Indirect ELISA (enzyme-linked immuno sorbent assay) detection method and detection kit based on pyruvate kinase protein of Mangnogonia maindroni
By establishing an indirect ELISA detection method based on the PK protein of Spirometra mansoni, using recombinant protein as an antigen, combining specific antibodies and enzyme-labeled detection, the sensitivity and accuracy problems of existing detection methods were solved, and efficient and specific detection of Spirometra mansoni was achieved.
Patent Information
- Application Number
- CN202510875059.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-27
- Publication Date
- 2025-09-12
AI Technical Summary
Existing methods for detecting Spirometra mansoni, such as fecal smear microscopy, have low sensitivity, are time-consuming and labor-intensive, and are prone to missed detections. There is a lack of efficient and accurate indirect ELISA detection methods.
An indirect ELISA detection method was established using the PK protein of Spirometra mansoni as the coating antigen. The recombinantly expressed PK protein of Spirometra mansoni was used as the coating antigen, combined with specific primary antibodies, secondary antibodies and triggering agents. The OD value of the serum sample was detected by a microplate reader to determine the infection status.
It achieves high sensitivity, specificity and good repeatability for the detection of Spirometra mansoni, provides a new detection target, and enriches the detection methods of Spirometra mansoni.
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Figure CN120629567A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of parasite detection, and in particular to an indirect ELISA detection method and a detection kit based on Spirometra mansoni PK protein. Background Art
[0002] Spirometra mansoni (Sm) is a foodborne and waterborne zoonotic parasite belonging to the genus Spirometra, order Diplochaetes, family Diplochaetidae, class Cestoda. It is widely distributed and can infect dogs, cats, and other carnivores. The tapeworm larvae (sparganosis) can cause sparganosis in humans, a serious health threat to both humans and animals.
[0003] Currently, the commonly used detection method for Spirometra mansoni is fecal smear microscopy. However, the positive detection rate of smear microscopy is affected by the technician's experience and professional knowledge, and it is time-consuming and labor-intensive, prone to missed detections, and has many limitations. The indirect ELISA detection method has the advantages of high sensitivity, simple and fast operation, and may become an effective diagnostic method for Spirometra mansoni antibody detection. Currently, there are few reports on ELISA detection methods for Spirometra mansoni. It is particularly important to establish a rapid, accurate, and efficient indirect ELISA detection method for Spirometra mansoni.
[0004] Pyruvate kinase (PK), a key rate-limiting enzyme in the glycolysis pathway, is referred to as PK protein. Spirometra mansoni pyruvate kinase 1 (SmPK1) is 1812 bp in length, encoding 603 amino acids with a molecular weight of 65237.82 Ku and an isoelectric point of 6.96. It lacks a transmembrane region or signal peptide, contains two PK domains, eight active sites, and possesses 61 phosphorylation sites. As a key gene influencing the growth and development of Spirometra mansoni, SmPK1 is an ideal candidate target for ELISA testing. Currently, there are no reports of indirect ELISA antibody detection methods based on PK protein. Developing an indirect ELISA method based on this protein could enrich the current detection targets for Spirometra mansoni. Summary of the Invention
[0005] In view of the shortcomings of existing detection technologies, the present invention provides a pyruvate kinase recombinant prokaryotic protein as a coating antigen to establish an indirect ELISA detection method and detection kit with strong specificity and high sensitivity.
[0006] The technical solutions of the present invention are as follows:
[0007] An indirect ELISA detection kit based on Spirometra mansoni PK protein comprises a coating antigen Spirometra mansoni PK protein, a primary antibody, a labeled secondary antibody, and an initiator. The primary antibody is a mouse anti- or cat anti-Sp. mansoni PK protein antibody to be detected; the labeled secondary antibody is a goat anti-mouse IgG with a detection marker, capable of binding to Spirometra mansoni PK protein, and the labeled secondary antibody carries a detection marker.
[0008] The initiator is TMB substrate color developing solution, and the marker is horseradish peroxidase, phosphatase or luciferase.
[0009] The coating concentration of the Spirometra mansoni PK protein is 5.0 μg / mL.
[0010] The preparation method of the Spirometra mansoni PK protein comprises the following steps: cloning the coding gene of the Spirometra mansoni PK protein into a PQE80L(+) expression vector to obtain a recombinant expression plasmid, transforming the recombinant expression plasmid into a host cell, expressing the plasmid, and isolating and purifying the recombinantly expressed Spirometra mansoni PK protein.
