CN116908459A - Immune test paper card for detecting brucella antigen and preparation method and application thereof - Google Patents
Immune test paper card for detecting brucella antigen and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biological detection, in particular to a brucella antigen quantum dot microsphere immune test paper card, a preparation method and application thereof. The immune test paper card comprises a PVC bottom plate, wherein a sample pad, a quantum dot combination pad, a nitrocellulose membrane and a water absorption pad are sequentially fixed on the PVC bottom plate; the quantum dot binding pad is internally coated with a quantum dot marked anti-brucella monoclonal antibody; the surface of the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is internally coated with an anti-Brucella monoclonal antibody, and the quality control line is internally coated with a goat anti-mouse IgG antibody. The immunity test paper card is suitable for detecting brucella in the swab with cow and sheep vaginal secretion and aborted fetal amniotic fluid, can diagnose brucella infection in a short time, is particularly suitable for on-site brucella infection diagnosis, epidemiological investigation and live cow/sheep international trade quarantine inspection, and has the technical advantages of convenient use, rapid and simple operation and high accuracy.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a brucella antigen quantum dot microsphere immune test paper card, a preparation method and application thereof.
Background
Brucellosis (brucellosis), also known as brucellosis, wavy heat, is an infectious disease of animal origin caused by brucellosis (Brucella), and humans are often ill by contact with the excreta of infected animals or by ingestion of foods made from infected or diseased animals.
The diagnosis of brucellosis is a key point and a difficult problem of the research of the brucellosis, the separation of pathogens is a gold standard for diagnosing the brucellosis, but in view of the fact that the separation rate of the pathogens is low, and the complete biological safety protection facility is required to be provided, and the diagnosis can not be completed by the operation of very experienced professionals, the technology cannot be widely applied, so that the exploration and the research of the diagnosis method of the brucellosis are very important for screening and diagnosing the brucellosis. The existing Brucella laboratory diagnosis technical standard (GB/T18646-2018) in China comprises bacterial culture, a serological detection method and an etiology detection method, wherein the serological detection method also comprises a tiger red plate test (RBT), an enzyme-linked immunosorbent assay (ELISA), a test tube agglutination test (SAT), a Complement Fixation Test (CFT) and the like. The tiger red plate agglutination test is easy to miss detection or false detection due to low sensitivity and low specificity, and can not distinguish immune antibodies and infectious antibodies, so that the tiger red plate agglutination test is only suitable for the primary screening of brucellosis. The test tube agglutination test has higher specificity, but the sensitivity is still low, the operation is complex, the time consumption is long, the test tube agglutination test is not suitable for mass detection, the application is inconvenient in the actual operation, and the detection method using RBT and SAT has the influence of human judgment factors and has certain limitation, so the SAT test is not claimed to be used in international trade. Although specific, complement fixation assays are more complex to operate than other methods, and positive events occur later, and are only useful in humans for people with difficult diagnosis. Because of the harsh conditions, the expensive reagents are difficult for primary veterinarians to develop, and their use is greatly limited. The enzyme-linked immunosorbent assay (ELISA) and the Polymerase Chain Reaction (PCR) detection method need relatively independent laboratory conditions, the technical level requirement of operators is high, the experiment time is too long, and the requirement of clinical rapid detection cannot be met. Therefore, development of a specific and sensitive method for rapidly detecting clinical diseases is still a urgent need.
Disclosure of Invention
In order to solve the technical problems, the invention provides an immune test paper card for detecting brucella antigen quantum dot microspheres, a preparation method and application thereof.
The invention provides an immunity test paper card for detecting Brucella antigen, which comprises a PVC bottom plate, wherein a sample pad, a quantum dot combination pad, a nitrocellulose membrane and a water absorption pad are sequentially fixed on the PVC bottom plate; quantum dot-labeled anti-Brucella monoclonal antibodies are coated in the quantum dot binding pad; the surface of the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is internally coated with an anti-Brucella monoclonal antibody, and the quality control line is internally coated with a goat anti-mouse IgG antibody.
Alternatively, anti-Brucella monoclonal antibodies were purchased from Beijing, middle, shake-in technology, inc.
Optionally, the concentration of the quantum dot marked anti-Brucella monoclonal antibody coating in the quantum dot binding pad is 10-100 mu L/cm 2 Preferably 50. Mu.L/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The coating quality of the anti-Brucella monoclonal antibody in the detection line is 0.5-3 mug, preferably 2 mug, of the coating in each centimeter of the detection line; the coating quality of the goat anti-mouse IgG antibody coating in the quality control line is 0.5-3 mug, preferably 2 mug, of coating in each centimeter detection line; the distance between the detection line and the quality control line is 4-8 mm, preferably 5mm.
