CN117347624A - Fluorescence quenching immunochromatography test strip and preparation method thereof - Google Patents
Fluorescence quenching immunochromatography test strip and preparation method thereof Download PDFInfo
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention belongs to the technical field of immunofluorescence detection, and discloses a fluorescence quenching immunochromatography test strip and a preparation method thereof, wherein the fluorescence quenching immunochromatography test strip consists of ACE2 coupled with green fluorescent microspheres ACE2-FNPs, RBD labeled nano-gold RBD-AuNPs, NC films, a PVC plate and a clamping shell. The preparation method of the fluorescence quenching immunochromatography test strip comprises the following steps: preparing a colloidal gold solution by using chloroauric acid and trisodium citrate; preparing RBD-AuNPs by using colloidal gold, recombinant RBD protein and BSA; and (3) respectively coupling ACE2 and BSA by using fluorescent microsphere FNPs and assembling the test strip. The invention develops a blocking method fluorescence quenching immunochromatography test strip for rapidly detecting the neutralizing antibody of the new coronavirus by utilizing a unique fluorescence resonance energy transfer-based fluorescence quenching immunochromatography platform, and can realize the accurate, rapid and high-sensitivity detection of the neutralizing antibody of the new coronavirus.
Description
Technical Field
The invention belongs to the technical field of immunofluorescence detection, and particularly relates to a fluorescence quenching immunochromatography test strip and a preparation method thereof.
Background
After vaccination, it is particularly important to evaluate the protective power of the human body, and advice whether vaccination or booster immunization is required can be given according to the evaluation result. In addition, the rapid detection of neutralizing antibodies of popular masses can establish a crowd protection admittance white list, has the crowd with equivalent neutralizing antibody level, and has important clinical significance and social value in consideration of normal life and work.
SARS-CoV-2 is an enveloped positive-strand RNA virus with four major structural proteins: s protein (spike protein), N protein (nucleocap protein), E protein (envelope protein) and M protein (membrane protein). The S protein is critical for the entry of SARS-COV-2 into cells, as it plays an important role in fusion with cellular receptors and membranes. The S protein comprises two functional subunits, S1 and S2, wherein the RBD domain on the S1 subunit is responsible for binding to the host cell receptor ACE2 (angiotenin-converting enzyme, ace2), and the S2 subunit is responsible for anchoring the S protein to the host cell membrane and mediating fusion of the viral envelope with the host cell membrane. When SARS-CoV-2 enters the body, immune response is activated to generate antibodies against SARS-CoV-2, wherein the antibodies against RBD can bind to RBD sites of viruses to block the combination of the viruses and ACE2, thereby preventing SARS-CoV-2 from infecting cells, therefore, RBD is the most main target of vaccine development, and the antibodies which can block RBD are neutralizing antibodies. From the infection mechanism of SARS-CoV-2, it is known that the key to detecting virus neutralizing antibodies is to detect antibodies in vivo that are anti-RBD and prevent RBD from binding to ACE2, and that this is also the key to the detection technology.
The laboratory gold standard for neutralizing antibody detection is live virus neutralization assay (conventional virus neutralization test, cVNT) which uses quantitative samples of live virus mixed with equal amounts of serum at different dilutions for plaque reduction neutralization assay (plaque reductionneutralization test, PRNT) for analysis by detecting the amount of cytopathic effect (cytopathic effect, CPE). Because of the high pathogenicity and infectivity, test procedures for assessing neutralizing antibodies using live viruses all have to be performed in a laboratory of biosafety level (BSL) 3, which makes it difficult to achieve large-scale routine laboratory tests and rapid tests. To solve this difficulty, pseudo-virus-based VNT (pnnt) was developed for neutralizing antibody detection, and even though pnnt can be performed in a laboratory of biosafety level (BSL) 2, complex operations and time consumption of 2 to 4 days are still required to complete, which cannot meet the need for extensive neutralization efficacy assessment by the era of mass vaccination. In this context, instead of virus neutralization assay (sVNT) developed for neutralizing antibody detection, the method simulates RBD binding to ACE2 in an ELISA plate, and can be performed in a common laboratory using an ELISA or chemiluminescent immunoassay procedure, and can be completed in 2 hours. However, the sVNT requires a large-scale detection instrument, and the operation time is relatively long, so that the result cannot be obtained immediately, and therefore, the sVNT is difficult to popularize in popular people and in antibody detection. In summary, the research on a rapid, simple and accurate detection method for neutralizing antibodies is used for monitoring neutralizing antibodies of large-scale population, so that the recognition of the immune barrier of the whole society has important value.
The immunochromatography test strip has become an in vitro diagnosis technology which is increasingly important in POCT instant detection and home self-detection due to the advantages of simple operation, rapid detection, portability, low cost and the like. Therefore, the kit is very suitable for developing a rapid detection reagent for virus neutralizing antibodies, and realizes the instant and on-site detection of the neutralizing antibodies of the population.
