A canine vector-borne disease five-in-one antibody test card and its preparation method and kit

Abstract

The present invention relates to the technical field of antibody detection, and discloses a canine vector-borne disease five-in-one antibody detection card and a preparation method and a kit thereof. The detection card includes the following a) to e) five detection antigens: a) Leishmania antigen: a fusion protein of rk39, rk26 and rk9; b) Babesia antigen: a fusion protein of TRAP and rBcMSA1 / rBcSA1; c) Anaplasma antigen: a fusion protein of P44 and OMP‑1X; d) Ehrlichia antigen: a gp19 protein; e) Lyme antigen: an outer membrane protein of Borrelia burgdorferi OspC. The detection card and kit of the present invention can realize the combined detection of IgG and IgM antibodies corresponding to canine ehrlichiosis, canine anaplasmosis, canine babesiosis, canine Lyme disease and canine leishmaniasis, and have high detection sensitivity and specificity.

Description

Technical Field

[0001] The present invention relates to the field of antibody detection technology, and in particular to a canine vector-borne disease five-in-one antibody detection card and a preparation method and a kit thereof. Background Technology

[0002] Insect-borne diseases are a type of infectious disease that is transmitted by arthropods. There are many species of insect-borne organisms with complex distribution, including mosquito-borne, tick-borne, mite-borne, louse-borne and flea-borne, which are transmitted to animals and human hosts through bites. Among them, the main diseases that seriously affect the health of dogs include Ehrlichia canis, Anaplasmosis (ANA), Babesia, Lyme disease and Leishmaniasis (LSH). Insect-borne diseases are highly contagious and destructive, especially these insects have strong adaptability and survival ability, usually hiding in hidden places such as grass and bushes. When pet dogs inadvertently pass through these areas, insects will quickly attach to their bodies, suck blood and spread diseases.

[0003] Most dogs that are bitten often do not show obvious symptoms in the early stage, and the symptoms will gradually appear after a few weeks or even months. In addition, due to the wide variety of insect-borne organisms, the distribution of insect-borne infectious diseases is diverse and complex, and the long-distance transmission of insect-borne infectious diseases is becoming more and more common, and the clinical symptoms are also very similar. Therefore, during the high-incidence season of insect-borne diseases, for physical abnormalities shown by dogs, highly specific and sensitive canine insect-borne disease IgG and IgM antibody test cards can be used for preliminary screening to determine whether they are currently or have been infected, which will help veterinarians to treat them symptomatically.

[0004] However, the combined detection of IgG and IgM antibodies for multiple vector-borne diseases requires that the different vector-borne disease antigens used have good binding properties with the corresponding IgG and IgM to achieve high-sensitivity detection, and that the binding between each antigen and the corresponding IgG and IgM has high specificity to avoid mutual interference between the detection of different vector-borne disease IgG and IgM antibodies, thus having a high degree of design difficulty. At present, there has been no report on the combined detection of IgG and IgM for the five common vector-borne diseases (ehrlichiosis, anaplasmosis, babesiosis, Lyme disease, and leishmaniasis) in dogs. SUMMARY OF THE INVENTION

[0005] In order to solve the above technical problems, the present invention provides a canine vector-borne disease five-in-one antibody detection card and its preparation method and kit. The detection card and kit can realize the combined detection of IgG and IgM antibodies corresponding to canine ehrlichiosis, canine anaplasmosis, canine babesiosis, canine Lyme disease and canine leishmaniasis, and have high detection sensitivity and specificity.

[0006] The specific technical solution of the present invention is:

[0007] In the first aspect, the present invention provides a canine vector-borne disease five-in-one antibody test card, comprising the following a) to e) five detection antigens:

[0008] a) Leishmania antigen: a fusion protein of rk39, rk26 and rk9;

[0009] b) Babesia antigen: a fusion protein of TRAP and rBcMSA1 / rBcSA1;

[0010] c) Anaplasma antigen: a fusion protein of P44 and OMP-1X;

[0011] d) Ehrlichia antigen: gp19 protein;

[0012] e) Lyme antigen: OspC outer membrane protein of Borrelia burgdorferi.

[0013] In the present invention, the "rBcMSA1 / rBcSA1" mentioned here refers to the fusion protein of rBcMSA1 and rBcSA1.

[0014] In the detection of IgG and IgM antibodies corresponding to Leishmaniasis, when only rK39 protein is used, some dogs with canine Leishmaniasis in Brazil and India will be missed. The use of rk39, rk26 and rk9 fusion proteins can solve the above problems, improve the detection rate of Leishmaniasis, and reduce false negatives. The common subtypes of Babesia that infect dogs are small Babesia, namely Babesia gibsoni, and large Babesia, namely Babesia canis. In the detection of IgG and IgM antibodies corresponding to Babesiasis, the use of TRAP and rBcMSA1 / rBcSA1 fusion proteins can take into account the detection of the above two subtypes of Babesia and improve the detection rate of Babesia. Canine Anaplasma includes two subtypes: Anaplasma phogocytophilum and Anaplasma platys. In the detection of IgG and IgM antibodies corresponding to anaplasmosis, the fusion protein of P44 and OMP-1X can detect both subtypes of canine Anaplasma.

[0015] In addition, the design of the detection antigen will affect the detection sensitivity. Among the three fusion proteins used in the present invention, by selecting specific monomer proteins for coordination, the fusion protein can still have good binding performance with the corresponding canine vector-borne disease antibodies after being constructed, thereby achieving a higher detection sensitivity, which helps to improve the detection rate and reduce false negatives.

[0016] In addition, IgM antibodies are mainly produced in the early stage of infection, IgG antibodies are mainly produced in the relatively late stage after infection, and the specific differences in different regions of anti-IgG antibodies and anti-IgM antibodies are very large. The 5 detection antigens used in the present invention can take into account the detection of IgM and IgG antibodies of 5 canine vector-borne diseases, enabling both IgM and IgG to be detected, and having high detection sensitivity and specificity for IgM and IgG antibodies of each canine vector-borne disease. Therefore, it helps to achieve high detection accuracy.

