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@#Objective To prepare rabbit polyclonal antibodies against pertussis toxin(PT) and develop a double antibody sandwich ELISA for quantitative determination of PT antigen,identify and apply the method.Methods The rabbit polyclonal antibody against PT was prepared by immunizing Chinchilla rabbit with PT using traditional method.The reaction conditions of ELISA system were optimized,the double antibody sandwich ELISA method for quantitative determi-nation of PT was developed,and the specificity,linearity,accuracy,precision and sensitivity were verified.The developed method was used to detect PT antigen content in fimbriae proteins(FIM) stock solution of samples during detoxification and other purification process of pertussis antigen.Results The working condition of double antibody sandwich ELISA for detection of PT antigen content was the coating concentration of PT rabbit polyclonal antibody of 1 μg/mL,and the enzyme-labeled antibody dilution of 1:8 000.This detection system showed specific reaction with PT purified protein,but had no cross reaction with filamentous hemagglutinin,diphtheria toxoid and tetanus toxoid;the linear detection range of the developed double antibody sandwich ELISA was within 25—400 ng/mL;the recovery rates of PT at high,moderate and low concentrations were 103.27%,91.48% and 103.52%,respectively;both the intra-and inter-coefficients of variation(CVs)were less than 10%;the sensitivity of the method was 20.719 ng/mL,and the detection limit was 41.438 ng/mL.Thirty-five batches of samples were detected under five different detoxification process conditions and at different sampling time points,and the changes of antigen content were all consistent with the trend of detoxification reaction.Conclusion The PT rabbit polyclonal antibody was successfully prepared,and a double antibody sandwich ELISA with high precision and accuracy was developed for the quantitative determination of PT antigen content,which can be used for the antigen content detection of chemically detoxified samples in the production process of component DPT vaccines.
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@#Objective To prepare murine and rabbit polyclonal antibodies against rabies virus(RV) matrix(M) protein and compare their reactivity.Methods The prokaryotic expression vector pET-28a-M was constructed by using the cDNA of cells infected with RV CVS-11 strain as template,then transformed into E.coli BL21(DE3),and the induced by IPTG to express M protein.After nickel column affinity chromatography and dialysis renaturation,female BALB/c mice and New Zealand white rabbits were immunized with the M protein,and the whole blood was taken to separate the serum.The titers of the murine and rabbit polyclonal antibodies were detected by ELISA,and the reactivity was measured by Western blot,indirect immunofluorescence assay(IFA) and immunoprecipitation(IP).Results The plasmid pET-28a-M was constructed correctly as identified by sequencing.The titers of murine and rabbit polyclonal antibodies were 1:100 and 1:256 000respectively,and the polyclonal antibodies had reactivity with different RV strains.Conclusion The murine and rabbit polyclonal antibodies against M protein were successfully prepared,which provides important biological tools for exploring the interaction between M protein and host protein as well as studying the pathogenesis of RV.
