ELISA diagnostic kit for cattle and sheep universal echinococcosis and application thereof

Abstract

The invention relates to the technical field of molecular biology detection, in particular to a cattle and sheep universal type echinococcosis ELISA diagnostic kit and application thereof.The cattle and sheep universal type echinococcosis ELISA diagnostic kit comprises an SPG-HRP conjugate, the SPG-HRP conjugate is obtained by coupling SPG recombinant protein and horse radish peroxidase, the SPG recombinant protein comprises r2CSPG and r3CSPG, the amino acid sequence of the r2CSPG recombinant protein is shown as SEQ ID NO.1, the amino acid sequence of the r3CSPG recombinant protein is shown as SEQ ID NO.2, and the amino acid sequence of the r3CSPG recombinant protein is shown as SEQ ID NO.2. The amino acid sequence of the r3CSPG recombinant protein is as shown in SEQ ID NO. 2. The problem of how to apply the streptococcus protein G to early screening and early diagnosis of echinococcosis is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology detection, and particularly relates to a general ELISA diagnostic kit for echinococcosis of cattle and sheep and application thereof. BACKGROUND

[0002] Echinococcosis, commonly known as echinococcosis, is a two-host parasitic disease caused by Echinococcus adult and larva, which is prevalent in the world, mainly in the grazing or semi-agricultural and pastoral areas. Human and animal parasitic tapeworm disease / cystic echinococcosis, cystic echinococcosis and alveolar echinococcosis are considered to be the first, second and third related foodborne parasitic diseases in the world.

[0003] Cattle and sheep are the main intermediate hosts of echinococcosis, therefore, early diagnosis of echinococcosis is the key to control the disease. Streptococcus Protein G (SPG) is a protein that can combine with IgG of various mammals, and is widely used for purification of recombinant proteins and antibodies.

[0004] In view of the above defects, the present inventors have finally obtained the present application after long-term research and practice. SUMMARY

[0005] The present application aims to solve the problem of how to apply Streptococcus Protein G to early screening and early diagnosis of echinococcosis, and provides a general ELISA diagnostic kit for echinococcosis of cattle and sheep and application thereof.

[0006] In order to achieve the above-mentioned purpose, the present application discloses a general ELISA diagnostic kit for echinococcosis of cattle and sheep, which comprises a SPG-HRP conjugate, wherein the SPG-HRP conjugate is obtained by coupling a SPG recombinant protein with horseradish peroxidase, the SPG recombinant protein comprises r2CSPG and r3CSPG, the amino acid sequence of the r2CSPG recombinant protein is shown as SEQ ID NO. 1, and the amino acid sequence of the r3CSPG recombinant protein is shown as SEQ ID NO. 2.

[0007] Amino acid sequence of r2CSPG recombinant protein (SEQ ID NO. 1):

[0008] GSTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKAFKQYANDNGVDGEWTYDDATKTFTVTE.

[0009] Amino acid sequence of r3CSPG recombinant protein (SEQ ID NO. 2):

[0010] GSTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKAFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTE.

[0011] Nucleotide sequence encoding r2CSPG recombinant protein (SEQ ID NO. 3):

[0012] GGATCCACGTATAAATTAGTTATTAATGGTAAAACATTAAAAGGAGAAACTACTACTGAAGCAGTTGATGCAGCAACAGCTGAAAAAGTTTTTAAACAATATGCTAATGATAATGGAGTAGATGGTGAATGGACATATGATGATGCTACTAAAACATTTACAGTAACTGAAAAACCAGAAGTAATAGATGCTAGTGAATTAACTCCTGCAGTAACAACATATAAATTAGTAATTAATGGTAAAACATTAAAAGGTGAAACAACAACAGAAGCTGTTGATGCTGCTACTGCTGAAAAAGCATTTAAACAATATGCTAATGATAATGGTGTTGATGGTGAATGGACATATGATGATGCAACAAAAACATTTACAGTAACTGAATAAGAATTC.

[0013] Nucleotide sequence encoding r2CSPG recombinant protein (SEQ ID NO. 4):

[0014] .