Optionally, the preparation method of the quantum dot labeled anti-brucella monoclonal antibody comprises the following steps:
s1, activating quantum dot microspheres, and adding MES buffer;
s2, adding an anti-Brucella monoclonal antibody into the activated quantum dot microsphere solution, uniformly mixing, and standing at 35-38 ℃ for 0.5-2 hours; the volume mass ratio of the quantum dot microsphere to the anti-Brucella monoclonal antibody is 4:3 to 4, preferably 4:3, a step of; the unit of the quantum dot microsphere is mu L, and the unit of the antibody is mu g; the diameter of the quantum dot microsphere is 150-350 nm, preferably 200nm;
s3, adding a blocking agent, standing at 35-38 ℃ for a period of time, centrifuging, adding MES buffer solution, and standing at 35-38 ℃ for a period of time to prepare a solution containing quantum dot labeled anti-Brucella monoclonal antibody;
the formula of MES buffer solution is as follows: 0.01M to 0.1M MES, and the pH value is 6.5;
the blocking agent was glycine buffer containing 1% BSA, and the concentration of glycine was 50mmol/L.
Optionally, in S1, activating by EDC and NHS solution, wherein the activating condition is oscillation reaction for 3-10 seconds; preferably, the mass ratio of the quantum dot microsphere to EDC and NHS is 1:20:20, a step of; 20-30 mL MES buffer solution, preferably 25mL MES buffer solution, is added to each 1mg quantum dot microsphere.
The invention also provides a preparation method of the immune test paper card, which comprises the following steps:
s1, coating treatment liquid on a bonding pad, and drying to obtain a pretreated bonding pad;
s2, coating a solution containing quantum dot labeled anti-Brucella monoclonal antibody on the pretreated bonding pad, and freeze-drying to obtain a quantum dot bonding pad;
s3, spraying a detection line and a quality control line on the surface of the nitrocellulose membrane; the detection line is internally coated with an anti-Brucella monoclonal antibody; the quality control line is internally coated with a goat anti-mouse IgG antibody;
s4, assembling the sample pad, the quantum dot binding pad, the nitrocellulose membrane and the water absorption pad to obtain the immunity test paper card for detecting the Brucella antigen.
Optionally, the composition of the treatment fluid is:
PEG20000: the mass percentage concentration is 0.2-1%;
BSA: the mass percentage concentration is 0.05 to 0.2 percent;
tween-20: the volume percentage concentration is 0.01 to 0.1 percent;
preferably, it is:
PEG20000: the mass percentage concentration is 0.5%;
BSA: the mass percentage concentration is 0.1%;
tween-20: the volume percentage concentration is 0.05%;
in S2, labeled quantum dot microsphere solution 1: after 100 dilution, the mixture was diluted to 50. Mu.L/cm 2 Uniformly coating on the pretreated quantum dot bonding pad;
in S3, the concentration of the Brucella monoclonal antibody is 0.5-2 mg/mL, preferably 1mg/mL; the concentration of the goat anti-mouse IgG antibody is 0.5-2 mg/mL, preferably 1mg/mL.
The invention also provides a kit for detecting the Brucella antigen, which comprises the immune test paper card and is characterized by further comprising sample diluent, wherein the composition of the sample diluent is as follows:
sodium chloride: the mass percentage concentration is 0.3-0.7%;
Na 2 HPO 4 ：0.08mmol～0.12mmol；
casein: the mass percentage concentration is 0.3-0.7%;
tween-20: the volume percentage concentration is 0.5-1.5%;
preferably, it is:
sodium chloride: the mass percentage concentration is 0.4%;
Na 2 HPO 4 ：0.1mmol；
casein: the mass percentage concentration is 0.3-0.7%;
tween-20: the volume percentage concentration is 0.5-1.5%.
The invention also provides a method for detecting Brucella antigen, which adopts the kit and at least comprises the following steps:
soaking a sampling swab into a sample diluent, adding 3-10 drops after fully and uniformly mixing, standing at room temperature for 15-20 minutes, inserting into a portable immunofluorescence analyzer, and converting a fluorescence signal detected by the immunofluorescence analyzer into a detection value by collecting and analyzing the fluorescence signal; if the detection value is more than 100, the result is judged to be positive, and the existence of Brucella antigen in the sample is indicated; if the detection value is less than or equal to 100, the result is judged to be negative, which indicates that no brucella antigen exists in the sample; the sampling swab is a cow or sheep vaginal secretion swab or a fetal amniotic fluid aborted swab.
The invention also provides an application of the immune test paper card or the kit in epidemiological investigation or international trade quarantine inspection of live cattle/sheep.
Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following advantages:
the quantum dot microsphere immune test paper card for detecting the brucella antigen is suitable for detecting the brucella in a swab with cow and sheep vaginal secretion and aborted fetal amniotic fluid, can diagnose the brucella infection in a short time, and is particularly suitable for on-site brucella infection diagnosis, epidemiological investigation and international trade quarantine inspection of live cows/sheep. The portable immunofluorescence analyzer is used cooperatively with the portable immunofluorescence analyzer, can directly display the detection result, is convenient to use, quick and simple to operate, high in accuracy, free from human factor interference and high in practicability.
Drawings
FIG. 1 is a schematic diagram of an assembly structure of an immune test paper card according to an embodiment of the invention;
FIG. 2 shows the sensitivity detection result of an immune test paper card according to an embodiment of the present invention;
wherein: 1-upper shell, 2-lower shell, 3-sample-adding hole, 4-visual window, 5-sample pad, 6-quantum dot combination pad, 7-nitrocellulose membrane, 8-water absorption pad, 9-detection line and 10-quality control line.
Detailed Description
In order that the above objects, features and advantages of the invention will be more clearly understood, a further description of the invention will be made. It should be noted that, without conflict, the embodiments of the present invention and features in the embodiments may be combined with each other.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the invention.
The embodiment of the invention provides an immunity test paper card for detecting brucella antigen, which adopts a quantum dot fluorescence immunochromatography technology to detect brucella virus, and the principle is as follows: the test strip prepares a combination pad by using quantum dot microspheres marked by anti-brucella protein, and the nitrocellulose membrane is respectively coated with the anti-brucella protein and goat anti-mouse IgG (C line). In the lateral chromatography process, when the sample to be detected contains Brucella virus, the Brucella virus can be specifically combined with the antibody marked on the quantum dot microsphere and captured to form immune complex. Redundant quantum dot antigen conjugates were captured by goat anti-mouse monoclonal antibodies on line C, immobilized on line C. The captured quantum dot microsphere can generate red fluorescent signals through ultraviolet (wavelength 365 nm) excitation. Specifically, the immunity test paper card for detecting Brucella antigen comprises: a PVC bottom plate, on which a sample pad, a quantum dot bonding pad, a nitrocellulose membrane and a water absorption pad are sequentially fixed; the quantum dot binding pad is internally coated with a quantum dot marked anti-brucella monoclonal antibody; the surface of the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is internally coated with an anti-Brucella monoclonal antibody, and the quality control line is internally coated with a goat anti-mouse IgG antibody.
As an improvement of the embodiment of the present invention, an anti-brucella monoclonal antibody was purchased from the company of sciences, limited, of the middle department of beijing, specification: 20mg (2 tubes), 0.5 mL/tube. In the embodiment of the invention, the anti-Brucella monoclonal antibody is required to be connected with the quantum dot labeled microsphere, so that the monoclonal antibody has higher requirement on the selection of the monoclonal antibody.
As an improvement of the embodiment of the invention, the concentration of the quantum dot marked anti-Brucella monoclonal antibody coating in the quantum dot binding pad is 10-100 mu L/cm 2 Preferably 50. Mu.L/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The coating quality of the anti-Brucella monoclonal antibody in the detection line is 0.5-3 mug, preferably 2 mug, of the coating in each centimeter of the detection line. The coating quality of the goat anti-mouse IgG antibody coating in the quality control line is 0.5-3 mug, preferably 2 mug, of the coating in each centimeter detection line. The sensitivity of detection can be well controlled by the coating concentration and the ratio. If the coating amount of the anti-Brucella monoclonal antibody is too large, a non-specific signal exists, and if the coating amount is too small, the signal becomes weak, and the sensitivity is reduced; and therefore needs to be controlled within a reasonable range. Meanwhile, it is also necessary to control the coating amounts in the detection line and the quality control line, if the coating amounts in the detection line and the quality control line are too large, non-specific signals are generated, and if the coating amounts are too small, the signals are weakened, and the sensitivity is lowered.
As an improvement of the embodiment of the invention, the distance between the detection line and the quality control line is 4-8 mm, preferably 5mm. If the distance between the two is too large or too small, the signal strength thereof is lowered, resulting in a decrease in sensitivity.
As an improvement of the embodiment of the invention, the implementation of the invention also provides a preparation method of the quantum dot labeled anti-Brucella monoclonal antibody, which specifically at least comprises the following steps:
s1, activating quantum dot microspheres, and adding a buffer solution;
s2, adding an anti-Brucella monoclonal antibody into the activated quantum dot microsphere solution, uniformly mixing, and standing at 35-38 ℃ for 0.5-2 hours;
wherein, the mass ratio of the quantum dot microsphere to the anti-Brucella monoclonal antibody is 1:1, a step of;
s3, adding a blocking agent, standing at 37 ℃ for 30min for blocking, centrifuging, adding MES buffer solution, and standing at 37 ℃ for 1h to obtain a solution containing quantum dot labeled anti-Brucella monoclonal antibody;
the formula of MES buffer solution is as follows: 0.01M to 0.1M MES, and the pH value is 6.5;
the blocking agent was glycine buffer containing 1wt% BSA, the concentration of glycine was 50mmol/L.
