Canine entomophilous disease quintuplet antibody detection card, preparation method thereof and kit

Abstract

The invention relates to the technical field of antibody detection, and discloses a canine entomophilous disease quintuplet antibody detection card, a preparation method thereof and a kit. The detection card comprises the following five detection antigens a)-e): a) a leishmania antigen which is fusion protein of rk39, rk26 and rk9; b) a babesia antigen which is a fusion protein of TRAP and rBcMSA1 / rBcSA1; c) an amorphous antigen which is a fusion protein of P44 and OMP-1X; the detection card and the kit have the advantages that joint detection of IgG (immunoglobulin G) and IgM (immunoglobulin M) antibodies corresponding to canine Ehrlichia, canine anaplasmosis, canine babesiosis, canine lyme disease and canine leishmania can be realized by the detection card and the kit, and the detection sensitivity and the specificity are high. The detection card and the kit can be used for detecting canine Ehrlichia, canine anaplasmosis, canine babesiosis, canine lyme disease and canine leishmania. The detection card and the kit have the advantages that the detection card and the kit can be used for detecting canine Ehrlichia, canine anaplasmosis, canine babesiosis and canine leishmania;

Description

Technical Field

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

[0002] Insect-borne diseases are a class of infectious diseases transmitted by arthropods. These vectors are numerous and complex, encompassing mosquitoes, ticks, mites, lice, and fleas, and are transmitted to animals and humans through bites. Among these, the most serious health threats to dogs include ehrlichiosis (Ehrlichia canis), anaplasmosis (ANA), babesiosis, Lyme disease, and leishmaniasis (LSH). Insect-borne diseases are highly contagious and destructive, especially given the resilience and resilience of these insects, which often hide in hidden areas like grass and bushes. When pet dogs inadvertently pass through these areas, these insects can quickly attach to their bodies, suck their blood, and transmit the disease.

[0003] Most dogs that are bitten don't show obvious symptoms initially, and symptoms only gradually become apparent weeks or even months later. Furthermore, due to the large variety and complex distribution of vector-borne organisms, and the increasing prevalence of long-distance transmission of vector-borne diseases, clinical symptoms can be very similar. Therefore, during peak season for vector-borne diseases, dogs exhibiting physical abnormalities can be screened using highly specific and sensitive canine vector-borne disease IgG and IgM antibody test cards to determine current or past infection, helping veterinarians provide targeted treatment.

[0004] However, the combined detection of IgG and IgM antibodies for multiple vector-borne diseases requires both good binding between the different vector-borne disease antigens and the corresponding IgG and IgM antibodies to achieve high sensitivity, and high specificity between the binding of each antigen and the corresponding IgG and IgM antibodies to avoid cross-interference between the detection of different vector-borne disease IgG and IgM antibodies, making the design of a test card quite challenging. Currently, no test card has been reported for the combined detection of IgG and IgM antibodies for the five common vector-borne diseases in dogs (ehrlichiosis, anaplasmosis, babesiosis, Lyme disease, and leishmaniasis). Summary of the Invention

[0005] To address the above technical issues, the present invention provides a five-in-one canine insect-borne disease antibody test card, its preparation method, and a test kit. The test card and kit enable the combined detection of IgG and IgM antibodies corresponding to canine ehrlichiosis, canine anaplasmosis, canine babesiosis, canine Lyme disease, and canine leishmaniasis, with high sensitivity and specificity.

[0006] The specific technical solutions of the present invention are: In a first aspect, the present invention provides a canine insect-borne disease five-in-one antibody test card, comprising the following five detection antigens a) to e): 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: gp19 protein; e) Lyme antigen: OspC outer membrane protein of Borrelia burgdorferi.

[0007] In the present invention, the “rBcMSA1 / rBcSA1” mentioned refers to the fusion protein of rBcMSA1 and rBcSA1.

[0008] When testing for IgG and IgM antibodies against leishmaniasis using only the rK39 protein, some dogs with canine leishmaniasis in Brazil and India may be missed. Using a fusion protein of rk39, rk26, and rk9 can address this issue, improving the detection rate and reducing false negatives. Common subtypes of Babesia infecting dogs are small Babesia gibsoni and large Babesia canis. Using a fusion protein of TRAP and rBcMSA1 / rBcSA1 in testing for IgG and IgM antibodies against babesiosis can detect both subtypes and improve the detection rate. 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 of the above-mentioned Anaplasma canine subtypes.

[0009] Furthermore, the design of the detection antigen can affect detection sensitivity. By selecting specific monomeric proteins for the three fusion proteins used in this invention, the resulting fusion proteins can still exhibit good binding properties with the corresponding canine vector-borne disease antibodies, thereby achieving higher detection sensitivity, helping to improve detection rates and reduce false negatives.