[0017] Preferably, the five-in-one antibody detection card for canine vector-borne diseases includes 5 reagent strips; each of the reagent strips includes a bottom plate and a sample pad, a gold-labeled conjugate pad, a nitrocellulose membrane, and a water-absorbing pad stacked in sequence on the bottom plate; the gold-labeled conjugate pads of the 5 reagent strips are respectively coated with conjugates of the 5 detection antigens and colloidal gold, and detection lines coated with anti-canine IgG antibodies and anti-canine IgM antibodies are provided on the nitrocellulose membranes.

[0018] Preferably, 2 detection lines coated with anti-canine IgG antibodies and anti-canine IgM antibodies respectively are provided on the nitrocellulose membranes of the 5 reagent strips.

[0019] By setting the above 2 detection lines, the detection of IgG and IgM antibodies can be distinguished, so as to more intuitively judge whether a dog has been recently infected or has been infected before, facilitating pet doctors to quickly take targeted treatment and reducing the transmission risk.

[0020] Taking leishmaniasis (LSH) as an example, the detection principle of its test strip is as follows: The gold-labeled conjugate pad is coated with a conjugate of Leishmania antigen and colloidal gold (hereinafter simply referred to as "gold-labeled LSH antigen"), and the conjugate is purple-red. When the test sample contains the corresponding IgG or IgM antibody of LSH, the antibody can specifically bind to the gold-labeled LSH antigen on the gold-labeled conjugate pad to form a gold-labeled LSH antigen-LSH IgG or IgM antibody complex. Using the nitrocellulose membrane as a carrier, the above complex diffuses and moves through capillary chromatography. When the test sample contains the corresponding IgG antibody of LSH, the gold-labeled LSH antigen-LSH IgG antibody complex binds to the anti-canine IgG antibody coated on the nitrocellulose membrane, and a purple-red band is shown on its corresponding detection line, indicating that the LSH IgG antibody is positive; when the test sample does not contain the corresponding IgG antibody of LSH, the detection line coated with the anti-canine IgG antibody does not show color, indicating that the LSH IgG antibody is negative. Similarly, when the test sample contains the corresponding IgM antibody of LSH, a purple-red band is shown on the detection line coated with the anti-canine IgM antibody, indicating that the LSH IgM antibody is positive; when the test sample does not contain the corresponding IgM antibody of LSH, the detection line coated with the anti-canine IgM antibody does not show color, indicating that the LSH IgM antibody is negative.

[0021] The detection principles of ehrlichiosis, anaplasmosis, babesiosis and Lyme disease are the same as those of the above-mentioned leishmaniasis.

[0022] Furthermore, the anti-canine IgG antibody is a rabbit anti-canine IgG antibody and / or a mouse anti-canine IgG antibody; the anti-canine IgM antibody is a rabbit anti-canine IgM antibody and / or a mouse anti-canine IgM antibody.

[0023] Preferably, the gold-labeled conjugate pads of the 5 test strips are all coated with the conjugate of chicken IgY and colloidal gold, and a quality control line coated with anti-chicken IgY antibody is provided on the nitrocellulose membrane.

[0024] The Fc segment of the structure of chicken IgY does not interact with mammalian Fc receptors, complement, rheumatoid factor, protein A, protein G and protein L. The phylogenetic distance from canine secondary antibodies (such as rabbit anti-canine IgG / IgM, mouse anti-canine IgG / IgM) is greater, and there is almost no cross-reaction with IgG and IgM. Therefore, using chicken IgY as a quality control can avoid the significant interference caused by the binding between mammalian immunoglobulins and reduce the occurrence of false positives.

[0025] Furthermore, the anti-chicken IgY antibody is a goat anti-chicken IgY antibody.

[0026] Preferably, a blood filtration membrane is provided between the sample pad and the gold-labeled conjugate pad.

[0027] The blood filtration membrane can intercept red blood cells in the test sample, which helps to ensure a clean background on the membrane surface during the chromatography process and avoid the situation that the membrane surface of the test strip turns red due to canine red blood cells. Therefore, when the test sample is serum, plasma or whole blood, the results can be accurately interpreted.

[0028] In a second aspect, the present invention provides a method for preparing the five-in-one antibody test card for canine vector-borne diseases, comprising the following steps:

[0029] S1: Coating the gold-labeled solutions containing the conjugates of the 5 test antigens and colloidal gold onto five gold-labeled conjugate pads respectively, and drying;

[0030] S2: Assembling the gold-labeled conjugate pads processed in step S1 with the sample pad, nitrocellulose membrane and absorbent pad into test strips.

[0031] Preferably, in step S1, the preparation method of the gold-labeled solution comprises the following steps: adding sodium citrate aqueous solution to chloroauric acid aqueous solution, adjusting the pH value to 7.5-8.5, adding the 5 test antigens respectively, then adding bovine serum albumin (BSA) and polyethylene glycol (PEG) in sequence, fully reacting, centrifuging to collect the precipitate, and dispersing it in water.

[0032] Preferably, in step S1, in the gold-labeled solution, the conjugate of Leishmania antigen and colloidal gold is used at a final concentration of OD 540 = 3 - 5, the conjugate of Babesia antigen and colloidal gold is used at a final concentration of OD 540 = 60 - 65, the conjugate of Anaplasma antigen and colloidal gold is used at a final concentration of OD 540 = 2.5 - 5.0, the conjugate of Ehrlichia antigen and colloidal gold is used at a final concentration of OD 540 = 3 - 5, the conjugate of Lyme antigen and colloidal gold is used at a final concentration of OD 540 = 50 - 55.

[0033] Preferably, in step S1, the gold-labeled solution further contains the conjugate of chicken IgY and colloidal gold; in the gold-labeled solution, the conjugate of chicken IgY and colloidal gold is used at a final concentration of OD 540 = 1.2 - 25.

[0034] In the present invention, the "OD 540 " all refers to the absorbance value at an absorption wavelength of 540 nm.

