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.

Get effective polyclonal antisera in one month

According to the traditional immunization procedure, after the first injection of the sample A (emulsion of aimed antigen and Freund's complete adjuvant) to immunize rabbit, successive injections of the sample B (emulsion of aimed antigen and Freund's incomplete adjuvant) were followed every 2-4 weeks. In general,high titer of the corresponding polyclonal antisera will be observed after 4-5 injections of sample B in 3-4months. This report presents a simply modified procedure that was able to stimulate the antisera formation in one month and achieve enough avidity to satisfy either Western blot or immunohistochemistry analysis.It just applied an additional injection of the sample A to the rabbit at the 3rd day after the primary immunization injection. You could gain the high titer of the antisera right after the first sample B injection in one month. This method has produced the desired results in three different recombinant antigens with different molecular weight (5.9 KD-55 KD) expressed from prokaryotic or eukaryotic cells.

Prokaryotic Expression, Ascitic Polyclonal Antibody Preparation and Identification of Cashmere Goat Izumo1

Izumol is a novel member of the immunoglobulin superfamily locating on sperm, and is indispensable for sperm-egg fusion. According to its immunoglobulin-like domain in the extracellular region, Izumol was fractionated into 6 fragments （F0-F5） which were ligated with pGEX-4T1 to construct the prokaryotic expression vectors pGEX-Fn. The recombinant plasmids were transformed into Escherichia coli BL21 （DE3） and the GST-Fn fusion proteins were expressed successfully by induction with IPTG. GST-F0, a recombinant fusion protein of GST with the full length of extracellular region of mature cashmere goat Izumol, was purified by polyacrylamide gel slicing method and was used as an antigen to immunize the Kunming mouse to generate anti-GST-Izumol ascetic polyclonal antibody with intraperitoneal injection of S 180 cells. Subsequently, the anti-GST-Izumol polyclonal antibody was purified with miscellaneous antigen by glutaraldehyde cross-linking method. Western blotting analysis showed that the purified ascetic polyclonal antibody had high affinity to all 6 GST-Izumol fragment fusion proteins. Immunohistochemical analysis with this antibody displayed that the cashmere goat Izumol proteins were at the equatorial segment of sperm head surface. These results indicate that this polyclonal antibody has high specificity and lays the foundations for further study on the expression pattern of Izumol in cashmere goat testis and binding abilities of each extra-membrane fragment of Izumol to the egg surface.

Development of Anti-Isoproturon Polyclonal Antibody

A competitive enzyme-linked immunosorbent assay （ELISA） suitable for the determination of the urea herbicide isoproturon, 3-（4-isopropylphenyl）-l,l-dimethylurea, in food and environmental samples was developed. Two haptens named 1-（3- carboxypropyl）-3-（4-isopropylphenyl）-1-methylurea （hapten 4C） and 1-（5-carboxypentyl）-3-（4-isopropylphenyl）-1- methylurea （hapten 6C） were synthesized. The haptens were coupled to bovine serum albumin （BSA） and ovalbumin （OVA）, respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen, with which a high-titer anti-isoproturon polyclonal antibody （pAb） was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36：1 and 46：1, respectively, as calculated by a UV spectrophotometry. The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6 × 10^5. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg·L^-1 and 1.0 × 10^5, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding （IC50） was 0.07 mg·mL^-1. The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.

Preparation of rabbit anti-rat LRRN3 polyclonal antibody and study of its expression

