Polyclonal Antibody Preparation Research Articles

High-Level Production of MSH2 from Arabidopsis thaliana: A DNA Mismatch Repair System Key Subunit

Biochemical and immunological information concerning DNA mismatch repair proteins from higher plants is currently limited, probably due to their low abundance in vivo. An initial analysis of AtMSH2 gene expression by quantitative real-time RT-PCR indicates that calli and seedlings contain 96.7 and 1.4 cDNA copies per ng RNA, respectively, confirming that this gene is predominantly expressed in rapidly dividing tissues. In order to obtain large quantities of AtMSH2, the protein was efficiently expressed in an Escherichia coli system. The expressed gene product has an in-frame N-terminal Trx-His(6)-S-tag. The fusion protein represents about 11% of the soluble protein from IPTG-induced E. coli cells. After a two-step purification procedure the final yield accounts for 0.7mg/g cells. Digestion of this electrophoretically homogeneous recombinant protein with enterokinase results in an intact protein with only one extra amino acid introduced at the N-terminal end. Purified intact protein was used to induce polyclonal antibodies in rabbits. These antibodies cross-react with a 110-kDa protein from cauliflower inflorescences. Together, our data describe the transcript level, cloning, expression, purification, and polyclonal antibody preparation of AtMSH2. This work will surely be useful for carrying out plant mismatch repair assays in vitro and analyzing protein expression after the exposure of plants to various stresses.

Cloning, expression and characterization of gE protein of Duck plague virus

BackgroundThe gE protein of duck plague virus is the important membrane glycoprotein, its protein characterization has not been reported. In this study, we expressed and presented the characterization of the DPV gE product.ResultsAccording to the sequence of the gE gene, a pair of primers were designed, and the DNA product with 1490bp in size was amplified by using the polymerase chain reaction (PCR). The PCR product was cloned into pMD18-T vector, and subcloned into pET32a(+), generating the recombinant plasmid pET32a/DPV-gE. SDS-PAGE analysis showed that the fusion pET32a/DPV-gE protein was highly expressed after induction by 0.2 mM IPTG at 30°C for 4.5 h in Rosseta host cells. Over expressed 6×His-gE fusion protein was purified by nickel affinity chromatography, and used to immunize the rabbits for the preparation of polyclonal antibody. The result of the intracellular localization revealed that the gE protein was appeared to be in the cytoplasm region. The real time PCR, RT-PCR analysis and Western blotting revealed that the gE gene was produced most abundantly during the late phase of replication in DPV-infected cells.ConclusionsIn this work, the DPV gE protein was successfully expressed in a prokaryotic expression system, and we presented the basic properties of the DPV gE product for the first time. These properties of the gE protein provided a prerequisite for further functional analysis of this gene.

Prokaryotic expression, purification, and polyclonal antibody production of a hydrophobin from <italic>Grifola frondosa</italic>

Hydrophobins are small fungal proteins that self-assemble spontaneously at hydrophilic-hydrophobic interfaces and change the polar nature of the surfaces to which they attach. A new hydrophobin gene hgfI was identified recently from the edible mushroom Grifola frondosa. In this paper, the cloning, expression, purification, and polyclonal antibody preparation of the HGFI were described. The hgfI gene was cloned into pET-28a expression plasmid at the EcoRI and NdeI restriction sites and then transformed into Escherichia coli BL21 strain. SDSPAGE analysis showed that recombinant HGFI protein was satisfactorily expressed by optimizing the concentration and induction time of IPTG. The expressed recombinant HGFI protein was purified by electroelution because its inclusion body was insoluble in traditional processing method. After a desalting procedure with Sephadex G-25, the recombinant HGFI protein was used to immunize adult rabbits following standard protocol. ELISA and western blot analysis indicated that the produced antiserum could detect both HGFI protein expressed in the prokaryotic (E. coli) and in the eukaryotic cells (G. frondosa). Furthermore, the antiserum was used to determine the localization of HGFI protein in G. frondosa cells using an immunofluorescence technique. The results demonstrated that HGFI protein was localized in the cell wall, especially at the budding position of hypha. The polyclonal antibody against HGFI will facilitate further production and functional study of HGFI protein.