[0035] Preferably, before step S2, a treatment solution is coated on the sample pad and then dried; the treatment solution includes components with the following concentrations: 8 - 10 g / L bovine serum albumin (BSA), 10 - 12 g / L tris (hydroxymethyl) aminomethane (Tris), 8 - 10 g / L polyvinylpyrrolidone (PVP), 5 - 8 g / L Tween 20, 8 - 11 g / L surfactant S9 (Tetronic 1307), 13 - 15 g / L casein, 0.01 - 0.05 wt% bacteriostatic agent.

[0036] Preferably, before step S2, the nitrocellulose membrane is treated as follows: a solution of anti-chicken IgY antibody at 0.8 - 1.0 mg / mL, a solution of anti-canine IgG antibody at 1.0 - 1.5 mg / mL, and a solution of anti-canine IgM antibody at 1.0 - 1.8 mg / mL are respectively spotted on the quality control line and two detection lines of the nitrocellulose membrane at a spraying amount of 0.9 μL / cm, and then dried.

[0037] In the third aspect, the present invention provides a kit for detecting five canine vector-borne disease antibodies, including the above-mentioned detection card for five canine vector-borne disease antibodies.

[0038] Preferably, the kit for detecting five canine vector-borne disease antibodies further includes a buffer solution.

[0039] Further, the pH of the buffer solution is 7.2 - 7.8, and it comprises components with the following concentrations: 4 - 6 g / L NaCl, 10 - 12 g / L EDTA, 6 - 7 g / L Casein, 20 - 30 mmol / L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 0.01 - 0.05 wt% bacteriostatic agent.

[0040] EDTA can form complexes with metal ions and can be used as a stabilizer for enzymes to ensure the accuracy and repeatability of detection results; Casein, as a blocking agent, can reduce the generation of non-specific signals; HEPES can endow the buffer solution with a low ionic strength and osmotic pressure, can provide a stable ion detection environment, and eliminate some interfering bindings.

[0041] Compared with the prior art, the present invention has the following advantages:

[0042] (1) In the detection card of the present invention, by adopting specific 5 detection antigens, it is possible to achieve the combined detection of canine ehrlichiosis, canine anaplasmosis, canine babesiosis, canine Lyme disease, and canine leishmaniasis, and it can take into account different subtypes in each canine vector-borne disease, as well as the detection of IgG and IgM antibodies, and has high detection sensitivity and specificity. Therefore, it can quickly screen and diagnose diseased dogs infected with vector-borne diseases in the initial stage and past infections, shorten the window period, and facilitate the timely adoption of effective preventive and treatment measures.

[0043] (2) The detection card of the present invention can distinguish the detection of IgG and IgM antibodies of each canine vector-borne disease, so as to more intuitively judge whether the dog is recently infected or has been infected, which is convenient for veterinarians to quickly take targeted treatment and reduce the transmission risk.

[0044] (3) In the detection card of the present invention, through the setting of the blood filtration membrane, it is possible to avoid the reagent strip membrane surface turning red due to the presence of canine red blood cells in the sample to be tested. Therefore, the detection card can be used for the detection of serum, plasma samples, and whole blood samples.

[0045] (4) In the detection card of the present invention, a quality control system composed of chicken IgY and anti-chicken IgY antibody can avoid the significant interference caused by the binding between mammalian immunoglobulins and reduce the occurrence of false positives. Description of the Drawings

[0046] Figure 1 is a schematic external structure diagram of the upper shell of the five-in-one antibody detection card for canine vector-borne diseases of the present invention.

[0047] Figure 2 is a schematic internal structure diagram of the upper shell of the five-in-one antibody detection card for canine vector-borne diseases of the present invention.

[0048] Figure 3It is a schematic diagram of the internal structure of the lower shell of the canine vector-borne disease penta-antibody detection card of the present invention.

[0049] The reference numerals are: upper shell 1, lower shell 2, result reading window 3, sample adding hole 4, reagent strip card slot 5, reagent strip 6, reagent strip baffle 7, reagent strip staple 8. Specific embodiments

[0050] The present invention will be further described below in conjunction with embodiments.

[0051] First, the present invention relates to a canine vector-borne disease penta-antibody detection card, which includes the following 5 detection antigens a) - e):

[0052] a) Leishmania antigen: a fusion protein of rk39, rk26 and rk9;

[0053] b) Babesia antigen: a fusion protein of TRAP and rBcMSA1 / rBcSA1;

[0054] c) Anaplasma antigen: a fusion protein of P44 and OMP-1X;

[0055] d) Ehrlichia antigen: gp19 protein;

[0056] e) Lyme antigen: Borrelia burgdorferi OspC outer membrane protein.

[0057] In some specific embodiments, the canine vector-borne disease penta-antibody detection card includes 5 reagent strips; each reagent strip includes a bottom plate and a sample pad, a gold-labeled conjugate pad, a nitrocellulose membrane and a water absorption pad stacked in sequence on the bottom plate; the gold-labeled conjugate pads of the 5 reagent strips are respectively coated with conjugates of the 5 detection antigens and colloidal gold, and detection lines coated with anti-canine IgG antibody and anti-canine IgM antibody are provided on the nitrocellulose membranes.

[0058] In the above specific embodiments, optionally or preferably:

[0059] 2 detection lines coated with anti-canine IgG antibody and anti-canine IgM antibody are provided on the nitrocellulose membranes of the 5 reagent strips; the anti-canine IgG antibody can be selected from rabbit anti-canine IgG antibody and / or mouse anti-canine IgG antibody; the anti-canine IgM antibody can be selected from rabbit anti-canine IgM antibody and / or mouse anti-canine IgM antibody;

[0060] The gold-labeled conjugate pads of the 5 reagent strips are all coated with conjugates of chicken IgY and colloidal gold, and a quality control line coated with anti-chicken IgY antibody is provided on the nitrocellulose membranes; the anti-chicken IgY antibody can be selected from goat anti-chicken IgY antibody;

[0061] A blood filtration membrane is provided between the sample pad and the gold-labeled conjugate pad.

