Immunoradiometric assay for in-vitro determination of prolactin hormone in human serum or plasma using solid phase anti-PRL cellulose particles

The objective of the present study was oriented to produce and purify polyclonal anti-PRL antibody as the main key store in immunoradiometric assay (IRMA) using solid phase cellulose particles for determination of PRL in human sera. The preparation of ¹²⁵I-PRL was carried out by lactoperoxidase method for estimation of the titre of antibody production. The preparation of standards was undertaken. The activation of cellulose particles using 1,1-carbonyl diimidazole (CDI) and coupling of these solid phase particles with purified Rabbit anti-PRL were carried out. Optimization and validation of the assay were carried out. Results revealed that the produced PRL polyclonal antibodies have high titre. Cellulose particles IRMA system was highly sensitive and specific. The intra- and inter-assay variations were satisfactory. The recovery and dilution tests indicated accurate calibration and appropriate matrix. The present technique agreed well with IRMA commercial kit. These cellulose particles retained their characteristics during storage for 6 months at 4 °C. In conclusion, this low cost assay could be used as a useful diagnostic tool for diagnosis and follow up of galactorrhea, infertility and pituitary adenoma.     

Purification and Characterization of PRL Protein Tyrosine Phosphatases

PRLs constitute a subfamily of protein tyrosine phosphatases(PTPs). In the present paper are reported the molecular cloning, expression, purification, and characterization of all the three members of the PRL enzymes in human and the only PRL in C. elegans. These enzymes were expressed as glutathione S-transferase (GST) fusion proteins in DE3pLysS E. coil cells, and the recombinant fusion proteins were purified on glutathione-Sepharose affinity columns. Having been cleaved with thrombin, GST-free enzymes were further purified on an S-100 Sepharose gel filtration column. The purified proteins show single polypeptide bands on SDS-polyacrylamide gel electrophoresis. With para-nitrophenyl phosphate(p-NPP) as a substrate, PRLs exhibit classical Michaelis-Menten kinetics with Vmax values two orders of magnitude smaller than those of classic PTPs. The responses of PRLs to ionic strength, metal ions and phosphatase inhibitors are similar to those of other characterized PTPs, but their optimal pH values are different. These data thus reveal distinct common biochemical properties of PRL subfamily PTPs as well.     

Catching protein antigens by antibody affinity electrophoresis

A new kind of affinity electrophoresis called antibody affinity electrophoresis is a technique used to capture protein antigens based on their interactions with specific monoclonal or polyclonal antibodies incorporated in the polyacrylamide gel. Polyclonal anti-glutathione-S-transferase (anti-GST), monoclonal anti-bovine serum albumin (anti-BSA), and polyclonal anti-human alpha-lactalbumin are embedded in distinct areas of a 7.5% native polyacrylamide gel. Some of the embedded antibodies get covalently and/or noncovalently incorporated into the gel matrix network. Under electrophoresis conditions, these antibodies do not show significant electrophoretic mobility, as compared to their specific protein antigen analytes. We observed that electrophoretic migration of GST, BSA, and protein G ceases when they encounter anti-GST, anti-BSA, and immunoglobulin G, respectively.     

Poly(o-phenylenediamine)-carried nanogold particles as signal tags for sensitive electrochemical immunoassay of prolactin