[0010] Furthermore, IgM antibodies are primarily produced early in infection, while IgG antibodies are primarily produced relatively late after infection. The specificity of different segments of anti-IgG and anti-IgM antibodies varies significantly. The five detection antigens employed in this invention can detect both IgM and IgG antibodies against five canine vector-borne diseases, enabling the detection of both IgM and IgG. Furthermore, the assay has high sensitivity and specificity for both IgM and IgG antibodies against each canine vector-borne disease, thereby contributing to high detection accuracy.

[0011] Preferably, the canine vector-borne disease five-in-one antibody test card includes 5 reagent strips; each of the reagent strips includes a base plate and a sample pad, a gold label binding pad, a nitrocellulose membrane and a water-absorbing pad stacked in sequence on the base 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.

[0012] Preferably, the nitrocellulose membranes of the five reagent strips are each provided with two detection lines coated with anti-canine IgG antibodies and anti-canine IgM antibodies, respectively.

[0013] By setting the above two detection lines, it is possible to distinguish between the detection of IgG and IgM antibodies, so as to more intuitively determine whether the dog has been infected recently or in the past, so that the pet doctor can quickly take targeted treatment and reduce the risk of transmission.

[0014] Taking Leishmaniasis (LSH) as an example, the test strip's detection principle is as follows: a gold-labeled conjugate of a Leishmania antigen and colloidal gold (hereinafter referred to as the "gold-labeled LSH antigen") is coated on a gold-labeled conjugate pad. This conjugate is purple-red. When the test sample contains IgG or IgM antibodies specific to LSH, these antibodies specifically bind to the gold-labeled LSH antigen on the gold-labeled conjugate pad, forming a gold-labeled LSH antigen-LSH IgG or IgM antibody complex. This complex diffuses through a nitrocellulose membrane, migrating via capillary chromatography. When the test sample contains IgG antibodies specific to LSH, the gold-labeled LSH antigen-LSH IgG antibody complex binds to the anti-canine IgG antibody coated on the nitrocellulose membrane, resulting in a purple-red band on the corresponding test line, indicating a positive LSH IgG antibody test. When the test sample does not contain IgG antibodies specific to LSH, no color develops on the test line coated with the anti-canine IgG antibody, indicating a negative LSH IgG test. Similarly, when the sample to be tested contains IgM antibodies corresponding to LSH, a purple-red band will appear on the test line coated with anti-canine IgM antibodies, indicating that the LSH IgM antibody is positive; when the sample to be tested does not contain IgM antibodies corresponding to LSH, no color will appear on the test line coated with anti-canine IgM antibodies, indicating that the LSH IgM antibody is negative.

[0015] The testing principles for ehrlichiosis, anaplasmosis, babesiosis, and Lyme disease are the same as those described above for leishmaniasis.

[0016] 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.

[0017] Preferably, 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 a quality control line coated with anti-chicken IgY antibodies.

[0018] The Fc region of chicken IgY does not interact with mammalian Fc receptors, complement, rheumatoid factor, protein A, protein G, and protein L. It is more distant from the germline of canine secondary antibodies (such as rabbit anti-canine IgG / IgM and mouse anti-canine IgG / IgM) and has almost no cross-reaction with IgG and IgM. Therefore, using chicken IgY as a quality control can avoid significant interference caused by binding with mammalian immunoglobulins and reduce the occurrence of false positives.

[0019] Furthermore, the anti-chicken IgY antibody is a goat anti-chicken IgY antibody.

[0020] Preferably, a blood filter membrane is provided between the sample pad and the gold label binding pad.

[0021] The blood filter membrane can intercept red blood cells in the sample to be tested, which helps to ensure a clean membrane background during chromatography and prevent canine red blood cells from turning the reagent strip membrane surface red. This allows accurate interpretation of results when the sample to be tested is serum, plasma, or whole blood.

[0022] In a second aspect, the present invention provides a method for preparing the canine insect-borne disease five-in-one antibody detection card, comprising the following steps: S1: applying gold conjugate solutions containing the five detection antigens and colloidal gold conjugates onto five gold conjugate pads and drying; S2: Assemble the gold-labeled conjugate pad treated in step S1 with the sample pad, nitrocellulose membrane, and absorbent pad to form a reagent strip.

[0023] Preferably, in step S1, the method for preparing the gold-labeled solution comprises the following steps: adding a trisodium citrate aqueous solution to a chloroauric acid aqueous solution, adjusting the pH value to 7.5-8.5, adding the five detection antigens respectively, and then adding bovine serum albumin (BSA) and polyethylene glycol (PEG) in sequence. After sufficient reaction, the precipitate is collected by centrifugation and dispersed in water.