[0062] Second, the present invention relates to a method for preparing the canine vector-borne disease five-in-one antibody detection card, comprising the following steps:

[0063] S1: Coating the gold-labeled solutions containing the conjugates of the 5 detection antigens and colloidal gold respectively onto five gold-labeled conjugate pads, and drying;

[0064] S2: Assembling the gold-labeled conjugate pads processed in step S1 with a sample pad, a nitrocellulose membrane and an absorbent pad into a test strip.

[0065] In some specific embodiments, in step S1, the method for preparing the gold-labeled solution comprises the following steps: adding an aqueous solution of trisodium citrate to an aqueous solution of chloroauric acid, adjusting the pH value to 7.5 - 8.5, adding the 5 detection antigens respectively, then successively adding bovine serum albumin (BSA) and polyethylene glycol (PEG), fully reacting, centrifuging to collect the precipitate, and dispersing it in water.

[0066] In some specific embodiments, in step S1, in the gold-labeled solution, the conjugate of Leishmania antigen and colloidal gold is used at a final concentration of OD 540 = 3 - 5, the conjugate of Babesia antigen and colloidal gold is used at a final concentration of OD 540 = 60 - 65, the conjugate of Anaplasma antigen and colloidal gold is used at a final concentration of OD 540 = 2.5 - 5.0, the conjugate of Ehrlichia antigen and colloidal gold is used at a final concentration of OD 540 = 3 - 5, and the conjugate of Borrelia burgdorferi antigen and colloidal gold is used at a final concentration of OD 540 = 50 - 55.

[0067] In some specific embodiments, in step S1, the gold-labeled solution further contains the conjugate of chicken IgY and colloidal gold; in the gold-labeled solution, the conjugate of chicken IgY and colloidal gold is used at a final concentration of OD 540 = 1.2 - 25.

[0068] In some specific embodiments, before step S2, coating a treatment solution on the sample pad and drying; the treatment solution comprises components with the following concentrations: 8 - 10 g / L bovine serum albumin (BSA), 10 - 12 g / L tris (Tris), 8 - 10 g / L polyvinylpyrrolidone (PVP), 5 - 8 g / L tween 20 (Tween20), 8 - 11 g / L surfactant S9 (Tetronic1307), 13 - 15 g / L casein, and 0.01 - 0.05 wt% bacteriostatic agent.

[0069] In some specific embodiments, before step S2, the nitrocellulose membrane is treated as follows: an anti-chicken IgY antibody solution at a concentration of 0.8 - 1.0 mg / mL, an anti-dog IgG antibody solution at a concentration of 1.0 - 1.5 mg / mL, and an anti-dog IgM antibody solution at a concentration of 1.0 - 1.8 mg / mL are spotted on the quality control line and two detection lines of the nitrocellulose membrane respectively at a spraying amount of 0.9 μL / cm, and then dried.

[0070] Thirdly, the present invention relates to a canine vector-borne disease five-antibody detection kit, which includes the canine vector-borne disease five-antibody detection card described above.

[0071] In some specific embodiments, the canine vector-borne disease five-antibody detection kit further includes a buffer solution. Optionally or preferably, the buffer solution has a pH of 7.2 - 7.8 and includes components with the following concentrations: 4 - 6 g / L NaCl, 10 - 12 g / L EDTA, 6 - 7 g / L casein, 20 - 30 mmol / L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 0.01 - 0.05 wt% bacteriostatic agent.

[0072] The present invention will be described below through specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0073] Example 1

[0074] A canine vector-borne disease five-antibody detection kit is composed of a buffer solution and a canine vector-borne disease five-antibody detection card. Among them, the buffer solution has a pH of 7.2, and the formula is as follows: 5 g / L NaCl, 10 g / L EDTA, 6.5 g / L casein, 20 mmol / L HEPES, 0.02 wt% Proclin300, and the solvent is deionized water.

[0075] The structure of the canine vector-borne disease five-antibody detection card is as Figures 1 to 3 shown, and it includes an upper shell 1 and a lower shell 2 connected by a snap structure. Five result reading windows 3 are provided on the upper shell 1, and a sample adding hole 4 is provided below each result reading window 3. Five reagent strip slots 5 are provided inside the lower shell 2, and a reagent strip 6 is placed in each reagent strip slot 5; two rows of reagent strip baffles 7 for fixing the reagent strip 6 are provided on the inner surface of the upper shell 1, and a reagent strip staple 8 for fixing the reagent strip 6 is provided on the lower shell 2.

[0076] The five reagent strips 6 are numbered as reagent strip I - reagent strip V respectively, and their uses and structures are as follows:

[0077] (1)Reagent strip I: Used to detect IgG and IgM antibodies corresponding to canine leishmaniasis. It is composed of a bottom plate I and a sample pad I, a blood filtration membrane I, a gold-labeled conjugate pad I, a nitrocellulose membrane I, and a water absorption pad I stacked in sequence on the bottom plate I. The gold-labeled conjugate pad I is coated with a conjugate of chicken IgY and colloidal gold and a conjugate of Leishmania antigen and colloidal gold. The Leishmania antigen is a fusion protein of rk39, rk26, and rk9. The nitrocellulose membrane I is provided with a test line T1, a test line T2, and a quality control line C1. The test line T1 is coated with rabbit anti-canine IgG antibody, the test line T2 is coated with rabbit anti-canine IgM antibody, and the quality control line C1 is coated with goat anti-chicken IgY antibody.

[0078] (2)Reagent strip II: Used to detect IgG and IgM antibodies corresponding to canine ehrlichiosis. It is composed of a bottom plate II and a sample pad II, a blood filtration membrane II, a gold-labeled conjugate pad II, a nitrocellulose membrane II, and a water absorption pad II stacked in sequence on the bottom plate II. The gold-labeled conjugate pad II is coated with a conjugate of chicken IgY and colloidal gold and a conjugate of Ehrlichia antigen and colloidal gold. The Ehrlichia antigen is the gp19 protein. The nitrocellulose membrane II is provided with a test line T3, a test line T4, and a quality control line C2. The test line T3 is coated with mouse anti-canine IgG antibody, the test line T4 is coated with mouse anti-canine IgM antibody, and the quality control line C2 is coated with goat anti-chicken IgY antibody.

