CN114252600B - Cat triple antibody detection test strip and preparation method and application thereof - Google Patents
Cat triple antibody detection test strip and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a test strip capable of simultaneously carrying out detection and test of feline herpesvirus, feline calicivirus and/or feline panleukopenia virus, which is found by researching the use proportion of a fluorescent microsphere marked rabbit anti-cat IgG polyclonal antibody and an internal reference goat anti-chicken antibody, when the mass percentages of the fluorescent microsphere marked rabbit anti-cat IgG polyclonal antibody and the fluorescent microsphere marked goat anti-chicken IgY antibody are (20-25): 1, the detection linear range of the test strip is better. Compared with the conventional cat antibody detection test strip, the test strip can obtain 3 project detection results by single card, single sample mixing and single sample adding, effectively reduces the detection cost, maintains good detection performance, ensures that the cat antibody detection is simpler, and is suitable for various detection scenes. The method is predicted to have wide clinical detection application prospects for felines.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a cat triple antibody detection test strip, a preparation method and application thereof.
Background
Feline calicivirus (Feline calicivirus, FCV) belongs to a member of the caliciviridae (Calicivirdae) genus of the water sore virus (Vesivirus), a highly pathogenic pathogen for most felines. FCV is widely prevalent in felines and is distributed worldwide, and it primarily infects cats, can infect adult cats and cause symptoms of hyperthermia, edema, head and limb ulcers, canker sores, jaundice, and the like, and presents a high mortality rate, and can infect felines wild animals including lions, tigers, leopards, and the like, causing significant hypochondriac health to cats and rare wild animals.
Feline parvovirus disease, also known as feline pandemic or feline panleukopenia, feline infectious enteritis, is an acute, highly contagious infectious disease in cats, one of the major diseases that endanger felines. Feline parvovirus (Feline parvovirus, FPV), also known as feline pestivirus, feline infectious enteritis virus, feline panleukopenia virus, naturally causes the onset of a variety of animals in the felines, raccoons, and weasel families, such as lions, tigers, leopards, cats, lynx, minks, raccoons, mice, and the like. The clinical manifestations are mainly characterized by sudden fever, refractory vomiting, diarrhea, severe dehydration and rapid decrease of leukocytes. Cats that are not immunized or failed are most susceptible to cat fever, especially young cats of 3-5 months of age, and often present endemic epidemics.
Feline herpesvirus type1 (Feline herpesvirus type, FHV 1) belongs to the herpesviridae, subfamily a herpesvirus, which is the causative agent of feline infectious rhinotracheitis. FHV1 can cause acute and high-contact upper respiratory tract infection of cats, has high incidence, and although most adult cats are healed after infection, the death rate is low, the symptoms of young cats are serious after infection, the death rate can reach 50%, even the life-long poison is discharged, and the cat can be vertically transmitted and has great harm.
At present, clinically, the three types of virus cats have higher infection risk, lower cure rate and higher cure cost after infection, and are mainly prevented by injection vaccines. Cat triple as one of the most common vaccination projects at present generally refers to a three-in-one vaccine which can simultaneously prevent feline herpesvirus, feline calicivirus and feline panleukopenia virus, i.e. a vaccine which can combat three viruses. The corresponding vaccine products on the market are currently mainly Miaosan Duo (inactivated vaccine) and Yingtei (attenuated vaccine) by using subcutaneous injection. 1ml (containing 1 part) of each cat is first immunized by 8 weeks or more, and 1 part is boosted after 3-4 weeks. Cats vaccinated below 12 weeks of age should be boosted with 1 serving at 12-16 weeks of age, with 1 serving repeated each year.
In the vaccination process, the vaccination interval time is too short or too long due to improper immunization procedures, and the effect is reduced; vaccine damage, which may be caused by mistransportation or by manual mispreservation; the problem of the cat itself is that if some of the cats have a defect in their own immune system, normal immune response to the vaccine cannot be generated, and the effectiveness of the injected vaccine is difficult to ensure when the vaccine is injected into the cat. Thus, effective antibody detection in time is necessary for vaccinated cats. And the immune effect evaluation can be carried out on cats after vaccination by detecting the antibody level regularly, so that the problem of excessive immunity is avoided, and scientific immunity is realized.
The multi-linked antibody detection technology is mostly used for immune effect evaluation or routine physical examination scene after multi-linked vaccination. The conventional antibody detection reagent cards are mostly used for single-card single-item detection, one card is used for each item, and mixed samples are required to be added once for each item to be tested; at present, single-card multi-item parallel detection exists in the market, and the combined card can mix samples and perform multi-item test 2-3 times, and each item is added with a sample once. Compared with the conventional antibody detection reagent card, the method has the advantages that the operation steps are simplified correspondingly, the sample size is reduced, and the sample adding step is simplified and the cost is reduced.
Disclosure of Invention
Based on the above, one of the purposes of the present invention is to provide a test strip for detecting a cat triplet antibody with good detection performance.