[0011] An indirect ELISA detection method using the kit comprises the following steps:
[0012] (1) Coating the PK protein of Spirometra mansoni onto an ELISA plate, washing, blocking, and then washing again;
[0013] (2) adding a serum sample to be tested that has been infected with Spirometra mansoni and incubating the sample, followed by washing;
[0014] (3) adding horseradish peroxidase-labeled goat anti-mouse secondary antibody, incubating, and washing after incubation;
[0015] (4) Adding an initiator, testing the serum sample, and using an enzyme marker to measure the OD value of the serum sample at a wavelength of 450 nm to determine whether the serum sample is infected with Spirometra mansoni.
[0016] The blocking condition is 5% skim milk powder at 37° C. for 60 minutes, and the coating concentration of the Spirometra mansoni PK protein is 5.0 μg / mL.
[0017] The sample to be tested is the serum of mice or cats infected with Spirometra mansoni; the serum is diluted at a ratio of 1:200 and incubated at 37°C for 60 minutes.
[0018] The dilution ratio of the labeled secondary antibody was 1:8000, and the cells were incubated at 37° C. for 30 min.
[0019] The initiator is TMB substrate colorimetric solution. After color development at 37°C for 15 minutes, the stop solution is added to terminate the reaction. The OD value of each serum sample at a wavelength of 450nm is measured by a microplate reader. When the OD value of the sample to be tested is <0.5212, the serum sample is negative; when the OD value is ≥0.5212, the serum sample is positive.
[0020] Beneficial effects of the present invention
[0021] The present invention cloned the pyruvate kinase protein gene from Spirometra mansoni into the prokaryotic expression vector PQE80L(+) to generate the recombinant expression plasmid PQE80L-PK. High-purity recombinant His-PK protein was obtained through IPTG-induced expression and purification. The recombinant protein was used as an antigen to coat an ELISA plate, and an indirect ELISA antibody detection method based on the PK protein was established.
[0022] The present invention optimizes the reaction conditions through step-by-step experiments and obtains the optimal process parameters for ELISA antibody detection, including coating concentration, serum dilution multiple, serum incubation and blocking time, secondary antibody dilution multiple and incubation time, substrate color development time and other parameters. The optimized process is experimentally verified, and an indirect ELISA detection method for PK protein antibodies of Spirometra mansoni is established.
[0023] The detection method of the present invention has the advantages of high sensitivity, strong specificity, good repeatability, etc. The method enriches the detection targets of Spirometra mansoni and provides a new idea for the detection of Spirometra mansoni. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 is the PCR amplification product of PK gene, where M: Marker; 1-2: PCR amplification product at annealing temperature of 58°C; 3: negative control;
[0025] Figure 2 SDS-PAGE analysis of His-PK recombinant protein before and after induction and ultrasound (A);
[0026] Where M: Marker; 1: bacterial protein before IPTG induction; 2: bacterial protein after IPTG induction; 3: supernatant after sonication; 4: precipitate after sonication; 5: protein after supernatant purification;
[0027] Figure 3 The reactivity of the recombinant protein with Sparganosis mansoni positive serum was identified, where (a) M: protein pre-stained marker; 1: rSmPK1 + anti-rSmPK1 serum; (b) M: protein pre-stained marker; 1: rSmPK1 + infected mouse serum; 2: rSmPK1 + blank mouse serum. DETAILED DESCRIPTION
[0028] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and implementation examples.
[0029] Example 1. Construction of prokaryotic expression plasmid of pyruvate kinase
[0030] (1) Primer Primer 5.0 software was used to design primers for the SmPK1 gene, and PCR amplification was performed using a Spirometra mansoni cDNA template. Recognition sites and protective bases for the BamH I and Pst I restriction endonucleases were added to the 5' end of the primers.
[0031] Upstream primer sequence: 5'-TA GGATCC ATGACTGGGGCTTTTGAAAGTTCTC-3' (SEQ ID NO. 1);
[0032] Downstream primer sequence: 5'-TA CTGCAG TCACTTGCAGACCTGAATCG-3'SEQ ID NO. 2).
[0033] PCR reaction system: 1 μL each of upstream and downstream primers, 10 μL Master Mix, 1 μL template, and 7 μL ddH2O.