[0079] (3)Reagent strip III: Used to detect IgG and IgM antibodies corresponding to canine babesiosis. It is composed of a bottom plate III and a sample pad III, a blood filtration membrane III, a gold-labeled conjugate pad III, a nitrocellulose membrane III, and a water absorption pad III stacked in sequence on the bottom plate III. The gold-labeled conjugate pad III is coated with a conjugate of chicken IgY and colloidal gold and a conjugate of Babesia antigen and colloidal gold. The Babesia antigen is a fusion protein of TRAP and rBcMSA1 / rBcSA1. The nitrocellulose membrane III is provided with a test line T5, a test line T6, and a quality control line C3. The test line T5 is coated with mouse anti-canine IgG antibody, the test line T6 is coated with mouse anti-canine IgM antibody, and the quality control line C3 is coated with goat anti-chicken IgY antibody.

[0080] (4)Reagent Strip IV: Used to detect IgG and IgM antibodies corresponding to canine anaplasmosis. It is composed of a bottom plate IV, and successively stacked on the bottom plate IV are a sample pad IV, a blood filtration membrane IV, a gold-labeled conjugate pad IV, a nitrocellulose membrane IV, and a water absorption pad IV. The gold-labeled conjugate pad IV is coated with a conjugate of chicken IgY and colloidal gold and a conjugate of anaplasma antigen and colloidal gold. The anaplasma antigen is a fusion protein of P44 and OMP-1X. The nitrocellulose membrane IV is provided with a test line T7, a test line T8, and a quality control line C4. The test line T7 is coated with a mouse anti-canine IgG antibody, the test line T8 is coated with a mouse anti-canine IgM antibody, and the quality control line C4 is coated with a goat anti-chicken IgY antibody.

[0081] (5)Reagent Strip V: Used to detect IgG and IgM antibodies corresponding to canine Lyme disease. It is composed of a bottom plate V, and successively stacked on the bottom plate V are a sample pad V, a blood filtration membrane V, a gold-labeled conjugate pad V, a nitrocellulose membrane V, and a water absorption pad V. The gold-labeled conjugate pad V is coated with a conjugate of chicken IgY and colloidal gold and a conjugate of Lyme antigen and colloidal gold. The Lyme antigen is the outer membrane protein OspC of Borrelia burgdorferi. The nitrocellulose membrane V is provided with a test line T9, a test line T10, and a quality control line C5. The test line T9 is coated with a rabbit anti-canine IgG antibody, the test line T10 is coated with a rabbit anti-canine IgM antibody, and the quality control line C5 is coated with a goat anti-chicken IgY antibody.

[0082] The five-in-one antibody detection card for canine vector-borne diseases in this embodiment is prepared through the following steps:

[0083] S1: Prepare Reagent Strip I

[0084] S1.1: Coat the treatment solution on the sample pad I and dry it. The formula of the treatment solution is as follows: 8 g / L BSA, 11 g / L Tris, 10 g / L PVP 10, 7 g / L Tween 20, 9 g / L surfactant S9, 13 g / L casein, 0.04 wt% Proclin300, and the solvent is deionized water.

[0085] S1.2: Heat 100 mL of 0.01 wt% chloroauric acid aqueous solution to boiling, add 2.5 mL of 1 wt% sodium citrate aqueous solution, react for 20 min and then cool to obtain a colloidal gold solution. Use 1 wt% K 2 CO 3Adjust the pH value of the solution to 8, then add Leishmania antigen (the fusion protein of rk39, rk26 and rk9) at a ratio of 17 μg / mL, and then add 2 wt% BSA solution and 0.1 wt% PEG20000 solution in turn (add 1 mL of PEG20000 solution and 1 mL of BSA solution to every 100 mL of colloidal gold solution). React fully at 96 °C for 15 min and then cool down. Centrifuge at 6 °C and 12000 rpm for 30 min, discard the supernatant and take the precipitate, disperse it in deionized water to prepare gold-labeled solution I, and measure its OD value at an absorption wavelength of 540 nm.

[0086] S1.3: Prepare chicken IgY gold-labeled solution according to the method in step S1.2 and measure its OD value at an absorption wavelength of 540 nm. The difference from step S1.2 is only that the Leishmania antigen is replaced with chicken IgY.

[0087] S1.4: Mix gold-labeled solution I and chicken IgY gold-labeled solution to obtain gold-labeled mixed solution I. The dosages of gold-labeled solution I and chicken IgY gold-labeled solution are calculated according to their OD 540 values, so that the final concentration OD 540 value of the conjugate of Leishmania antigen and colloidal gold in the gold-labeled mixed solution I is 3, and the final concentration OD 540 value of the conjugate of chicken IgY and colloidal gold is 1.2. Make up the insufficient part with PBS buffer. Coat the gold-labeled mixed solution on the gold-labeled conjugate pad I (polyester film) at a coating amount of 2 μL / cm, and freeze-dry at -50 °C for 2 h.

[0088] S1.5: Dilute the 3.6 mg / mL rabbit anti-dog IgG antibody solution to 1 mg / mL, the 2.4 mg / mL rabbit anti-dog IgM antibody solution to 1.2 mg / mL, and the 5 mg / mL goat anti-chicken IgY antibody solution to 0.8 mg / mL with 1×PBS buffer. Then, spray the above-diluted rabbit anti-dog IgG antibody solution, rabbit anti-dog IgM antibody solution, and goat anti-chicken IgY antibody solution on the test line T1, test line T2, and quality control line C1 on the nitrocellulose membrane I at a spraying amount of 0.9 μL / cm, and dry at 37 °C for 12 h.

[0089] S1.6: Connect and assemble the sample pad I, blood filter membrane I, gold-labeled conjugate pad I processed in step S1.4, nitrocellulose membrane I processed in step S1.5, and absorbent pad I in sequence on the bottom plate I (made of polyvinyl chloride) and cut to obtain test strip I.

