Use of Polyclonal Antibody for the Diagnosis of Human African Trypanosomiasis

Human African trypanosomiasis (HAT), commonly known as sleeping sickness is one of the neglected tropical diseases (NTDs), which is fatal if left untreated. Its diagnosis is a challenge since the signs and symptoms of the primary phase are not specific, the existing diagnostic methods have low sensitivity and specificity, and the available drugs have some toxicity. New, robust, and cost-effective techniques are needed for the early identification of parasites. This study aimed to assess the sensitivity and specificity of two different types of polyclonal antibodies against T. b. gambiense using antigen detection ELISA. Polyclonal antibodies against the expressed proteins Tbg I2 and Tbg I17 were produced using New Zealand white rabbits. The antibody titer measured was greater than 32 g/L after the 3rd immunization for the expressed protein Tbg I2. For the expressed protein Tbg I17, the antibody titer measured was greater than 32 g/L after the 4th immunization. The sensitivity and specificity of the Tbg I2 polyclonal antibody confirmed with Polymerase Chain Reaction (PCR) as gold standard were respectively 89.5% and 80.6%, while for the Tbg I17 polyclonal antibody, the sensitivity and specificity were respectively 92.1% and 88.9%. The area under the curve for the Tbg I2 polyclonal antibody was 0.90 ± 0.032, while for the Tbg I17 polyclonal antibody, the area under the curve was 0.92 ± 0.0. The Tbg I17 polyclonal antibody produced in New Zealand white rabbits has good sensitivity and good specificity;it can be successfully used in the diagnosis of HAT.

Polyclonal antibody preparation and expression in liver tissues of transactivated protein 5 of hepatitis C virus nonstructural 5A

Objective To prepare polyclonal antibody of transactivated protein 5 of hepatitis C virus nonstructural 5A (NA5ATP5) and to explore its expression in the liver tissues. Methods In Escherichia coli BL21,the prokaryotic expression vector pET32a(+)-NS5ATP5 was induced by isopropyl-β-D-thiogalactoside (IPTG),and it was analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. And the purified protein was used to immunize the rabbit to prepare polyclonal antibody,with which we studied the function of NS5ATP5 by determining the different liver tissues with the streptavidin-perosidase (SP) immunohistochemistry method. Results Recombinant NS5ATP5 (molecular weight:65 kD) and polyclonal antibody were successfully prepared. NS5ATP5 expression in the liver of patients with chronic HCV infection was much higher than that of a normal person,and it was detected mainly in the cytoplasm. Conclusion The findings of the expression difference between HCV patients and normal people led to a novel diagnostic marker to detect HCV infection.

Production of Rabbit Polyclonal Antibody Using hAPOA1 Protein Expressed in Prokaryotic Cells

The open reading frame(ORF)of hAPOA1 was inserted into the prokaryotic expression vector pGEX-4T-1to construct the recombinant plasmid pGEX-4T-1-hAPOA1,which was then transformed into Escherichiacolistrain BL21.The expression of target fusion protein was induced with isopropylβ-D-1-thiogalactopyranoside(IPTG).The purified fusion protein in inclusion bodies was used to immunize New Zealand white rabbits to prepare hAPOA1 antiserum and the antibody titer was detected with indirect enzyme-linked immunosorbent assay(ID-ELISA).ID-ELISA and Western Blot proved that rabbit polyclonal antibody with a high titer of 1∶40 000 was produced,which may bring considerable economic benefits.

ELISA of polyclonal antibody to fish MT and study on heavy metal tolerance in fish

On the basis of the purification of metallohioncin(MT)from fish,the purified polyclonal antibody of rabbit against fish MT was prepared.Antibody was labeled with horseradish peroxide(HRP)and then the enzyme linked immunusorbent(ELISA)for MT in fish tissucs was cstablished,the minimal,detection level being 0.1-1.0 ng/g FW.Induction of fish tolerance against heavy metals indicated that the contents of MT,SHI-group and Cd increase with the accumulation doses of Cd in the water.The investigations from 7 species of fresh-water fish showed that Cd and MT amounts in their liver and kidney are very low and,particularly,the muscle is lowest.approximately to be zero.So,it is considered that the degree of the heavy metal pollu-tion in water body in which fish live is light and fish is permission to consume.