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@#Objective To prepare rabbit polyclonal antibodies against pertussis toxin(PT) and develop a double antibody sandwich ELISA for quantitative determination of PT antigen,identify and apply the method.Methods The rabbit polyclonal antibody against PT was prepared by immunizing Chinchilla rabbit with PT using traditional method.The reaction conditions of ELISA system were optimized,the double antibody sandwich ELISA method for quantitative determi-nation of PT was developed,and the specificity,linearity,accuracy,precision and sensitivity were verified.The developed method was used to detect PT antigen content in fimbriae proteins(FIM) stock solution of samples during detoxification and other purification process of pertussis antigen.Results The working condition of double antibody sandwich ELISA for detection of PT antigen content was the coating concentration of PT rabbit polyclonal antibody of 1 μg/mL,and the enzyme-labeled antibody dilution of 1:8 000.This detection system showed specific reaction with PT purified protein,but had no cross reaction with filamentous hemagglutinin,diphtheria toxoid and tetanus toxoid;the linear detection range of the developed double antibody sandwich ELISA was within 25—400 ng/mL;the recovery rates of PT at high,moderate and low concentrations were 103.27%,91.48% and 103.52%,respectively;both the intra-and inter-coefficients of variation(CVs)were less than 10%;the sensitivity of the method was 20.719 ng/mL,and the detection limit was 41.438 ng/mL.Thirty-five batches of samples were detected under five different detoxification process conditions and at different sampling time points,and the changes of antigen content were all consistent with the trend of detoxification reaction.Conclusion The PT rabbit polyclonal antibody was successfully prepared,and a double antibody sandwich ELISA with high precision and accuracy was developed for the quantitative determination of PT antigen content,which can be used for the antigen content detection of chemically detoxified samples in the production process of component DPT vaccines
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@#Objective To express and purify the protein of variable region of adeno-associated virus 9(AAV9) capsid in prokaryotic cells,and prepare rabbit polyclonal antibody against it.Methods DNA sequence encoding variable region of AAV9 capsid protein was designed,synthesized and inserted into prokaryotic expression vector pET-30a. The obtained plasmid pET-30a-AAV-VR was transformed to E.coli BL21(DE3),induced by IPTG to express the multivalent antigenic peptide and purified by Ni-NTA resin under denaturation conditions. Male Japanese large-eared white rabbits were immunized with the AAV9 variable region protein after dialysis and renaturation to prepare polyclonal antibody,which was determined for the antibody potency by indirect ELISA,and for the specificity by Western blot and cellular immunofluorescence.Results The recombinant prokaryotic expression plasmid pET-30a-AAV-VR expressing the variable region of AAV9 capsid was constructed correctly as identified by XhoⅠ and BglⅡ digestion. The expressed protein was recognized by His tag antibody after purification with a relative molecular mass of about 20 000. The potency of rabbit polyclonal antibody was 1∶10 240 000,which specifically recognize AAV9 capsid protein.Conclusion The capsid variable region protein of AAV9 was successfully expressed and rabbit polyclonal antibody with high potency was prepared,which laid a foundation of the subsequent development of AAV vector and the research of AAV biological function.
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Objective To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of Toxoplasma gondii, and explore its preliminary applications. Methods The GRA24 coding sequences of different T. gondii strains were aligned using the MEGA-X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET-28a vector, and the generated GRA24 truncated protein was transformed into Escherichia coli BL21. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA). Results SDS-PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high-purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1:208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different T. gondii strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following T. gondii invasion in host cells. Conclusions The polyclonal antibody against the T. gondii GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.
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@#Objective To prepare polyclonal antibodies against the serum albumin of human,cattle,sheep,pig and horse,and evaluate their efficacy in the identification of human serum albumin(HSA). Methods The specific polypeptides of human,cattle,sheep,pig and horse serum albumin were prepared by bioinformatics and polypeptide synthesis method,which were coupled with keyhole limpet hemocyanin(KLH)to prepare the peptide antigen after the purity was identified by high performance liquid chromatography(HPLC). Male New Zealand white rabbits were immunized with polypeptide antigens of five species subcutaneously,with 2 for each kind of antigen. The antiserum was then obtained and purified by Protein A affinity chromatography to prepare the polyclonal antibody. The titers and specificity of the polyclonal antibodies were determined by ELISA and Western blot respectively,and the prepared five species of serum albumin were used to identify HSA products. Results The synthetic peptides of human,cattle,sheep,pig and horse serum albumin had a purity of over 91%,and the corresponding polyclonal antibodies all had the titer of 1∶160 000,which showed specific binding with the corresponding antigens and effectively identified the HSA products. Conclusion The polyclonal antibodies of human cattle,sheep,pig and horse serum albumin prepared in this study have good specificity and the preparation process is simple and rapid,suitable for the mass production,which lays a foundation of the development of HSA rapid identification kit.