[0090] S2: Prepare reagent strip II

[0091] Prepare reagent strip II according to the method in step S1. The difference from step S1 is only that:

[0092] In step S1.2, replace the Leishmania antigen with Ehrlichia antigen (gp19 protein).

[0093] In step S1.5, replace the 3.6 mg / mL rabbit anti-dog IgG antibody solution with a 7 mg / mL mouse anti-dog IgG antibody solution, and the diluted concentration is 1.5 mg / mL; replace the 2.4 mg / mL rabbit anti-dog IgM antibody solution with an 8.1 mg / mL mouse anti-dog IgM antibody solution, and the diluted concentration is 1.8 mg / mL.

[0094] S3: Prepare test strip III

[0095] Prepare test strip III according to the method in step S1, and the difference from step S1 is only that:

[0096] In step S1.2, replace the Leishmania antigen with Babesia antigen (the fusion protein of TRAP and rBcMSA1 / rBcSA1) to obtain gold-labeled solution III.

[0097] In step S1.4, mix gold-labeled solution III and chicken IgY gold-labeled solution to obtain gold-labeled mixed solution III. The final concentration OD of the conjugate of Babesia antigen and colloidal gold in gold-labeled mixed solution III 540 value is 60, and the final concentration OD of the conjugate of chicken IgY and colloidal gold 540 value is 25; after the coating of gold-labeled mixed solution III is completed, replace freeze-drying with drying at 37 °C for 12 h.

[0098] In step S1.5, replace the 3.6 mg / mL rabbit anti-dog IgG antibody solution with a 7 mg / mL mouse anti-dog IgG antibody solution, and the diluted concentration is 1.5 mg / mL; replace the 2.4 mg / mL rabbit anti-dog IgM antibody solution with an 8.1 mg / mL mouse anti-dog IgM antibody solution, and the diluted concentration is 1.8 mg / mL.

[0099] S4: Prepare test strip IV

[0100] Prepare test strip IV according to the method in step S1, and the difference from step S1 is only that:

[0101] In step S1.2, replace the Leishmania antigen with Anaplasma antigen (the fusion protein of P44 and OMP-1X) to obtain gold-labeled solution IV.

[0102] In step S1.4, mix gold-labeled solution IV and chicken IgY gold-labeled solution to obtain gold-labeled mixed solution IV. The final concentration OD of the conjugate of Anaplasma antigen and colloidal gold in gold-labeled mixed solution IV 540 value is 2.5;

[0103] In step S1.5, replace the 3.6 mg / mL rabbit anti-dog IgG antibody solution with a 7 mg / mL mouse anti-dog IgG antibody solution, and the diluted concentration is 1.5 mg / mL; replace the 2.4 mg / mL rabbit anti-dog IgM antibody solution with an 8.1 mg / mL mouse anti-dog IgM antibody solution, and the diluted concentration is 1.8 mg / mL.

[0104] S5: Prepare test strip V

[0105] Prepare test strip V according to the method in step S1, and the difference from step S1 is only that:

[0106] In step S1.2, replace the Leishmania antigen with Lyme antigen (Borrelia burgdorferi OspC outer membrane protein) to obtain gold-labeled solution V;

[0107] In step S1.4, mix gold-labeled solution V and chicken IgY gold-labeled solution to obtain gold-labeled mixed solution V. The conjugate of Lyme antigen and colloidal gold in gold-labeled mixed solution V is used at a final concentration OD 540 value of 50, and the conjugate of chicken IgY and colloidal gold is used at a final concentration OD 540 value of 25; after the coating of gold-labeled mixed solution V is completed, replace freeze-drying with drying at 37 °C for 12 h.

[0108] S6: Assemble the test card

[0109] Assemble test strips I - V into the reagent strip slots in the lower shell in sequence, and fasten the upper shell and the lower shell to obtain a five-in-one antibody test card for canine vector-borne diseases.

[0110] Use the five-in-one antibody test kit for canine vector-borne diseases in this embodiment to detect the test sample. The steps are as follows: After dropping 10 μL of the test sample into each of the five sample addition holes, drop two drops of buffer into each sample addition hole, and observe the color development of the test line and the control line in the result reading window.

[0111] According to the above steps, the samples to be tested in Table 1 (obtained from dogs, and the negative and positive conditions of canine vector-borne disease-related antibodies have been determined) are tested to verify the detection accuracy of the kit of this embodiment. The test results of each sample to be tested show that purple-red stripes appear at the quality control lines C1, C2, C3, C4 and C5, indicating that the test card is effective; the color development at each test line is shown in Table 1 (in Table 1, "+" indicates the appearance of purple-red stripes, the more "+"s there are, the darker the strip color, and "-" indicates that no purple-red stripes appear), which is consistent with the actual situation of each sample, indicating that the kit of this embodiment has high detection accuracy, high detection rate, and high specificity, and the detection of five canine vector-borne diseases will not interfere with each other; in the detection of each whole blood sample, plasma sample and serum sample, the membrane surface is clean, and the results are read clearly and effectively, indicating that the kit of this embodiment can prevent the presence of canine red blood cells in the sample to be tested from causing the membrane surface of the reagent strip to turn red.

[0112] Table 1 Test results of the canine vector-borne disease five-in-one antibody detection kit of Example 1

[0113]

[0114] Example 2

[0115] The difference between this embodiment and embodiment 1 is that: in the reagent strips I to V, no blood filter membranes I to V are provided. The rest is the same as embodiment 1.

[0116] When the kit of this example was used to test canine plasma and canine serum samples, the membrane surface was clean and the results were clear and effective; however, when testing canine whole blood samples, after adding buffer and letting it stand for 10 minutes, it was clearly observed that the membrane background of the five result reading windows all appeared red, affecting the accuracy of the judgment results.

[0117] Comparative Example 1

[0118] The difference between this comparative example and Example 1 is that in the reagent strips I to V, the detection lines T2, T4, T6, T8 and T10 are not provided. The rest are the same as Example 1.