[0015] The preparation method of the SPG-HRP conjugate includes the following steps:

[0016] S1, dissolve horseradish peroxide in acetate buffer, add anhydrous ethanol solution, stir gently at room temperature, add NaIO4, place at 4℃ for 30 min, then add ethylene glycol, and stir gently at room temperature to carry out the reaction.

[0017] S2, then add carbonate buffer for the SPG recombinant protein to be labeled, mix well, and incubate at 4°C overnight;

[0018] S3, add sodium borohydride solution, mix well, place at 4℃ for 3h, dialyze, centrifuge at 3000r / min for 30min, remove precipitate and collect supernatant to obtain SPG-HRP conjugate.

[0019] This invention also discloses the application of the above-mentioned universal echinococcosis ELISA diagnostic kit for cattle and sheep in the preparation of reagents for detecting echinococcosis in cattle and sheep.

[0020] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention modifies the C region of the SPG gene using genetic engineering technology to construct two recombinant proteins, r2CSPG and r3CSPG, and optimizes detection conditions by combining them with laboratory-preserved rEgSeverin antigen. Ultimately, it demonstrates excellent performance in specificity, sensitivity, and repeatability, providing a new technical means for early screening and epidemiological monitoring of echinococcosis in cattle and sheep. The widespread application of this method is expected to improve the detection rate of echinococcosis in pastoral areas, facilitate the implementation of the integrated prevention and control strategy of "livestock deworming - human protection," and is of great significance for reducing the prevalence of echinococcosis and ensuring the economic and public health security of animal husbandry. Attached Figure Description

[0021] Figure 1 The results are as follows: A represents the PCR identification result of the recombinant plasmid pET-28a(+)-r2CSPG, B represents the PCR identification result of the recombinant plasmid pET-28a(+)-r3CSPG, C represents the double enzyme digestion identification result of the recombinant plasmid pET-28a(+)-r2CSPG, and D represents the double enzyme digestion identification result of the recombinant plasmid pET-28a(+)-r3CSPG.

[0022] Figure 2 SDS-PAGE was used to identify r2CSPG and r3CSPG proteins. A: r2CSPG protein induction results: 1: no IPTG induction; 2-7: IPTG induction for 1h, 2h, 3h, 4h, 6h, and 8h; B: r3CSPG protein induction results: 1: no IPTG induction; 2-7: IPTG induction for 1h, 2h, 3h, 4h, 6h, and 8h; C: r2CSPG protein purification results: 1-6: recombinant protein eluted with 20mM, 40mM, 80mM, 100mM, 120mM, 150mM, and 200mM imidazole; D: r3CSPG protein purification results: 1-6: recombinant protein eluted with 20mM, 40mM, 80mM, 100mM, 120mM, 150mM, and 200mM imidazole.

[0023] Figure 3 Results of Western blot analysis of r2CSPG and r3CSPG;

[0024] Figure 4 The results are for indirect ELISA sensitivity and specificity analysis, where A is the sensitivity and specificity analysis of sheep serum rEgSeverin-ELISA, and B is the sensitivity and specificity analysis of bovine serum rEgSeverin-ELISA.

[0025] Figure 5The results of positive serological cross-reactivity tests for Echinococcus aureus, Toxoplasma gondii, Cryptosporidium, Coccidia, and Sarcocystis. Detailed Implementation

[0026] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0027] The reagents and experimental materials used in the experiment were sourced from the following sources:

[0028] Escherichia coli (E. coli) BL21 (DE3) was purchased from Beijing TransGen Biotech Co., Ltd.; horseradish peroxidase was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.; trichrome prestained protein molecular weight standard and BCA protein concentration assay kit were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; TMB substrate solution and stop solution were purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0029] Standard positive serum, negative serum, sample serum, and recombinant EgSeverin protein from the echinococcosis control group were all preserved in the Animal-Derived Food Safety Laboratory of the Shanghai Veterinary Research Institute.