The quantum dot labeled anti-Brucella monoclonal antibody prepared by the embodiment of the invention can obviously improve the stability of the test strip.
Specifically, in the preparation method, the quantum dot microsphere is selected from quantum dot microspheres with the diameter of 150-350 nm, preferably 200nm; if the particle size of the quantum dot microsphere is too large, the quantum dot microsphere can not run under the NC film or has a nonspecific signal, and if the particle size of the quantum dot microsphere is too small, the signal can be weakened, and the sensitivity is reduced.
Specifically, in the preparation method, the volume-mass ratio of the quantum dot microsphere to the monoclonal antibody is 4:3 to 4, preferably 4:3, a step of; the unit of the quantum dot microsphere is mu L, and the unit of the antibody is mu g; if the ratio is too large, there is a nonspecific signal, and if the ratio is too small, the signal becomes weak and the sensitivity decreases.
Specifically, in the preparation method, EDC and NHS solution are adopted for activation, and the condition of activation is oscillation reaction for 3-10 seconds; the mass ratio of the quantum dot microsphere to EDC and NHS is 1:20:20, a step of; 20-30 mL, preferably 25mL, of buffer solution is added to each 1mg of quantum dot microsphere.
The embodiment of the invention also relates to a preparation method of the immune test paper card, which comprises the following steps:
s1, coating treatment liquid on a bonding pad, and drying to obtain a pretreated bonding pad;
s2, coating a solution containing quantum dot labeled anti-Brucella monoclonal antibody on the pretreated bonding pad, and freeze-drying to obtain a quantum dot bonding pad;
s3, spraying a detection line and a quality control line on the surface of the nitrocellulose membrane;
s4, assembling the sample pad, the quantum dot binding pad, the nitrocellulose membrane and the water absorption pad to obtain the immunity test paper card for detecting the Brucella antigen.
The composition of the treatment fluid is as follows:
PEG20000: the mass percentage concentration is 0.2-1%;
BSA: the mass percentage concentration is 0.05 to 0.2 percent;
tween-20: the volume percentage concentration is 0.01 to 0.1 percent;
preferably, it is:
PEG20000: the mass percentage concentration is 0.5%;
BSA: the mass percentage concentration is 0.1%;
tween-20: the volume percentage concentration is 0.05%;
in S2, labeled quantum dot microsphere solution 1: after 100 dilution, the mixture was diluted to 50. Mu.L/cm 2 Uniformly coating on the pretreated quantum dot bonding pad.
The embodiment of the invention also relates to a kit for detecting Brucella antigen, which comprises the immune test paper card, and the kit also comprises sample diluent, wherein the composition of the sample diluent is as follows: sodium chloride: the mass percentage concentration is 0.3-0.7%; na (Na) 2 HPO 4 :0.08mmol to 0.12mmol; casein: the mass percentage concentration is 0.3-0.7%; tween-20: the volume percentage concentration is 0.5-1.5%; preferably, it is: sodium chloride: concentration by mass percent0.4%; na (Na) 2 HPO 4 :0.1mmol; casein: the mass percentage concentration is 0.3-0.7%; tween-20: the volume percentage concentration is 0.5-1.5%.
According to the invention, through the sample diluent, the swab with the vaginal secretion of the cattle and sheep and the amniotic fluid of the aborted fetus can be used for detection, so that the bacterial antigen in the swab can be rapidly extracted, the blood sampling operation is avoided, and the pathogen diffusion and pollution in the sampling process are reduced.
The embodiment of the invention also relates to a method for detecting Brucella antigen, which adopts the kit and at least comprises the following steps: soaking a sampling swab into a sample diluent, adding 3-10 drops after fully and uniformly mixing, standing at room temperature for 15-20 minutes, inserting into a portable immunofluorescence analyzer, and converting a fluorescence signal detected by the immunofluorescence analyzer into a detection value by collecting and analyzing the fluorescence signal; if the detection value is more than 100, the result is judged to be positive, and the existence of Brucella antigen in the sample is indicated; if the detection value is less than or equal to 100, the result is judged to be negative, which indicates that no brucella antigen exists in the sample; the sampling swab is a cow or sheep vaginal secretion swab or a fetal amniotic fluid aborted swab.
The embodiment of the invention also relates to application of the immune test paper card kit in epidemiological investigation or international trade quarantine inspection of live cattle/sheep.
The reagents and biological sources used in the following examples are commercially available.