Prokaryotic expression, purification and polyclonal antibody preparation of Cyclophilin A

Prokaryotic expression, purification and polyclonal antibody preparation of Cyclophilin A

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

Izumo1 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, Izumo1 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 Izumo1, was purified by polyacrylamide gel slicing method and was used as an antigen to immunize the Kunming mouse to generate anti-GST-Izumo1 ascetic polyclonal antibody with intraperitoneal injection of S180 cells. Subsequently, the anti-GST-Izumo1 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-Izumo1 fragment fusion proteins. Immunohistochemical analysis with this antibody displayed that the cashmere goat Izumo1 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 Izumo1 in cashmere goat testis and binding abilities of each extra-membrane fragment of Izumo1 to the egg surface.

Preparation of Polyclonal Antibody against Human MxA Protein and Its Specificity to Diversified Myxovirus Resistant Protein A

To study the human myxovirus resistant protein A (MxA), a specifically induced peptide by interferon I, and to use its level as a diagnostic criterion for viral infections. Anti-MxA antisera from immunized mice were prepared with the expressed MxA protein of pET32a-MxA in E. coli BL-21(DE3). To confirm the antiserum activity and specificity, the expression product of BL21, wild type MxA pEGFP-C1-wMxA and site-directed mutant MxA pEGFP-C1-mMxA(N589S) stably transfected 3T3 cells and induced A549 cells were detected by Western blot with the antisera using non-MxA transfected or non-IFN-beta induced cells, intact A549, NIH 3T3 cells transfected with pEGFP-C1 and pET32a (+)-transformed BL-21 as controls. The antisera had specific positive immunoreactivity to the NIH3T3 cells transformed with pEGFP-C1-wMxA and pEGFP-C1-mMxA, INF-beta induced A549 cells and BL21 proteins expressed with pET32a (+)-MxA. The hybridization signals from IFN-beta induced A549 cells depended on the IFN-beta inducing concentrations. Meanwhile, immunohistochemical assay showed that NIH 3T3 cells with pEGFP-C1-wMxA and pEGFP-C1-mMxA had > 98% of positive cells at 1:50 dilution of the serum and A549 cells induced by 20 ng/mL IFN-beta for 48 h showed 95% positive cells. pEGFP-C1-transfected NIH 3T3 cells were all negative. Anti-sera are highly specific to diversified MxAs. The antibody is detectable by Western blot, immunocytochemistry and immunofluorescence assay.

Sensitive Plasma Protein Analysis by Microparticle-based Proximity Ligation Assays

Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.

Autoantibody potential of cancer therapeutic monoclonal antibodies

We and others have reported that multiple autoantibodies are unmasked in human polyclonal antibody preparations after exposure to physiological oxidizing agents (hemin) or electromotive force. We now have asked if oxidation unmasks autoantibody reactivities in monoclonal antibodies (mAb). To do this, we have studied 9 FDA approved mAb used therapeutically, including 4 chimeric, 4 humanized and 1 chemically modified chimeric Fab that were exposed to the physiological oxidizing agent hemin at 36 degrees C for 20 hr. These mAb were studied for autoantibody activity to phospholipids and DNA before and after oxidation with hemin and found to develop autoantibody activities after oxidation, while retaining their original specificity as measured by mAb anti-glycophorin A binding of erythrocytes, CD 19 binding to B lymphocytes and anti-HLA-A29 binding to A29-positive lymphocytes. The finding that certain mAb have the potential to unmask autoantibody activities as a consequence of exposure to physiological redox reactions in vitro gives pause to our present understanding of the immunological basis of tolerance and concern for potential autoimmune side effects in patients receiving mAb for diagnosis or treatment.

Cloning, Expression, Purification and Polyclonal Antibody Preparation of Human Apolipoprotein A5

Apolipoprotein A5 (ApoA5) gene has been shown to play an important role in determining plasma triglyceride concentrations in humans, and is a major independent risk factor of cardiovascular disease. It is important to obtain the purified ApoA5 and antiserum. In this paper, full-length ApoA5 gene was amplified from human hepatoma cell line 7721 cDNA, and cloned into expression plasmid pThioHisD named as pTh-ApoA5. The pTh-ApoA5 was introduced into Escherichia coli BL21 (DE3). After optimizing its fermentation condition, recombinant ApoA5 was highly expressed. The recombinant ApoA5 protein that contained a 6-His sequence was separated by Ni2+ affinity column and the purity of recombinant ApoA5 was more than 95%. The purified recombinant ApoA5 protein was employed for immunizing rabbits and the antiserum was obtained. Western blot showed that the antibody was specific.