A novel class of redox-active molecular tags, poly(o-phenylenediamine)-carried nanogold particles (GPPDs), was first synthesized and functionalized with horseradish peroxidase-anti-prolactin conjugates (HRP-anti-PRL). Thereafter, a specific sandwich-type electrochemical immunoassay was designed for determination of prolactin (PRL) by using GPPD-labeled HRP-anti-PRL conjugates as molecular tags on anti-PRL antibody-modified glassy carbon electrode. Compared with pure gold nanoparticles and poly(o-phenylenediamine) microspheres, the as-prepared GPPDs increased the surface coverage of the nanostructures, and enhanced the immobilization amount of biomolecules. Several labeling protocols compromising GPPD-labeled HRP-anti-PRL, nanogold particles-labeled HRP-anti-PRL and poly(o-phenylenediamine) microspheres-labeled HRP-anti-PRL, were investigated for detection of PRL, and improved analytical features were obtained with the GPPD-based strategy. With the GPPD labeling method, dependence of the electrochemical signals on the incubation time and pH of the assay solution were also studied. The strong attachment of HRP-anti-PRL to the GPPDs resulted in a good repeatability and intermediate reproducibility down to 9.8%. The dynamic concentration range spanned from 0.5 to 180 ng mL⁻¹ PRL with a detection limit of 0.1 ng mL⁻¹ at the 3S_blank level. No significant differences at the 95% confidence level were encountered in the analysis of 10 spiked blank cattle serum samples between the developed immunoassay and enzyme-linked immunosorbent assay method for determination of PRL.     

A selective Seoul-Fluor-based bioprobe, SfBP, for vaccinia H1-related phosphatase--a dual-specific protein tyrosine phosphatase

We report a Seoul-Fluor-based bioprobe, SfBP, for selective monitoring of protein tyrosine phosphatases (PTPs). A rational design based on the structures at the active site of dual-specific PTPs can enable SfBP to selectively monitor the activity of these PTPs with a 93-fold change in brightness. Moreover, screening results of SfBP against 30 classical PTPs and 35 dual-specific PTPs show that it is selective toward vaccinia H1-related (VHR) phosphatase, a dual-specific PTP (DUSP-3).     

Kinetics of Phosphatase of Regenerating Liver-3 （PRL-3） Inhibition by Small-molecular Inhibitors

Introduction Proteintyrosinephosphatases(PTPs)playakey roleinsignaltransductionpathways,whichregulatethe growth,differentiationandmalignanttransformationof cells[1].PhosphataseofRegeneratingLiver3(PRL3)belongstoarecentlydiscoveredfamilyofproteinphos phata…     

抗体酶催化Oxy-Cope重排反应的研究

经七步反应合成了Oxy-Cope重排反应的过渡态类似物-2-对羧丙氧基苯基环己醇(3),并以此为半抗原联接载体蛋白BSA制成抗原后免疫动物,从动物血清中分离纯化得到多克隆抗体。在200mmol/L的MES(4-吗啉乙磺酸)和200mmol/L的NaCl缓冲液中(pH=6.0),底物和抗体的摩尔比为100:1及37℃条件下,该抗体能催化3-对甲氧苯基-4-羟基-1,5-已二烯(1)重排生成6-对甲氧苯基-5-烯-已醛(2),ν~m~a~x=15μmol/L·min,K~M=1025μmol/L,K~c~a~t~.=10min^-^1.     

Interaction between chicken protein tyrosine phosphatase 1 (CPTP1)-like rat protein phosphatase 1 (PTP1) and p60(v-src) in v-src-transformed Rat-1 fibroblasts

CPTP1 is a nontransmembrane chicken protein tyrosine phosphatase having 92% sequence homology to the corresponding 321 amino acids of human protein tyrosine phosphatase 1B (HPTP1B). Using anti-CPTP1 antibody, we identified CPTP1-like rat PTP1 of 51 kDa in Rat-1 and v-src-transformed Rat-1 fibroblasts. Here we show that CPTP1-like rat PTP1 binds to p60(v-src) in vivo and CPTP1 also can associate with p60(v-src) in cell lysate of v-src- transformed Rat-1 fibroblasts. Interaction between HPTP1B-type PTPs, CPTP1-like rat PTP1 and CPTP1, and p60(v-src) was reduced by vanadate treatment for 13 h due to down regulation of the protein level of p60(v-src) in vivo. Interestingly, CPTP1-like rat PTP1 was coimmunoprecipitated with a 70-kDa protein which has a possibility to be tyrosine- phosphorylated by p60(v-src) in v-src-transformed Rat-1 fibroblasts. These results suggest that HPTP1B-type PTPs may play an important role in p60(src) dependent signal pathway in eucaryotic cells.     