Example 1:
this example is used to illustrate the preparation of an immunoassay paper card for detecting Brucella antigen.
The preparation method of the immunity test paper card for detecting the brucella antigen comprises the following steps:
1. preparation of quantum dot labeled anti-brucella monoclonal antibody:
1.1, adding 20mg/mL EDC solution and 20mg/mL NHS solution into 20 mu L quantum dot microspheres, oscillating for 5 seconds by a vortex instrument, and preserving heat at 37 ℃ for 15 minutes to obtain activated quantum dot microspheres; the particle size of the quantum dot microsphere is 200nm;
1.2, centrifuging the activated quantum dot microsphere buffer solution, discarding the supernatant, and adding 25 mu L of buffer solution (0.1M MES pH=6.5) to obtain the activated quantum dot microsphere buffer solution;
1.3, after ultrasonic dispersion of the activated quantum dot microsphere buffer solution, adding 15 mug of Brucella monoclonal antibody, uniformly mixing, and standing at 37 ℃ for 1 hour;
1.4, adding 5 mu L of blocking solution (glycine buffer containing 1wt% BSA and having the concentration of 50 mmol/L), standing at the constant temperature of 37 ℃ for 30min for blocking, centrifuging after 30min, and adding 25 mu L of 0.1M MES (pH 6.5) buffer to prepare a marked quantum dot microsphere solution;
2. preparation of a quantum dot conjugate pad labeled with a quantum dot labeled monoclonal antibody against brucella:
2.1, uniformly coating untreated combined optical pads (Shanghai gold mark biotechnology Co., ltd.) with a treatment liquid, wherein the composition of the treatment liquid is as follows: 0.5wt% PEG20000, 0.1wt% BSA, 0.05wt% Tween-20, and a drying temperature of 55 ℃;
2.2, adding the marked quantum dot microsphere solution 1: after 100 dilution, the mixture was diluted to 50. Mu.L/cm 2 Uniformly coating on the pretreated quantum dot bonding pad, placing into a freeze dryer cold trap at a temperature below-50 ℃, pre-freezing for 30min, and pumping for more than 3 hours.
3. Spraying a nitrocellulose membrane detection line and a quality control line:
spraying a detection line (T) and a quality control line (C) on the surface of the nitrocellulose membrane by using a metal spraying and membrane drawing integrated machine, wherein the detection line is a Brucella monoclonal antibody, and the quality control line is a goat anti-mouse IgG antibody;
the coating concentration of the Brucella monoclonal antibody is 1.0mg/mL, the coating concentration of the goat anti-mouse IgG antibody is 1.0mg/mL, the spraying amount is 1 mu L/cm when the Brucella monoclonal antibody and the goat anti-mouse IgG antibody are used for drawing films, and the distance between the detection line and the quality control line is 5mm.
4. Assembly of quantum dot microsphere immune test paper card
The PVC bottom plate (Shanghai gold mark biotechnology Co., ltd.) is stuck upwards, a nitrocellulose membrane is stuck in the middle of the PVC plate according to the structural schematic diagram of the immune test paper card shown in figure 1, and then a quantum dot bonding pad is overlapped by 2mm before being stuck on the nitrocellulose membrane; and then respectively adhering a sample pad (Shanghai gold mark biotechnology Co., ltd.) and a water absorption pad (Shanghai gold mark biotechnology Co., ltd.) on the PVC bottom plate before the quantum dot bonding pad and after the nitrocellulose membrane, wherein the sample pad and the water absorption pad are respectively overlapped with the quantum dot bonding pad and the nitrocellulose membrane by 2mm. Finally cutting into test paper strips with the width of 3.5mm, and pressing a plastic shell outside to assemble the test paper card.
The plastic shell consists of a lower shell and an upper shell; the inner side of the lower shell is provided with a plurality of positioning columns for test strips, and the upper shell is provided with a sample adding hole which is opposite to the sample pad and a visual window which is opposite to the nitrocellulose membrane.
The using method of the prepared immune test paper card comprises the following steps:
1. if the sample is refrigerated or frozen for storage, the sample to be tested and the required reagent are taken out from the storage condition and are balanced to the room temperature (15-30 ℃).
2. In preparation for detection, the aluminum foil pouch is opened from the tear. And taking out the detection card and placing the detection card on a horizontal tabletop.
3. The sample number is marked on the test card.
4. The swab sample is placed in the buffer and the swab is rotated for about 10 seconds while the swab head is pressed against the inner wall of the sampling tube to release the antigen from the swab.
5. The swab is removed and discarded following a biohazard waste treatment protocol.
6. The bottle cap is screwed onto the sampling tube, and the sampling tube is shaken forcefully to fully mix the sample with the buffer solution.
7. 3 drops of the solution (about 100. Mu.L) were added to the sample wells and the timing was started. The results were interpreted at 15-20 minutes.