CDNA Cloning, Prokaryotic Expression, Polyclonal Antibody Preparation of the Auxin-Binding Protein 1 Gene from Grape Berry

Auxin-binding protein 1 (ABP1) plays an important role in the growth and development of plants. In this study, a novel grape ABP1 cDNA was isolated by reverse transcription polymerase chain reaction, using in silico cloning strategy based on grape dbEST. The obtained grape ABP1 cDNA is 756 bp containing a 567 bp open reading frame, which encodes a 188 amino acid protein. The deduced amino acid sequence possessed all of the three typical domains of plant ABP1s, an amino-terminal signal peptide and a carboxyl-terminal sorting signal, KDEL. A full-length ABP1 cDNA was introduced into an expressed plasmid pET-30a(+) vector at the EcoR I and Xho I restriction sites. The pET-ABP1 was found to be highly expressed in Escherichia coli BL21(DE3) pLysS cells with isopropyl-β-d-thiogalactoside induction. A fusion protein was purified and used as the antigen to immunize a New Zealand rabbit. The resulting antiserum was then further purified by HiTrap rProtein A FF Affinity Purification Kit to obtain the immunoglobulin G fraction. Western blot analysis indicated that ABP1 was regulated in fruits depending on the developmental stage. Our work represented a first step towards a better understanding of function analysis of grape ABP1.

Development of a Sandwich ELISA to Detect Hepcidin in Human Serum.

Abstract 2000Poster Board I-1022Hepcidin is a 25 amino acid peptide that is the central mediator of iron metabolism. Iron excess, deficiency and maldistribution have been implicated in the etiology of many diseases including atherosclerosis, diabetes, neurodegeneration and the anemia of inflammation. Determination of hepcidin levels may be useful in diagnosis and treatment decisions for some or all of these diseases. Serum hepcidin measurement has so far been limited to a prohepcidin (60 amino acid hepcidin precursor) ELISA, mass spectrometry (MS)-based assays or competition ELISAs using polyclonal anti-hepcidin antibodies. The current work describes the generation of a sandwich ELISA using monoclonal antibodies to detect human hepcidin (hHepc) and optimization of assay conditions to resolve inconsistencies between MS- and ELISA-based detection.The ability of two anti-hHepc antibodies to sandwich (bind simultaneously) with hHepc was demonstrated using a rabbit polyclonal antibody preparation from hHepc-immunized animals. The same polyclonal antibody preparation was used for both hHepc capture and detection. The limit of detection achieved with this assay was O.D.450<1, suggesting that only a small proportion of the total antibodies could bind concurrently. To improve hHepc detection, a panel of monoclonal antibodies was screened for the ability to sandwich. Antibody epitope characterization studies using purified antibodies and >1000 hybridoma supernatants identified three classes of antibodies: classes 1 and 2 each recognized epitopes found in both full length mature hHepc (hHepc 25) and a shorter version (hHepc 20); class 3 bound a different epitope and demonstrated an increased affinity for hHepc 25 over hHepc 20. The majority of antibodies characterized were in class 1 while antibodies in classes 2 and 3 were rare (∼1% of antibody panel) highlighting the difficulty in achieving a sandwiching event.Antibodies 19D12 (class 1) and 23F11 (class 2) were identified as the optimal sandwich pair with a detection range of approximately 0.2-1000 ng/ml using synthetic hHepc. Initial comparisons of data generated using the sandwich ELISA and a fully-quantitative MS-based assay demonstrated a lack of consistent agreement. This issue was somewhat addressed by introduction of an alkaline treatment step to dissociate any protein/hHepc complexes in serum. Subsequent comparison of the two assays using sera from several different patient populations (anemia of cancer, chemotherapy-induced anemia, kidney disease) as well as healthy donors demonstrated good correlation (R2 range = 0.83-0.92; n=237). This sandwich ELISA may represent a tool for aligning the MS and ELISA-generated results in a format that has the potential to be high throughput and widely available. Disclosures:Arvedson:Amgen: Employment. Doellgast:Amgen: Employment. Salimi-Moosavi:Amgen: Employment. King:Amgen: Employment. Foltz:Amgen: Employment. Chen:Amgen: Employment. Li:Amgen: Employment. Sasu:Amgen: Employment.

