations of CD44 and FKBPL, and by comparing the CD44/FKBPL concentration ratio, a preeclamptic patient can be rapidly screened, and the diagnosis result is consistent with the diagnosis result of a gynecologist having clinical experience of more than 15 years, thereby indicating that the immunochromatographic test card provided in the present invention is very accurate in the diagnosis result of preeclampsia. In addition, as can be seen from table 1, since the serum concentrations of FKBPL in normal pregnant women and preeclamptic patients are low, the immunochromatographic test card prepared using colloidal gold as a marker can detect only CD44 at a high concentration, and cannot detect FKBPL at a low concentration. Although the color of the strip is dark and light, the content of CD44 or FKBPL cannot be estimated by naked eyes through the dark and light of the color, namely, CD44 and FKBPL cannot be quantified, so that the concentration ratio of CD44 to FKBPL cannot be compared, and the strip cannot be used for diagnosing and evaluating preeclampsia.
(2) Results of sensitivity
TABLE 2 test results for known concentrations of CD44 and FKBPL samples
Note: -indicates that CD44 or FKBPL cannot be detected.
As can be seen from the sample detection results in table 2, when the immunochromatographic detection card of embodiments 1-2 of the present invention is used to detect a serum sample with a known concentration, the detection result is substantially the same as the original concentration, and there is no significant difference, mainly because of the unique luminescence property of the upconversion luminescent nanoparticle and the stable luminescence property, the detection sensitivity is high, the stability is good, and the detection result is accurate and reliable. The immunochromatographic detection card of example 1-2 of the present invention had a minimum detection limit of 0.1ng/mL, and was able to detect both CD44 and FKBPL at concentrations above 0.1 ng/mL. The immunochromatographic card provided by the invention can be used for more accurately detecting CD44 and FKBPL, and can be used for more accurately quantifying CD44 and FKBPL at a low concentration level, so that the immunochromatographic card has an extremely important significance in the rapid detection and diagnosis of preeclamptic patients by using the concentration ratio of CD44 to FKBPL.
Although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. An immunochromatography detection card for rapidly detecting preeclampsia is characterized in that: the detection card comprises a test strip, the test strip comprises a sample pad, a binding pad and a reaction membrane, a first detection line, a second detection line and a quality control line are arranged on the reaction membrane, the first detection line is coated with a first anti-FKBPL monoclonal antibody, the second detection line is coated with a first anti-CD 44 monoclonal antibody, or the first detection line is coated with a first anti-CD 44 monoclonal antibody, the second detection line is coated with a first anti-FKBPL monoclonal antibody, and the quality control line is coated with a first negative control antibody;
the conjugate pad is coated with an up-conversion luminescent nanoparticle-anti-CD 44 second monoclonal antibody, an up-conversion luminescent nanoparticle-anti-FKBPL second monoclonal antibody, and an up-conversion luminescent nanoparticle-second negative control antibody.
2. The immunochromatographic test card for rapid detection of preeclampsia according to claim 1, wherein: the particle size of the up-conversion luminescence nano-particles is 25-200 nm.
3. The immunochromatographic test card for rapid detection of preeclampsia according to claim 1, wherein: the particle size of the up-conversion luminescence nano-particles is 50 nm.
4. The immunochromatographic test card for rapid detection of preeclampsia according to claim 1, wherein: the up-conversion luminescent nano-particles are obtained by carboxyl modification.
5. The immunochromatographic test card for rapid detection of preeclampsia according to claim 1, wherein the method for preparing the second monoclonal antibody of the up-conversion luminescent nanoparticle anti-CD 44 comprises the following steps: preparing a suspension by using the up-conversion luminescent nano particles and a PB buffer solution, and sequentially adding NHS and EDC into the suspension for reaction; centrifuging to remove supernatant, adding PB buffer solution, and mixing; adding a second anti-CD 44 monoclonal antibody into the system for reaction, adding BSA for blocking after the reaction is finished, and performing centrifugal washing to obtain the upconversion luminescent nanoparticle-anti-CD 44 second monoclonal antibody;
the preparation method of the upconversion luminescent nanoparticle-anti-FKBPL second monoclonal antibody comprises the following steps: preparing a suspension by using the up-conversion luminescent nano particles and a PB buffer solution, and sequentially adding NHS and EDC into the suspension for reaction; centrifuging to remove supernatant, adding PB buffer solution, and mixing; adding a second anti-FKBPL monoclonal antibody into the system for reaction, adding BSA for blocking after the reaction is finished, and centrifuging and washing to obtain the upconversion luminescent nanoparticle-anti-FKBPL second monoclonal antibody;
the preparation method of the up-conversion luminescent nanoparticle-second negative control antibody comprises the following steps: preparing a suspension by using the up-conversion luminescent nano particles and a PB buffer solution, and sequentially adding NHS and EDC into the suspension for reaction; centrifuging to remove supernatant, adding PB buffer solution, and mixing; and adding a second negative control antibody into the system for reaction, adding BSA (bovine serum albumin) for blocking after the reaction is finished, and performing centrifugal washing to obtain the upconversion luminescent nanoparticle-second negative control antibody.
6. The immunochromatographic test card for rapid detection of preeclampsia according to claim 5, wherein: the mass ratio of the NHS to the up-conversion luminescent nanoparticles is 1:2-5, and the mass ratio of the EDC to the up-conversion luminescent nanoparticles is 1: 2-5.
7. The immunochromatographic test card for rapid detection of preeclampsia according to claim 5, wherein: the mass ratio of the second anti-CD 44 monoclonal antibody, the second anti-FKBPL monoclonal antibody, the second negative control antibody to the up-conversion luminescent nanoparticle is 1: 100-1000.
8. The immunochromatographic test card for rapid detection of preeclampsia according to claim 5, wherein: the PB buffer had a pH of 7.2 and a concentration of 20 mM.
9. A method for simultaneously and quantitatively detecting the concentration of CD44 and FKBPL, which is characterized by comprising the following steps: dropping a sample to be detected on the immunochromatography detection card for rapidly detecting preeclampsia according to any one of claims 1 to 8, irradiating the detection card by using a laser diode under the action of a hemispherical lens, and collecting signals by using a signal collector.
10. The method of claim 9 for simultaneous quantitative determination of CD44 and FKBPL concentration, wherein: the laser diode is selected from one of 790nm, 980nm and 1550nm laser diodes.
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