[0024] Preferably, in step S1, the conjugate of Leishmania antigen and colloidal gold in the gold conjugate solution 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.

[0025] Preferably, in step S1, the gold-labeled solution further contains a 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.

[0026] In the present invention, the "OD 540 ” refers to the absorbance value at an absorption wavelength of 540 nm.

[0027] Preferably, before step S2, a treatment solution is applied to the sample pad and then dried; the treatment solution comprises the following components in the following concentrations: 8-10 g / L bovine serum albumin (BSA), 10-12 g / L tromethamine (Tris), 8-10 g / L polyvinylpyrrolidone (PVP), 5-8 g / L Tween 20 (Tween 20), 8-11 g / L surfactant S9 (Tetronic 1307), 13-15 g / L casein (Casein), and 0.01-0.05 wt% antibacterial agent.

[0028] Preferably, before step S2, the nitrocellulose membrane is treated as follows: 0.8-1.0 mg / mL anti-chicken IgY antibody solution, 1.0-1.5 mg / mL anti-dog IgG antibody solution, and 1.0-1.8 mg / mL anti-dog IgM antibody solution are sprayed onto the quality control line and two test lines of the nitrocellulose membrane at a spray volume of 0.9 μL / cm, respectively, and dried.

[0029] In a third aspect, the present invention provides a canine insect-borne disease five-in-one antibody detection kit, comprising the canine insect-borne disease five-in-one antibody detection card.

[0030] Preferably, the canine vector-borne disease five-in-one antibody detection kit further comprises a buffer solution.

[0031] Furthermore, the pH of the buffer solution is 7.2-7.8, and the buffer solution comprises the following components at the following concentrations: 4-6 g / L NaCl, 10-12 g / L EDTA, 6-7 g / L casein, 20-30 mmol / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), and 0.01-0.05 wt% antibacterial agent.

[0032] EDTA can form complexes with metal ions and can serve as an enzyme stabilizer to ensure the accuracy and reproducibility of test results; casein, as a blocking agent, can reduce the generation of nonspecific signals; HEPES can give the buffer a lower ionic strength and osmotic pressure, providing a stable ion detection environment and eliminating some interfering binding.

[0033] Compared with the prior art, the present invention has the following advantages: (1) The test card of the present invention, by adopting five specific detection antigens, can achieve the combined detection of canine ehrlichiosis, canine anaplasmosis, canine babesiosis, canine Lyme disease and canine leishmaniasis, and can take into account the detection of different subtypes of canine vector-borne diseases, as well as IgG and IgM antibodies, and has high detection sensitivity and specificity. Therefore, it can quickly screen and diagnose sick dogs in the early stage of infection or those previously infected with vector-borne diseases, shorten the window period, and facilitate timely and effective prevention and treatment measures.

[0034] (2) The test card of the present invention can distinguish the detection of IgG and IgM antibodies for various canine insect-borne diseases, thereby more intuitively judging whether the dog has been infected recently or in the past, making it easier for veterinarians to quickly take targeted treatment and reduce the risk of transmission.

[0035] (3) In the test card of the present invention, the blood filter membrane is provided to 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, so that the test card can be used for the detection of serum and plasma samples as well as whole blood samples.

[0036] (4) The quality control system composed of chicken IgY and anti-chicken IgY antibodies used in the test card of the present invention can avoid significant interference caused by binding with mammalian immunoglobulins and reduce the occurrence of false positives. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a schematic diagram of the external structure of an upper shell of the canine insect-borne disease five-in-one antibody test card of the present invention.

[0038] Figure 2 This is a schematic diagram of the internal structure of an upper shell of the canine insect-borne disease five-in-one antibody test card of the present invention.

[0039] Figure 3This is a schematic diagram of the internal structure of a lower shell of the canine insect-borne disease five-in-one antibody detection card of the present invention.

[0040] The reference numerals are: upper shell 1 , lower shell 2 , result reading window 3 , sample addition hole 4 , reagent strip slot 5 , reagent strip 6 , reagent strip baffle 7 , reagent strip staple 8 . DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to the embodiments.

[0042] First, the present invention relates to a canine insect-borne disease five-in-one antibody test card, comprising the following five detection antigens a) to e): 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: gp19 protein; e) Lyme antigen: OspC outer membrane protein of Borrelia burgdorferi.

[0043] In some specific embodiments, the canine vector-borne disease five-in-one antibody test card includes 5 reagent strips; each of the reagent strips includes a base plate and a sample pad, a gold label conjugate pad, a nitrocellulose membrane and a water-absorbing pad stacked in sequence on the base plate; the gold label conjugate pads of the 5 reagent strips are respectively coated with conjugates of the 5 detection antigens and colloidal gold, and the nitrocellulose membrane is provided with detection lines coated with anti-dog IgG antibodies and anti-dog IgM antibodies.