The detection of antibodies is usually done using an indirect method, so that an indirect method test strip was first developed for rapid detection of virus-neutralizing antibodies, such as the covd-19 macro NP/RBD test (sensitivity 71.6%, specificity 89.3%) from cornis BioConcept, belgium) and the InfectCheck COVID-19NAb test (sensitivity 98.4%, specificity 58.3%) from condile GmbH, germany, however, the indirect method has a higher cross-reactivity, and has a lower sensitivity and specificity, resulting in the susceptibility to false positives and missed detection. In addition, the indirect method has higher requirements on the secondary antibody raw material, so that the cost is increased, and the popularization to the masses is not facilitated.
Another common method for detecting antibodies is a double antigen sandwich test strip, which can be established by using recombinant RBD proteins, has a short development period and low cost, and is therefore applied to the detection of neutralizing antibodies. However, the double antigen sandwich method utilizes the bivalent characteristic of the antibody, so long as the antibody aiming at RBD can be detected, but a plurality of RBD antibodies do not block the combined action of RBD and ACE2, so false positives are easily caused, the total coincidence rate of the result of detecting the neutralizing antibody by the double antigen sandwich test strip and the result of sVNT is 92%, wherein the negative coincidence rate is only 50%, the false positives rate is higher, and the protection effect of people cannot be truly reflected.
Along with the development of recombinant protein expression technology, a blocking method for simulating the in vivo infection process of a neutralizing antibody to block viruses is widely used for detecting the neutralizing antibody, wherein the blocking method is to fix recombinant ACE2 protein on a detection line (T line) of a membrane, RBD (RBD) marker signals (colloidal gold, latex microspheres, fluorescent microspheres and the like) are sprayed on a binding pad, when the neutralizing antibody does not exist, RBD is combined with ACE2, the signals are displayed on the membrane, and when the neutralizing antibody exists, the neutralizing antibody is combined with RBD first, so that the RBD is blocked from being combined with ACE2, and no signals are displayed. The blocking method can simulate the real condition in the body, so that the detected neutralizing antibody is more real and reliable, and the false positive rate is lower. However, according to the principle, the test strip is a negative signal, namely, a neutralizing antibody exists and no signal exists, and the neutralizing antibody does not exist, so that the analysis sensitivity is low, and false negative is easy to cause misjudgment.
It has been shown that adding a line (T2 line) based on the blocking method (T1 line) using the indirect method or the double antigen sandwich method to increase the analysis sensitivity can effectively reduce false negative, however, this method uses more protein and antibody raw materials, has higher industrialization requirements, and in addition, if used for on-site instant detection, the interpretation result is complex and difficult to understand.
Summary of the inventionit is not difficult to find out that the rapid and accurate test paper for rapidly detecting the virus neutralizing antibodies is developed by using a simple and readable means with low cost, is a research trend in the field of evaluating the virus protection force, and is also an important subject in the era.
Through the above analysis, the problems and defects existing in the prior art are as follows:
(1) The existing neutralization test conditions of live viruses or pseudoviruses are harsh, the operation is complex and the time is long, and the requirement of a large amount of neutralization efficacy evaluation caused by the time of large-scale vaccination cannot be met.
(2) The substitution virus neutralization test needs a large-scale detection instrument, the operation time is long, and the result cannot be obtained immediately, so that the substitution virus neutralization test is difficult to popularize in the popular population and in the detection of the neutralizing antibody.
(3) The sensitivity and the specificity of the indirect immunochromatography test strip are low, so that false positive and detection omission are easy to occur; and the indirect method has higher requirements on the secondary antibody raw material, so that the cost is increased, and the popularization to the masses is not facilitated.
(4) Because many antibodies of RBD do not block the combined action of RBD and ACE2, the false positive rate of the double-antigen sandwich test strip is high, and the protective efficacy of people cannot be truly reflected.
(5) The detection result in the test strip of the blocking method is a negative signal, namely, a neutralizing antibody exists and no signal exists, and the neutralizing antibody does not exist, so that the analysis sensitivity is low, and false negative is easy to cause misjudgment.
(6) The blocking method is used to increase the number of protein and antibody raw materials used in the method of using the indirect method or the double antigen sandwich method, the industrialization requirement is higher, the interpretation result is complex, and the interpretation is difficult to understand in some cases.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a fluorescence quenching immunochromatography test strip and a preparation method thereof.