Purification and characterization of a high specificity polyclonal antibody to the adenomatous polyposis coli tumour suppressor protein

Recombinant proteins, commonly expressed in fusion with an affinity tag to facilitate purification, are often used as immunogens for polyclonal antibody production. Careful immunopurification of the antibody product is often the key to obtaining a high-specificity polyclonal antibody against the protein domain of interest. This study describes the purification and characterization of such an antibody directed against the adenomatous polyposis coli (APC) tumour suppressor. We used a combination of affinity chromatography and biosensor analysis to optimize and monitor antibody purification. This antibody was then characterized by immunoprecipitation, proteomic analyses and immunofluorescence staining and shown to be a valuable reagent for the study of APC biology. Using this antibody we successfully isolated and identified APC, using MS/MS, from transfected cell lines. A novel phosphorylation site on APC was identified at ser 1436. Similar strategies involving multiple immuno-affinity steps coupled with surface plasmon resonance (SPR), immunoprecipitation proteomic and immunofluorescence analyses should be generally applicable for the purification and characterization of other polyclonal antibodies.     

The biological buffer bicarbonate/CO2 potentiates H2O2-mediated inactivation of protein tyrosine phosphatases

Hydrogen peroxide is a cell signaling agent that inactivates protein tyrosine phosphatases (PTPs) via oxidation of their catalytic cysteine residue. PTPs are inactivated rapidly during H(2)O(2)-mediated cellular signal transduction processes, but, paradoxically, hydrogen peroxide is a rather sluggish PTP inactivator in vitro. Here we present evidence that the biological buffer bicarbonate/CO(2) potentiates the ability of H(2)O(2) to inactivate PTPs. The results of biochemical experiments and high-resolution crystallographic analysis are consistent with a mechanism involving oxidation of the catalytic cysteine residue by peroxymonocarbonate generated via the reaction of H(2)O(2) with HCO(3)(-)/CO(2).     

Preparation of highly specific anti-zearalenone antibodies by using the cationic protein conjugate and development of an indirect competitive enzyme-linked immunosorbent assay

Although anti-zearalenone (ZEN) antibodies have been widely prepared, these antibodies cross-react with α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL). To overcome this problem and improve the specificity of immunoassays, we produced anti-ZEN antibodies based on a ZEN-cationic protein conjugate. In this study, ZEN was coupled with cationic bovine serum albumin (cBSA) via a Mannich reaction. After BALB/c mice were immunized with ZEN-cBSA, an immunological response was rapidly induced. The titers of the polyclonal antisera and monoclonal antibody were 30,000 and 20,000, respectively. Cross-reactivity (CR) values of the anti-ZEN polyclonal antisera and monoclonal antibody with the 5 analogs were <7% and <2%, respectively. An indirect competitive enzyme-linked immunosorbent assay based on the monoclonal anti-ZEN antibody was established. The recovery rates of ZEN in spiked cereal and feed were in the range of 80%-120% with coefficients of variation <15%. The intra-assay variation and inter-assay variation in assay buffer were both <5%. Therefore, the results demonstrated a feasible approach for preparing highly specific, higher titer and more rapidly induced antibodies against ZEN by using a ZEN-cBSA conjugate as the immunogen instead of currently used immunogens.     

Inhibitors of Protein Tyrosine Phosphatases: Next‐Generation Drugs?

The protein tyrosine phosphatases (PTPs) constitute a family of closely related key regulatory enzymes that dephosphorylate phosphotyrosine residues in their protein substrates. Malfunctions in PTP activity are linked to various diseases, ranging from cancer to neurological disorders and diabetes. Consequently, PTPs have emerged as promising targets for therapeutic intervention in recent years. In this review, general aspects of PTPs and the development of small-molecule inhibitors of PTPs by both academic research groups and pharmaceutical companies are discussed. Different strategies have been successfully applied to identify potent and selective inhibitors. These studies constitute the basis for the future development of PTP inhibitors as drugs.     