Preparation and identification of polyclonal antibody against cysteinyl leukotriene receptor 2

To prepare and identify a polyclonal antibody against cysteinyl leukotriene receptor (CysLT(2)receptor). Rabbits were immunized with KLH-coupled CysLT(2) receptor peptide to prepare the polyclonal antibody (pAb). The titer of the pAb in rabbit plasma was detected by indirect ELISA, and the specificity of the pAb was tested by antigen blockade. The tissue distribution of CysLT(2) receptor was detected by Western blot and immunohistochemistry with the prepared pAb. The pAb showed a titer higher than 1/1047296, and was specific to CysLT(2) receptor, without cross-reaction with the antigens of CysLT(1) receptor and GPR17. A higher expression of CysLT(2) receptor in kidney, brain and lung of rats and mice was detected by Western blot analysis using the prepared pAb. The molecular weight of CysLT(2) receptor protein was about 40 kD. Immunohistochemical examination showed that CysLT(2) receptor was expressed mainly in the neuron, and partly in astrocytes in rat brain. The prepared CysLT(2) receptor pAb has high sensitivity and specificity, and can be used in Western blot and immunohistochemistry.

Production of and applications for a polyclonal IgY diagnostic reagent specific for Mycobacterium avium subsp. paratuberculosis

Antibodies specific to the cell surface antigens of Mycobacterium avium subsp. paratuberculosis (MAP) have multiple useful applications, e.g. organism detection, immunoconcentration, and cell visualization. The aim of this study was to produce and compare polyclonal antibodies for such research and diagnostic purposes. Three polyclonal antibodies to MAP were produced using sera from immunized rabbits and chickens plus naturally infected cows. Cross-reactive antibodies in each MAP antibody preparation were removed by absorption with heterologous mycobacterial and non-mycobacterial cells. The specificity of each resulting polyclonal antibody preparation was evaluated by ELISA to multiple bacterial cell wall extract antigens. After absorption, chicken anti-MAP IgY had the highest specificity of the three antibody preparations. FITC-la-beled anti-MAP IgY was used to effectively locate MAP in macrophages 12 h post-infection. Also, immunomagnetic beads coated with anti-MAP IgY enhanced recovery of MAP from bacterial suspensions in comparison with non-antibody coated beads. Anti-MAP IgY provides a novel new reagent with broad diagnostic and research applications requiring specific concentration, detection, and quantification of MAP.

Preparation,identification and application of polyclonal antibodies against human PIWIL2 protein

Objective:To prepare rabbit polyclonal antibody against human P-element-induced wimpy testis like 2 (HIWI2) protein,identify its properties and investigate its distribution in normal and tumor tissues by means of tissue chip. Methods: PIWIL2 peptide was synthesized chemically and conjugated to Keyhole limpet hemocyanin (KLH) as an immunogen. Then the PIWIL2-KLH conjugation was injected into rabbits subcutaneously to produce polyclonal antibodies. The specificity and sensitivity of antibodies were identified by ELISA and Western blotting after purification by affinity chromatography. PIWIL2 was then immuno-stained on the tissue chip to study its distribution in the normal and tumor tissues. Results: Rabbit’s antibodies against human PIWIL2 were prepared after the injection of PIWIL2-KLH conjugation subcutaneously. These antibodies were identified as PIWIL2 peptides by ELISA and Western blotting assay. PIWIL2 protein expression was tissue-specific in tumor tissues,with PIWIL2 protein found in the cytoplasm of smooth muscle cells of most normal and tumor tissues. Conclusion: The polyclonal antibodies against human PIWIL2 protein have been successfully prepared,which provides a basis for further study on the role of PIWIL2 in the pathway of miRNA/RNA.

Effect Mechanism Of Immunosupressive Drugs

Effective immunosuppression is a key to successful organ transplantation. This study will provide an overview of different immunosuppressive agents used in solid organ transplantation. An increasing number of immunosuppressive agents are available and these agents target different steps of the immunological response to an allograft. These immunosuppressive agents are steroids, anti- proliferative agents (azathioprine and mycophenolate), calcineurin inhibitors (cyclosporine and tacrolimus), TOR inhibitors (sirolimus and everolimus), polyclonal and monoclonal antibody preparations.

Preparation of the polyclonal antibody against human SLC35F2 and detection of the expression of SLC35F2 in non-small-cell lung cancer