[0044] In the above specific implementation manner, optionally or preferably: The nitrocellulose membranes of the five test strips are each provided with two detection lines coated with anti-dog IgG antibodies and anti-dog IgM antibodies, respectively; the anti-dog IgG antibodies can be selected from rabbit anti-dog IgG antibodies and / or mouse anti-dog IgG antibodies; the anti-dog IgM antibodies can be selected from rabbit anti-dog IgM antibodies and / or mouse anti-dog IgM antibodies; 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 a quality control line coated with an anti-chicken IgY antibody; the anti-chicken IgY antibody can be selected from a goat anti-chicken IgY antibody; A blood filter membrane is provided between the sample pad and the gold label binding pad.

[0045] Second, the present invention relates to a method for preparing the canine insect-borne disease five-in-one antibody detection card, comprising the following steps: S1: applying gold conjugate solutions containing the five detection antigens and colloidal gold conjugates onto five gold conjugate pads and drying; S2: Assemble the gold-labeled conjugate pad treated in step S1 with the sample pad, nitrocellulose membrane, and absorbent pad to form a reagent strip.

[0046] In some specific embodiments, in step S1, the method for preparing the gold conjugate solution includes the following steps: adding a trisodium citrate aqueous solution to a chloroauric acid aqueous solution, adjusting the pH value to 7.5-8.5, adding the five detection antigens respectively, and then adding bovine serum albumin (BSA) and polyethylene glycol (PEG) in sequence. After sufficient reaction, centrifugation is performed to collect the precipitate and disperse it in water.

[0047] In some embodiments, in step S1, the conjugate of Leishmania antigen and colloidal gold in the gold conjugate solution 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.

[0048] In some specific embodiments, in step S1, the gold conjugate solution further contains a conjugate of chicken IgY and colloidal gold; in the gold conjugate solution, the conjugate of chicken IgY and colloidal gold is used at a final concentration of OD 540 =1.2~25.

[0049] In some specific embodiments, before step S2, a treatment solution is applied to the sample pad and then dried; the treatment solution comprises the following components in the following concentrations: 8-10 g / L bovine serum albumin (BSA), 10-12 g / L tromethamine (Tris), 8-10 g / L polyvinylpyrrolidone (PVP), 5-8 g / L Tween 20 (Tween 20), 8-11 g / L surfactant S9 (Tetronic 1307), 13-15 g / L casein (Casein), and 0.01-0.05 wt% antibacterial agent.

[0050] In some specific embodiments, before step S2, the nitrocellulose membrane is treated as follows: 0.8-1.0 mg / mL anti-chicken IgY antibody solution, 1.0-1.5 mg / mL anti-dog IgG antibody solution, and 1.0-1.8 mg / mL anti-dog IgM antibody solution are sprayed onto the control line and two test lines of the nitrocellulose membrane at a spray volume of 0.9 μL / cm, respectively, and dried.

[0051] Third, the present invention relates to a canine insect-borne disease five-in-one antibody detection kit, including the canine insect-borne disease five-in-one antibody detection card.

[0052] In some embodiments, the canine vector-borne disease five-in-one antibody detection kit further includes a buffer. Optionally or preferably, the buffer has a pH of 7.2-7.8 and includes the following components at the following concentrations: 4-6 g / L NaCl, 10-12 g / L EDTA, 6-7 g / L casein, 20-30 mmol / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), and 0.01-0.05 wt% antibacterial agent.

[0053] The present invention is described below by way of specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, any changes and advantages that can be imagined 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.

[0054] Example 1 A five-antibody test kit for canine vector-borne diseases consists of a buffer solution and a five-antibody test card. The buffer solution has a pH of 7.2 and a formula of 5g / L NaCl, 10g / L EDTA, 6.5g / L casein, 20mmol / L HEPES, and 0.02wt% Proclin 300. The solvent is deionized water.

[0055] The structure of the canine vector-borne disease five-in-one antibody test card is as follows: Figures 1 to 3 As shown, the device comprises an upper shell 1 and a lower shell 2 connected by a snap-fit ​​structure. The upper shell 1 is provided with five result reading windows 3, each with a sample loading well 4 below it. The lower shell 2 is provided with five reagent strip slots 5, each containing a reagent strip 6. Two rows of reagent strip baffles 7 are provided on the inner surface of the upper shell 1 for securing the reagent strips 6, and the lower shell 2 is provided with reagent strip staples 8 for securing the reagent strips 6.