Studies on the Affinity‐based Biosensors for Electrochemical Detection of HER2 Cancer Biomarker

As cancer diseases are the second main cause of death it is necessary to elaborate fast and efficient early diagnosis methods for their detection. One of the possibilities is the analysis of protein biomarkers, which abnormal concentration in physiological fluids might be an indication of cancer disease progression. Herein, we present the studies on the development of affinity‐based biosensors for electrochemical detection of HER2 protein, which is a common biomarker of breast cancer. The main objective was to verify the possibility of fabrication of HER2‐specific hybrid aptamer‐polyclonal antibody and antibody‐based sandwich sensing layer on gold electrode surface. The effectiveness of each electrode modification step was confirmed using voltammetric and impedimetric techniques in the presence of ferri/ferrocyanide redox couple. It was observed that hybrid construct was unlikely to be formed on the gold electrode due to a higher affinity of secondary polyclonal antibody towards target protein, which resulted in the separation of HER2‐antibody complex from the electrode surface. On the contrary, an antibody‐based sandwich receptor layer allowed for protein discrimination in the range from 1 to 100 ng mL^?1 by the application of TMB/H₂O₂ system and chronoamperometry detection technique. Though, the occurrence of interactions between interfering proteins and antibody‐based layer was noted, it led to at least two times smaller current responses than for HER2 protein.     

Structural basis for inhibition of protein tyrosine phosphatases by Keggin compounds phosphomolybdate and phosphotungstate

Protein-tyrosine phosphatases (PTPs) constitute a family of receptor-like, and cytoplasmic enzymes, which catalyze the dephosphorylation of phosphotyrosine residues in a variety of receptors and signaling molecules. Together with protein tyrosine kinases (PTKs), PTPs are critically involved in regulating many cellular signaling processes. In this study, diverse compounds were screened for PTP inhibition and selectively screened for inhibitors with the end product inhibition properties. Among phosphate analogues and their derivatives for PTP inhibition, Keggin compounds phosphomolybdate (PM) and phosphotungstate (PT) strongly inhibited both PTP-1B and SHP-1, with K(i) values of 0.06-1.2 micromM in the presence of EDTA. Unlike the vanadium compounds, inhibition potencies of PM and PT were not significantly affected by EDTA. PM and PT were potent, competitive inhibitors for PTPs, but relatively poor inhibitors of Ser/Thr phosphatase. Interestingly, PM and PT did not inhibit alkaline phosphatase at all. The crystal structure of PTP-1B in complex with PM, at 2.0 A resolution, reveals that MoO(3), derived from PM by hydrolysis, binds at the active site. The molybdenium atom of the inhibitor is coordinated with six ligands: three oxo-ligands, two apical water molecules and a S atom of the catalytic cysteine residue. In support of the crystallographic finding, we observed that molybdenium oxides (MoO(3), MoO(2), and MoO(2)Cl(2)) inhibited PTP-1B with IC(50) in the range 5-15 micromM.     

Recombinant polyclonal antibody libraries

We describe a technology for generating recombinant polyclonal antibody libraries (PCALs) that enables the creation and perpetuation of standardized mixtures of polyclonal whole antibodies specific for a multiantigen (or polyantigen). Therefore, this technology combines the advantages of targeting multiple antigenic determinants -- high avidity, low likelihood of antigen 'escape variants', and efficient mediation of effector functions, with the advantages of using monoclonal antibodies -- unlimited supply of standardized reagents and the availability of the genetic material for desired manipulations. The technology for generating recombinant polyclonal antibody libraries begins with the creation of phage display Fab (antibody) libraries. This is followed by selection of sublibraries with desired antigen specificities, and mass transfer of the variable region gene pairs of the selected sublibraries to a mammalian expression vector for generation of libraries of polyclonal whole antibodies. We review here our experiments for selection of phage display antibody libraries against microbes and tumor cells, as well as the recent literature on the selection of phage display antibody libraries to multiantigen targets.     