BACKGROUND： Studies have shown that the Repeat superfamily, could be related to neural LRRN3, a member of the Neuron Leucine-Rich development, differentiation, information transmission, and other functions, but most studies have focused on nucleic acid levels and few have reported on LRRN3 protein levels. OBJECTIVE： To prepare rabbit anti-rat LRRN3 polyclonal antibody and to observe protein tissue expression profiles. DESIGN, TIME AND SEI-rlNG： In vitro, molecular, biological experiments were performed from October 2007 to April 2009 in Laboratory of Neurobiology at Xiangya School of Medicine, Central South University. MATERIALS： Immunization antigen, namely rat MaI-LRRN3C-His recombinant protein, was provided by the Laboratory of Neurobiology at Xiangya School of Medicine, Central South University. METHODS： Rat Mal-LRRN3C-His recombinant protein was used to immunize male, New Zealand rabbits, and rabbit anti-rat LRRN3 polyclonal antibody was prepared. MAIN OUTCOME MEASURES： Antibody purification was conducted using Protein A affinity chromatography, and the LRRN3 anti-serum titer was identified using enzyme-linked immunosorbent assay. Immunohistochemical techniques and Western blot preliminary tests were used to determine LRRN3 protein expression profiles in adult rats. RESULTS： A highly purified rabbit anti-rat LRRN3 polyclonal antibody was obtained. Western Blot results from rat brain total protein revealed a band at 79 kD, which was consistent with the size of LRRN3. Immunohistochemistry results showed that protein was mainly expressed in the central nervous system, and no significant positive signals were observed in other tissues. Positive cells included neurons of cerebral cortex and hippocampal dentate gyrus granule cell layer, and cerebellar Purkinje cells. There was no positive expression in glial cells. CONCLUSION： Rabbit anti-rat LRRN3 polyclonal antibody was successfully prepared at a high purity from the prokaryotic-expressed MaI-LRRN3C-His recombinant protein, which served as an antigen. Rat LRRN3 protein was primarily expressed in cerebral cortex neurons, hippocampal dentate gyrus granule cell layer neurons, and cerebellar Purkinje cells.

Effects of the Sheep Polyclonal Antibodies Against the Porcine Adipocyte Plasma Membrane Proteins on Porcine Carcass Composition and Meat Quality

To detect the effects of the polyclonal antibodies raised in sheep against porcine adipocyte plasma membranes on the porcine carcass composition and meat quality, 30 pigs assigned into 6 treatment groups were given intraperitoneal injections of sheep antipig adipocyte plasma membrane immunoglobulin （ASIg） or sheep nonimmune serum immunoglobulin （NSIg）. At the end of the experiment, the pigs were slaughtered at 90 kg body weight, and carcasses and meat quality were evaluated. The results showed that when pigs intraperitoneally immunized with 20 or 30 mg ASIg at 15 kg body weight, 20 mg purified ASIg twice at 15 and 60 kg body weight, or 20 mg purified ASIg at 60 kg body weight, respectively, their lean meat percentage, fat meat percentage, backfat thickness, loin eye area leaf fat weight, caul fat weight, heart weight, liver weight, and kidney weight were significantly affected. However, the kidney weight, lurrg weight, dressing percentage, and spleen weight did not remarkably change. Our results indicated that pigs intraperitoneally immunized with 20 or 30 mg ASIg at 15 kg body weight, and 20 mg ASIg twice at 15 and 60 kg body weight, have significantly different drip loss rate, cooked meat ratio, tenderness, storage loss rate, muscle fiber diameter, moisture content, dry matter content, crude protein content, and crude fat content from the control group that received 20 mg NSIg at 15 kg body weight. However, meat pH, meat color value, meat marbling score, inosinate, and myohemoglobin were not significantly affected. Our results indicated ASIg could not significantly affect the content of most muscular amino acids and intramuscular fatty acids.

A Novel Scheme for Production of Polyclonal Antibody against Estrogenic Bisphenols

A polyclonal antibody against the currently concerned estrogenic bisphenol compounds was produced according to a new scheme. 4,4-Bis (4-hydroxyphenyl) valeric acid was used to synthesize the complete antigen in which the characteristic bisphenol structure was exposed to the largest extent. The produced polyclonal antibody showed high specificity and affinity for bisphenol A.

Preparation of Polyclonal Antibodies Against Testis-specific Protease 50 and Characterization of Antibody Specificity

Testis-specific protease 50 （TSP50） is a testis-specific oncogene, which is abnormally activated in most tested patients with breast cancer. This property makes it an attractive molecular marker and a promising target for the diagnosis and therapy of breast cancer. In order to obtain the protective and specific polyclonal antibodies for further research, TSPS0 cDNA was amplified by RT-PCR from normal human testicular tissue, and inserted into eukaryotic expression vector PeDNA3.1. Rabbit anti-TSPS0 polyclonal antibodies were prepared by means of intramuscular injection of peDNA3.1-TSPS0 into the rabbits. Titem of the anti-sera were measured by ELISA and Western blotting with the E. coli cell lysate containing the induced GST-TSPS0 fusion protein as an antigen. In addition, we examined the expression of TSPS0 in both breast cancer cell line MCF-7 and breast cancer tissue by immunofluorescent and immunohistochemistry analysis.