[0056] The five reagent strips 6 are numbered as reagent strip I to reagent strip V, and their uses and structures are as follows: (1) Reagent strip I: used to detect IgG and IgM antibodies corresponding to canine leishmaniasis. It consists of a base plate I and a sample pad I, a blood filter membrane I, a gold-labeled binding pad I, a nitrocellulose membrane I, and a water-absorbing pad I stacked on the base plate I in sequence. The gold-labeled binding 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 sheep anti-chicken IgY antibody.

[0057] (2) Reagent strip II: used to detect IgG and IgM antibodies corresponding to canine ehrlichiosis. It consists of a base plate II and a sample pad II, a blood filter membrane II, a gold-labeled binding pad II, a nitrocellulose membrane II, and a water-absorbing pad II stacked on the base plate II. The gold-labeled binding 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 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-dog IgG antibody, the test line T4 is coated with mouse anti-dog IgM antibody, and the quality control line C2 is coated with sheep anti-chicken IgY antibody.

[0058] (3) Reagent strip III: used to detect IgG and IgM antibodies corresponding to canine babesiosis. It consists of a base plate III and a sample pad III, a blood filter membrane III, a gold-labeled binding pad III, a nitrocellulose membrane III, and a water-absorbing pad III stacked on the base plate III. The gold-labeled binding 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-dog IgG antibody, the test line T6 is coated with mouse anti-dog IgM antibody, and the quality control line C3 is coated with sheep anti-chicken IgY antibody.

[0059] (4) Reagent strip IV: used to detect IgG and IgM antibodies corresponding to canine anaplasmosis. It consists of a base plate IV and a sample pad IV, a blood filter membrane IV, a gold-labeled binding pad IV, a nitrocellulose membrane IV, and a water-absorbing pad IV stacked on the base plate IV. The gold-labeled binding pad IV is coated with a conjugate of chicken IgY and colloidal gold and a conjugate of anaplasmosis antigen and colloidal gold. The anaplasmosis 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 mouse anti-dog IgG antibody, the test line T8 is coated with mouse anti-dog IgM antibody, and the quality control line C4 is coated with sheep anti-chicken IgY antibody.

[0060] (5) Reagent strip V: used to detect IgG and IgM antibodies corresponding to canine Lyme disease. It consists of a base plate V and a sample pad V, a blood filter membrane V, a gold-labeled conjugate pad V, a nitrocellulose membrane V, and a water-absorbing pad V stacked on the base plate V in sequence. 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 rabbit anti-dog IgG antibody, the test line T10 is coated with rabbit anti-dog IgM antibody, and the quality control line C5 is coated with sheep anti-chicken IgY antibody.

[0061] The canine vector-borne disease five-in-one antibody test card in this embodiment is prepared by the following steps: S1: Prepare reagent strip I S1.1: Apply the treatment solution to sample pad I and dry. The treatment solution formula 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% Proclin 300, and the solvent is deionized water.

[0062] S1.2: Heat 100 mL of 0.01 wt% chloroauric acid aqueous solution to boiling, then add 2.5 mL of 1 wt% trisodium citrate aqueous solution. Let react for 20 minutes, then cool to obtain a colloidal gold solution. Adjust the pH to 8 with 1 wt% K2CO3 solution, then add 17 μg / mL of Leishmania antigen (a fusion protein of rk39, rk26, and rk9). Then, add 2 wt% BSA solution and 0.1 wt% PEG20000 solution (1 mL of PEG20000 solution and 1 mL of BSA solution per 100 mL of colloidal gold solution). Incubate at 96°C for 15 minutes, then cool. Centrifuge at 6°C and 12,000 rpm for 30 minutes. Discard the supernatant, collect the precipitate, and disperse it in deionized water to prepare gold conjugate solution I. Measure the OD value at an absorption wavelength of 540 nm.

[0063] 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 only difference from step S1.2 is that the Leishmania antigen is replaced with chicken IgY.

[0064] S1.4: Mix the gold labeled solution I and the chicken IgY gold labeled solution to obtain the gold labeled mixed solution I. The amount of gold labeled solution I and chicken IgY gold labeled solution is determined by the OD values ​​of the two solutions. 540 The final concentration of the conjugate of Leishmania antigen and colloidal gold in the gold label mixture I was OD 540 The value is 3, the conjugate of chicken IgY and colloidal gold uses a final concentration OD 540 The value was 1.2, and the insufficient amount was supplemented with PBS buffer. The gold-labeled mixture was coated onto the gold-labeled conjugate pad I (polyester film) at a coating volume of 2 μL / cm and freeze-dried at -50°C for 2 h.