The method comprises the following technical scheme:
the cat triplet antibody detection test paper strip is characterized by comprising a mark binding pad, an analysis film and a water absorption pad which are sequentially connected according to the flowing direction of a sample; the marking combination pad is treated by marking pad working solution, and the marking pad working solution comprises a fluorescent microsphere marked detection antibody and a fluorescent microsphere marked internal reference antibody; the detection antibody is a rabbit anti-cat IgG polyclonal antibody or a sheep anti-cat IgG polyclonal antibody; the analytical membrane is sequentially provided with a quality control line, a first detection line, a second detection line and a third detection line along the same direction as the sample flow; the quality control line is coated with an internal reference quality control antibody which is specifically combined with the internal reference antibody marked by the fluorescent microsphere; the first detection line is coated with a specific first antigen which is matched and combined with the detection antibody, the second detection line is coated with a specific second antigen which is matched and combined with the detection antibody, and the third detection line is coated with a specific third antigen which is matched and combined with the detection antibody; the first antigen of the first detection line, the second antigen of the second detection line and the third antigen of the third detection line are respectively selected from cat plague antigen, cat herpes antigen and cat cup antigen.
The invention also aims to provide the application of the cat triplet antibody detection test strip in cat antibody detection.
The invention also aims to provide an application of the cat triplet antibody detection test strip in detection of feline herpesvirus, feline calicivirus and/or feline pestivirus antibodies.
The invention further provides a cat triplet antibody detection reagent card, which comprises a shell and the cat triplet antibody detection test strip assembled in the shell, wherein the shell is formed by buckling an upper shell and a lower shell, the upper shell is provided with a sample adding hole and a display window, the sample adding hole corresponds to a combination pad of the cat triplet antibody detection test strip, and the display window corresponds to a quality control line, a first detection line, a second detection line and a third detection line of the cat triplet antibody detection test strip.
The invention also provides a cat triple antibody detection kit, which comprises the cat triple antibody detection kit.
The invention also aims at providing a cat antibody detection method.
The method comprises the following technical scheme:
obtaining a biological sample;
adding a biological sample into a sample diluent for dilution, wherein the sample diluent comprises 0.01-0.04mM Tris-HCl, 0.8-2.2% S9 and 0.8-1.0% NaCl;
and detecting the diluted and uniformly mixed sample by adopting the cat triplet antibody detection test strip or the cat triplet antibody detection reagent card: after reacting for 5min-20min at room temperature, the fluorescence signal intensity is detected.
The inventor of the invention is based on the intensive study of veterinary multi-vaccine and antibody immunochromatography, and invents a test strip capable of simultaneously detecting feline herpesvirus, feline calicivirus and/or feline pestivirus antibodies, and combines the inventor to find out that the study experience of the test strip is combined, and the invention combines the study of the use proportion of the fluorescent microsphere marked rabbit anti-cat IgG polyclonal antibody and the internal reference goat anti-chicken antibody by specifically adjusting the quality control line of the test strip and the position sequence of three detection lines of the items to be detected. The detection test strip is prepared when the quality control line of the test strip is arranged in front of the detection lines of three items, the first detection line is a cat pestivirus antibody detection item, the content of the rabbit anti-cat IgG polyclonal antibody marked by the fluorescent microsphere on the marked binding pad is 15% -25% by mass percent, interference among detection lines of different items can be reduced, good detection performance of the test strip can be guaranteed, and meanwhile, the detection linear range is better.
Compared with the conventional cat antibody detection test strip, the test strip provided by the invention can obtain 3 project detection results by single card, single sample mixing and single sample adding, has excellent detection stability and consistency while effectively reducing the detection cost, has good detection performance, enables the detection of the cat antibody to be simpler, and is suitable for various detection scenes. The method is predicted to have wide clinical detection application prospects for felines.
Drawings
FIG. 1 is a side view of a test strip structure of the present invention, wherein 1 is a bottom plate, 2 is a bonding pad, 3 is an analytical membrane, and 4 is absorbent paper.
Fig. 2 is a schematic diagram of positions of a detection line and a quality control line in a test strip according to the present invention, wherein 5 is a quality control line (C line), 6 is a first detection line, 7 is a second detection line, and 8 is a third detection line.
FIG. 3 is a standard curve of the test strip of the invention in example 1 for the detection of the Magnaporthe grisea antibody project.
FIG. 4 is a standard curve of the test strip of the present invention for the detection of feline herpesvirus antibody project in example 1.
FIG. 5 is a standard curve of the test strip of the present invention for detecting feline calicivirus antibody project in example 1.
FIG. 6 is a graph showing the results of analysis of the effect of the ratio of the use of the fluorescent microsphere conjugate for the rabbit anti-feline IgG polyclonal antibody on the detection of feline pestivirus antibody project in example 2.
FIG. 7 is a graph showing the results of analysis of the effect of the ratio of the use of the fluorescent microsphere conjugate of the rabbit anti-feline IgG polyclonal antibody on the detection of feline herpesvirus antibody items in example 2.
FIG. 8 is a graph showing the results of analysis of the effect of the ratio of the use of the fluorescent microsphere conjugate of the rabbit anti-feline IgG polyclonal antibody on the detection of feline calicivirus antibody in example 2.