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Objective:To prokaryotically express a peptide fragment of 660 - 1468 amino acids in Neisseria gonorrhoeae NGO2105 protein, and to prepare and identify its polyclonal antibody. Methods:The pCold TF-NGO2105 660-1468 aa recombinant plasmid was transformed into the bacterium Escherichia coli BL21 (DE3) for protein expression. After the inclusion body protein was denatured and renatured, the target protein was purified. Then, BALB/c mice were immunized with the target protein to prepare a polyclonal antiserum; the antibody potency was evaluated by enzyme-linked immunosorbent assay, the specificity of the antibody against NGO2105 protein in Neisseria gonorrhoeae was analyzed by Western blot analysis, the affinity of the antiserum with Neisseria gonorrhoeae was analyzed by flow cytometry, and adhesion inhibition assay was performed to evaluate the inhibitory effect of anti-NGO2105 660-1468 aa antibody on the adhesion of Neisseria gonorrhoeae to human cervical epithelial ME-180 cells. Comparisons between different groups were performed by using t test. Results:The NGO2105 660-1468 aa protein was expressed as the inclusion body, and the soluble target protein was obtained by denaturation, renaturation, and purification. After immunization of mice with the target protein, the antiserum titer was 5.12 × 10 6, and flow cytometry showed that the antibody bound well to the Neisseria gonorrhoeae NGO2105 660-1468 aa. Adhesion inhibition assay showed that the anti-NGO2105 660-1468 aa antibody significantly inhibited the adhesion of Neisseria gonorrhoeae to ME-180 cells, and the inhibitory effect was concentration-dependent to some extent, with the adhesion rates of Neisseria gonorrhoeae treated with 20- and 40-fold dilutions of the anti-NGO2105 660-1468 aa antibody being 52.9% and 79.2% respectively, significantly lower than the adhesion rate in the untreated group (100%, t = 8.40, 5.29, P < 0.001, = 0.006, respectively) . Conclusion:The NGO2105 660-1468 aa protein was successfully expressed and purified, and a highly potent polyclonal antibody was prepared, which had a good affinity with Neisseria gonorrhoeae and an adhesion inhibition ability.
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@#Objective To culture human sapovirus(HuSaV) GⅠ.1 in vitro and prepare polyclonal antibody against the capsid protein VP1.Methods HuSaV GⅠ.1 positive stool specimens preserved in diarrhea department of National Institute for Viral Disease Control and Prevention were inoculated with HuTu-80 cells supplemented with different bile acid salts[glycine chenodeoxycholic acid(GCDCA) and glycine cholic acid(GCA)],and the infection,proliferation and passage of the virus were determined by PCR and RT-qPCR.The VP1 gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-6P-1.The constructed recombinant expression plasmid pGEX-6P-1-VP1 was transformed into E.coli BL21(DE3) and induced by IPTG.Two female New Zealand white rabbits were immunized with the purified recombinant VP1 protein for 4 times.The blood samples were collected 18,28,38 and 48 d after immunization,and the serum titers were detected by ELISA.Results HuTu-80 cells were effectively infected by HuSaV GⅠ.1 in the presence of bile acid salt GCA,and the proliferated virus were stably and continuously transmitted for three generations in HuTu-80 cells.The expressed recom-binant GST-VP1 protein showed a relative molecular mass of about 86 000,and about 60 000 after purification(GST tag excision).The titer of polyclonal antibody against HuSaV VP1 protein was over 1:12 800.Conclusion HuSaV was successfully isolated and cultured in vitro using HuTu-80 cells supplemented with bile acid salt,and polyclonal antibody with high titer against HuSaV VP1 protein was prepared,which laid a foundation of in-depth research of HuSaV identification,infection and pathogenesis.