Development and optimization of a double antibody sandwich ELISA for the detection of goose T cell surface CD8α molecule

CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains. Of these, the CD8α chain was chosen for the detection because it involved in both the CD8αα homodimer and the CD8αβ heterodimer. Here, we established a double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA) for specific detection of goose CD8α(go CD8α). The results showed that the optimal coated antibody and antigen dilutions were 1:50(the antibody titer was 1:12 800) and 1:32(0.3 ng m L^(–1)), respectively, while the optimal capture antibody and horseradish peroxidase(HRP)-labelled goat anti-rabbit Ig G dilutions were 1:50(the antibody titer was 1:51 200) and 1:4 000(the antibody titer was 1:5 000), respectively. The optimal blocking buffer was 5% bovine serum albumin(BSA). The best incubating condition was overnight at 4°C, the best blocking time was 120 min and the best anti-capture antibody working time was 150 min. In addition, the minimum dose detectable by DAS-ELISA was 5×10^(–3) ng m L^(–1). Most importantly, go CD8α expression levels in goose spleen mononuclear cells(MNCs) post-Goose parvoviruse(GPV) infection were found to be significantly up-regulated using the DAS-ELISA method, which was consistent with previous results obtained using real-time quantitative PCR. In conclusion, the DAS-ELISA method reported here is a novel, specific technique for the clinical detection of go CD8α.

Comparative analysis of clinicopathological correlations of cyclooxygenase-2 expression in resectable pancreatic cancer

AIM:To perform a comparative analysis of clinicopathological correlations of cyclooxygenase2 (COX2) expression in pancreatic cancer, examined by monoclonal and polyclonal antibodies.METHODS: The COX2 expression in 85 resection specimens of pancreatic ductal adenocarcinoma was immunohistochemically examined using both monoclonal and polyclonal antibodies. The final immunoscores were obtained by multiplying the percentage of positive cells with the numeric score reflecting the staining intensity.COX2 expression levels were classified into three categories (0, 1+, and 2+) and the clinicopathological correlations were statistically evaluated and analyzed.RESULTS: The positive tumor expression rates of COX2 were 80.5% using monoclonal antibody and 69.4% using polyclonal antibody. In the KaplanMeier analysis, no significant correlations were found between levels of COX2 expression and overall survival (OS), but trends to longer OS were found in COX2 negative cases using monoclonal antibody. Significantly longer disease free survival was revealed in COX2 negative cases using monoclonal antibody (P = 0.019). No correlations between COX2 expression levels and grade (G), tumor (T) status and nodal (N) status were demonstrated. Low histological grade showed a strong association with a longer OS (P < 0.001). Correlation of survival and T status revealed a shorter OS in T3 tumors, but the results reached only marginal statistical significance (P = 0.070). In the multivariate Cox proportional hazards regression model, histological grade, T and N status remained valuable predictors of a worse survival with borderline significance for T [hazards ratio (HR) = 4.18 for G (if G = 3, P < 0.001); HR = 1.64 for T (if T = 3, P = 0.065); HR = 2.53 for N (if N = 1, P = 0.006)]. Higher grade, T or N status was associated with a worse OS. CONCLUSION: The immunohistochemically assessed level of COX2 expression does not seem to represent a valuable independent prognostic factor and is not superior to the conventional prognostic factors.

Antiserum Preparation and Specific Detection of Banana RING-E3 Ubiquitin-Protein Ligase

According to the data of banana transcriptome sequencing, an E3 ubiquitin-protein ligase gene was cloned by RT-PCR method using the cDNA sample of banana leaves. The complete ORF of E3 ubiquitin-protein ligase is 681 bp long and its encoded protein showed 100% sequence identity to homologue RING-H2 finger protein (XP_009407047.1) of Musa_acuminata. Bioinformatic analysis indicated that E3 ubiquitin-protein ligase contains the Ring finger domain in C terminus and eight cross-brace motifs are found in the domain. The target gene was digested by EcoR V and EcoR I, and was inserted into prokaryotic expression vector pET-32a of the same digestions to obtain the plasmid pET32a-E3 ubiquitin-protein ligase. The recombinant plasmid was introduced into Escherichia coli strain BL21 (DE3), and induced at 25°C with 0.4 mmol/L IPTG for 6 hours. The soluble fusion protein was expressed and high purity fusion protein was obtained by Ni2+-NTA agarose affinity chromatography purification. The fusion protein was injected into mice 3 times to prepare the antiserum. Western blot analysis showed a specific protein band was detected in total protein sample of banana leaves, but not for the samples of wild-type Nicotiana benthamiana (N.B.) and wild-type Arabidopsis thaliana (A.T.), implying the antiserum was specific to banana E3 ubiquitin-protein ligase.