FIG. 9 is a graph showing the results of analysis of the effect of different positions in the Magnaporthe grisea detection line treatment strip on the detection of Magnaporthe grisea antibody project in example 2.
Detailed Description
The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Throughout the specification and claims, the following terms have the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used in the present invention does not necessarily refer to the same embodiment, although it may. Furthermore, the phrase "in another embodiment" as used in the present invention does not necessarily refer to a different embodiment, although it may. Accordingly, as described below, various embodiments of the present invention may be readily combined without departing from the scope or spirit of the present invention.
Furthermore, as used herein, the term "or" is an inclusive "or" symbol and is equivalent to the term "and/or" unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout the specification, the meaning of "a", "an", and "the" include plural referents. The meaning of "in" is included "in" and "on".
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The present invention will be described in further detail with reference to specific examples.
Some embodiments of the invention provide a cat triplet antibody test strip comprising a label binding pad, an analytical membrane and a water absorbing pad connected in sequence according to the sample flow direction; the marking combination pad is treated by marking pad working solution, and the marking pad working solution comprises a fluorescent microsphere marked detection antibody and a fluorescent microsphere marked internal reference antibody; the detection antibody is a rabbit anti-cat IgG polyclonal antibody or a sheep anti-cat IgG polyclonal antibody;
the analysis membrane comprises a quality control line and a detection line, wherein the quality control line is close to the mark combination pad, and the detection line is close to the absorbent paper;
the analytical membrane is sequentially provided with a quality control line, a first detection line, a second detection line and a third detection line along the same direction as the sample flow;
the quality control line is coated with an internal reference quality control antibody which is specifically combined with the internal reference antibody marked by the fluorescent microsphere;
the first detection line is coated with a specific first antigen which is matched and combined with the detection antibody, the second detection line is coated with a specific second antigen which is matched and combined with the detection antibody, and the third detection line is coated with a specific third antigen which is matched and combined with the detection antibody; the first antigen of the first detection line, the second antigen of the second detection line and the third antigen of the third detection line are respectively selected from cat plague antigen, cat herpes antigen and cat cup antigen.
In some embodiments, the feline triple antibody test strip detects feline pestivirus, feline herpesvirus, and feline calicivirus coated on the line, and the feline calicivirus, and the feline herpesvirus, and the feline calicivirus are preferably synthetic virus antigens, so that the infectivity is reduced, and the detection safety is improved.
In some embodiments, the first antigen coated on the detection line of the cat triple antibody detection test strip is a cat plague antigen, the second antigen is a cat herpes antigen, and the third antigen is a cat cup antigen, so that the detection performance of the cat triple antibody detection test strip is the best.
In some embodiments, the working solution of the marking pad is sprayed on the binding pad according to the ratio of 0.1-0.17 μl/2.8cm×1cm, and the content of the fluorescent microsphere marked detection antibody in the working solution of the marking pad is 10% -30% by mass and volume percent.
In some embodiments, the working solution of the labeling pad contains 15% -25% by mass and volume of fluorescent microsphere labeled detection antibody, and further, the detection antibody is rabbit anti-cat IgG polyclonal antibody.
In some embodiments, the antigen or antibody coated in the quality control line, the first detection line, the second detection line and the third detection line is diluted by an analysis membrane buffer solution, and further, the analysis membrane buffer solution comprises 2% -4% of methanol, 0.05% -0.15% of BSA and 10-15mM of phosphate buffer solution with the following mass percentage contents; it is further preferable that the analytical membrane buffer is a phosphate buffer comprising 2.5% -3.5% methanol, 0.08% -0.12% BSA and 12-14 mM; more preferably, the assay membrane buffer is a phosphate buffer containing 3% methanol, 0.1% BSA and 13mM by mass. The analytical membrane buffer can provide proper isoelectric points for the coating proteins in the test strip, has strong hydrophobic effect, and can stably coat antibodies on an analytical membrane.
In some embodiments, the reference antibody is selected from any one of chicken IgY, sheep anti-chicken IgY, biotin, avidin, rabbit IgG, sheep anti-rabbit IgG.
In some embodiments, the reference antibody is a goat anti-chicken IgY antibody, and the quality control line comprises a chicken IgY antibody specifically combined with the goat anti-chicken IgY antibody.
In some embodiments, the marking pad working solution further comprises, by mass, 10% -30% of trehalose, 1% -2% of casein sodium salt, 0.5% -1.5% of BSA and 0.005% -0.035M of PBS; it is further preferable that the labeled antibody working solution further comprises 15-25% trehalose, 1.2-1.8% casein sodium salt, 0.7-1.3% BSA and 0.008-0.03M PBS by mass percent; more preferably, the labeled antibody working solution further comprises 20% trehalose, 1.5% casein sodium salt, 1% bsa and 0.02M PBS by mass.
In some embodiments, the concentration of the fluorescent microsphere-labeled rabbit anti-cat IgG polyclonal antibody in the labeled antibody working solution is 0.2mg/ml-1.0mg/ml, and the concentration of the fluorescent microsphere-labeled goat anti-chicken IgY antibody is 0.05mg/ml-0.15mg/ml.