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@#Objective To express and purify the E protein Domain Ⅲ(ED Ⅲ) of tick-borne encephalitis virus(TBEV) in tandem and prepare the corresponding polyclonal antibody.Methods The TBEV RNA was extracted by Trizol method,and then reversely transcribed into cDNA,which was used as template to amplify ED Ⅲ gene fragment by PCR.Two ED Ⅲ gene fragments were ligated into fusion gene by the hydrophobic flexible polypeptide(G_4S)_3 using overlapping PCR,which was then linked to prokaryotic expression vector pET-28a(+) to construct the recombinant expression plasmid pET-28a-2ED Ⅲ.After sequencing,pET-28a-2ED Ⅲ was transformed into E.coli BL21(DE3) competent cells,induced by IPTG and purified by Ni~(2+) affinity chromatography.Female New Zealand white rabbits were immunized with the renatured recombinant protein to prepare polyclonal antibody.The antibody titer was detected by indirect ELISA and the specificity was identified by Western blot.The homology of ED Ⅲ amino acid sequence between TBEV and other flaviviruses was analyzed by DNAMAN software.Results The recombinant plasmid pET-28a-2ED Ⅲ was identified by sequencing,and the amplified sequence contained two genes consistent with the E sequence of TBEV "Senzhang" strain(JQ650523.1) included on GenBank,indicating that the recombinant plasmid was constructed correctly.The recombinant 2ED Ⅲ protein was expressed mainly in the form of inclusion bodies,with a relative molecular mass of about 21 000 and a purity of 97.5%.The titer of rabbit anti-2ED Ⅲ serum polyclonal antibody was 1:10~7,which reacted specifically with TBEV whole virus.DNAMAN software alignment showed that the homology of ED Ⅲ amino acid sequences between TBEV and Japanese encephalitis virus(JEV),yellow fever virus(YFV) and Dengue virus(DENY) was 36.56%,9.28% and 30.77%,respectively.Conclusion The TBEV envelope ED Ⅲ tandem recombinant expression plasmid pET-28a-2ED Ⅲ was successfully constructed.The expressed recombinant 2ED Ⅲ protein had good reactivity and immunogenicity,and the prepared polyclonal antibody had high titer.
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Objective:To compare the detection performance of serum free light chain (sFLC) in two platforms and evaluate the comparability of serum free light chain results in patients with multiple myeloma (MM).Methods:To evaluate the detection performance (repeatability, accuracy, linear range, reference range, interfering substances, etc.) of sFLC kit based on polyclonal antibodies. Spearman correlation analysis and Bland-Altman were used to analyze 214 sFLC results obtained on two detection platforms at the same time to evaluate the correlation between the results of the two methods and analyze the causes of methodological bias. 119 cases with aMM and 23 cases of disease control group (AL, WM, POEMS syndrome, MGUS, diffuse large B-cell lymphoma) initially diagnosed in the hematology department of Zhongshan Hospital of Fudan University from March 2020 to March 2021 were all included. A retrospective analysis was conducted to calculate the area under the curve of receiver operating characteristic (AUC-ROC) and obtain the optimal sensitivity and specificity cut-off points for the diagnosis of MM patients on monoclonal antibody platform.Results:Repeatability, accuracy, linear range, reference interval and anti-interfering capacity of the detection platform based on polyclonal antibodies were verified to meet clinical needs. The overall consistency of FLC/κ, FLC/λ and κ/λ ratios in two methods was 89.3%, 84.1% and 77.1% respectively; but the correlation results were highly heterogeneous. The correlation coefficient of FLC/κ R 2 was 0.922( P<0.001), while the correlation coefficients R 2of the FLC/λ and κ/λ ratios were only 0.349 and 0.441( P<0.001). After segment analysis, it was found that the correlation of FLC/λ was improved within the linear range and R 2 could rise to 0.78( P<0.001). Compared with monoclonal antibody platform, the vast majority points of FLC/κ fell within the 95% limit by Bland Altman analysis. While the results of FLC/λ on polyclonal antibody platform showed significant positive bias. The AUC of MM diagnosis on monoclonal antibody platform was 0.751 ( P=0.001), and the optimal cutoff value was 24.67. Conclusion:The overall consistency between the two platforms was good, but there were significant differences between the results, so they were not comparable and could not be interchanged. For monitoring the prognosis of patients with multiple myeloma, the same platform should be selected for testing.