The invention is realized in such a way that the fluorescence quenching immunochromatographic test strip consists of a card shell, a PVC bottom plate, a sample pad, a bonding pad, a Nitrocellulose (NC) membrane and a water absorption pad, wherein the sample pad, the bonding pad, the Nitrocellulose (NC) membrane and the water absorption pad are sequentially crossed and overlapped and stuck on the PVC bottom plate, the test strip is arranged in the card shell, RBD marked nano gold (RBD-AuNPs) is sprayed on the bonding pad, RBD is marked on the nano gold through electrostatic adsorption, a T line area on the NC membrane is coated with ACE2 coupled green fluorescent microspheres (ACE 2-FNPs), ACE2 is coupled with FNPs through a covalent bonding mode, and a C line area on the NC membrane is coated with BSA coupled green fluorescent microspheres (BSA-FNPs). When the sample dripped on the sample pad is a negative sample, namely no neutralizing antibody exists, the sample flows towards the water absorption pad under the siphon action of the test strip, RBD-AuNPs on the binding pad flow along with the liquid and flow to the T line area of the NC membrane when the sample flows to the binding pad, RBD is combined with ACE2 to form an RBD-AuNPs-ACE2-FNPs structure, the AuNPs and FNPs generate fluorescence resonance energy transfer, fluorescence of the FNPs is quenched by the AuNPs, no fluorescence signal flows to the C line area of the NC membrane, the RBD-AuNPs is not combined with BSA-FNPs, and the FNPs emit fluorescence signals; when the sample dripped on the sample pad is a positive sample, namely, a neutralizing antibody exists, the sample flows to the direction of the water absorption pad under the siphon action of the test paper strip, when the sample flows to the binding pad, the neutralizing antibody in the sample is combined with RBD-AuNPs on the binding pad firstly, and flows to the T line area of the NC film along with the liquid flow, the RBD cannot be combined with ACE2 to form an RBD-AuNPs-ACE2-FNPS structure, the FNPs normally emit fluorescent signals, the RBD-AuNPs cannot be combined with BSA-FNPs, and the FNPs emit fluorescent signals. In other words, when the neutralizing antibody is negative, the T-line has no fluorescence and the C-line has fluorescence; when the neutralizing antibody is positive, the T line has fluorescence and the C line has fluorescence, so that the reading of a forward signal is realized.
The invention also aims at providing a preparation method for implementing the fluorescence quenching immunochromatography test strip, which comprises the following steps:
step one, preparing a colloidal gold solution by using chloroauric acid and trisodium citrate;
preparing RBD-AuNPs by using colloidal gold, recombinant RBD protein and BSA;
step three, respectively coupling ACE2 and BSA by using fluorescent microsphere FNPs;
and step four, preparing a gold-labeled pad and a sample pad respectively and assembling the test paper strips.
Further, the preparation method of the colloidal gold solution in the first step comprises the following steps:
cleaning the conical flask, measuring 100mL of ultrapure water, placing the conical flask in a magnetic stirrer, heating to boil, adding 2mL of chloroauric acid, rapidly adding 3.2mL of trisodium citrate, and keeping boiling for 15min; after cooling to room temperature, adding ultrapure water to complement 100mL to prepare colloidal gold solution with the particle size of 20nm, and storing at the temperature of 4 ℃.
Further, chloroauric acid concentration was 1%, and trisodium citrate concentration was 1%.
Further, the preparation method of the RBD-AuNPs in the second step comprises the following steps:
cleaning small beaker, weighing 5mL of colloidal gold solution, placing in magnetic stirrer, adding appropriate amount of K 2 CO 3 Adjusting the pH of the colloidal gold solution to 8.7; adding 3 mu L of recombinant RBD proteins with different concentrations, stirring for 15min, and standing for 15min; adding 500 mu L BSA solution, stirring for 15min, and standing for 15min; centrifuging at 10000rpm at 4deg.C for 15min, collecting supernatant, adding 240 μl Jin Biaofu solution, dissolving, and storing at 4deg.C.
Further, K 2 CO 3 The concentration was 250mM and the BSA solution concentration was 10% w/v.
Further, the fluorescent microsphere FNPs in step three coupled with ACE2 and BSA comprises:
100. Mu.L of FNPS was dissolved in 400. Mu.L of ultrapure water, and 13.4. Mu.L of EDC and 8.05. Mu.L of NHS were added thereto to carry out a spin reaction from light for 30 minutes; adding 7 mu L of ACE2 with different concentrations, carrying out light-proof rotary reaction for 2 hours, adding 10 mu L of 10% BSA, carrying out light-proof rotary reaction for 1 hour, and centrifuging at 13000rpm at 4 ℃ for 30 minutes; the supernatant was aspirated and reconstituted with 200. Mu.L of PBS pH7.4, and the particle size was measured and stored at 4 ℃.
Further, EDC concentration was 1% w/v, and NHS concentration was 1% w/v.