In some embodiments, the concentration of the fluorescent microsphere-labeled rabbit anti-cat IgG polyclonal antibody in the labeled antibody working solution is 1mg/ml, and the concentration of the fluorescent microsphere-labeled goat anti-chicken IgY antibody is 0.1mg/ml.
In some embodiments, the concentration of the cat litter antigen coated on the first detection line is 0.5mg/ml to 1.5mg/ml, and the cat litter antigen is sprayed on the analysis film according to 0.5 μl to 1.5 μl/cm; the concentration of the cat herpesantigen coated on the second detection line is 0.5mg/ml-1.5mg/ml, and the cat herpesantigen is sprayed on the analysis film according to 0.5 mu l-1.5 mu l/cm; the concentration of the cat cup antigen coated on the third detection line is 0.5mg/ml-1.5mg/ml, and the cat cup antigen is sprayed on the analysis film according to 0.5 mu l-1.5 mu l/cm; the concentration of the chicken IgY antibody coated on the quality control line is 0.25mg/ml-0.75mg/ml, and the chicken IgY antibody is sprayed on an analysis film according to 0.5 mu l-1.5 mu l/cm.
In some embodiments, the concentration of the cat-plague antigen coated on the first detection line is preferably 1.0mg/ml, the concentration of the cat-herpes antigen coated on the second detection line is preferably 1.0mg/ml, the concentration of the cat-cup antigen coated on the third detection line is preferably 1.0mg/ml, and the concentration of the chicken IgY antibody coated on the quality control line is preferably 0.5mg/ml.
In some embodiments, the detection line and the quality control line are each preferably sprayed onto the analytical membrane at 1 μl/cm.
In some embodiments, the interval between the quality control line and the first detection line is 0.3cm to 0.5cm, and the interval between adjacent detection lines is 0.3cm to 0.5cm; further preferably, the distance between the quality control line and the first detection line is 0.4cm, and the distance between adjacent detection lines is 0.4cm.
Some embodiments of the invention provide for the use of the above-described feline triplet antibody test strip in the detection of feline antibodies.
Some embodiments of the invention provide for the use of the above-described feline triplet antibody test strip in the detection of feline herpesvirus, feline calicivirus and/or feline pestivirus antibodies.
Some embodiments of the invention provide a cat triplet antibody detection reagent card, which comprises a shell and the cat triplet antibody detection test strip assembled in the shell, wherein the card shell is formed by buckling an upper shell and a lower shell, the upper shell is provided with a sample adding hole and a display window, the sample adding hole corresponds to a combination pad of the cat triplet antibody detection test strip, and the display window corresponds to a quality control line, a first detection line, a second detection line and a third detection line of the cat triplet antibody detection test strip.
Some embodiments of the invention provide a cat triple antibody detection kit comprising the triple antibody detection kit described above.
In some embodiments, the above kit further comprises a sample diluent comprising 0.01-0.04mM Tris-HCl,0.8% -2.2% S9,0.8% -1.0% NaCl; further preferably, 0.01-0.02mM Tris-HCl,1% -2% S9,0.9% NaCl.
Some embodiments of the present invention provide a method for detecting a cat antibody, comprising the steps of:
obtaining a biological sample;
adding a biological sample into a sample diluent for dilution, wherein the sample diluent comprises 0.01-0.04mM Tris-HCl, 0.8-2.2% S9 and 0.8-1.0% NaCl;
the diluted and mixed sample is detected by adopting the cat triplet antibody detection test strip according to any one of claims 1 to 10 or the cat triplet antibody detection reagent card according to claim 13:
and taking out the cat triple antibody detection reagent card, adding the diluted and mixed sample into a sample adding hole of the detection card, reacting for 5-20 min at room temperature, and detecting the fluorescence signal intensity.
In some embodiments, the biological sample is selected from whole blood, serum, and plasma.
In some embodiments, the dilution volume ratio of the sample diluent to the biological sample is (1-50): 1, more preferably (20 to 40): 1, a step of; more preferably 30:1.
In some of these embodiments, the reaction time is 7min to 15min, more preferably 10min.
Example 1 preparation of cat triple antibody fluorescence immunochromatography detection card
The cat triple antibody immunochromatography test paper is formed by mutually lapping a base plate (such as number 1 in figure 1) with an adhesive through a coupling protein which is a rabbit anti-cat IgG polyclonal antibody 1#, a goat anti-chicken (GAC) antibody, a label bonding pad (such as number 2 in figure 1) with a fluorescent microsphere as a luminescent substance, an analysis membrane (such as number 3 in figure 1) with three detection T lines (such as number 6,7 and 8 in figure 2) and a quality control C line (such as number 5 in figure 2) sequentially, and a water absorbing paper (such as number 4 in figure 1), wherein the label bonding pad is a glass cellulose membrane, the analysis membrane is a nitrocellulose membrane (NC membrane), and the base plate is a PVC plate (such as shown in figure 1 and number 1) with the adhesive.