Cloning, Expression and Polyclonal Antibody Preparation of the Asialoglycoprotein Receptor of Marmota Himalayan

The objective of this study is to express the carbohydrate recognition domain （CRD） of the asialoglycoprotein receptor （ASGPR） H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E.coli BL21（DE3）plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay （ELISA）, Western blotting and immunohistochemical staining （IHC）. The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marrnota himalayan—— for the research of infectious diseases of hepatitis viruses and liver cancer treatment.

GST Fusion Protein Based Specific Polyclonal Antibody Preparation of Mouse Aquaporin 1

Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C-terminus of Aquaporin 1（AQP1） as an unique antigen.The long C-terminus of mouse AQP1 was overexpressed in the Glutathione S-tansferase Gene Fusion System.On the basis of the resonable amounts of soluable membrane protein peptides,we prepared the specific antibody.To pursure this object,we constructed pGEX-4T-1/mAQP1（DNA sequence from 700 to 801 bp） recombinant plasmid and transformed it into Escherichia coli BL21 cells.The GST-AQP1 C-terminal hydrophilic peptide fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain the right size fusion protein.Then we immunized the New Zealand rabbits to prepare the antiserum.The purified AQP1 antibody showed high sensitivity by ELISA assay and high specificity by Western blot with AQP1 null mice served as negative control.Finally,we also checked the AQP1 localization in the mouse renal tissues in wild type of mice and AQP1 null mice served as negative control.We demonstrated that AQP1 was highly expressed at the descending limb of Henle tube using our purified AQP1 antibody,which was consistent with previous report.The successful design and preparation of AQP1 antibody through GST technique is an example as making antibodies against a specific membrane protein.

Production of human polyclonal antibodies by transgenic animals

Polyclonal antibodies collected from the blood of animals and humans experimentally immunised or spontaneously immunised respectively can be injected into patients to protect them against pathogens, toxins, tumours etc. This approach is severely limited by the availability of human polyclonal antibodies of interest. Moreover, polyclonal antibodies from animals are recognised as antigens by patients and are thus rapidly rejected and inactivated. To circumvent this problem, animals (essentially rabbits, chicken, pigs and cows) are being genetically engineered. Their immunoglobulin genes are being inactivated and the corresponding human immunoglobulin genes are being transferred to them. These animals will be immunized and it is expected that large amounts of pure human polyclonal antibodies will be extracted from their blood to be administered to patients. The possible acceptability problem of this approach is under a case study of the European Union Pegasus project.

Preparation of Polyclonal Antibodies against NS3 Protein of Japanese Encephalitis Virus

Japanese encephalitis （JE） is a central nervous system disease caused by Japanese encephalitis virus （JEV）, which can infect human and a variety of animals and cause irreversible nerve damages. NS3 protein plays an important role in the process of JEV polyprotein hydrolysis, which is essential for JEV replication. Therefore, NS3 protein may be used as a potential drug target to treat Japanese encephalitis. In this study, the pET-28a-NS3 plasmid was successfully constructed and expressed in E. coli BL21 （ DE3 ） under IPTG induction. The molecular weight of the expressed recombinant protein was 55 ku, which was consistent with the expected result. The positive serum was prepared by immunizing BALB/c mice with NS3 protein and identified by indirect immunofluorescence （IFA）. The results showed that there was a fluorescence reaction between the prepared positive serum of NS3 protein and cells infected with JEV.

SERUM IMMUNOGLOBULIN OF THE MANDARIN FISH,SINIPERCA CHUATSI WITH DEVELOPMENT OF POLYCLONAL ANTIBODY

Serum immunoglobulin from the mandarin fish, or the so called Chinese perch, Siniperca chuatsi (Basilewsky), was successfully purified using affinity chromatography. Heavy and light chains were detected on electrophoresis gel, with molecular weights being estimated at 72 and 29 kDa, respectively. The tetrameric IgM of S. chuatsi was calculated to be 808 kDa. The rabbit polyclonal antisera against the purifed immunoglobulin were developed and tested by Western blot analysis. The antisera reacted strongly with the heavy chains of S. chuatsi immunoglobulin. Humoral immune responses of the mandarin fish can then be examined using the developed polyclonal antibody.