[0065] S1.5: Using 1× PBS buffer, dilute 3.6 mg / mL rabbit anti-canine IgG antibody solution to 1 mg / mL, 2.4 mg / mL rabbit anti-canine IgM antibody solution to 1.2 mg / mL, and 5 mg / mL goat anti-chicken IgY antibody solution to 0.8 mg / mL. Then, spray the diluted rabbit anti-canine IgG antibody solution, rabbit anti-canine IgM antibody solution, and goat anti-chicken IgY antibody solution onto nitrocellulose membrane I at test line T1, test line T2, and control line C1, respectively, at a spray volume of 0.9 μL / cm. Dry at 37°C for 12 h.

[0066] S1.6: Assemble and cut the sample pad I treated in step S1.1, the blood filter membrane I, the gold label conjugate pad I treated in step S1.4, the nitrocellulose membrane I treated in step S1.5, and the absorbent pad I in sequence on the base plate I (made of polyvinyl chloride) to obtain a test strip I.

[0067] S2: Prepare reagent strip II Prepare the reagent strip II according to the method in step S1, the only difference from step S1 is: In step S1.2, the Leishmania antigen was replaced with an Ehrlichia antigen (gp19 protein); In step S1.5, the 3.6 mg / mL rabbit anti-canine IgG antibody solution was replaced with a 7 mg / mL mouse anti-canine IgG antibody solution, and the concentration after dilution was 1.5 mg / mL; the 2.4 mg / mL rabbit anti-canine IgM antibody solution was replaced with an 8.1 mg / mL mouse anti-canine IgM antibody solution, and the concentration after dilution was 1.8 mg / mL.

[0068] S3: Prepare reagent strips III Prepare the reagent strip III according to the method in step S1, the only difference from step S1 is: In step S1.2, the Leishmania antigen was replaced with a Babesia antigen (a fusion protein of TRAP and rBcMSA1 / rBcSA1) to obtain gold conjugate solution III; In step S1.4, the gold label solution III and the chicken IgY gold label solution were mixed to obtain the gold label mixed solution III. The conjugate of Babesia antigen and colloidal gold in the gold label mixed solution III had a final concentration of OD 540 The value is 60, and the conjugate of chicken IgY and colloidal gold uses a final concentration OD 540 The value is 25; after the coating of the gold label mixture III is completed, the freeze-drying is changed to drying at 37 ° C for 12 h; In step S1.5, the 3.6 mg / mL rabbit anti-canine IgG antibody solution was replaced with a 7 mg / mL mouse anti-canine IgG antibody solution, and the concentration after dilution was 1.5 mg / mL; the 2.4 mg / mL rabbit anti-canine IgM antibody solution was replaced with an 8.1 mg / mL mouse anti-canine IgM antibody solution, and the concentration after dilution was 1.8 mg / mL.

[0069] S4: Prepare reagent strip IV Prepare the reagent strip IV according to the method in step S1, the only difference from step S1 is: In step S1.2, the Leishmania antigen was replaced with an Anaplasma antigen (a fusion protein of P44 and OMP-1X) to obtain gold conjugate IV; In step S1.4, the gold labeled solution IV and the chicken IgY gold labeled solution were mixed to obtain the gold labeled mixed solution IV. The conjugate of the Anaplasma antigen and colloidal gold in the gold labeled mixed solution IV had a final concentration of OD 540 The value is 2.5; In step S1.5, the 3.6 mg / mL rabbit anti-canine IgG antibody solution was replaced with a 7 mg / mL mouse anti-canine IgG antibody solution, and the concentration after dilution was 1.5 mg / mL; the 2.4 mg / mL rabbit anti-canine IgM antibody solution was replaced with an 8.1 mg / mL mouse anti-canine IgM antibody solution, and the concentration after dilution was 1.8 mg / mL.

[0070] S5: Prepare reagent strips V Prepare the reagent strip V according to the method in step S1, the only difference from step S1 is: In step S1.2, the Leishmania antigen was replaced with the Lyme antigen (OspC outer membrane protein of Borrelia burgdorferi) to obtain the gold conjugate solution V; In step S1.4, gold conjugate solution V and chicken IgY gold conjugate solution were mixed to obtain gold conjugate solution V. The conjugate of Lyme antigen and colloidal gold in gold conjugate solution V had a final concentration of OD 540The value is 50, and the conjugate of chicken IgY and colloidal gold uses a final concentration OD 540 The value is 25; after the coating of the gold-labeled mixed solution V is completed, the freeze-drying is changed to drying at 37°C for 12 hours.

[0071] S6: Assemble the test card Assemble test strips I to V into the reagent strip slot in the lower shell in sequence, fasten the upper shell and the lower shell to obtain the canine insect-borne disease five-in-one antibody test card.