(1) Preparation of the label conjugate pad:
carboxyl modified fluorescent microsphere (Siemens F8811, using concentration is 10%), EDC of 5.5mg/mL and NHS of 5.5mg/mL are added to react for 30min at room temperature, rabbit anti-cat IgG polyclonal antibody 1# (Siemens A18879) of 1.0mg/mL is added, shaking is carried out at room temperature for 2 hours, a fluorescent microsphere-rabbit anti-cat IgG polyclonal antibody 1# conjugate is formed, and finally pH7.4 containing 1% BSA and 0.02M PBS solution are used for storage at 4 ℃. Fluorescent microsphere-GAC conjugates were prepared using the same procedure, wherein goat anti-chicken IgY antibody (Boersi 0.1 mg/ml).
Preparation of marking pad working solution: the rabbit anti-cat IgG polyclonal antibody 1# fluorescent microsphere conjugate and the GAC fluorescent microsphere conjugate are respectively and uniformly mixed according to the proportion of 20 percent and 1 percent, and then are prepared into a working solution of the marking pad in 0.01M PBS solution containing 20 percent trehalose, 1.5 percent casein sodium salt and 1 percent BSA.
Spraying the three lines at a spray rate of 4 μl/cm, centering, and drying at 40-45deg.C for 12 hr to obtain label conjugate pad (shown in figure 1 and number 2).
(2) Preparation of analytical films containing T-line and C-line (as shown in FIG. 1, number 3):
cat fever antigen # 1 (eastcoastbrio LA 221), cat herpesantigen # 1 (VrioStaT 8950), cat calico antigen # 1 (eastcoastbrio LA 223), and GAC antibody (bolxi) were diluted to the desired coating concentrations, respectively, with analytical membrane buffers. Wherein the assay membrane buffer is 10-15mM phosphate buffer, pH7-7.5, comprising 0.1% BSA, 2% -4% methanol.
The spraying speed of the film drawing instrument is 1 mu L/cm, the T1 line is cat plague antigen 1#, the concentration is 1.0mg/mL, the T2 line is cat herpes antigen 1#, the concentration is 1.0mg/mL, the T3 line is cat cup antigen 1#, the concentration is 1.0mg/mL, the C line is GAC antibody, and the concentration is 0.5mg/mL. The position of TC line on the analysis membrane is shown in fig. 2, the quality control C line is respectively used as the first position (shown in fig. 2, the serial number 5), the cat plague antigen T line (T1 line) is used as the second position (shown in fig. 2, the serial number 6), the cat herpes antigen T line (T2) is used as the third position (shown in fig. 2, the serial number 7), and the cat cup antigen T line (T3) is used as the fourth position (shown in fig. 2, the serial number 8).
After the preparation, the NC film is dried for 12 hours at 40-45 ℃.
Preparation of analytical membrane buffer: 12mM pH7-7.5 phosphate buffer, 2.5% methanol, 0.1% BSA.
Assembling into a reagent strip: the dried label bonding pad (shown in figure 1 and with the number 2), NC film (shown in figure 1 and with the number 3) and absorbent paper (shown in figure 1 and with the number 4) are sequentially stuck and assembled, a test strip with the width of 4mm is cut by a strip cutting machine, and a test strip card is arranged, so that the detection card is prepared.
Detection principle: the specific combination of the feline fever IgG antibody/feline herpes IgG antibody/feline calicivirus IgG antibody in the sample and the rabbit anti-feline IgG polyclonal antibody coupled with fluorescent microspheres on a marked binding pad forms a fluorescent microsphere-rabbit anti-feline IgG polyclonal antibody-feline fever IgG antibody/feline herpes IgG antibody/feline calicivirus IgG antibody complex on the binding pad. Under the capillary force of NC film, GAC fluorescent microsphere conjugate flows to the end of water absorption paper, when chromatography reaches C line, it is captured by chicken IgY antibody coated by C line, forming fluorescent microsphere-GAC antibody-chicken IgY antibody complex fixed at C line of NC film; the fluorescent microsphere-rabbit anti-cat IgG polyclonal antibody-cat fever IgG antibody complex flows to the end of the absorbent paper, is captured by the cat fever antigen coated by the T1 line when the chromatography reaches the T1 line, and is fixed at the T1 line of the NC membrane by an antigen-antibody sandwich method; the fluorescent microsphere-rabbit anti-cat IgG polyclonal antibody-cat herpesIgG antibody complex flows to the end of the absorbent paper, is captured by the cat herpesantigen coated by the T2 line when the antibody is chromatographed to the T2 line, and is fixed at the T2 line of the NC membrane by an antigen-antibody sandwich method; the fluorescent microsphere-rabbit anti-cat IgG polyclonal antibody-cat calicivirus antibody complex flows to the end of the absorbent paper, is captured by the cat calicivirus antigen coated by the T3 line when chromatography reaches the T3 line, is immobilized at the T3 line of the NC membrane by the "antigen-antibody sandwich method", and the more cat plague/cat herpes/cat calicivirus IgG antibodies in the sample are captured. Fluorescent microsphere conjugates not captured by the C line or the T line directly pass through the detection line and reach the end of the absorbent paper.