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Objective:To construct a recombinant plasmid pET30a-leucine-rich repeat (LRR) containing 15 (LRRC15) of Taenia solium, prokaryotically express and purify the LRRC15 recombinant protein, and prepare a rabbit polyclonal antibody. Methods:The LRRC15 protein encoding gene of Taenia solium was obtained by whole gene synthesis; it was cloned into pET30a vector, and the recombinant plasmid pET30a-LRRC15 was constructed and identified by double-enzyme PCR; the recombinant plasmid was transformed into competent cells of Escherichia coli BL21 (DE3), and the recombinant protein LRRC15 was induced to express by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression product was analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); the LRRC15 recombinant protein was purified by Ni-IDA affinity columns, the purified recombinant protein was analyzed and identified by SDS-PAGE, and the specificity of the purified recombinant protein was identified by Western blot (WB); the New Zealand rabbits were immunized with purified LRRC15 recombinant protein to prepare polyclonal antibodies against LRRC15, and the potency of the purified polyclonal antibody was determined by indirect enzyme-linked immunosorbent assay (ELISA). Results:After PCR identification, a band with a length of 1 506 bp was amplified, which was consistent with the LRRC15 gene; after SDS-PAGE and WB identification, the LRRC15 target protein with a relative molecular mass ( Mr) of about 55.36 × 10 3 was obtained; after immunizing New Zealand rabbits with purified LRRC15 recombinant protein, a polyclonal antibody against LRRC15 was obtained, and its potency was 1∶1 587 200. Conclusion:The recombinant plasmid pET30a-LRRC15 is successfully constructed, the LRRC15 recombinant protein of Taenia solium is prepared, and a high purity and high potency rabbit anti polyclonal antibody against LRRC15 recombinant protein is obtained.
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@#ObjectiveTo obtain recombinant H.pylori adhesin A(rHpaA)by molecular cloning,protein expression and purification,immunize BALB/c mice to prepare anti-HpaA polyclonal antibody,and analyze its antibody specificity.MethodsThe three-dimensional structure and antigenic properties of rHpaA were analyzed by bioinformatics softwares such as Phyre2 and DNAstar;Adhesin HpaA gene was obtained by PAS(PCR-based accurate synthesis)and inserted into plasmid pCzn1.The prepared recombinant plasmid pCzn1-rHpaA was transformed to E.coli Artic Express(DE3),induced by IPTG and purified by Ni-IDA affinity chromatography to obtain rHpaA protein,which was identified for reactivity by Western blot.Six male BALB/c mice were immunized with rHpaA plus Freund's adjuvant to prepare anti-HpaA polyclonal anti-body,and the antibody specificity was identified by ELISA.ResultsrHpaA showed good three-dimensional structure and antigenic properties.Restriction analysis and gene sequencing showed that the recombinant plasmid pCzn1-rHpaA contained completely correct HpaA gene sequence.The recombinant strain pCzn1-rHpaA/Arctic Express expressed the soluble target protein rHpaA,which accounted for about 68.3% of total protein in the supernatant,with a purity of 98.1%.rHpaA bound to anti-His antibodies and anti-H.pylori antibodies;The anti-HpaA polyclonal antibody specifically recognized rHpaA and H.pylori lysates.ConclusionrHpaA protein with high purity can be obtained by induction at low temperature and purification.The prepared anti-HpaA polyclonal antibody had good specificity,which laid an experimental foundation of the development of H.pylori-related diagnostic reagents.