[0034] PCR reaction conditions: pre-denaturation for 5 min (95°C), denaturation for 50 s (95°C), annealing for 50 s (58°C), extension for 50 s (72°C), denaturation, annealing, and extension for 30 cycles, followed by extension at 70°C for 10 min and termination at 20°C.
[0035] (2) The PCR products were electrophoresed on 1% agarose gel at 120V for 30 min for identification.
[0036] The electrophoresis results are shown in Figure 1 ,The figure shows a single clear band at about 1812 bp, which is consistent with the expected size of the SmPK1 gene, indicating that the target band was successfully amplified.
[0037] Ligate the target product recovered from the gel with the cloning vector pMD19-T. Transform the ligation product into DH5α competent cells using the heat shock method. The ligation system is as follows: 1.5 μL of the target gene, 1 μL of the pMD19-T cloning vector, and 2.5 μL of T4 DNA ligase Solution I. Mix these components by pipetting and centrifuging briefly. Incubate in a metal bath at 16°C for 60 minutes to obtain the ligation product.
[0038] (3) The plasmid was extracted and double enzyme digestion was performed for verification. The target gene was then recovered by gel and connected to the expression vector PQE80L(+). The connection system was: 1.5 μL of target gene, 1 μL of PQE80L expression vector, and 2.5 μL of Solution I. The above components were mixed and pipetted to mix well, centrifuged instantly, and placed in a metal bath at 16°C for 60 min to obtain the PQE80L-PK recombinant plasmid.
[0039] The recombinant plasmid pQE80L-PK was transformed into BL21 (DE3) competent cells, and single colonies were picked to LB medium containing corresponding antibiotics. After culture, the cells were sent to a biotechnology company for sequencing verification. Sequencing was performed using M13 universal sequencing primers. The correctness of the recombinant plasmid construction was confirmed by comparing the sequencing results with the expected SmPK1 gene sequence.
[0040] Example 2. Inducible expression and solubility analysis of recombinant protein
[0041] (1) Induced expression of recombinant protein: The pQE80L-PK recombinant plasmid obtained in Example 1 was transformed into E. coli BL21 (DE3) competent cells, and the transformed cell suspension was evenly spread on the surface of LB solid medium containing ampicillin and inverted cultured at 37°C for 18 h; a single colony was picked and expanded in ampicillin-selective LB liquid medium at 37°C and 200 rpm with shaking;
[0042] When the OD value of the bacterial solution reached 0.6, IPTG was added at a final concentration of 0.2 mmol / L and induced at 25°C for 13 h. 1 mL of the bacterial solution before and after induction was centrifuged at 10,000 × g for 10 min, the cells were collected, and the precipitate was resuspended in 50 μL of Milli-Q water. 4× protein loading buffer was then added and the cells were heated at 100°C for 10 min for SDS-PAGE identification.
[0043] (2) Recombinant protein expression identification and solubility analysis: 100 mL of induced bacterial solution was centrifuged at 10,000 × g for 10 min, the supernatant was discarded, and the suspension was resuspended in 10 mL of Ni-Native-0 and ultrasonically disrupted in an ice bath. The ultrasonication program was as follows: ultrasonication for 5 s with an interval of 7 s, ultrasonication for 10 min each time, and rest for 5 to 10 min, for a total of 6 times.
[0044] The sonicated bacterial solution was centrifuged at 10,000 × g for 10 min at 4°C to separate the insoluble components. The supernatant was added with 4× protein loading buffer and incubated at 100°C in a metal bath for 10 min. 50 μL of Milli-Q water was added to resuspend the precipitate. 4× protein loading buffer was added and incubated at 100°C in a metal bath for 10 min.
[0045] The bacterial samples before and after induction, as well as the supernatant and precipitate samples after ultrasonication were identified by SDS-PAGE. Figure 2 , indicating that the His-PK recombinant protein with a size of 65.24 kDa was successfully induced and distributed in both the supernatant and the precipitate.