Further, the method for assembling the test strip in the fourth step comprises the following steps:
adhering an NC film to the center of a PVC plate, and storing in a dry environment for later use; diluting ACE2-FNPs and BSA-FNPs 400 times, coating the diluted ACE2-FNPs and BSA-FNPs on T-line and C-line of NC membrane with different membrane dividing amounts by using a membrane dividing instrument, and drying at 37deg.C overnight; the RBD-AuNPs are sprayed on the polyester film with different spraying amounts by a metal spraying film drawing instrument to prepare a bonding pad, and the bonding pad is dried for 2 days at 37 ℃. Cutting the bonding pad to enable the upper and lower parts to be left with 1mm blank; bonding the bonding pad and the sample pad to the NC film in a mutually overlapped manner; cutting the pasted plate into strips with the width of 4mm by using a strip cutting machine, and filling the strips into a card shell to prepare the test paper strip.
Further, the dividing amount of ACE2-FNPs and BSA-FNPs was 2.0. Mu.L/cm, and the spraying amount of RBD-AuNPs was 2.0. Mu.L/cm; the overlap of the conjugate pad and the sample pad was 1mm.
In combination with the technical scheme and the technical problems to be solved, the technical scheme to be protected has the following advantages and positive effects:
first, aiming at the technical problems in the prior art and the difficulty of solving the problems, the technical problems solved by the technical proposal of the invention are analyzed in detail and deeply by tightly combining the technical proposal to be protected, the results and data in the research and development process, and the like, and some technical effects brought after the problems are solved have creative technical effects. The specific description is as follows:
the invention provides a fluorescence quenching immunochromatography test strip for detecting virus neutralizing antibodies based on a Fluorescence Resonance Energy Transfer (FRET) blocking method. FRET means that in two different fluorescent substances, if the emission spectrum of one fluorescent substance (donor) overlaps with the absorption spectrum of the other substance (acceptor) to some extent, when the distance between the two fluorescent substances is appropriate (generally smaller than) The transfer of fluorescence energy from the donor to the acceptor is observed, i.e. the fluorescence emitted by the latter species is observed when excited at the excitation wavelength of the former species. Therefore, the invention uses ACE2 coupled green fluorescent microspheres (ACE 2-FNPs) and is coated on the T line of NC membrane, while RBD labeled nano-gold (RBD-AuNPs) is sprayed in the bonding pad; if there is no neutralizing antibody in the sample, RBD-AuNPs bind to ACE2-FNPs on the T line, and fluorescent signals are not visible when the FNPs are excited by excitation light, because the emitted light of the FNPs and the absorbed light of the AuNPs are mostly overlapped, the FNPs and the AuNPs generate FRET, energy is transferred to the AuNPs, and the AuNPs are non-luminescent substances; if neutralizing antibodies are present in the sample, the neutralizing antibodies bind to RBD-AuNPs first,the combination of RBD-AuNPs and ACE2-FNPs is blocked, when the FNPs are excited by using excitation light, the FNPs emit corresponding fluorescence, so that a fluorescence signal exists, in other words, the fluorescence signal is enhanced along with the increase of the concentration of the neutralizing antibody, thereby achieving the purpose of detecting a forward signal.
In addition, because fluorescence belongs to light penetration, the analysis sensitivity is higher than that of the traditional blocking method LFIA, so that the false negative rate is reduced. The fluorescence quenching immunochromatography test strip with the blocking method is used, and the analysis sensitivity is improved by 80 times compared with that of the fluorescence immunochromatography test strip with the blocking method; the negative signal of the common fluorescent immunochromatographic test strip is changed into a positive signal, and the result is easy to interpret.
Secondly, the technical scheme is regarded as a whole or from the perspective of products, and the technical scheme to be protected has the following technical effects and advantages:
the invention develops a blocking method fluorescence quenching immunochromatography test strip for rapidly detecting virus neutralizing antibodies by utilizing a unique Fluorescence Resonance Energy Transfer (FRET) based fluorescence quenching immunochromatography platform, and can realize the accurate, rapid and high-sensitivity detection of the neutralizing antibodies. The scheme of the invention can change the signal reading mode of the blocking method immunochromatographic test paper from negative to positive, thereby being more in line with humanized reading habit and having higher accuracy; because fluorescence belongs to light penetration, fluorescence is used as a signal of the immunochromatographic test paper, so that the sensitivity of the scheme of the invention is higher than that of other blocking-method immunochromatographic test papers; the detection range of the scheme of the invention is wider than that of the existing product, and the detection of the neutralizing antibody is more accurate.