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Objective:To establish and validate a fluorescence focus assay (FFA) for rapid titration of Japanese encephalitis virus (JEV) and to evaluate its application in the production of Japanese encephalitis vaccine.Methods:Recombinant JEV non-structural protein 1 (NS1) was expressed in a prokaryotic expression system. After purification, JEV-NS1 was used to immunize rabbits to induce polyclonal antibody. FFA was established with the polyclonal antibody to titer JEV. The accuracy of FFA was validated by comparing with plaque assay, and the specificity, precision, linearity, range and robustness of FFA were also validated. Twenty-eight batches of live-attenuated Japanese encephalitis vaccine were titrated with FFA and plaque assay to analyze the relationship between the two assays.Results:FFA established with polyclonal antibody against JEV-NS1 could be used to titrate JEV, and there was no cross reaction with other viruses (tick-borne encephalitis virus, yellow fever virus, coxsackievirus A2, coxsackievirus A4). Results of the validation tests showed that FFA met the requirement of quality control for live-attenuated Japanese encephalitis vaccine. FFA was more consistency than plaque assay.Conclusions:The established FFA could be used for virus titration in the production of live-attenuated Japanese encephalitis vaccine.
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Objective:To express the head domain of influenza A virus hemagglutinin (HA) in a prokaryotic expression system and to evaluate its immunogenicity.Methods:The genes encoding the HA head domains of H1N1 and H3N2 influenza viruses were cloned into pET-22b(+ ) prokaryotic expression plasmid. After the induction with IPTG, the fusion proteins rH1N1-HA and rH3N2-HA containing HA head domain and His-tag were expressed and obtained from E. coli BL21. SDS-PAGE and Western blot was used to verify the expression of the recombinant proteins. Rabbits were immunized with multiple doses of the purified recombinant proteins to obtain polyclonal antibodies against the HA head domains of H1N1 and H3N2. The immunogenicity of the recombinant proteins was evaluated in BALB/c mice. Results:rH1N1-HA and rH3N2-HA induced protective antibodies (geometric mean titer ≥40) in mice and could be used as protective antigens. Polyclonal antibodies against rH1N1-HA and rH3N2-HA could be used as important materials for Western blot, ELISA and other immunological assays.Conclusions:The HA head domains prepared in this study could be used as protective antigens to induce protective antibodies in mice. Polyclonal antibodies against the HA head domains could be used for immunological and serological studies of influenza A viruses.
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The risk of early acute rejection after kidney transplantation is relatively high, which severely affects the quality of life of the recipients. In 2009, Kidney Disease: Improving Global Outcomes (KDIGO) recommended that immune inducers should be included in the immune-inducing regime before kidney transplantation, aiming to provide certain strength of immunosuppression during this critical phase and effectively reduce the incidence of acute rejection following kidney transplantation. At present, the selection, efficacy and safety of immune inducers remain controversial among transplantation centers around the world. In this article, clinical efficacy of monoclonal antibodies including interleukin-2 receptor antagonist, alemtuzumab, rituximab and polyclonal antibody antithymocyte globulin in immune induction therapy before kidney transplantation were compared and literature review was performed at home and abroad, aiming to provide reference for promoting the individualized selection of immune inducers for kidney transplantation and improving the quality of life of recipients.