[0046] Example 3. Affinity purification and identification of recombinant protein
[0047] The supernatant obtained by ultrasonic centrifugation in Example 2 was filtered through a 0.22 μm filter, the original preservation solution in the nickel column was emptied, and 10 mL of Ni-Native-0 solution was added to balance the column. The filtered supernatant solution was repeatedly passed through the column 5 times, and 10 mL of Ni-Native-0 solution was added to elute non-specific proteins; then 6 times the column volume of Ni-Native-20, Ni-Native-50, and Ni-Native-100 solutions were applied to the column for single-tube collection at 1 mL / tube. The nickel column was washed with 10 times the column volume of Ni-Native-0 solution; the nickel column was sealed with 30% ethanol and stored at 4°C.
[0048] The obtained washing and elution solutions were added with 4× protein loading buffer, and then placed in a metal bath at 100°C for 10 min. The results were identified by SDS-PAGE. Figure 2 , indicating that the His-PK recombinant protein was mainly in the Ni-Native-50 eluted and purified product and had a single band.
[0049] Table 1. Protein purification reagent formula
[0050]
[0051] At the same time, the antigenicity of His-PK protein was analyzed by Western Blot using the laboratory-stored Sparganosis positive serum as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody.
[0052] The molecular weight of PK protein is 65.24KDa. Figure 3 a shows that the rSmPK1+anti-rSmPK1 serum group (i.e., the primary antibody is SmPK1 protein immune serum) has a single and clear band at the position below 70 KDa, indicating that the recombinant protein can be recognized by the SmPK1 protein immune serum. Figure 3 b shows that the samples of the rSmPK1+ infected mouse serum group (i.e., the primary antibody is Sparganosis mansoni positive serum) have a single band, while the samples of the rSmPK1+ blank mouse serum group have no colored band, indicating that the His-PK recombinant protein has good reactivity with the antibody.
[0053] Figure 3a is the identification result of the reaction between serum recombinant protein rSmPK1+anti-rSmPK1 and Sparganosis mansoni positive serum;
[0054] Figure 3 b shows the results of the reaction between serum from mice infected with the recombinant protein rSmPK1+ and the positive serum of Sparganosis mansoni.
[0055] Example 4. Indirect ELISA detection kit based on Spirometra mansoni PK protein
[0056] The Spirometra mansoni PK protein prepared in Example 3 was used in an indirect ELISA kit. The kit included a coating antigen (S. mansoni PK protein), a primary antibody (mouse or cat anti-S. mansoni PK protein antibody to be detected), a labeled secondary antibody, and an initiator. The primary antibody was a mouse or cat anti-S. mansoni PK protein antibody to be detected; the labeled secondary antibody was capable of binding to the S. mansoni PK protein and carried a detection marker, which was peroxidase. The initiator was a TMB substrate developer, and the peroxidase was horseradish peroxidase. The coating concentration of the S. mansoni PK protein was 5.0 μg / mL.
[0057] Example 5. Establishment of the optimal reaction conditions based on the indirect ELISA antibody detection method for PK protein
[0058] 1. Determination of the optimal coating concentration and dilution multiple of the serum to be tested
[0059] The experiment was designed using the checkerboard method. The recombinant PK protein obtained in Example 1 was prepared into 8 different working concentrations of 0.25 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 1.5 μg / mL, 2.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, and 10.0 μg / mL using ELISA coating solution. 100 μl was added to each well of a 96-well plate. The corresponding antigen coating amount per well was 0.025 μg, 0.05 μg, 0.1 μg, 0.15 μg, 0.2 μg, 0.25 μg, 0.5 μg, and 1 μg, respectively. At the same time, the serum to be tested was diluted according to the ratio of 1:100, 1:200, 1:400, 1:800, and 1:1600.
[0060] The OD value of the serum to be tested was read at a wavelength of 450 nm. The optimal antigen coating concentration was determined based on the principle that the OD value was greater than 1 and the P / N value (P / N = positive mean / negative mean) was maximized.
[0061] The results are shown in Table 2, which shows the changes in OD and P / N values at different combinations of antigen concentrations and serum dilutions. Ultimately, 5.0 μg / mL was determined to be the optimal antigen coating concentration, and the P / N value was maximized when the serum dilution was 1:200.
[0062] Table 2. OD values and P / N values of samples at different antigen concentrations and serum dilutions
[0063]
[0064]
[0065] 2. Determination of optimal sealing conditions
[0066] To determine the optimal blocking conditions, according to the determined conditions (antigen coating concentration 5.0 μg / mL, serum dilution multiple 1:200), the blocking time was selected as 30 min, 60 min, 90 min, and 120 min, respectively, and the blocking solution was 5% skim milk powder or 1% BSA for blocking treatment of the serum to be tested.