[0072] The canine insect-borne disease five-in-one antibody detection kit in this embodiment is used to detect the test sample. The steps are as follows: after adding 10 μL of the test sample to each of the five sample wells, two drops of buffer solution are added to each sample well, and the color development of the test line and the quality control line is observed in the result reading window.

[0073] Following the above steps, the test samples listed in Table 1 (sampled from dogs, with negative and positive results for canine vector-borne disease-related antibodies) were tested to verify the accuracy of the test kit of this embodiment. The test results for each test sample showed the appearance of purple-red bands at control lines C1, C2, C3, C4, and C5, indicating that the test card was valid. The color development at each test line is shown in Table 1 (where "+" indicates the appearance of a purple-red band, a greater number of "+"s indicates a darker band color, and "-" indicates the absence of a purple-red band). This is consistent with the actual results of each sample, demonstrating that the test kit of this embodiment has high detection accuracy, a high detection rate, and high specificity, and that the detection of the five canine vector-borne diseases does not interfere with each other. In the testing of whole blood, plasma, and serum samples, the membrane surface was clean and the results were clear and effective, indicating that the test kit of this embodiment can prevent the presence of canine red blood cells in the test sample from causing the reagent strip membrane surface to turn red.

[0074] Table 1 Test results of the canine vector-borne disease five-in-one antibody detection kit of Example 1

[0075] Example 2

[0076] The only difference between this embodiment and embodiment 1 is that the blood filter membranes I to V are not provided in the reagent strips I to V. The rest is the same as in embodiment 1.

[0077] When the kit of this example was used to test canine plasma and serum samples, the membrane surface was clean and the results were clearly and effectively read. However, when testing canine whole blood samples, after adding the buffer solution and letting it stand for 10 minutes, it was clearly observed that the membrane background of all five result reading windows appeared red, affecting the accuracy of the interpretation results.

[0078] Comparative Example 1 The only difference between this comparative example and Example 1 is that the detection lines T2, T4, T6, T8, and T10 are not provided in the reagent strips I to V. The rest are the same as in Example 1.

[0079] When verifying the detection accuracy of the kit of this comparative example, the test samples used were the same as those in Example 1. The test results of each test sample 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 any test line, indicating that this comparative example had false negative results in the detection of IgG-positive samples.

[0080] Comparative Example 2 The only difference between this comparative example and Example 1 is that the detection lines T1, T3, T5, T7, and T9 are not provided in the reagent strips I to V. The rest are the same as in Example 1.

[0081] When verifying the detection accuracy of the kit of this comparative example, the test samples used were the same as those in Example 1. The test results of each test sample showed that when testing 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 purple-red bands appeared at any of the test lines, indicating that this comparative example had false negative results in the detection of IgM-positive samples.

[0082] Comparative Example 3 This comparative example differs from Example 1 only in 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; and in test strip IV, the fusion protein of P44 and OMP-1X is replaced with OMP-1X protein. All other procedures are the same as in Example 1.

[0083] When verifying the detection accuracy of the kit of this comparative example, the test samples used were the 12 samples corresponding to canine leishmaniasis, canine babesiosis, and canine anaplasmosis in Table 1. The color development at each test line in the test results of each test sample is shown in Table 2.

[0084] Table 2 Test results of the canine vector-borne disease five-in-one antibody detection kit of Comparative Example 3

[0085] Table 2 shows that this comparative example exhibited false-negative results when testing for antibodies corresponding to canine leishmaniasis, canine babesiosis, and canine anaplasmosis. This is due to the following: when using the rK39 protein as a detection antigen for canine leishmaniasis, some dogs with canine leishmaniasis in Brazil and India were missed; the common subtypes of Babesia infecting dogs are Babesia microphylla (Babesia gibbsii) and Babesia macrophylla (Babesia canis); using only the detection proteins rBcMSA1 / rBcSA1 for Babesia canis would not detect Babesia gibbsii; and Anaplasma infecting dogs primarily includes two subtypes, Anaplasma phagocytophilum and Anaplasma planulum; using only the detection protein OMP-1X for Anaplasma planulum would reduce the detection of Anaplasma phagocytophilum. Therefore, compared to using a single antigen, using the fusion protein of the present invention can improve the detection rate.

[0086] Comparative Examples 4 to 6 The only difference between Comparative Examples 4 to 6 and Example 1 is that in test strip 1, the fusion proteins of rk39, rk26, and rk9 are replaced with the fusion protein of rk39 and rk9, the KMP11 protein, and the fusion protein of rk26 and KMP11, respectively. The rest are the same as in Example 1.

[0087] When verifying the detection accuracy of the test kits of Comparative Examples 4 to 6, the test samples used were the four samples corresponding to canine leishmaniasis in Table 1. The color development at the detection lines T1 and T2 on the reagent strip I in the test results of each test sample is shown in Table 3.