Thirdly, as inventive supplementary evidence of the claims of the present invention, the following important aspects are also presented:
the technical scheme of the invention solves the technical problems that people are always desirous of solving but are not successful all the time: the technical scheme of the invention solves the technical problem of negative reading of signals in the detection neutralizing antibodies of the blocking immunochromatographic test paper, the blocking immunochromatographic test paper only can use reverse signals, which is inconsistent with the cognition habit of people, but has no solution for technical development, the invention skillfully utilizes Fluorescence Resonance Energy Transfer (FRET), uses FRET on the blocking immunochromatographic test paper, develops the blocking fluorescence quenching immunochromatographic test paper for the rapid detection of virus neutralizing antibodies, and solves the technical problem that the detection neutralizing antibodies are not successful all the time.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a preparation method of a fluorescence quenching immunochromatographic test strip provided by an embodiment of the invention;
FIG. 2 is a schematic diagram of a fluorescence quenching immunochromatographic strip for blocking method based on Fluorescence Resonance Energy Transfer (FRET) according to an embodiment of the present invention;
FIG. 3A is a graph showing the sensitivity contrast of a fluorescence immunochromatographic test strip by a blocking method according to an embodiment of the present invention;
FIG. 3B is a graph showing the sensitivity contrast of a fluorescence quenching immunochromatographic test strip by a blocking method according to an embodiment of the present invention;
FIG. 4 is a picture of the detection effect of the present invention provided by the embodiment of the present invention;
fig. 5 is a picture of the detection effect of Tianjin Hongyu tai biotechnology limited company provided by the embodiment of the invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Aiming at the problems existing in the prior art, the invention provides a fluorescence quenching immunochromatography test strip and a preparation method thereof, and the invention is described in detail below with reference to the accompanying drawings.
The fluorescence quenching immunochromatographic test strip provided by the embodiment of the invention comprises a card shell, a PVC bottom plate, a sample pad, a bonding pad, a Nitrocellulose (NC) membrane and a water absorption pad, wherein RBD-AuNPs are sprayed on the bonding pad, ACE2-FNPs are coated on a NC membrane T line area, and BSA-FNPs are coated on a C line area.
As shown in FIG. 1, the preparation method of the fluorescence quenching immunochromatographic test strip provided by the embodiment of the invention comprises the following steps:
s101, preparing a colloidal gold solution by using chloroauric acid and trisodium citrate;
s102, preparing RBD-AuNPs by using colloidal gold, recombinant RBD protein and BSA;
s103, respectively coupling ACE2 and BSA by using fluorescent microsphere FNPs;
s104, preparing a gold-labeled pad and a sample pad respectively and assembling the test paper strips.
As a preferred embodiment, as shown in FIG. 2, the preparation method of the fluorescence quenching immunochromatographic test strip provided by the embodiment of the invention specifically comprises the following steps:
(1) Preparation of colloidal gold solution
Cleaning a conical flask, measuring 100mL of ultrapure water, placing the conical flask in a magnetic stirrer, heating to boil, adding 2mL of 1% chloroauric acid, then rapidly adding 3.2mL of 1% trisodium citrate, keeping boiling for 15min, cooling to room temperature, adding ultrapure water to complement 100mL, preparing a colloidal gold solution with the particle size of about 20nm, and storing at 4 ℃.
(2) Preparation of RBD-AuNPs
Cleaning the small beaker, weighing 5mL of colloidal gold solution, placing in a magnetic stirrer, adding a proper amount of 250mM K 2 CO 3 Adjusting the pH of the colloidal gold solution to be approximately equal to 8.7, adding 3 mu L of recombinant RBD proteins with different concentrations, stirring for 15min, standing for 15min, adding 500 mu L of 10% (w/v) BSA solution, stirring for 15min, standing for 15min, centrifuging at 10000rpm at 4 ℃ for 15min, sucking the supernatant clean, adding 240 mu L of Jin Biaofu solution for redissolution, and storing at 4 ℃.
(3) Fluorescent microspheres (FNPs) coupled ACE2 and BSA
Taking 100 mu L of FNPs, dissolving in 400 mu L of ultrapure water, adding 13.4 mu L of 1 permillage (w/v) EDC and 8.05 mu L of 1 permillage (w/v) NHS, carrying out light-proof rotary reaction for 30min, adding 7 mu L of ACE2 with different concentrations, carrying out light-proof rotary reaction for 2h, adding 10 mu L of 10% BSA, carrying out light-proof rotary reaction for 1h, centrifuging at 13000rpm for 30min at 4 ℃, sucking the supernatant, adding 200 mu L of PBS with pH=7.4, re-dissolving, measuring the particle size, and storing at 4 ℃. The FNPs are coupled with BSA by the same method as described above, and the ACE2 is replaced by BSA.
(4) Test strip assembly
NC film was adhered to the center of PVC plate, stored in dry environment for use, diluted ACE2-FNPs and BSA-FNPs 400 times, coated with different dividing amounts of 2.0. Mu.L/cm on the T-line and C-line of NC film, respectively, with a dividing film instrument, and dried at 37deg.C overnight. RBD-AuNPs were sprayed onto polyester films with different spray levels of 2.0. Mu.L/cm by a metal spraying and film drawing instrument to prepare bonding pads, and dried at 37℃for 2 days.