[0030] I. Expression plasmid construction

[0031] The C region gene fragment encoding SPG was obtained from GenBank. Regions C1, C2, C3, and D were identified. The gene fragments in regions C1 and C2 were replaced with the gene fragment in region C3, resulting in the C3DC3DC3 (r3CSPG) gene sequence. Simultaneously, the gene fragment in region C2 was replaced with the gene fragment in region C3, resulting in the C1DC3DC3 (r2CSPG) gene sequence. The recombinant gene sequences were synthesized by Sangon Biotech Co., Ltd. The synthesized r2CSPG and r3CSPG gene sequences were subjected to PCR reaction and gel extraction. The purified fragments were ligated with the digested pET-28a(+) empty vector overnight at 16°C using ligase to construct the pET-28a(+)-r2CSPG and pET-28a(+)-r3CSPG recombinant plasmids. These plasmids were then transformed into E. coli BL21 and plated onto LB solid medium containing kanamycin and cultured overnight at 37°C. Several single colonies were then picked for expansion culture, followed by bacterial PCR and double enzyme digestion identification, and finally sent to the company for sequencing.

[0032] The two recombinant plasmids were verified by PCR and identified by double enzyme digestion. Figure 1 The sequencing results showed that the sequence was consistent with the designed sequence. The sizes of the target gene fragments were approximately 390 bp and 600 bp, respectively.

[0033] II. Protein Expression and Purification

[0034] Three mL of bacterial cultures containing the correctly sequenced pET-28a(+)-r2CSPG and pET-28a(+)-r3CSPG recombinant plasmids, respectively, were inoculated into 300 mL of LB broth containing Kan+ and incubated at 37°C with shaking. When the culture reached the logarithmic growth phase, IPTG was added to a final concentration of 1 mmol / L for induction. One mL of bacterial culture was collected before induction and at 1 h, 2 h, 3 h, 4 h, 6 h, and 8 h after induction. The optimal induction time was analyzed by SDS-PAGE electrophoresis. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in PBS. The precipitate was sonicated in an ice-water bath for 30 min, then centrifuged again, and the precipitate and supernatant were collected. The precipitate was resuspended in 8 mol of urea, and the centrifugation process was repeated, collecting the supernatant. Finally, the supernatant after sonication and the supernatant after precipitate resuspending were added to equal volumes of protein electrophoresis buffer, and the solubility of the expression product was analyzed by SDS-PAGE electrophoresis. The protein was purified using the Ni-TED 6FF His-tagged protein purification kit. After purification, the protein was stained with Coomassie Brilliant Blue, and the results showed a clear molecular weight band. (See attached image). Figure 2 .

[0035] III. Western blot experiment

[0036] After transferring r2CSPG and r3CSPG proteins onto a PVDF membrane, it was blocked with 5% skim milk powder and incubated at room temperature for 2 hours. Then, HRP-labeled goat anti-mouse IgG was added as an antibody, and the membrane was incubated overnight at 4 °C. After incubation, the membrane was washed three times with PBST solution, followed by three more washes with PBST solution. The PVDF membrane was then treated with HRP substrate chromogenic solution and developed in a developer.

[0037] Western blotting analysis showed that both His-r2CSPG and His-r3CSPG reacted with HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:5000 (see [link to Western blotting]). Figure 3 Recognition bands were observed at 26 kDa and 35 kDa, demonstrating that both proteins have the ability to bind to IgG.

[0038] IV. Preparation of SPG-HRP Conjugates

[0039] Dissolve 5 mg of HRP in 1 mL of 0.2 mol / L acetate buffer, add 0.1 mL of anhydrous ethanol solution, and stir gently at room temperature for 1 h. Add 0.5 mL of freshly prepared 0.1 mol / L NaIO4. The solution changes from brown to dark green. Incubate at 4°C for 30 min. Add 1 mL of ethylene glycol and stir gently at room temperature for 1 h to terminate the reaction. Add 5-10 mg of the SPG to be labeled, mix with carbonate buffer, and incubate at 4°C overnight. Add 0.1 mL of sodium borohydride solution, mix, and incubate at 4°C for 3 h. Dialyze, centrifuge at 3000 rpm for 30 min, remove the precipitate, and collect the supernatant as the SPG-HRP conjugate (store with 30-50% glycerol).