Inserting the portable immunofluorescence analyzer, and converting the fluorescence signal detected by the immunofluorescence analyzer into a detection value by collecting and analyzing the fluorescence signal;
inserting the fluorescent signal into a socket of a portable immunofluorescence analyzer, and converting the fluorescent signal into a detection value A by collecting and analyzing the fluorescent signal detected by the immunofluorescence analyzer:
if the detection value A is larger than the critical value B, the result is judged to be positive, and the existence of Brucella antigen in the sample is indicated;
if the detection value A is less than or equal to the critical value B, the result is judged to be negative, which indicates that no brucella antigen exists in the sample.
Example 2
The present example is a kit for detecting brucella antigen, comprising the immunoassay paper card of example 1, further comprising a sample diluent having the following composition:
sodium chloride: the mass percentage concentration is 0.4%;
Na 2 HPO 4 ：0.1mmol；
casein: the mass percentage concentration is 0.3-0.7%;
tween-20: the volume percentage concentration is 0.5-1.5%.
Experimental example 1:
1. determination of detection critical value of quantum dot microsphere immune test paper card
Using the brucella antigen quantum dot microsphere immune test paper card prepared in example 1, 150 serum samples confirmed to be negative by brucella were detected according to the above method, and an immunofluorescence analyzer read each sample detection value (table 1), and a detection threshold of the brucella antigen quantum dot microsphere immune test paper card was calculated, the threshold=average detection value+2sd (standard deviation) =9.99+2×9.75=29.49.
2. Sensitivity determination of quantum dot microsphere immune test paper card
The recombinant protein of brucella lipopolysaccharide expressed and purified in escherichia coli is diluted into 50ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL and 1ng/mL by a sterile PBS gradient, and is detected by using a brucella antigen quantum dot microsphere immunoassay paper card, each sample is repeatedly tested for 10 times, and the maximum dilution of the recombinant lipopolysaccharide protein with positive detection results of 10 times is taken as the lowest detection limit of the detection card, and the results are shown in table 1.
Table 1: brucella quantum dot microsphere immune test paper card sensitivity detection result
From the experimental results, the sensitivity of the test strip in the embodiment of the invention is 1ng/mL.
3. Quantum dot microsphere immune test paper card coincidence rate determination
And detecting 120 collected clinical samples by using a brucella fluorescent PCR detection kit, detecting the brucella antigen quantum dot microsphere immune test paper card, and determining the detection sensitivity, the specificity and the total coincidence rate of the brucella antigen quantum dot microsphere immune test paper card to the clinical samples by using the detection result of the brucella fluorescent PCR detection kit as a standard.
The result is detected by a brucella fluorescence PCR detection kit, the brucella antigen quantum dot microsphere immune test paper card,
sensitivity= (number of positive detection/number of positive) x 100% = (44/46) x 100% = 95.65%;
specificity= (number of negative detections/number of negative) ×100% = (74/74) ×100% = 100%;
total coincidence= (correct detection number/total number of samples) ×100% = (118/120) ×100% = 98.33%.
4. And (3) measuring the card specificity of the quantum dot microsphere immune test paper:
cross reaction detection is carried out on escherichia coli O157, yersinia O9, tularemia, pseudomonas, vibrio cholerae and salmonella enteritidis, and cross test results show that the test strip does not cross react with the escherichia coli O157, the yersinia O9, the tularemia, the pseudomonas, the vibrio cholerae and the salmonella enteritidis.
Experimental example 2:
test paper cards were prepared by the method of example 1, except that the sources of the monoclonal antibodies were different. The prepared test paper card detects sensitivity according to the method of example 2, and the obtained experimental results are shown in table 2.
TABLE 2
Wherein, the monoclonal antibody 1 is monoclonal antibody (5 mL/bottle) of animal brucellosis competition ELISA antibody detection kit, CVCC number is Z242, the monoclonal antibody 2 is monoclonal antibody (1 mL/bottle) of brucellosis antibody detection test strip, and CVCC number is Z249.
The monoclonal antibody of the Sharpness science and technology limited liability company in Beijing is found to have the best effect through screening.
Experimental example 3
Test paper cards were prepared by the method of example 1, except that: the coating amount of the anti-Brucella monoclonal antibody is different. The prepared test paper card was tested for sensitivity according to the method of example 2, and the test results obtained are shown in table 3.
Table 3:
experimental example 4
Test paper cards were prepared by the method of example 1, except that: the coating amount of the goat anti-mouse IgG antibody is different. The prepared test paper card was tested for sensitivity according to the method of example 2, and the test results obtained are shown in table 4.
TABLE 4 Table 4
As shown in Table 4, the concentrations of the goat anti-mouse IgG antibodies were 0.8 to 1.2mg/mL, and the detection requirements were satisfied, preferably 1mg/mL.