The detection method of the cat triple antibody fluorescence immunochromatography detection card prepared by the preparation method is specifically as follows:
establishment of a Standard Curve
The feline fever, feline herpes, feline calicivirus IgG antibody reference (feline pestivirus IgG monoclonal antibody: eastCoast Bio HM790; the cat herpesvirus IgG antibodies EastCoast Bio HM550, cat calicivirus IgG antibodies EastCoast Bio HM 515) were formulated into working references of gradient concentration (wherein cat pestivirus IgG antibody concentrations were 0U, 4.0U, 8.3U, 12.8U, 22.3U, 36.5U, 63.0U, 156.0U, 220.0U, cat herpesvirus IgG antibody concentrations were 0.0U, 8.0U, 16.5U, 26.3U, 63.0U, 80.0U, 99.0U, 153.0U, 198.0U, 260.0U, cat calicivirus IgG antibody concentrations were 0.0U, 3.2U, 6.8U, 9.5U, 17.0U, 29.0U, 39.0U, 78.0U, 115.0U, 200.0, and the cat herpesvirus IgG antibody concentrations were measured as the values of the T-cell signal line, and the T-cell signal line was plotted as the T-cell signal line and the T-cell signal line was plotted as the fluorescence signal line and the T-cell signal line plotted as the values.
TABLE 1-1
The data of the fluorescence detection values obtained in the above table 1-1 are processed, the T/C value is taken as an ordinate, the concentration value is taken as an abscissa, and the data are fitted as standard curves of the kit aiming at different projects, and the standard curves are specifically shown in fig. 3-5, wherein the fitted curve function aiming at the detection of the IgG antibody of the Magnaporthe grisea: y=6e-07 x 3 -0.0003x 2 +0.067x+0.4656; fitted curve function for feline herpesvirus IgG antibody detection: y=6e—10x 4 -5E-07x 3 +0.0001x 2 +0.0105x+0.4223; fitted curve function for feline calicivirus IgG antibody detection: y= -7E-09x 4 +4E-06x 3 -0.0007x 2 +0.0684x+0.3146。
The actual sample detection method to be detected specifically comprises the following steps:
taking out the detection card, using a pipette and a corresponding suction head in the kit to pipette 10 mu L of serum samples (or whole blood and plasma samples) to be detected, adding the serum samples into a centrifuge tube which is respectively filled with 300 mu L of sample diluent (0.01-0.02 mM Tris-HCL,1% -2% S9 and 0.9% NaCl), and uniformly mixing the serum samples with the whole blood and the plasma samples for 5-10 times. And then adding 75 mu L of the uniformly mixed sample into the sample adding hole, reacting for 10min at room temperature, and detecting the fluorescence signal intensity of the C line and the T1, T2 and T3 line areas by using a fluorescence quantitative detection analyzer.
Sample results: and detecting according to the detection method, substituting the fluorescent signal value measured by the fluorescent quantitative analyzer into the established standard curve, and obtaining a corresponding sample concentration value. The fluorescence signal values at different T lines are positively correlated with the content of the corresponding to-be-detected object in the sample.
The determination criteria of the detection results are shown in the following tables 1-2:
TABLE 1-2
Example 2 optimization of preparation of cat triple antibody fluorescence immunochromatography detection card
(1) Rabbit anti-cat IgG polyclonal antibody 1# fluorescent microsphere conjugate use ratio
The rabbit anti-cat IgG polyclonal antibody marked by the fluorescent microsphere in the triplet antibody detection test strip is a marked antibody shared by three detection items, so that excessive addition is needed, but the actual situation is not considered to be as much as possible. In order to study the influence of the usage amount of the fluorescent microsphere marked rabbit anti-cat IgG polyclonal antibody on the detection effect of the test strip, 3 marking pad working solutions are respectively prepared according to the following proportion: wherein the mass and volume percentage content of the rabbit anti-cat IgG polyclonal antibody marked by the fluorescent microsphere is 15%,20% and 25% respectively. Other components of the working fluid for the marking pad were composed, and the test strips were prepared in the same manner as described in example 1.
The 3 prepared test cards were used to test 5 calibrator samples of different concentrations of feline fever, feline herpes, feline calicivirus IgG antibodies, respectively, with reference to the test method described in example 1. The detection results are shown in the following tables 2-1 and FIGS. 6-8.
TABLE 2-1
As can be seen from the above table and fig. 6 to 8, when the addition ratio of the fluorescent microsphere-labeled rabbit anti-cat IgG polyclonal antibody is 20%, the gradient of the test calibrator is better than 15% to meet the performance requirement, the gradient of the test calibrator is better, and the gradient of the 25% ratio test calibrator is not significantly improved, so that 20% is selected.