[0040] V. SPG-HRP Binding Viability Detection

[0041] IgG from different species was diluted 1:100 and coated onto ELISA plates, 100 μL per well, with three replicates, and incubated overnight at 4°C. The conjugated antibodies 2CSPG and 3CSPG were serially diluted from 1:100 to 1:204800 and subjected to direct ELISA assays to obtain OD values ​​for different species, comparing their binding affinity to IgG from different species.

[0042] The binding capacity of SPG-HRP to human serum is shown in Table 1: the binding capacity of 3CSPG-HRP is higher than that of 2CSPG-HRP. When the human serum dilution ratio is 1:25,600, the OD value of 2CSPG-HRP begins to be less than 1, while when the dilution ratio is 1:204,800, the OD value of 3CSPG-HRP is still greater than 2.

[0043] Table 1. Binding ability of SPG-HRP to human serum

[0044]

[0045] Note: "***" indicates that the OD value is outside the reading range of the microplate reader.

[0046] The binding ability of SPG-HRP to bovine serum is shown in Table 2: the binding ability of 3CSPG-HRP is slightly better than that of 2CSPG-HRP. When the bovine serum dilution ratio is 1:51,200, the OD value of 2CSPG-HRP begins to be less than 1, while when the dilution ratio is 1:102,400, the OD value of 3CSPG-HRP begins to be less than 1.

[0047] Table 2. Binding ability of SPG-HRP to bovine serum

[0048]

[0049] Note: "***" indicates that the OD value is outside the reading range of the microplate reader.

[0050] The binding ability of SPG-HRP to sheep serum is shown in Table 3: the binding ability of 3CSPG-HRP is better than that of 2CSPG-HRP. When the sheep serum dilution ratio is 1:25,600, the OD value of 2CSPG-HRP begins to be less than 1, while when the dilution ratio is 1:102,400, the OD value of 3CSPG-HRP begins to be less than 1.

[0051] Table 3. Binding ability of SPG-HRP to sheep serum

[0052]

[0053] Note: "***" indicates that the OD value is outside the reading range of the microplate reader.

[0054] The binding ability of SPG-HRP to canine serum is shown in Table 4: the binding ability of 3CSPG-HRP is better than that of 2CSPG-HRP. When the canine serum is diluted at a ratio of 1:800, the OD value of 2CSPG-HRP begins to be less than 1, while when the dilution ratio is 1:3,200, the OD value of 3CSPG-HRP begins to be less than 1.

[0055] Table 4. Binding ability of SPG-HRP to canine serum

[0056]

[0057] The binding ability of SPG-HRP to rabbit serum is shown in Table 5: the binding ability of 3CSPG-HRP is better than that of 2CSPG-HRP. When the rabbit serum dilution ratio is 1:25,600, the OD value of 2CSPG-HRP begins to be less than 1, while when the dilution ratio is 1:102,400, the OD value of 3CSPG-HRP begins to be less than 1.

[0058] Table 5. Binding ability of SPG-HRP to rabbit serum

[0059]

[0060] Note: "***" indicates that the OD value is outside the reading range of the microplate reader.

[0061] The binding ability of SPG-HRP to mouse serum is shown in Table 6: the binding ability of 3CSPG-HRP is better than that of 2CSPG-HRP. When the mouse serum dilution ratio is 1:200, the OD value of 2CSPG-HRP begins to be less than 1, while when the dilution ratio is 1:800, the OD value of 3CSPG-HRP begins to be less than 1.

[0062] Table 6. Binding ability of SPG-HRP to mouse serum

[0063]

[0064] In summary, 3CSPG-HRP has a better binding capacity than 2CSPG-HRP. SPG-HRP has a high binding capacity with human, rabbit, bovine, and sheep serum, but a poor binding capacity with dog and mouse serum.

[0065] VI. Determination of Optimal Antigen Coating Concentration and Serum Dilution

[0066] Recombinant antigen EgSeverin was diluted to 1.0, 2.0, 4.0, and 6.0 μg / mL with antigen coating buffer, 100 μL per well, with three replicates, and incubated overnight at 4°C. Serum was diluted with PBS at 1:50, 1:100, 1:200, and 1:400, 100 μL per well to obtain positive and negative serum OD. 450nm The values ​​of the two in each group are compared, and the condition with the largest P / N value is the best condition.