[0067] During the experiment, the blocking effect of each blocking solution (5% skim milk powder, 1% BSA) was tested at 37°C for 30, 60, 90, and 120 minutes. The data were analyzed by ELISA, and the OD values of the test serum were read at a wavelength of 450 nm. The optimal blocking conditions were determined based on the principle of maximizing the P / N ratio (P / N = mean positive / mean negative).
[0068] The experimental results are shown in Table 3. It can be seen that the maximum P / N value was obtained when 5% skim milk powder was used as the blocking solution and the blocking time was 60 minutes at 37°C. Therefore, 5% skim milk powder, 37°C, and 60 minutes of blocking were selected as the optimal blocking conditions.
[0069] Table 3. OD values and P / N values of samples in different blocking solutions and blocking times
[0070]
[0071] 3. Determination of the optimal incubation time for the serum to be tested
[0072] According to the determined conditions (coating concentration 5.0 μg / mL, serum dilution 1:200, 5% skim milk powder, 37°C, blocking for 60 min), four schemes were selected for incubation of the serum to be tested at 37°C for 30 min, 37°C for 60 min, 37°C for 90 min, and 37°C for 120 min, respectively, and the OD value of the serum to be tested was read at a wavelength of 450 nm.
[0073] The optimal incubation time for the test serum was determined based on the principle of maximizing the P / N value (P / N = mean positive value / mean negative value). Data analysis is shown in Table 4. The results show that the P / N value is maximized when the test serum is incubated at 37°C for 60 minutes.
[0074] Table 4. OD values and P / N values of the serum to be tested at 37°C and different incubation times
[0075]
[0076] 4. Determination of the optimal secondary antibody dilution ratio and incubation conditions
[0077] According to the established conditions (coating concentration 5.0 μg / mL, serum dilution 1:200, incubation at 37°C for 60 min, 5% skim milk powder, 37°C, serum blocking for 60 min), the HRP-labeled goat anti-mouse IgG secondary antibody was diluted at 6 ratios of 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, and 1:8000 for parallel optimization experiments, and the OD value of the serum to be tested was read at a wavelength of 450 nm.
[0078] Based on the principle of maximizing the P / N value, the optimal secondary antibody dilution factor for the serum to be tested was determined. The experimental data are shown in Table 5. The results show that the P / N value is maximized when .
[0079] According to the determined conditions (coating concentration 5.0 μg / mL, serum dilution multiple 1:200, serum incubation conditions 37 ° C for 60 min and blocking conditions 5% skim milk powder, 37 ° C, blocking 60 min, secondary antibody dilution ratio of 1:8000), the experiment was carried out, and the secondary antibody conditions to be tested were optimized according to four schemes of 37 ° C for 30 min, 37 ° C for 60 min, 37 ° C for 90 min, and 37 ° C for 120 min. The OD value of the serum to be tested was read at a wavelength of 450 nm. Based on the principle of maximum P / N value, the optimal secondary antibody incubation time for the serum to be tested was determined. The experimental data are shown in Table 6. The results show that the P / N value is the largest when incubated at 37 ° C for 30 min.
[0080] Table 5. OD values and P / N values of the secondary antibodies tested at different dilutions at 37°C
[0081]
[0082] Table 6. OD values and P / N values of the secondary antibodies tested at 37°C and different reaction times
[0083]
[0084] 5. Determination of optimal color development temperature and time
[0085] According to the determined conditions (coating concentration 5.0 μg / mL, serum dilution ratio 1:200, serum incubation at 37°C for 60 min, 5% skim milk powder, 37°C, blocking for 60 min, secondary antibody dilution ratio 1:8000, secondary antibody incubation at 37°C for 30 min), three schemes of 37°C for 5 min, 37°C for 10 min, and 37°C for 15 min were selected for the optimization experiment of substrate color development time, and the OD value of the serum to be tested was read at a wavelength of 450 nm.
[0086] Based on the principle of maximizing the P / N value, the optimal color development time for the serum to be tested was determined. The experimental data are shown in Table 7. The results show that the P / N value is maximized when the color development time is 15 minutes at 37°C.