The bonding pad was cut so that 1mm of space remained up and down. The combination pad and the sample pad are adhered to the NC film in a mutually overlapped mode, the overlapped part is about 1mm, the adhered plate is cut into strips with the width of 4mm by a cutting machine, and the strips are put into a card case to prepare the test strip.
In order to prove the inventive and technical value of the technical solution of the present invention, this section is an application example on specific products or related technologies of the claim technical solution.
1. As shown in figures 3A-3B, the fluorescence quenching immunochromatographic test strip with the blocking method is used, and the analytical sensitivity is improved by 80 times compared with that of the fluorescence quenching immunochromatographic test strip with the blocking method; the negative signal of the common fluorescent immunochromatographic test strip is changed into a positive signal, and the result is easy to interpret.
(1) The preparation steps of the fluorescence quenching immunochromatography test strip by the blocking method are as follows: firstly, preparing a colloidal gold solution by using chloroauric acid and trisodium citrate; then preparing RBD-AuNPs by using colloidal gold, recombinant RBD protein and BSA; then, respectively coupling ACE2 and BSA by using fluorescent microsphere FNPs; finally, preparing a gold label pad, coating an NC film, preparing a sample pad and assembling a test strip.
The method for preparing the fluorescence quenching immunochromatographic test strip according to claim 2, wherein the method for preparing the colloidal gold solution in the step one comprises the following steps:
1) Cleaning a conical flask, measuring 100mL of ultrapure water, placing the conical flask in a magnetic stirrer, heating to boil, adding 2mL of 1% chloroauric acid, rapidly adding 3.2mL of 1% trisodium citrate, and keeping boiling for 15min; after cooling to room temperature, adding ultrapure water to complement 100mL to prepare colloidal gold solution with the particle size of 20nm, and storing at the temperature of 4 ℃.
2) The method for preparing a fluorescence quenching immunochromatographic test strip according to claim 2, wherein the method for preparing RBD-AuNPs in the second step comprises the following steps:
cleaning the small beaker, weighing 5mL of colloidal gold solution, placing the solution in a magnetic stirrer, and adding a proper amount of 500mM K 2 CO 3 Adjusting the pH of the colloidal gold solution to 8.7; adding 3 mu L of recombinant RBD proteins with different concentrations, stirring for 15min, and standing for 15min; adding 500 mu L of 10% w/v BSA solution, stirring for 15min, and standing for 15min; centrifuging at 10000rpm at 4deg.C for 15min, collecting supernatant, adding 240 μl Jin Biaofu solution, dissolving, and storing at 4deg.C.
3) The method for preparing the fluorescence quenching immunochromatographic test strip according to claim 2, wherein the fluorescent microsphere FNPs in the third step are coupled with ACE2 and BSA, and the method comprises the following steps:
100. Mu.L of FNPs are dissolved in 400. Mu.L of ultrapure water, and 13.4. Mu.L of 1%w/v EDC and 8.05. Mu.L of 1%w/v NHS are added for light-shielding rotary reaction for 30min; adding 7 mu L of ACE2 with different concentrations, carrying out light-proof rotary reaction for 2 hours, adding 10 mu L of 10% w/v BSA, carrying out light-proof rotary reaction for 1 hour, and centrifuging at 13000rpm at 4 ℃ for 30 minutes; the supernatant was aspirated and reconstituted with 200. Mu.L of PBS pH7.4, and the particle size was measured and stored at 4 ℃.
4) The method for preparing a fluorescence quenching immunochromatographic test strip according to claim 2, wherein the method for assembling the test strip in the fourth step comprises the following steps:
adhering an NC film to the center of a PVC plate, and storing in a dry environment for later use; diluting ACE2-FNPs and BSA-FNPs 400 times, coating the diluted ACE2-FNPs and BSA-FNPs on T-line and C-line of NC membrane with a membrane dividing amount of 2.0 mu L/cm respectively by using a membrane dividing instrument, and drying at 37deg.C overnight; spraying RBD-AuNPs onto the polyester film by using a metal spraying film drawing instrument according to the spraying amount of 2.0 mu L/cm to prepare a gold mark pad, and drying at 37 ℃ for 2 days; cutting the gold mark pad to enable the upper and lower parts to be left with 1mm blank; adhering the gold mark pad and the sample pad to the NC film in a mutually overlapped mode; cutting the stuck plate into strips with the width of 4mm by using a cutting machine, and filling the strips into a card shell to prepare the test paper strip.
2. Clinical sample detection
The clinical detection sample types of the fluorescence quenching immunochromatography test strip by the blocking method are human serum and plasma. Clinical sample testing includes two steps, sample collection processing and sample testing.
(1) Clinical samples were collected using different types of blood collection tubes:
1) Human venous whole blood was collected using a vacuum blood collection tube containing an anticoagulant. After sufficient blood was drawn, the tube was gently inverted multiple times and placed in a pre-chill centrifuge for centrifugation at 3000rpm for 10min to collect plasma. The collected plasma was stored at 4℃for a short period (within one week) and at-20℃for more than one week.