[0067] Different concentrations of recombinant antigen EgSeverin and different serum dilutions were used to coat the sheep serum. The results showed that the optimal coating concentration of rEgSeverin in sheep serum was 2.0 μg / mL, and the optimal serum dilution was 1:50 (Table 7). The optimal coating concentration of rEgSeverin in bovine serum was 1.0 μg / mL, and the optimal serum dilution was 1:50 (Table 8).

[0068] Table 7 Determination of optimal rEgSeverin antigen coating concentration and sheep serum dilution

[0069]

[0070] Table 8 Determination of optimal rEgSeverin antigen coating concentration and bovine serum dilution

[0071]

[0072] VII. Determination of the optimal action time of serum

[0073] The procedure was performed under the optimal conditions determined above. 100 μL of serum was added per well, and the experiment was repeated three times in parallel. The serum was incubated at 37°C for 1 h, 1.5 h, and 2 h, respectively, and the OD was measured. 450nm The condition with the largest P / N value is the optimal condition.

[0074] According to the experimental results, the optimal treatment time for rEgSeverin with sheep serum is 2 h at 37℃ (Table 9), and the optimal treatment time with bovine serum is 1.5 h at 37℃ (Table 10).

[0075] Table 9 Optimal Treatment Time of rEgSeverin in Sheep Serum

[0076]

[0077] Table 10 Optimal Action Time of EgSeverin from Bovine Serum

[0078]

[0079] VIII. Determination of the optimal incubation time for conjugated enzyme-labeled secondary antibodies

[0080] The operation was performed under the optimal conditions determined above. The incubation time for the conjugated enzyme-labeled secondary antibody was set to 0.5 h, 1 h, and 1.5 h at 37°C, and the OD was measured. 450nm The condition with the largest P / N value is the optimal condition.

[0081] According to the experimental results, the optimal incubation time for rEgSeverin-conjugated enzyme-labeled secondary antibodies against bovine and ovine serum was 37℃ for 1 h (Tables 11 and 12).

[0082] Table 11 Optimal Induction Time of rEgSeverin Conjugated with Secondary Antibody (Sheep Serum)

[0083]

[0084] Table 12 Optimal Induction Time of rEgSeverin Conjugated with Secondary Antibody (Bovine Serum)

[0085]

[0086] IX. Determination of Critical Values

[0087] Under the established optimal conditions, the cutoff value was determined using 25 healthy sheep negative serum samples, with the results replicated in triplicate, and OD was measured. 450nm Value. Calculate serum OD. 450nm average value( The cut-off value and standard deviation (SD) are calculated using the formula: Cut-off = Sample Mean / Standard Deviation. +3 standard deviations, when OD 450nm Value ≥ At +3S, it is judged as positive; when OD 450nm Value < At +2S, it is judged as negative, and this is used as the positive / negative cut-off value for the indirect ELISA method.

[0088] The cutoff value was determined by testing 20 sheep negative serum samples. The results showed (Table 13) that the mean value of rEgSeverin-ELISA was 0.218, the standard deviation was 0.055, and the cutoff value was ( The +3S (+3S) value was 0.383. Theoretically, a serum OD450nm value ≥ 0.383 is considered positive, OD450nm < 0.328 is considered negative, and 0.328 ≤ OD450 ≤ 0.383 is considered suspected. The results of 20 bovine negative serum samples (Table 14) showed that the mean rEgSeverin-ELISA value was 0.338, the standard deviation was 0.045, and the cut-off (+3S) value was... The +3S value is 0.473. Theoretically, when the serum OD450nm is ≥0.473, it can be judged as positive; when OD450nm <0.428, it can be judged as negative; and when 0.428≤OD450≤0.473, it is suspected.

[0089] Table 13 ELISA results of 20 sheep negative serum samples

[0090]

[0091] Table 14 ELISA results of 20 bovine negative serum samples

[0092]

[0093] 10. Specificity and Sensitivity Tests

[0094] The established indirect ELISA method was used to detect the specificity and sensitivity of 25 positive sheep sera infected with Echinococcus granulosus and 30 negative sheep sera. The calculation formulas were: Sensitivity = True positive (above the cutoff value) / (True positive + False negative) × 100%; Specificity = True negative (below the cutoff value) / (True negative + False positive) × 100%.