[0087] Table 7. OD values and P / N values of substrates at 37°C and different color development times
[0088]
[0089] Example 6. Detection method based on indirect ELISA antibody of PK protein
[0090] In combination with the above embodiments, the indirect ELISA detection method of the present invention is obtained, comprising the following steps:
[0091] (1) The PK protein of Spirometra mansoni was coated onto an ELISA plate at a coating concentration of 5.0 μg / mL at 4°C overnight, and then washed three times to remove impurities such as unadsorbed protein. The plate was then blocked with 5% skim milk powder at 37°C for 60 min, and then washed again three times to remove unbound blocking substances.
[0092] (2) The samples to be tested are mouse serum and cat serum. The serum of mice and cats infected with Spirometra mansoni was taken respectively, diluted at a dilution ratio of 1:200, and then added to the ELISA plate. The plate was incubated at 37°C for 60 minutes to allow the specific antibodies in the sample to bind to the coated Spirometra mansoni PK protein. After the incubation, the plate was washed three times to remove the unbound sample components.
[0093] Serum collection method: Blood was collected from the tail vein of mice or cats. The blood samples were placed in a 37°C incubator for 2 hours, then centrifuged at 3000 rpm for 15 minutes to collect serum, and the serum was stored at -20°C.
[0094] (3) Add labeled secondary antibody, the label on the secondary antibody is horseradish peroxidase, the dilution ratio of the labeled secondary antibody is 1:8000, and incubate at 37°C for 30 minutes; after incubation, wash again three times to remove unbound labeled secondary antibody;
[0095] (4) Add the initiator TMB substrate colorimetric solution and develop the color at 37°C for 15 min. Then add the stop solution (TMB colorimetric solution purchased from Solebol, the stop solution is 2M H2SO4) to terminate the color development reaction. At this time, the color of the solution changes from blue to yellow. Use a microplate reader to measure the OD value of the sample at a wavelength of 450 nm.
[0096] Example 7. Application of indirect ELISA antibody detection method based on PK protein
[0097] 1. Determination of critical value
[0098] Based on the ELISA detection method established in Example 6, the samples were standardized and 30 serum samples negative for Sparganosis mansoni were randomly selected for testing. The OD value of each serum sample at a wavelength of 450 nm was measured by a microplate reader. To ensure the accuracy and reliability of the experimental results, the results were analyzed according to statistical principles. As shown in Table 8, the average reading of the 30 serum samples was 0.1722, the standard deviation (SD) was 0.1163, and the critical value was calculated. It is 0.5212.
[0099] Therefore, the criteria for the indirect ELISA detection method of PK protein were as follows: 450nm When OD < 0.5212, the sample was judged as negative; 450nm When the value was ≥0.5212, the sample was judged as positive.
[0100] Table 8. OD values of serum samples at 450 nm wavelength
[0101]
[0102] 2. Sensitivity test
[0103] To evaluate the sensitivity of the optimized ELISA method, the positive serum was diluted in two-fold ratios starting from 1:100 to 1:12800. The OD value of the serum at each dilution was measured at a wavelength of 450 nm according to the indirect ELISA method of Example 5. The maximum dilution factor at which the OD value was greater than the critical value was used as the maximum dilution factor of the serum to be tested.
[0104] The results are shown in Table 9. When the serum dilution ratio is 1:800, the OD value is still greater than the predetermined critical value. When the dilution ratio is greater than 1:800, the OD value is less than the critical value, indicating that the optimized ELISA method has a sensitivity of 1:800 and has good sensitivity.
[0105] Table 9. OD values of serum at different dilutions at 450 nm
[0106]
[0107] 3. Specificity test
[0108] Based on the PK protein indirect ELISA method of Example 6, a specific experiment was conducted, and the negative and positive serum of Sparganum mansoni were used as controls. The positive serum of Sparganum mansoni, Spirometra mansoni, Trichinella spiralis, Hymenolepis, Clonorchis sinensis, Schistosoma japonicum, Cryptosporidium parvum, Giardia lamblia, Toxoplasma gondii, and Leishmania donovani was tested respectively, and the OD values were read. 450nm and compare it with the established critical value to determine its positive or negative nature.
[0109] The experimental data are shown in Table 10, which shows the OD of 7 serum samples. 450nm The reading was lower than the critical value of 0.5212, indicating negative, indicating that the indirect ELISA method optimized based on PK protein had good specificity and could distinguish the above 7 sera from the positive serum of Sparganosis mansoni.