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Objective To compare the clinical efficacy of different T lymphocyte polyclonal antibodies in renal transplantation from donor kidney of organ donation after citizen's death. Methods Clinical data of 691 donors and recipients undergoing renal transplantation from donor kidney of organ donation after citizen's death were retrospectively analyzed. According to different T lymphocyte polyclonal antibodies used for induction, all recipients were divided into the rabbit anti human T lymphocyte immunoglobulin (rALG) group (n=414) and rabbit anti human thymocyte immunoglobulin (rATG) group (n=277). The recovery of renal graft function in recipients of the two groups were collected, including the incidence of delayed graft function (DGF) and acute rejection (AR), and the changes of serum creatinine level after renal transplantation. The 1-year survival rate of the recipients and renal grafts was collected. The incidence of adverse effects within 1 year after operation was calculated. According to the DGF risk score of donors, all recipients were divided into 5 groups. The use proportion of rALG and rATG in the recipients of each group was calculated. Results The incidence of DGF in the recipients of rALG and rATG groups was 14.5% (60/414) and 11.9% (33/277), respectively. The duration of DGF in the recipients of rALG and rATG groups was (7±4) d and (12±7) d respectively, with no statistically significant difference between two groups (P > 0.05). The incidence of AR in the rALG group was 7.5% (31/414), significantly higher than 4.0% (11/277) in the rATG group (P < 0.05). The serum creatinine levels of recipients within 6 months after renal transplantation tended to gradually decline in both groups. In renal transplantation for donor kidney with a DGF risk score of 0-15, the use proportion of rALG was significantly higher than that of rATG. However, the use proportion of rATG was significantly higher than that of rALG in renal transplantation for donor kidney with a DGF risk score over 16 (P < 0.05). The 1-year survival rates of the recipients and renal grafts in the rALG and rATG groups were 99.8% and 99.6%, 98.1% and 98.2%, respectively. There was no significant difference between two groups (both P > 0.05). The incidence of acute pulmonary edema and leukopenia in the recipients of rATG group was significantly higher than that in the rALG group (both P < 0.05). Conclusions Both rALG and rATG can effectively reduce the incidence of DGF and AR and achieve good clinical efficacy after renal transplantation from donor kidney of organ donation after citizen's death. The incidence of leukopenia and acute pulmonary edema induced by rATG is higher than that by rALG in the renal transplant recipients.
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Objective: To provide a method for preparing a specific polyclonal antibody of ARG. Methods: Firstly, the technology of polymerase chain reaction(PCR)was adopted to amplify the AbArg gene encoding arginase in Agaricus bisporus. Then the recombinant plasmid pET-28a(+)-Arg was constructed and transformed into E. coli BL21 for the expression of fusion protein. Afterwards, the purified protein was used to immunize New Zealand white rabbits, and the serum samples were collected. Results: The prokaryotic expression plasmid pET-28a(+)-Arg was constructed and the results of SDS-PAGE analysis showed that the fusion protein existing in form of inclusion body was successfully induced and expressed. After purification, the fusion protein with high purity (above 90%)was obtained. The immunity serum titer was 51 200 and Western blotting results showed that the polyclonal antibody could specifically recognize ARG protein in A. bisporus. Conclusion: Polyclonal antibody anti-ARG was prepared successfully, which lays a foundation for the further study on the mechanism of arginine metabolism.
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OBJECTIVE@#To prepare the recombinant peptide MVF-HER3 I composed of the 183-227aa peptide segment of human epidermal growth factor receptor 3 (HER3 I) and the measles virus protein 288-302 peptide segment (MVF), and prepare polyclonal antibodies (PcAb) against this recombinant peptide.@*METHODS@#The MVF-HER3 I gene was synthesized chemically and subcloned into pET21b or pET32a plasmid containing Thioredoxin (Trx) tag gene. The recombinant plasmids were identified by endonuclease digestion. MVF-HER3 I was expressed in BL21(DE3) cells under an optimal bacterial expression condition. The fusion protein Trx-MVF-HER3 I was purified using nickel ion affinity chromatography, and the purified protein was digested by enterokinase to remove Trx tag. The digested mixture underwent further nickel ion affinity chromatography to obtain purified MVF-HER3 I. The purified MVF-HER3 I was used to immunize SD rats subcutaneously for preparing anti-MVF-HER3 I PcAb. The titer of PcAb was determined using ELISA. The bindings of anti-MVF-HER3 I PcAb to MVF-HER3 I, native HER3 and MCF7 cells were analyzed using immunoblotting, immunoprecipitation and laser confocal microscopy. The growth inhibition effect of the antibodies on MCF7 cells cultured in the absence or presence of NRG was assessed using sulforhodamine B.@*RESULTS@#The recombinant peptide gene could not be expressed alone, but could be efficiently expressed after fusion with Trx gene under optimized conditions. The fusion peptide MVF-HER3 I was successfully prepared from Trx-MVF-HER3 I. The anti-MVF-HER3 I PcAb, with a titer reaching 1: 512 000, specifically bound to MVF-HER3 I, recognized native HER3 and bound to the membrane of MCF7 cells. The obtained PcAb could dose-dependently inhibit the growth of MCF7 cells irrespective of the presence or absence of NRG.@*CONCLUSIONS@#We successfully obtained the recombinant peptide MVF-HER3 I and prepared its PcAb, which can facilitate further functional analysis of HER3 signaling pathway.