Identification of Human Hepatocyte Proliferation Related Gene C2orf69

Objective To construct the prokaryotic expression vector p ET-32a(+)-C2orf69 and induce the expression of recombinant proteins in vitro. Then the possible effects of recombinant protein on cell proliferation was observed and rabbit-anti-C2orf69 protein polyclonal antibodies was obtained.Methods Gene fragment of C2orf69 was amplified by PCR and then prokaryotic expression plasmid pE T-32a(+)-C2orf69 was constructed. Recombinant protein C2orf69 expression was identified by SDS-PAGE and Western blot. The white-ear rabbits were immunized with purified recombinant protein C2orf69, and the potency and specificity of polyclonal antibody were evaluated by enzyme-linked immunosorbent assay(ELISA) and Western blot. Also, different liver cells were incubated with recombinant protein C2orf69 in vitro. Results C2orf69 gene fragment was successfully amplified, results of gene sequencing were consistent with the sequence in Gen Bank. Recombinant protein of C2orf69 was successfully induced and expressed. The polyclonal antibody titer was up to 1︰1 280 000 through enzyme-linked immunosorbent assay. Results of cell proliferation showed that the recombinant protein could inhibit the proliferation of different liver cells. Conclusions The recombinant protein C2orf69 could inhibit the proliferation of different liver cells, and we speculated that it may be a widely roled inhibitor of hepatocyte proliferation. Our experiment showed that the proliferation inhibition of cells may be realized by G1 phase extending and S phase shortening.

Identification of serum immunoglobulin M(IgM)of crucian carp(Carassius auratus gibelio)and immune response to cyprinid herpesvirus 2 infection

Crucian carp(Carassius auratus gibelio),an extensively cultivated freshwater fish,was one of the model species for the study of fish immunology.Polyclonal antibodies were advantageous molecular tools for studying teleost immune system.Specifically,polyclonal antibodies reacting with immunoglobulins(Ig)were used successfully in studies of the teleost fishes.In the present study,we produced polyclonal antibody against CH2 domains of crucian carp IgM,and measured the in vivo dynamics of IgM mRNA caused by CyHV-2 infection.The recombinant protein IgM with relative molecular weight about 53 KD was correctly expressed in prokaryotic cells.The specificity of the polyclonal antibody was evaluated by Western blotting and results revealed that the antibody not only specifically recognized crucian carp serum but also cross-reacted with grass carp serum.Quantitative RT-PCR analysis demonstrated the expression of IgM mRNA changed significantly after CyHV-2 infection.The expression of IgM in the kidney increased and reached a maximum at 6 h post-infection(hpi),while dropped to a low level at 5 days post-infection(dpi).In conclusion,the expression of IgM was significantly upregulated in the kidney of crucian carp infected with CyHV-2,indicating that IgM played a potential role in systemic immunity against viral infection.Polyclonal antibody against crucian carp IgM had certain clinical relevance,which might provide insight into the early stage of virus infection and prevention of the disease.

Synthesis of haptens and production of antibodies to bisphenol A

Three immunizing haptens of bisphenol A(BPA), including two new haptens, were used to produce highly sensitive and specific polyclonal antibodies. The spacer arms of haptens for coupling to the protein carrier were located at different positions in BPA, and different length spacer arms were tested. Highly sensitive polyclonal antibodies were obtained and characterized using indirect competitive enzyme-linked immunosorbent assay(ic ELISA). Under optimized conditions, the half maximal inhibitory concentration(IC50) value of the best polyclonal antibody was 2.1 mg·L^(-1), based on coating heterogeneous antigens, and this optimal polyclonal antibody was highly sensitive toward BPA and displayed negligible crossreactivity with bisphenol B and bisphenol E. A sensitive ic ELISA method utilizing the polyclonal antibody was developed for the determination of BPA in milk. In spiked samples(5, 10 and 20 mg·L^(-1)), the recovery ranged from 80% to 102% with a coefficient of variation(CV) value below 15.8%. The limit of detection of ic ELISA was1.95 mg·L^(-1). These results indicate that the ic ELISA method is suitable for the detection of BPA in milk.