[0110] Table 10. OD of the serum to be tested 450nm and judgment results
[0111]
[0112] 4. Repeatability test
[0113] In order to evaluate the reproducibility of the indirect ELISA method based on PK protein, the intra-assay and inter-assay reproducibility were investigated.
[0114] Five sera positive for S. mansoni (P1-P5) and three sera negative for S. mansoni (N1-N3) were taken, and four replicate wells were set for each serum. The serum samples were tested by the PK protein indirect ELISA method of Example 5, and the OD value of each well was read. 450nm The values were repeated within the batch and the mean and standard deviation of the readings of each serum sample were calculated. The coefficient of variation was calculated according to the statistical principle = (standard deviation / mean value) × 100%.
[0115] The experimental results are shown in Table 11, which show that the maximum value of the coefficient of variation is 5.77%, indicating good intra-batch repeatability.
[0116] Table 11. Intra-batch repeatability test results
[0117]
[0118] For the inter-batch reproducibility test, 5 sera positive for S. mansoni and 3 sera negative for S. mansoni were taken, and 2 replicate wells were set for each serum. The experiment was repeated three times independently by different experimenters at different times, and the OD of each replicate well was read. 450nmThe values were calculated and the average of two replicate wells was calculated for each experiment. The average and standard deviation of three experiments with the same serum were calculated, and the coefficient of variation was calculated. The results showed that the maximum coefficient of variation was 14.70%, indicating good inter-assay reproducibility (Table 12).
[0119] Table 12. Results of inter-batch repeatability test
[0120]
Claims
1. An indirect ELISA detection kit based on Spirometra mansoni PK protein, characterized in that: The method comprises a coating antigen, PK protein of Spirometra mansoni, a primary antibody, a labeled secondary antibody, and a trigger. The primary antibody is a mouse anti- or cat anti-PK protein antibody of Spirometra mansoni to be detected; the labeled secondary antibody is a goat anti-mouse IgG with a detection marker, which can bind to the PK protein of Spirometra mansoni, and the labeled secondary antibody carries a detection marker.
2. The indirect ELISA detection kit according to claim 1, wherein The initiator is TMB substrate color developing solution, and the marker is horseradish peroxidase, phosphatase or luciferase.
3. The indirect ELISA detection kit according to claim 1, wherein The coating concentration of the Spirometra mansoni PK protein is 5.0 μg / mL.
4. The indirect ELISA detection kit according to claim 1, wherein The preparation method of the Spirometra mansoni PK protein comprises the following steps: cloning the coding gene of the Spirometra mansoni PK protein into a PQE80L(+) expression vector to obtain a recombinant expression plasmid, transforming the recombinant expression plasmid into a host cell, expressing the plasmid, and isolating and purifying the recombinantly expressed Spirometra mansoni PK protein.
5. An indirect ELISA detection method using the kit according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Coating the PK protein of Spirometra mansoni onto an ELISA plate, washing, blocking, and then washing again; (2) adding a serum sample to be tested that has been infected with Spirometra mansoni and incubating the sample, followed by washing; (3) adding horseradish peroxidase-labeled goat anti-mouse secondary antibody, incubating, and washing after incubation; (4) Adding an initiator, testing the serum sample, and using an enzyme marker to measure the OD value of the serum sample at a wavelength of 450 nm to determine whether the serum sample is infected with Spirometra mansoni.
6. The indirect ELISA detection method according to claim 5, wherein The blocking conditions were 5% skim milk powder at 37°C for 60 min, and the coating concentration of Spirometra mansoni PK protein was 5.0 μg / mL.
7. The indirect ELISA detection method according to claim 5, wherein The sample to be tested is the serum of mice or cats infected with Spirometra mansoni; the serum is diluted at a ratio of 1:200 and incubated at 37°C for 60 minutes.
8. The indirect ELISA detection method according to claim 5, wherein The dilution ratio of the labeled secondary antibody was 1:8000, and the cells were incubated at 37° C. for 30 min.
9. The indirect ELISA detection method according to claim 5, wherein The initiator is TMB substrate colorimetric solution. After color development at 37°C for 15 minutes, the stop solution is added to terminate the reaction. The OD value of each serum sample at a wavelength of 450 nm is measured by a microplate reader. When the OD value of the sample to be tested is <0.5212, the serum sample is negative; when the OD value is ≥0.5212, the serum sample is positive.