[0119] When verifying the detection accuracy of the kit of this comparative example, the samples to be tested were the same as those in Example 1. The test results of each sample to be tested showed that when testing LSH IgM-positive serum samples, Ehrlichia IgM-positive serum samples, Babesia IgM-positive serum samples, ANA IgM-positive serum samples and Lyme IgM-positive plasma samples, no purple-red bands appeared at each test line, indicating that this comparative example had false negative results in the detection of IgG-positive samples.

[0120] Comparative Example 2

[0121] The difference between this comparative example and Example 1 is only that: in Test Strips I to V, test lines T1, T3, T5, T7, and T9 are not provided. The rest are the same as in Example 1.

[0122] When verifying the detection accuracy of the test kit of this comparative example, the test samples to be measured are the same as those in Example 1. The detection results of each test sample show that: when detecting LSH IgG positive plasma samples, Ehrlichia IgG positive plasma samples, Babesia IgG positive whole blood samples, ANA IgG positive whole blood samples, and Lyme IgG positive serum samples, no purplish red bands appear at each test line, indicating that there are false negative results in the detection of IgM positive samples in this comparative example.

[0123] Comparative Example 3

[0124] The difference between this comparative example and Example 1 is only that: in Test Strip I, the fusion protein of rk39, rk26, and rk9 is replaced with rk39 protein; in Test Strip III, the fusion protein of TRAP and rBcMSA1 / rBcSA1 is replaced with rBcMSA1 / rBcSA1 protein; in Test Strip IV, the fusion protein of P44 and OMP-1X is replaced with OMP-1X protein. The rest are the same as in Example 1.

[0125] When verifying the detection accuracy of the test kit of this comparative example, the test samples to be measured are the 12 samples corresponding to canine leishmaniasis, canine babesiosis, and canine anaplasmosis in Table 1. In the detection results of each test sample, the color development at each test line is shown in Table 2.

[0126] Table 2 Detection Results of the Five-in-one Antibody Test Kit for Canine Vector-borne Diseases in Comparative Example 3

[0127]

[0128] It can be seen from Table 2 that: there are false negative results in the detection of antibodies corresponding to canine leishmaniasis, canine babesiosis, and canine anaplasmosis in this comparative example. The reason for the analysis is that: when using rK39 protein as the detection antigen for canine leishmaniasis, there are missed detections for some dogs with canine leishmaniasis in Brazil and India; the common subtypes of Babesia infecting dogs are Babesia gibsoni (Babesia microti) and Babesia canis (Babesia bigemina), and only using the detection protein rBcMSA1 / rBcSA1 for Babesia canis will not be able to detect Babesia gibsoni; the main subtypes of Anaplasma infecting dogs include Anaplasma phagocytophilum and Anaplasma platys, and only using the detection protein OMP-1X for Anaplasma platys will reduce the detection rate of Anaplasma phagocytophilum. Therefore, compared with using a single antigen, using the fusion protein in the present invention can improve the detection rate.

[0129] Comparative Examples 4 to 6

[0130] The differences between Comparative Examples 4 to 6 and Example 1 are only as follows: In test strip I, the fusion proteins of rk39, rk26, and rk9 are respectively replaced with the fusion protein of rk39 and rk9, KMP11 protein, and the fusion protein of rk26 and KMP11. The rest are the same as in Example 1.

[0131] When verifying the detection accuracy of the kits of Comparative Examples 4 to 6, the test samples to be measured are the 4 corresponding samples of canine leishmaniasis in Table 1. In the detection results of each test sample, the color development conditions at the test lines T1 and T2 on test strip I are shown in Table 3.

[0132] Table 3 Detection Results of the Canine Vector-Borne Disease Five-Antibody Detection Kits of Comparative Examples 4 to 6

[0133]

[0134] It can be seen from Table 1 and Table 3 that: When using the fusion protein of rk39 and rk9 (Comparative Example 4) or KMP11 protein (Comparative Example 5), there are certain degrees of false negative results in different positive samples; When using KMP11 to replace rk39 and rk9 in the fusion protein of rk39, rk26, and rk9 (Comparative Example 6), although there are no false negative results in the detection of 3 positive samples, the color development is lighter compared to Example 1, indicating a decrease in detection sensitivity.

[0135] Comparative Examples 7 to 9

[0136] The differences between Comparative Examples 7 to 9 and Example 1 are only as follows: In test strip III, the fusion proteins of TRAP and rBcMSA1 / rBcSA1 are respectively replaced with TRAP protein, HSP70 protein, and the fusion protein of HSP70 and rBcMSA1 / rBcSA1. The rest are the same as in Example 1.

[0137] When verifying the detection accuracy of the kits of Comparative Examples 7 to 9, the test samples to be measured are the 4 corresponding samples of canine babesiosis in Table 1. In the detection results of each test sample, the color development conditions at the test lines T5 and T6 on test strip III are shown in Table 4.

[0138] Table 4 Detection Results of the Canine Vector-Borne Disease Five-Antibody Detection Kits of Comparative Examples 7 to 9

[0139]

[0140] As can be seen from Table 1 and Table 4: When using a single TRAP protein (Comparative Example 7) or HSP70 protein (Comparative Example 8), false negative results exist, and the detection sensitivity of the TRAP protein is lower than that of the HSP70 protein; but after forming a fusion protein with rBcMSA1 / rBcSA1, the detection sensitivity of the fusion protein of TRAP and rBcMSA1 / rBcSA1 (Example 1) is higher than that of the fusion protein of HSP7 and rBcMSA1 / rBcSA1 (Comparative Example 9). The above results show that: After a single antigen forms a fusion protein, its detection sensitivity will be affected, and the detection sensitivity of the fusion protein is not necessarily the same as that of the single antigen.

[0141] Comparative Examples 10 to 12

[0142] The differences between Comparative Examples 10 to 12 and Example 1 are only as follows: In Test Strip IV, the fusion proteins of P44 and OMP-1X are respectively replaced with Tr1 protein, P44 protein, and the fusion protein of Tr1 and OMP-1X. The rest are the same as in Example 1.