Objective To prepare, purify and characterize the polyclonal antibody against SLC35F2,and detect the expression of SLC35F2 protein in non-small-cell lung cancer (NSCLC).Methods Four polypeptides named peptide 1,2,3 and 4 were synthesized based on the bioinformatics analysis of SLC35F2.The mixed polypeptides were injected into New Zealand rabbits.The polyclonal antibody was purified by immunoaffinity chromatography, and identified by Western blot and immunohistochemistry.The expression of SLC35F2 protein in 129 cases of NSCLC and corresponding adjacent normal lung tissues was detected by tissue microarray with immunohistochemistry.Results The titers of polyclonal antibody were 1:105 detected by ELISA assay.Western blot confirmed its high specific 41 KD band.The expression of SLC35F2 protein was strongly positive (++ to +++) in90.7% (117/129) of the NSCLC samples.On the contrast, only 17.1% (22/129) adjacent normal lung tissues were strongly positive (++).The expression of SLC35F2 protein was significantly higher in NSCLC than in the adjacent normal lung tissues (P <0.01).Correlation analysis revealed that pathological stage had a low correlation with the expression of SLC35F2 protein.Factor analyses showed that 93.3% expression of SLC35F2 protein in NSCLC tissues were correlated with tumor differentiation and stage.Conclusion The specific antibody against human SLC35F2 was obtained.SLC35F2 protein was highly expressed in most of the NSCLC. Key words: Non small celllung cancer; Homo sapiens solute carrier family 35 member F2; Polyclonal antibody

PP-024 Expression of human X box binding protein 1-u and preparation of polyclonal antibody against protein

PP-024 Expression of human X box binding protein 1-u and preparation of polyclonal antibody against protein

Expression, purification and polyclonal antibody preparation for a novel gene BC097361

To express and purify of the BC097361 recombinant protein, and to prepare the BC097361 specific rabbit polyclonal antibody. BC097361 cDNA was ligated into the prokaryotic expressive vector pET-32a (+), and the resulting plasmid was transformed into E.coli BL21 (DE3). The protein expression was induced with IPTG and the protein was analyzed with SDS-PAGE and western blotting. The expressed product was purified using Ni+ affinity column chromatography.Then the purified pET-32a (+) -BC097361 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and potency of polyclonal antibody were evaluated by Western blot and ELISA. The BC097361 fusion protein was highly expressed.The protein was mainly in the inclusion body. ELISA indicated the titer of polyclonal antibody more than 1:320000. The high specificity was confirmed with Western blot. The recombinant BC097361 fusion protein and the BC097361 specific polyclonal antibody will be valuable tools for the investigation on the biological function of BC097361.

Preparation and Characterization of Olaquindox Polyclonal Antibody

Abstract2‐[N‐(2‐Hydroxyethyl)‐carbamoyl]‐3‐methyl‐oxoquinoxaline‐1,4‐oxides (OLA) was first mono‐esterificated by succinic acid anhydride, and was then turned into a derivative with carboxyl. The synthetic product was purified by recrystallization with a yield of 52.47%. Its chemical structure was determined by NMR, IR and MS spectra. The determined structure was used to synthese OLA hapten, which had the molecular structural characteristics of OLA. The OLA hapten and bovine serum albumin (BSA) were conjugated using the activated ester method, while the hapten and ovalbumin (OVA) were coupled by the mixed anhydride method. The UV scanning and infrared spectrum results showed that the hapten and the carrier protein were successfully coupled to BSA and OVA, with the combined ratios of 3.8:1 and 5:1, respectively. OLA‐BSA was used as the immunogen to immunize four New Zealand white rabbits, and the high titer anti‐serum produced relatively. The titers were 1:6400, 1:1600, 1:12800 and 1:6400, respectively, determined by indirect ELISA OLA‐OVA as the coating antigen. The intermediate inhibition concentration (IC50) of OLA in the indirect ELISA test was 743.3 ng/mL. The lowest detection limit (IC20) was 5.71 ng/mL. High‐purity OLA polyclonal antibody has been obtained by saturated ammonium sulfate and ion‐exchange chromatography.

Preparation and preliminary application of rabbit polyclonal antibodies against intracellular peptides of human platelet glycoprotein GPIbalpha

To prepare rabbit polyclonal antibodies against intracellular peptides of human platelet glycoprotein GPIbalpha. Two peptides corresponding to human platelet GPIbalpha C-terminus were synthesized and purified by high-performance liquid chromatography (HPLC). The peptides were cross-linked with keyhole limpet hemocyanin (KLH). Two New Zealand white rabbits were immunized with conjugated peptides for 3 times. The polyclonal antibodies were purified by Ammonium Sulfate Precipitation and identified by dot blotting and ELISA. GPIbalpha intracellular peptides phosphorylation was tested with these polyclonal antibodies by ELISA. The titers of the two polyclonal antibodies against the GPIbalpha C-terminus peptides were 1:32 000 and 1:64 000 respectively and both of these antibodies reacted with purified GPIbalpha. Two rabbit polyclonal antibodies against C-terminal peptides of human platelet GPIbalpha have been prepared successfully, providing a way for the preparation of these kinds of antibody. Both phosphorylation and dephosphorylation states exist in the intracellular peptide of human platelets.