[0095] In rEgSeverin-ELISA ( Figure 4 From 25 known positive sheep serum samples, 23 were positive; from 30 negative sheep serum samples, 28 were negative. Therefore, the sensitivity was 92% (23 / 25) and the specificity was 93.33% (28 / 30). From 20 known positive bovine serum samples, 19 were positive; from 30 negative sheep serum samples, 28 were negative. Therefore, the sensitivity was 96% (24 / 25) and the specificity was 93.33% (28 / 30).

[0096] XI. Repeatability Test

[0097] Under the established indirect ELISA conditions, six serum samples were randomly selected from bovine and ovine samples, with eight replicates. Simultaneously, six serum samples were randomly selected from bovine and ovine samples and coated with three different batches of purified recombinant protein, with three replicates. The CV was calculated using the formula: CV = [Standard deviation of serum OD450nm (SD) / Mean of serum OD450nm (SD)]. ] ×100%, calculate the coefficient of variance (CV) within the batch.

[0098] The results of the repeatability test in sheep serum (Table 15) showed that the highest intra-batch coefficient of variation for rEgSeverin was 9.17%, and the highest inter-batch coefficient of variation was 9.46%. The results of the repeatability test in bovine serum (Table 16) showed that the highest intra-batch coefficient of variation for rEgSeverin was 6.23%, and the highest inter-batch coefficient of variation was 8.34%, both less than 10%. This indicates that the established rEgSeverin-ELISA method has high repeatability.

[0099] Table 15 Repeatability Tests of rEgSeverin Sheep Serum

[0100]

[0101] Table 16 Repeatability Tests of rEgSeverin Bovine Serum

[0102]

[0103] 12. Cross-reactivity test

[0104] Under established conditions, rEgSeverin was used to detect positive sera for Toxoplasma gondii infection, Cryptosporidium microsporum infection, Sarcocystis infection, Trichinella infection, Coccidia infection, and Echinococcus multilocularis infection. Positive sera from Echinococcus granulosus infection and negative (healthy) sera were used as controls to observe whether there was cross-reactivity.

[0105] Cross-reactivity tests were performed with positive sera infected with *Echinococcus multilocularis*, *Toxoplasma gondii*, *Cryptospora*, *Coccidioidomyces*, and *Sarcocystis*, respectively. The results showed that ( Figure 5 The OD450nm values ​​detected by rEgSeverin-ELISA, except for the positive control, were below the cutoff value for all serum samples. These results indicate that rEgSeverin-ELISA does not exhibit cross-reactivity with serum samples from other parasites.

[0106] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. A universal ELISA diagnostic kit for echinococcosis in cattle and sheep, characterized in that, The invention includes an SPG-HRP conjugate, which is obtained by conjugating SPG recombinant protein with horseradish peroxidase. The SPG recombinant protein includes r2CSPG and r3CSPG. The amino acid sequence of the r2CSPG recombinant protein is shown in SEQ ID NO.1, and the amino acid sequence of the r3CSPG recombinant protein is shown in SEQ ID NO.

2.

2. The universal ELISA diagnostic kit for echinococcosis in cattle and sheep as described in claim 1, characterized in that, The preparation method of the SPG-HRP conjugate includes the following steps: S1, dissolve horseradish peroxide in acetate buffer, add anhydrous ethanol solution, stir gently at room temperature, add NaIO4, place at 4℃ for 30 min, then add ethylene glycol, and stir gently at room temperature to carry out the reaction. S2, then add carbonate buffer for the SPG recombinant protein to be labeled, mix well, and incubate at 4°C overnight; S3, add sodium borohydride solution, mix well, place at 4℃ for 3h, dialyze, centrifuge at 3000r / min for 30min, remove precipitate and collect supernatant to obtain SPG-HRP conjugate.

3. The application of the universal echinococcosis ELISA diagnostic kit for cattle and sheep as described in claim 1 or 2 in the preparation of reagents for detecting echinococcosis in cattle and sheep.

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