Development of a Polyclonal Antibody-based AC-ELISA and Its Comparison with PCR for Diagnosis of Canine Parvovirus Infection

A polyclonal antibody-based antigen-capture ELISA (AC-ELISA) has been developed for detection of Canine parvovirus (CPV) antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1:1 600 and 1:400, respectively, in the check-board titration. In this study, a total of 152 samples (129 faecal samples and 23 cell culture supernatant) were tested both by AC-ELISA and by polymerase chain reaction (PCR). Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 102.8 TCID50/mL whereas PCR sensitivity was 100.8 TCID50/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.

Development and characterization of new polyclonal antibodies specific for three polychlorinated biphenyls

三 polychlorinated 联本基(印刷电路板) 同种的物和他们的相应幸运十适用四碳长度能为提起抗体被连接到蛋白质的羧基的组被综合。三张结果的免疫情报被制作并且过去常在兔子刺激有免疫力的回答调查幸运的特征十。三结果的 polyclonal 抗体(Pabs ) 被获得。抗血清展出了相对高的抗体乳头物件(1:32 &#x2013； 64 ) 在两倍琼脂散开。

Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody

Survivin, a novel member of inhibitor of apoptosis（IAP） protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. The cancer-specific expression of survivin, coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B. The recombinant plasmid pRSET-B-Surv was expressed in E.coli BL21, and the relative molecule mass（Mr） of expressed fusion protein was approximately 21000. The recombinant protein was purified through Ni^2＋ affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified recombinant protein was then injected into rabbits, and antisurvivin polyclonal antibody with a high titer was obtained.

Polyclonal antibody production and expression of CREG protein in human vascular smooth muscle cells

Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.

Preparation of Adenovirus 5 E1A Polyclonal Antibody

The adenovirus E1A proteins are primarily concerned with modulating cellular metabolism to make the cell more susceptible to virus replication. In this study, adenovirus E1A gene was amplified via PCR on the HEK-293 cells genome and cloned into prokaryotic expression vector pRsetB. The recombinant plasmid pRsetB-E1A was expressed in E.coli BL21 and the relative molecular mass（Mr） of expressed fusion protein was approximately 36000. The recombinant protein was purified on a Ni-NIA agarose column and detected by SDS-PAGE and Westem blot. The purified recombinant protein was then injected into rabbits and anti-E1A polyclonal antibody with high titer was obtained.

Prokaryotic Expression and Polyclonal Antibody Preparation of PRL3

Phosphatase of regenerating liver 3 （ PRL3 ）, which belongs to the superfamily of protein tyrosine phosphatases （PTPs）, represents a group of low molecular weight PTPs that participate in tumorigenesis and metastasis processes. Presented here are the results of cloning, prokaryotic expression, purification, and polyclonal antibody preparation of PRL3. To obtain a specific polyclonal antibody against PRL3, the authors have prepared GST-PRL3 to immunize rabbits and purify an anti-PRL3 polyclonal antibody by negative selection affinity columns. Western blot analysis shows that the anti-PRL3 polyclonal antibody has a specific binding ability with PRL3 protein. The anti-PRL3 polyclonal antibody provides a good tool to further study the function of PRL3.

Production and Characterization of Polyclonal Antibody for the N-Methylcarbamate Insecticide Metolcarb

The hapten, 3-{ [1-（3-（methyl）phenyloxy）-carbonyl]amino}propanoic acid （HOM）, mimicking the analyte metolcarb, was synthesized and verified by mass spectrometry （MS） and 1H-nuclear magnetic resonance spectrometry （1H-NMR）. Then, HOM was conjugated with the carrier proteins bovine serum albumin （BSA） and ovalbumin （OVA） with stoichiometric amounts of N-hydroxysuccinimide/dicyclohexylcarbodimide （NHS/DCC） using the activated ester method. Polyclonal antibodies were raised against the conjugate of HOM-BSA in rabbits. Antiserum titres were determined by noncompetitive indirect ELISA procedures and the titer of pAb01 reached 1.28 × 10^6. The cross-reactivities of the structurally related Nmethylcarbamate insecticides were 0.0% except for dimethacarb. These results indicate that the antibody pAb01 with strong affinity and high specificity can be used to develop a sensitive and rapid detection protocol for metolcarb residue.