[0088] Table 3 Test results of the canine insect-borne disease five-in-one antibody detection kit for comparative examples 4 to 6

[0089] It can be seen from Tables 1 and 3 that when the fusion protein of rk39 and rk9 (Comparative Example 4) or KMP11 protein (Comparative Example 5) was used, there were a certain degree of false negative results in different positive samples; when KMP11 was used to replace rk39 and rk9 in the fusion protein of rk39, rk26 and rk9 (Comparative Example 6), although no false negative results occurred in the detection of 3 positive samples, the color was lighter than that in Example 1, indicating that the detection sensitivity was reduced.

[0090] Comparative Examples 7 to 9 Comparative Examples 7 to 9 differ from Example 1 only in that in test strip III, the fusion protein of TRAP and rBcMSA1 / rBcSA1 is replaced with a fusion protein of TRAP protein, HSP70 protein, and HSP70 and rBcMSA1 / rBcSA1, respectively. The rest of the steps are the same as in Example 1.

[0091] When verifying the accuracy of the test kits in Comparative Examples 7 to 9, the test samples used were the four canine babesiosis samples listed in Table 1. The color development at test lines T5 and T6 on test strip III for each test sample is shown in Table 4.

[0092] Table 4 Test results of the canine vector-borne disease five-in-one antibody detection kit for comparative examples 7 to 9

[0093] As shown in Tables 1 and 4, false negative results were observed when using either TRAP protein (Comparative Example 7) or HSP70 protein (Comparative Example 8), and the detection sensitivity of TRAP protein was lower than that of HSP70 protein. However, when fused with rBcMSA1 / rBcSA1, the detection sensitivity of the TRAP / rBcMSA1 / rBcSA1 fusion protein (Example 1) was higher than that of the HSP7 / rBcMSA1 / rBcSA1 fusion protein (Comparative Example 9). These results demonstrate that the detection sensitivity of a single antigen can be affected when it is used as a fusion protein, and the detection sensitivity of a fusion protein is not necessarily consistent with that of a single antigen.

[0094] Comparative Example 10~Comparative Example 12 Comparative Examples 10 to 12 differ from Example 1 only in that, in test strip IV, the fusion proteins of P44 and OMP-1X are replaced with Tr1 protein, P44 protein, and fusion proteins of Tr1 and OMP-1X, respectively. The rest are the same as in Example 1.

[0095] When verifying the detection accuracy of the test kits of Comparative Examples 10 to 12, the test samples used were the four samples corresponding to canine anaplasmosis in Table 1. The color development at the detection lines T7 and T8 on the test strip IV in the test results of each test sample is shown in Table 5.

[0096] Table 5 Test results of the canine vector-borne disease five-in-one antibody detection kit for comparative examples 10 to 12

[0097] As shown in Tables 1 and 5, false negative results were observed when using either the Tr1 protein (Comparative Example 10) or the P44 protein (Comparative Example 11) alone, and the detection sensitivity of the two proteins was similar. However, when fused with OMP-1X, the P44 / OMP-1X fusion protein (Example 1) exhibited higher detection sensitivity than the Tr1 / OMP-1X fusion protein (Comparative Example 12). These results also demonstrate that the detection sensitivity of a single antigen can be affected when it is used to form a fusion protein, and the detection sensitivity of a fusion protein is not necessarily consistent with that of a single antigen.

[0098] Comparative Example 13 This comparative example differs from Example 1 only in that chicken IgY is replaced with mouse IgG on the five gold-labeled conjugate pads (Gold-labeled conjugate pads I through V); goat anti-chicken IgY antibody is replaced with goat anti-mouse IgG on the five control lines (Control lines C1 through C5); and 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, resulting in a 1 mg / mL concentration after dilution. All other procedures are the same as in Example 1.

[0099] When verifying the detection accuracy of the kit of this comparative example, the test samples used were the five negative whole blood samples in Table 1. In the test results of each test sample, the color development at each test line is shown in Table 6.

[0100] Table 6 Test results of the canine vector-borne disease five-in-one antibody detection kit of Comparative Example 13

[0101] As can be seen from Table 6, in this comparative example, when reagent strip I tested negative whole blood sample -2 and reagent strip V tested negative whole blood sample -4, false positive readings were generated, indicating that the nonspecific binding of the mouse IgG-goat anti-mouse IgG quality control system to the antibody interfered with the detection line T, resulting in erroneous results.

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this disclosure pertains. Unless otherwise specified, the raw materials and equipment used herein are conventional in the art and can be obtained from conventional commercial sources. The methods used herein are conventional in the art, unless otherwise specified.

[0103] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution 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 and rBcMSA1 / 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.

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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