Fast purification of trace vitellogenin from Chinese rare minnow using protein A-immobilized antibody

Vitellogenin (Vtg) is a highly responsive biomarker for environmental exposure to various estrogenically active compounds. Here we present a simple, fast, mild, and stable immobilization of anti-Vtg antibody, and demonstrate its powerful applications for preconcentration and purification of fish Vtg proteins, allowing for the monitoring and screening of environmental exposure to estrogenically active compounds. In this immobilization method, rabbit antiserum containing a specific polyclonal antibody against Vtg was directly immobilized on an antibody-binding Staphylococcal protein A matrix (SpA) without the need for prior purification. Under the unique elution conditions, the Vtg protein can be eluted out alone without any leaked specific antibody. The developed method was further used to purify Vtg from whole-body homogenate of Chinese rare minnow. Compared with previous purification methods, the isolated Vtg fraction by this method displays higher purity and well-preserved structure integrity. Moreover, our method is eight times faster. The simple one-step protein A-based specific antibody immobilization and its associated elution strategy may be extended to a number of antibodies for various application purposes, highlighting the paramount advantages over traditional immunoprecipitation and covalent immobilization of antibodies, and suggesting a wide range of promising applications in environmental monitoring and proteome analysis.     

用多克隆抗体构建的夹心免疫传感器检测心肌肌钙蛋白I

用表面等离子体子共振生物传感器构建对心肌肌钙蛋白I特异性的免疫传感器检测心肌肌钙蛋白I,并建立两种检测方法:直接法的最低检测限为2.5μg/L,基于传感膜上的夹心免疫法的灵敏度为0.5μg/L,检测范围为0.5～20μg/L,批内及批间精密度分别为3.5%～4.9%,6.1%～7.4%;用夹心法及国外试剂盒对40名健康献血者和20例急性心肌梗死患者血清心肌肌钙蛋白I水平进行检测,两者符合率为95%.     

Polystyrene beads as an alternative support material for epitope identification of a prion-antibody interaction using proteolytic excision–mass spectrometry

The binding epitope structure of a protein specifically recognized by an antibody provides key information to prevent and treat diseases with therapeutic antibodies and to develop antibody-based diagnostics. Epitope structures of antigens can be effectively identified by the proteolytic epitope excision–mass spectrometry (MS) method, which involves (1) immobilization of monoclonal or polyclonal antibodies, e.g., on N-hydroxysuccinimide-activated sepharose, (2) affinity binding of the antigen followed by limited proteolytic digestion of the immobilized immune complex, and (3) elution and mass spectrometric analysis of the remaining affinity-bound peptide(s). In the epitope analysis of recombinant cellular bovine prion protein (bPrP^C) to a monoclonal antibody (mAb3E7), we found that epitope excision experiments resulted in extensive nonspecific binding of bPrP to a standard sepharose matrix employed. Here, we show that the use of amino-modified polystyrene beads with aldehyde functionality is an efficient alternative support for antibody immobilization, suitable for epitope excision–MS, with complete suppression of nonspecific bPrP binding.     

Detection of immune complexes by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to detect an immune complex formed between beta-lactoglobulin and polyclonal anti-beta-lactoglobulin antibody in the gas phase. The most important experimental parameters to detect such a specific antibody-antigen complex by MALDI were the use of solutions at near-neutral pH and of sinapinic acid matrix prepared by the dried-droplet method. Under such conditions, predominantly one but also two molecules of antigen protein were complexed by the antibody. Specific formation of the antibody-antigen complex was confirmed by performing competitive reactions. Addition of antibody to a 1:1 mixture of beta-lactoglobulin and one control protein resulted not only in the appearance of the expected antibody-antigen complex, but also in a strong decrease in the free beta-lactoglobulin signal, while the abundance of the control protein was not influenced.