(2) Position comparison of quality control lines
In conventional chromatographic test paper card detection systems, the calibration zone is typically located in the downstream region of the sample flow direction, i.e., the quality control line is near the absorbent paper end of the detection card. Since the invention relates to a plurality of detection items, in order to explore a method for improving detection performance, detection lines are respectively arranged near the end of the water absorption paper and the end of the marking pad, and the preparation methods of test strips are consistent and refer to the embodiment 1.
Using the 2 kinds of test cards prepared above, 6 samples (clinically known samples obtained from a certain pet hospital in Guangzhou city, which had been subjected to qualitative detection) were each tested by referring to the test method described in example 1. The results of the detection are shown in the following tables 2-2.
TABLE 2-2
According to the detection results of the table, the detection results of the cat triple antibody fluorescence immunochromatography detection card and the clinical significance comparison analysis of the samples after Baiaogao detection of the israel inlet antibody detection reagent show that when the quality control line is the first, namely is close to the end of the marking pad, the interference of the detection line caused by detection of different projects can be reduced, so that the effectiveness and consistency of the detection results are effectively improved.
(3) Comparison of the position of the detection lines
In order to study the influence of the sequence of the positions of the detection lines of different detection items in the triple antibody detection test strip on the detection effect of the test strip, the detection of the Magnaporthe grisea antibody is taken as an example, the positions of the detection lines of the Magnaporthe grisea antibody are respectively set to be T1, T2 and T3, and the preparation methods of the test strips are consistent and refer to the embodiment 1.
The 3 prepared detection cards are used for respectively detecting 5 calibrator samples with different concentrations of the feline pestivirus IgG antibody according to the detection method described in the example 1. The results of the detection are shown in tables 2 to 3 below and FIG. 9.
Tables 2 to 3
The influence experiments of the positions of the detection lines of the detection items of the feline herpes and feline calicivirus IgG antibodies are consistent with the above, and the influence of different positions on the feline pestivirus antibodies in the three items is greatly influenced by the combination of the experimental results, so that the influence of different positions on the detection results of the feline herpes and feline calicivirus IgG antibodies is less.
Furthermore, as can be seen from the above table and fig. 9, when the position of the cat pestivirus antibody detection line is the T1 position, the linear range of the detection curve of the item is better, and the inventor is combined to further research the positions of the detection lines of the cat herpes and cat calicivirus IgG antibody detection items, so as to ensure that all 3 detection items have good performance, the front and rear positions of the three items mainly depend on the low-end gradient of a single item, the better the low-end gradient of the item is, and the more suitable and rear positions of the T line are, therefore, the optimal sequence of the position sequences of the detection lines of different detection items in the triple antibody test strip is that the T1-T3 are sequentially the cat pestivirus, the cat herpes and the cat calicivirus IgG antibody detection lines; namely, the detection lines T1-T3 are sequentially coated with the feline fever antigen, the feline herpesantigen and the feline calicivilin antigen.
Example 3 comparison of the tests for different sample types
In order to compare whether the detection results of the test strip are different for different sample types, the homologous serum, the plasma and the whole blood are tested simultaneously, the difference of the detection results of the three sample types is compared, 10 cases of homologous serum, plasma and whole blood samples are tested simultaneously by combining the test results of the following table 3-1, and the clinical test consistency is higher.
TABLE 3-1
Example 4 stability detection of cat triple antibody detection reagent card
The test strip is placed for 2 years at 25 ℃, the measured values of 3 months, 6 months, 12 months, 18 months and 24 months are observed in the placing period, and the detection results are shown in the following table 4-1, and the test strip is still stable after being placed for 24 months at room temperature, namely the test strip can be stored for 2 years at normal temperature.
TABLE 4-1
Example 5 comparison of test results of cat triple antibody detection reagent card and Single detection card
10 samples (acquired from a pet hospital in Guangzhou city) were tested for consistency between three project test results and a single test result of the cat triple antibody test reagent card of the invention, and the test results of the following table 5-1 were combined, and 10 serum samples were tested by using a single card (Mo Dekang cat plague antibody test paper card, mo Dekang cat herpesvirus antibody test paper card, mo Dekang cat cup virus antibody test paper card) and a combined card (cat triple antibody test reagent card of the invention) at the same time, and the consistency of clinical test values was higher. The cat triple antibody detection reagent card has good detection performance.