Experimental example 5
Test paper cards were prepared by the method of example 1, except that: the distance between the detection line and the quality control line is different. The prepared test paper card was tested for sensitivity according to the method of example 2, and the test results obtained are shown in table 5.
TABLE 5
As shown in the experimental results in table 5, when the distance between the detection line and the quality control line is too large or too small, both the sensitivity and the signal strength of the detection are adversely affected.
Experimental example 6
Test paper cards were prepared by the method of example 1, except that: the ratio of the quantum dot microsphere volume to the monoclonal antibody mass is different. The prepared test paper card was tested for sensitivity according to the method of example 2, and the test results obtained are shown in Table 6.
TABLE 6
From the experimental results shown in table 6, when the ratio of the volume of the quantum dot microsphere to the mass of the monoclonal antibody is too large or too small, the sensitivity and the signal strength of the detection are adversely affected.
Experimental example 7
The kit was prepared by the method of example 2, except that: the compositions of the treatment solutions are different, and are shown in Table 7. The prepared test paper card was tested for sensitivity according to the method of example 2, and the test results obtained are shown in Table 7.
TABLE 7
As is clear from the experimental results shown in Table 6, too high a concentration of the treatment solution suppresses the fluorescence signal, and too low a concentration thereof is nonspecific and reduces the detection specificity thereof.
Experimental example 7
The test paper card is prepared by adopting the method of the embodiment 1, the test paper strips in the same batch are dried and stored in a dark place at room temperature, the test paper strips are randomly extracted at 1 month, 6 months, 12 months and 18 months respectively to detect 0.5ng/mL of Brucella standard substance, the test paper card is repeated three times, and the coefficient of variation is calculated by using a formula C.V = (SD/MN) multiplied by 100%, so as to check the stability of the test paper strips. The experimental results are shown in table 8.
TABLE 8
As shown in the experimental results of Table 8, the fluorescence intensity value does not obviously decrease within 1 year and the variation coefficient does not exceed 10%, which indicates that the quantum dot immunochromatographic test strip of the present invention has good repeatability in detecting Brucella and good stability within a shelf life within 1 year.
Experimental example 8
The method of example 2 is adopted to prepare other monoclonal antibody test paper cards, the test paper strips of the same batch are dried and stored in a dark place at room temperature, the test paper strips are randomly extracted at 1 month, 6 months and 12 months to detect 0.5ng/mL of Brucella standard substance, the test paper strips are repeated three times, and the coefficient of variation is calculated by using a formula C.V = (SD/MN) multiplied by 100%, so that the stability of the test paper strips is examined. The experimental results are shown in table 9.
TABLE 9
Other monoclonal antibodies 1 are monoclonal antibodies (5 mL/bottle) of animal brucellosis competition ELISA antibody detection kits, and CVCC number is Z242; other monoclonal antibodies 2 are monoclonal antibodies (1 mL/bottle) of Brucella antibody detection test strips, and CVCC number is Z249.
As can be seen from the experimental results in Table 9, the fluorescence intensity values are significantly reduced within 1 year, and the variation coefficients are all over 10%, which indicates that the quantum dot immunochromatography test strip invented by other antibodies is poor in repeatability when used for detecting Brucella and poor in stability within 1 year of storage period.
The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown and described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. An immunity test paper card for detecting Brucella antigen is characterized by comprising a PVC bottom plate, wherein a sample pad, a quantum dot combination pad, a nitrocellulose membrane and a water absorption pad are sequentially fixed on the PVC bottom plate;
the invention also provides an anti-Brucella monoclonal antibody with quantum dot marks coated in the quantum dot binding pad;
the surface of the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is internally coated with an anti-Brucella monoclonal antibody, and the quality control line is internally coated with a goat anti-mouse IgG antibody.
2. The immunoassay test paper card of claim 1, wherein said anti-brucella monoclonal antibody is purchased from the company of science and technology, ltd, of the middle of beijing.
3. The immunoassay test paper card of claim 1, wherein:
the concentration of the quantum dot marked anti-Brucella monoclonal antibody coating in the quantum dot binding pad is 10-100 mu L/cm 2 Preferably 50. Mu.L/cm 2 ；
The coating quality of the anti-Brucella monoclonal antibody in the detection line is 0.5-3 mug, preferably 2 mug, of the coating in each centimeter of the detection line; the coating quality of the goat anti-mouse IgG antibody coating in the quality control line is 0.5-3 mug, preferably 2 mug, of coating in each centimeter detection line; the distance between the detection line and the quality control line is 4-8 mm, preferably 5mm.