2) Human venous whole blood was collected using a vacuum biochemical tube (without anticoagulant). After sufficient blood is extracted, the mixture is placed at room temperature for 1 to 2 hours or at 4 ℃ until the blood is completely coagulated, and the mixture is placed in a precooling centrifuge for centrifugation at 3000rpm for 10 minutes to collect serum. The collected serum was stored at 4℃for a short period (within one week) and at-20℃for more than one week.
(2) Detection of samples
1) Sample dilution preparation and sample dilution: preparing a sample diluent by using TWEEN-20 and 0.015M PBS with pH of 7.4, adding 1mL TWEEN-20 into 100mL 0.015M PH7.4 PBS, and fully and uniformly mixing to obtain the sample diluent. According to 1:4 (1 volume sample: 4 volume dilution) ratio 5-fold dilution serum or plasma sample.
2) And (3) detecting a sample: placing a fluorescence quenching immunochromatographic test strip by a blocking method on a clean and tidy table top, taking 100 mu L of diluted sample, adding the diluted sample into a sample application hole for 15min, and reading a fluorescence value by using a fluorescence reader.
The Tianjin Hongyu biosciences limited applied for the name: novel coronavirus antibody detection method and its kit invention patent, application number: CN202210089203.7. The method utilizes a colloidal gold immunochromatography competition method, wherein the colloidal gold immunochromatography competition method is to label ACE2 protein with colloidal gold, and utilizes a neutralizing antibody to compete with the colloidal gold-labeled ACE2 protein to combine with the COVID-19 recombinant RBD protein coated on a detection line. Has the effect of simultaneously detecting novel coronavirus IgG, igM and neutralizing antibodies in organisms. However, the result judgment mode of the invention is traditional visual observation, which can cause deviation of judgment of the result at the critical value and can only carry out qualitative detection. The linear range of the application is 0.5 IU/mL-10 IU/mL, the S/CO value is 20, and the linear range is narrow, so that the determination of the neutralizing antibody level in a sample is not facilitated.
Compared with the method, the method has advantages in the aspects of signal acquisition and linear range. And for the detection result, a fluorescent reader is used for reading a fluorescent value, and the concentration of the neutralizing antibody in the sample can be accurately calculated by substituting the value into a standard curve. Secondly, the detection sensitivity is 125ng/mL, the linear range is 125 ng/mL-10,000 ng/mL, the S/CO value is 80, the linear range is wider, and the neutralizing antibody level in the sample can be well determined.
In conclusion, the invention has the advantages of wide linear range and capability of accurately and quantitatively detecting the neutralizing antibody level in the sample.
As shown in fig. 4, the detection effect picture of the present invention:
as shown in FIG. 5, the Tianjin Hongyu biological technology Co., ltd.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (10)
1. The fluorescence quenching immunochromatography test strip is characterized by comprising a card shell, a PVC bottom plate, a sample pad, a combination pad, a nitrocellulose NC film and a water absorption pad; the sample pad, the combination pad, the nitrocellulose NC film and the water absorption pad are sequentially and alternately stuck on the PVC bottom plate and are arranged in the test strip clamping shell, and RBD marked nano gold RBD-AuNPs are sprayed on the combination pad.
2. The fluorescence quenching immunochromatographic test strip according to claim 1, wherein RBD-labeled nano-gold RBD-AuNPs are sprayed on the binding pad, wherein RBD is labeled on nano-gold by electrostatic adsorption, a T line area on the NC membrane is coated with ACE2 coupled green fluorescent microspheres ACE2-FNPs, wherein ACE2 is coupled with FNPs by covalent bonding, and a C line area on the NC membrane is coated with BSA coupled green fluorescent microspheres BSA-FNPs.
3. The fluorescence quenching immunochromatographic test strip of claim 1, wherein when the sample dropped onto the sample pad is a negative sample, i.e., no neutralizing antibody is present, the sample flows toward the water absorbing pad under the siphoning action of the test strip, and when the sample flows to the binding pad, RBD-AuNPs on the binding pad follow the liquid flow, flow to the T-line region of the NC membrane, RBD binds with ACE2 to form RBD-AuNPs-ACE2-FNPs structure, auNPs and FNPs undergo fluorescence resonance energy transfer, fluorescence of FNPs is quenched by AuNPs, no fluorescent signal flows to the C-line region of the NC membrane, RBD-AuNPs does not bind with BSA-FNPs, and FNPs emit fluorescent signals.