TABLE 5-1
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The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (12)
1. The cat triplet antibody detection test paper strip is characterized by comprising a mark binding pad, an analysis film and a water absorption pad which are sequentially connected according to the flowing direction of a sample;
the marking combination pad is treated by marking pad working solution, and the marking pad working solution comprises a fluorescent microsphere marked detection antibody and a fluorescent microsphere marked internal reference antibody; the detection antibody is a rabbit anti-cat IgG polyclonal antibody or a sheep anti-cat IgG polyclonal antibody;
the analytical membrane is sequentially provided with a quality control line, a first detection line, a second detection line and a third detection line along the same direction as the sample flow;
the quality control line is coated with an internal reference quality control antibody which is specifically combined with the internal reference antibody marked by the fluorescent microsphere;
the first detection line is coated with a specific first antigen which is matched and combined with the detection antibody, the second detection line is coated with a specific second antigen which is matched and combined with the detection antibody, and the third detection line is coated with a specific third antigen which is matched and combined with the detection antibody; the first antigen of the first detection line, the second antigen of the second detection line and the third antigen of the third detection line are respectively selected from cat plague antigen, cat herpes antigen and cat cup antigen;
the marking pad working solution is sprayed onto the bonding pad according to the ratio of 0.1-0.17 mu l/2.8cm multiplied by 1cm, and the content of the detection antibody marked by the fluorescent microsphere in the marking pad working solution is 10% -30% by mass and volume percent; the working solution of the marking pad contains 15-25% of fluorescent microsphere marked detection antibody by mass and volume percent;
the antigen or antibody coated in the quality control line, the first detection line, the second detection line and the third detection line is diluted by an analysis membrane buffer solution, wherein the analysis membrane buffer solution comprises 2% -4% of methanol, 0.05% -0.15% of BSA and 10-15mM of phosphoric acid buffer solution in percentage by mass.
2. The test strip of claim 1, wherein the detection antibody is a rabbit anti-cat IgG polyclonal antibody.
3. The test strip for detecting a triplet antibody according to claim 1, wherein the reference antibody is selected from any one of chicken IgY, sheep anti-chicken IgY, biotin, avidin, rabbit IgG, sheep anti-rabbit IgG.
4. The test strip of claim 3, wherein the reference antibody is a goat anti-chicken IgY antibody, and the quality control line comprises a chicken IgY antibody specifically bound to the goat anti-chicken IgY antibody.
5. The triplet antibody test strip according to any one of claims 1 to 4, wherein the marking pad working liquid further comprises 10% -30% trehalose, 1% -2% casein sodium salt, 0.5% -1.5% bsa and 0.005% -0.035 m PBS by mass.
6. The test strip for detecting a triplet antibody according to claim 5, wherein the concentration of the fluorescent microsphere-labeled rabbit anti-cat IgG polyclonal antibody in the working solution of the labeling pad is 0.2mg/ml to 1.0mg/ml, and the concentration of the fluorescent microsphere-labeled goat anti-chicken IgY antibody is 0.05mg/ml to 0.15mg/ml.
7. The triplet antibody test strip according to claim 6, wherein the concentration of the cat fever antigen coated on the first detection line is 0.5mg/ml to 1.5mg/ml, and the cat fever antigen is sprayed on the analysis film according to 0.5 μl to 1.5 μl/cm;
the concentration of the cat herpesantigen coated on the second detection line is 0.5mg/ml-1.5mg/ml, and the cat herpesantigen is sprayed on the analysis film according to 0.5 mu l-1.5 mu l/cm;
the concentration of the cat cup antigen coated on the third detection line is 0.5mg/ml-1.5mg/ml, and the cat cup antigen is sprayed on the analysis film according to 0.5 mu l-1.5 mu l/cm;
the concentration of the chicken IgY antibody coated on the quality control line is 0.25mg/ml-0.75mg/ml, and the chicken IgY antibody is sprayed on an analysis film according to 0.5 mu l-1.5 mu l/cm.
8. Use of the feline triplet antibody test strip of any one of claims 1-7 for detection of a feline antibody for non-disease diagnostic purposes.
9. Use of the test strip for detection of feline triplet antibody according to any one of claims 1 to 7 for the detection of feline herpesvirus, feline calicivirus and/or feline pestivirus antibodies for non-disease diagnostic purposes.
10. The cat trigeminy antibody detection reagent card is characterized by comprising a shell and the cat trigeminy antibody detection test strip according to any one of claims 1-7 assembled in the shell, wherein the shell is formed by buckling an upper shell and a lower shell, the upper shell is provided with a sample adding hole and a display window, the sample adding hole corresponds to a combination pad of the cat trigeminy antibody detection test strip, and the display window corresponds to a quality control line, a first detection line, a second detection line and a third detection line of the cat trigeminy antibody detection test strip.
11. A kit for detecting a cat triple antibody, comprising the cat triple antibody detection kit of claim 10.
12. The cat triplex antibody test kit according to claim 11, further comprising a sample diluent comprising 0.01-0.04mM Tris-HCL,0.8% -2.2% S9,0.8% -1.0% NaCl.
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| CN114634564B (en) * | 2022-04-18 | 2023-04-04 | 北京华驰千盛生物科技有限公司 | Triple egg yolk antibody for cat, preparation method and application |
| CN114839378B (en) * | 2022-07-04 | 2022-09-06 | 山东康华生物医疗科技股份有限公司 | Kit for joint detection of feline herpes and feline calicivirus |
| CN116183915A (en) * | 2022-12-27 | 2023-05-30 | 深圳粒影生物科技有限公司 | Triple antibody detection test strip for feline panleukopenia virus, feline herpesvirus and feline calicivirus and application thereof |
| CN116735866A (en) * | 2023-02-01 | 2023-09-12 | 深圳粒影生物科技有限公司 | Test strip for triple detection of feline panleukopenia virus, feline herpesvirus and feline calicivirus antigen and preparation method thereof |
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