4. The immunoassay paper card according to claim 1, wherein the preparation method of the quantum dot labeled anti-brucella monoclonal antibody comprises the following steps:
s1, activating quantum dot microspheres, and adding MES buffer;
s2, adding the anti-Brucella monoclonal antibody into the activated quantum dot microsphere solution, uniformly mixing, and standing at 35-38 ℃ for 0.5-2 hours; the volume mass ratio of the quantum dot microsphere to the anti-Brucella monoclonal antibody is 4:3 to 4, preferably 4:3, a step of; the unit of the quantum dot microsphere is mu L, and the unit of the antibody is mu g;
the diameter of the quantum dot microsphere is 150-350 nm, preferably 200nm;
s3, adding a blocking agent, standing at 35-38 ℃ for a period of time, centrifuging, adding MES buffer solution, and standing at 35-38 ℃ for a period of time to prepare a solution containing the quantum dot labeled anti-Brucella monoclonal antibody;
the formula of the MES buffer solution is as follows: 0.01M to 0.1M MES, and the pH value is 6.5;
the blocking agent is glycine buffer solution containing 1% BSA, and the concentration of glycine is 50mmol/L.
5. The immunoassay test paper card according to claim 4, wherein in S1, the activation is performed by EDC and NHS solution under the condition of shaking reaction for 3-10 seconds; preferably, the mass ratio of the quantum dot microsphere to EDC and NHS is 1:20:20, a step of;
20-30 mL MES buffer solution, preferably 25mL MES buffer solution, is added to each 1mg quantum dot microsphere.
6. The method for manufacturing an immunoassay paper card according to any one of claims 1 to 5, comprising the steps of:
s1, coating treatment liquid on a bonding pad, and drying to obtain a pretreated bonding pad;
s2, coating a solution containing the quantum dot labeled anti-Brucella monoclonal antibody on the pretreated bonding pad, and freeze-drying to obtain the quantum dot bonding pad;
s3, spraying a detection line and a quality control line on the surface of the nitrocellulose membrane; the detection line is internally coated with an anti-Brucella monoclonal antibody; the quality control line is internally coated with a goat anti-mouse IgG antibody;
s4, assembling the sample pad, the quantum dot binding pad, the nitrocellulose membrane and the water absorption pad to obtain the immunity test paper card for detecting the Brucella antigen.
7. The method according to claim 6, wherein the composition of the treatment liquid is:
PEG20000: the mass percentage concentration is 0.2-1%;
BSA: the mass percentage concentration is 0.05 to 0.2 percent;
tween-20: the volume percentage concentration is 0.01 to 0.1 percent;
preferably, it is:
PEG20000: the mass percentage concentration is 0.5%;
BSA: the mass percentage concentration is 0.1%;
tween-20: the volume percentage concentration is 0.05%;
in S2, labeled quantum dot microsphere solution 1: after 100 dilution, the mixture was diluted to 50. Mu.L/cm 2 Uniformly coating on the pretreated quantum dot bonding pad;
in S3, the concentration of the Brucella monoclonal antibody is 0.5-2 mg/mL, preferably 1mg/mL; the concentration of the goat anti-mouse IgG antibody is 0.5-2 mg/mL, preferably 1mg/mL.
8. A kit for detecting brucella antigen, comprising the immunoassay paper card according to any one of claims 1 to 7, wherein the kit further comprises a sample diluent, and the sample diluent comprises the following components:
sodium chloride: the mass percentage concentration is 0.3-0.7%;
Na 2 HPO 4 ：0.08mmol～0.12mmol；
casein: the mass percentage concentration is 0.3-0.7%;
tween-20: the volume percentage concentration is 0.5-1.5%;
preferably, it is:
sodium chloride: the mass percentage concentration is 0.4%;
Na 2 HPO 4 ：0.1mmol；
casein: the mass percentage concentration is 0.3-0.7%;
tween-20: the volume percentage concentration is 0.5-1.5%.
9. A method for detecting brucella antigen, wherein the kit of claim 8 is used, and the method comprises at least the following steps:
soaking a sampling swab into a sample diluent, adding 3-10 drops after fully and uniformly mixing, standing at room temperature for 15-20 minutes, inserting into a portable immunofluorescence analyzer, and converting a fluorescence signal detected by the immunofluorescence analyzer into a detection value by collecting and analyzing the fluorescence signal; if the detection value is more than 100, the result is judged to be positive, and the existence of Brucella antigen in the sample is indicated; if the detection value is less than or equal to 100, the result is judged to be negative, which indicates that no brucella antigen exists in the sample;
the sampling swab is a cow or sheep vaginal secretion swab or a fetal amniotic fluid aborted swab.
10. Use of an immunoassay paper card according to any one of claims 1 to 7 or a kit according to claim 8 for epidemiological investigation or international trade quarantine inspection of live cattle/sheep.
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