Sujets)
Animaux , Humains , Rats , Anticorps , Test ELISA , Escherichia coli , Plasmides , Rat Sprague-Dawley , Récepteur ErbB-3 , Allergie et immunologie , Protéines de fusion recombinantesRésumé
Aims@#The current gold standard method for the detection of Campylobacter jejuni is the culturing method followed up by immuno-based detection method, of which, the ELISA is the most often used. Many commercial detection methods based on ELISA use monoclonal antibody preparations although polyclonal antibody can be more sensitive and cheaper to produce. In this study, a comparison of indirect and sandwich ELISA-based detection methods for the detection of C. jejuni using a commercial monoclonal and polyclonal antibody preparations was explored. @*Methodology and results@#An indirect and sandwich ELISA-based methods for the detection of C. jejuni was carried out using the same concentration of antibody (5 μg/mL) and the same concentration of the bacterium at 1×109 CFU/mL. At the pre-screening for optimum concentration of antibody to be used for both assay formats, the commercial monoclonal preparation gave a poor absorbance value of about 0.112 compared to 1.582 for the polyclonal antibody preparation. Hence, the use of the monoclonal antibody was not pursued further. Using the polyclonal antibody, the calculated Limits of Detection (LOD) value obtained for the indirect and sandwich ELISA methods were at 1.6×104 CFU/mL and at 1.29×104 CFU/mL, respectively, which are more sensitive than commercially used methods. The results of the specificity test obtained from the developed polyclonal antibody were then tested against other common food borne bacterial pathogens such as Salmonella Typhimurium, Listeria monocytogenes and Escherichia coli tested using the sandwich ELISA format indicated that the responses by other bacterial genus were relatively low with the translated cross-reactivity percentages of 1.78, 2.36, and 6.87%, respectively. @*Conclusion, significance and impact of study@#The results indicated that the developed system using a polyclonal antibody preparation can be more sensitive than monoclonal preparation. In addition, it is also specific towards Campylobacter while the monoclonal antibody preparation fares poorly.
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To prepare polyclonal antibody (PcAb) against Escherichia coli filamentous thermosensitive protein Z (Ec-FtsZ), the artificially synthesized gene fragment coding Ec-FtsZ was subcloned into pET-22b(+) plasmid, and Ec-FtsZ protein was expressed in E. coli BL21(DE3) cell under an optimal bacterial expression condition. Then Ec-FtsZ protein was purified by HisTrap affinity chromatography, and the GTPase (Guanosine triphosphatase) activity of purified Ec-FtsZ protein was further analyzed by malachite green assay. Subsequently, the purified Ec-FtsZ protein was used to immunize rat subcutaneously for preparation of anti-Ec-FtsZ PcAb. The results of enzyme-linked immunosorbent assay (ELISA), Western blotting analysis and immunofluorescence assay showed that the titer of PcAb was 1:256 000, and PcAb exhibited a perfect antigenic specificity against purified and endogenous Ec-FtsZ protein. All these data indicated that the anti-Ec-FtsZ PcAb is successfully prepared, which can be used for further cellular function study and biochemical analysis of Ec-FtsZ protein in vivo.