4. The fluorescence quenching immunochromatographic test strip of claim 1, wherein when the sample dropped onto the sample pad is a positive sample, i.e., a neutralizing antibody is present, the sample flows toward the absorbent pad under the siphoning action of the test strip, and when the sample flows to the binding pad, the neutralizing antibody in the sample is first bound to RBD-AuNPs on the binding pad, and then flows to the T-line region of the NC membrane, RBD cannot bind to ACE2, cannot form RBD-AuNPs-ACE2-FNPs structure, FNPs normally emit fluorescent signals, flows to the C-line region of the NC membrane, RBD-AuNPs does not bind to BSA-FNPs, and FNPs emit fluorescent signals. In other words, when the neutralizing antibody is negative, the T-line has no fluorescence and the C-line has fluorescence; when the neutralizing antibody is positive, the T line has fluorescence and the C line has fluorescence, so that the reading of a forward signal is realized.
5. A method for preparing a fluorescence quenching immunochromatographic test strip according to any one of claims 1 to 4, which is characterized by comprising the following steps:
step one, preparing a colloidal gold solution by using chloroauric acid and trisodium citrate;
preparing RBD-AuNPs by using colloidal gold, recombinant RBD protein and BSA;
step three, respectively coupling ACE2 and BSA by using fluorescent microsphere FNPs;
and step four, preparing a gold-labeled pad and a sample pad respectively and assembling the test paper strips.
6. The method for preparing a fluorescence quenching immunochromatographic test strip according to claim 5, wherein the method for preparing the colloidal gold solution in the step one comprises the following steps:
cleaning the conical flask, measuring 100mL of ultrapure water, placing the conical flask in a magnetic stirrer, heating to boil, adding 2mL of chloroauric acid, rapidly adding 3.2mL of trisodium citrate, and keeping boiling for 15min; after cooling to room temperature, adding ultrapure water to complement 100mL to prepare colloidal gold solution with the particle size of 20nm, and storing at 4 ℃;
the concentration of chloroauric acid is 1%, and the concentration of trisodium citrate is 1%.
7. The method for preparing a fluorescence quenching immunochromatographic test strip according to claim 5, wherein the preparation method of RBD-AuNPs in the second step comprises the following steps:
cleaning small beaker, weighing 5mL of colloidal gold solution, placing in magnetic stirrer, adding appropriate amount of K 2 CO 3 Adjusting the pH of the colloidal gold solution to 8.7; adding 3 mu L of recombinant RBD proteins with different concentrations, stirring for 15min, and standing for 15min; adding 500 mu LBSA solution, stirring for 15min, and standing for 15min; centrifuging at 10000rpm at 4deg.C for 15min, collecting supernatant, adding 240 μL Jin Biaofu solution, dissolving again, and storing at 4deg.C；
K 2 CO 3 The concentration was 250mM and the BSA solution concentration was 10% w/v.
8. The method for preparing a fluorescence quenching immunochromatographic test strip according to claim 5, wherein the fluorescent microsphere FNPs in the third step are coupled with ACE2 and BSA, and the method comprises the following steps:
dissolving 100 mu LFNPs in 400 mu L of ultrapure water, adding 13.4 mu LEDC and 8.05 mu LNHS, and performing light-proof rotary reaction for 30min; adding 7 mu L of ACE2 with different concentrations, carrying out light-proof rotary reaction for 2 hours, adding 10 mu L of 10% BSA, carrying out light-proof rotary reaction for 1 hour, and centrifuging at 13000rpm at 4 ℃ for 30 minutes; sucking the supernatant, adding 200 mu L PBS with pH7.4 for re-dissolving, measuring the particle size, and storing at 4 ℃;
EDC concentration is 1%w/v, NHS concentration is 1%w/v.
9. The method for preparing a fluorescence quenching immunochromatographic test strip according to claim 5, wherein the method for assembling the test strip in the fourth step comprises the following steps:
adhering an NC film to the center of a PVC plate, and storing in a dry environment for later use; diluting ACE2-FNPs and BSA-FNPs 400 times, coating the diluted ACE2-FNPs and BSA-FNPs on T-line and C-line of NC membrane with different membrane dividing amounts by using a membrane dividing instrument, and drying at 37deg.C overnight; the RBD-AuNPs are sprayed on the polyester film with different spraying amounts by a metal spraying film drawing instrument to prepare a bonding pad, and the bonding pad is dried for 2 days at 37 ℃. Cutting the bonding pad to enable the upper and lower parts to be left with 1mm blank; bonding the bonding pad and the sample pad to the NC film in a mutually overlapped manner; cutting the pasted plate into strips with the width of 4mm by using a strip cutting machine, and filling the strips into a card shell to prepare the test paper strip.
10. The method for preparing a fluorescence quenching immunochromatographic test strip according to claim 9, further comprising the steps of dividing the ACE2-FNPs and the BSA-FNPs into 2.0 mu L/cm and spraying the RBD-AuNPs into 2.0 mu L/cm; the overlap of the conjugate pad and the sample pad was 1mm.
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