[0143] When verifying the detection accuracy of the test kits of Comparative Examples 10 to 12, the test samples used are the 4 corresponding samples of canine anaplasmosis in Table 1. In the detection results of each test sample, the color development conditions at the test lines T7 and T8 on Test Strip IV are shown in Table 5.

[0144] Table 5 Detection Results of the Five-in-one Antibody Test Kit for Canine Vector-borne Diseases in Comparative Examples 10 to 12

[0145]

[0146] As can be seen from Table 1 and Table 5: When using a single Tr1 protein (Comparative Example 10) or P44 protein (Comparative Example 11), false negative results exist, and the detection sensitivities of the two proteins are similar; but after forming a fusion protein with OMP-1X, the detection sensitivity of the fusion protein of P44 and OMP-1X (Example 1) is higher than that of the fusion protein of Tr1 and OMP-1X (Comparative Example 12). The above results can also show that: After a single antigen forms a fusion protein, its detection sensitivity will be affected, and the detection sensitivity of the fusion protein is not necessarily the same as that of the single antigen.

[0147] Comparative Example 13

[0148] The difference between this comparative example and Example 1 is only as follows: on the five gold label conjugate pads (Gold Label Conjugate Pad I - Gold Label Conjugate Pad V), chicken IgY is replaced with mouse IgG; on the five control lines (Control Line C1 - Control Line C5), goat anti-chicken IgY antibody is replaced with goat anti-mouse IgG; in step S1.5, the 5 mg / mL goat anti-chicken IgY antibody solution is replaced with a 6.9 mg / mL goat anti-mouse IgG antibody solution, and the diluted concentration is 1 mg / mL. The rest is the same as in Example 1.

[0149] When verifying the detection accuracy of the kit of this comparative example, the 5 negative whole blood samples in Table 1 were used as the test samples to be detected. In the detection results of each test sample, the color development at each test line is shown in Table 6.

[0150] Table 6 Detection Results of the Canine Vector-Borne Disease Five-Antibody Detection Kit of Comparative Example 13

[0151]

[0152] It can be seen from Table 6 that in this comparative example, when Test Strip I tested the negative whole blood sample - 2 and Test Strip V tested the negative whole blood sample - 4, false positive readings occurred, indicating that due to the non-specific binding of the mouse IgG - goat anti-mouse IgG quality control system and the antibody, there was interference with the test line T, resulting in incorrect results.

[0153] Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the art to which the present disclosure belongs. The raw materials and equipment used in the present invention are conventional raw materials and equipment in the art and can be obtained from conventional commercial channels without special instructions; the methods used in the present invention are conventional methods in the art without special instructions.

[0154] The above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent transformations made to the above embodiments based on the technical essence of the present invention still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A canine vector-borne disease five-in-one antibody test card, characterized in that: Including the following 5 detection antigens a)~e): a) Leishmania antigen: a fusion protein of rk39, rk26 and rk9; b) Babesia antigen: a fusion protein of TRAP, rBcMSA1 and rBcSA1; c) Anaplasma antigen: a fusion protein of P44 and OMP-1X; d) Ehrlichia antigen: gp19 protein; e) Lyme antigen: OspC outer membrane protein of Borrelia burgdorferi; The antibodies are IgG and IgM antibodies.

2. The canine vector-borne disease five-in-one antibody test card according to claim 1, characterized in that: It comprises 5 reagent strips; each of the reagent strips comprises a bottom plate and a sample pad, a gold label binding pad, a nitrocellulose membrane and a water absorbent pad stacked in sequence on the bottom plate; the gold label binding pads of the 5 reagent strips are respectively coated with conjugates of the 5 detection antigens and colloidal gold, and the nitrocellulose membranes are each provided with detection lines coated with anti-dog IgG antibodies and anti-dog IgM antibodies.

3. The canine vector-borne disease five-in-one antibody test card according to claim 2, characterized in that: The nitrocellulose membranes of the five test strips are provided with two detection lines coated with anti-canine IgG antibody and anti-canine IgM antibody respectively.

4. The canine vector-borne disease five-in-one antibody test card according to claim 2, characterized in that: The gold-labeled conjugate pads of the five reagent strips are all coated with a conjugate of chicken IgY and colloidal gold, and the nitrocellulose membranes are all provided with quality control lines coated with anti-chicken IgY antibodies.

5. The canine vector-borne disease five-in-one antibody test card according to claim 2, characterized in that: A blood filter membrane is arranged between the sample pad and the gold label binding pad.

6. A method for preparing the canine vector-borne disease five-in-one antibody test card according to any one of claims 2 to 5, characterized in that: The following steps are involved: S1: applying gold-labeled solutions containing the conjugates of the five detection antigens and colloidal gold respectively onto five gold-labeled binding pads and drying; S2: Assemble the gold-labeled binding pad treated in step S1 with the sample pad, nitrocellulose membrane and absorbent pad into a reagent strip.

7. The preparation method according to claim 6, characterized in that: In step S1, in the gold label solution, the conjugate of Leishmania antigen and colloidal gold is used at a final concentration of OD 540 =3~5, the final concentration of the conjugate of Babesia antigen and colloidal gold is OD 540 =60~65, the final concentration of the conjugate of Anaplasma antigen and colloidal gold is OD 540 =2.5~5.0, the final concentration of the conjugate of Ehrlichia antigen and colloidal gold is OD 540 =3~5, the final concentration of Lyme antigen and colloidal gold conjugate is OD 540 =50~55.

8. A canine vector-borne disease five-in-one antibody detection kit, characterized in that: It comprises the canine vector-borne disease five-in-one antibody detection card according to any one of claims 1 to 5.

9. The canine vector-borne disease five-in-one antibody detection kit according to claim 8, characterized in that: Buffer is also included.

10. The canine vector-borne disease five-in-one antibody detection kit according to claim 9, characterized in that: The pH of the buffer solution is 7.2-7.8, and the buffer solution comprises the following components in concentrations: 4-6 g / L NaCl, 10-12 g / L EDTA, 6-7 g / L casein, 20-30 mmol / L 4-hydroxyethylpiperazineethanesulfonic acid, and 0.01-0.05 wt % of an antibacterial agent.

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