Immunochemical Reagents Research Articles

Production and Characterization of F(Ab')2 Fragments Obtained by Enzymatic Digestion from Murine Anti-MRSA PBP2a Monoclonal Antibodies.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a worldwide health problem. In a previous study, a murine monoclonal antibody (mMAB), capable of binding to PBP2a within MRSA strains, was generated. F(ab')2 antibody fragments are widely described in the literature as immunochemical tools and reagents for diagnostics and therapeutics, particularly because of their low immunogenicity and rapid pharmacokinetics. In this study, F(ab')2 fragments from mMAB were generated by enzymatic digestion, using pepsin. They were purified by affinity chromatography using protein A and concentrated by a MWCO 50kDa filtration unit. The results indicate that it is possible to obtain F(ab')2 fragments by pepsin digestion. ELISA, western blotting, and fluorescence microscopy data demonstrated that F(ab')2 affinity for PBP2a is not lost even after the enzymatic digestion process. As expected, in the pharmacokinetics tests, F(ab')2 presented a faster elimination (between 12 and 18h) compared to IgG. These F(ab')2 fragments could be used in future immunodiagnostic applications, including in vitro or in situ radiolabeling and in the treatment of infections caused by this important pathogen.

A novel chemiluminescent immunoassay for detection of Toxoplasma gondii IgG in human sera

This study describes Toxoplasma gondii IgG chemiluminescent immunoassay (CLIA) based on the use of a novel immunochemical reagents in the form of the conjugates of original acridinium ester (AE) labels attached to antibodies and SAG2-GRA1-ROP1L chimeric antigen and shows that this test is useful for diagnostic purposes.

Development of an immunochromatographic test system for the detection of Helicobacter pylori antigens

A test system based on immunochromatography in the sandwich format and intended for express detection of Helicobacter pylori antigens has been developed. Contact of a sample with a test strip coated with immunochemical reagents triggers the movement of the liquid along the membrane components of the test strip, immunochemical interactions, and the formation of detection zones stained by gold nanoparticles. The concentration and kinetic dependences of the immunochemical interactions have been characterized. The reagent and membrane composition of the test system has been selected to provide a minimal detection limit. The detection of H. pylori cell wall antigens at concentrations as low as 0.3 μg/mL in aqueous solution and a suspension of a clinical sample of feces has been demonstrated; the assay duration was 10 minutes. Staining enhancement by the addition of silver salts allowed for a further reduction of the detection limit to 0.03 μg/mL. The developed test system can be used for field diagnostics.

Electrochemical biosensors based on dendrimers

The main features of work of electrochemical biosensors including dendrimers of various structures in the biosensing layer are considered. The role and importance of dendrimers as matrixes for the immobilization of biological components of biosensors and an increase in the density of receptors and mediators of electron transfer are revealed. Examples of application of various types of biosensors based on enzymes, immunochemical reagents, and nucleic acids are presented.

Detection of drugs of abuse in exhaled breath using a device for rapid collection: comparison with plasma, urine and self-reporting in 47 drug users

Exhaled breath has recently been identified as a matrix for the detection of drugs of abuse. This work aims to further document this application using a new and simple collection device in patients following recovery from acute intoxication. Breath, plasma and urine samples were collected from 47 patients (38 males, age range 25–74) together with interview data. Analysis of breath and plasma samples was done by liquid chromatography–mass spectrometry methods. Urine was screened using immunochemical reagents and positive findings confirmed with liquid chromatography–mass spectrometry methods. The 12 analytes investigated were: methadone, amphetamine, methamphetamine, 6-acetylmorphine, morphine, benzoylecgonine, cocaine, diazepam, oxazepam, alprazolam, buprenorphine and tetrahydrocannabinol. In all 47 cases, recent intake of an abused substance prior to admission was reported, but in one case the substance (ketobemidone) was not investigated. In 40 of the remaining cases (87%) breath analysis gave a positive finding of any of the substances that were part of the analytical investigation. Identifications were based on correct chromatographic retention time and product ion ratios obtained in selected reaction monitoring mode. In general, data from breath, plasma, urine and self-reporting were in good agreement, but in 23% of the cases substances were detected that had not been self-reported. All substances covered were detected in a number of breath samples. Considering that breath sampling was often done about 24 h after intake, the detection rate was considered to be high for most substances. Analytes with low detection rates were benzodiazepines, and a further increase in analytical sensitivity is needed to overcome this. This study further supports use of exhaled breath as a new matrix in clinical toxicology.

Detection of Drugs of Abuse in Exhaled Breath from Users Following Recovery from Intoxication

It has recently been demonstrated that amphetamine, methadone and tetrahydrocannabinol are detectable in exhaled breath following intake. Exhaled breath, therefore, constitutes a new possible matrix for drugs-of-abuse testing. The present work aims to further explore this possibility by a study on patients treated for acute intoxication with abused drugs. Fifty-nine patients (44 males, age range 24-74) were included in the study, and breath, plasma and urine samples were collected following recovery, together with interview data. Analyses of breath and plasma samples were conducted with liquid chromatography-mass spectrometry methods. Urine was screened using immunochemical reagents and positive findings confirmed with liquid chromatography-mass spectrometry methods. The following analytes were investigated: methadone, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, codeine, 6-acetylmorphine, diazepam, oxazepam, morphine, benzoylecgonine, cocaine, buprenorphine and tetrahydrocannabinol. In 53 of the studied cases, recent intake of an abused substance prior to admission was reported. In 35 of these (66%), the breath analysis gave a positive finding. Identifications were based on correct chromatographic retention time and product ion ratios obtained in selected reaction monitoring mode. Generally, data from breath, plasma, urine and self-report were in agreement. Detected substances in breath included amphetamine, methamphetamine, buprenorphine, 6-acetylmorphine, morphine, codeine, methadone, tetrahydrocannabinol, diazepam, oxazepam and cocaine. Problem analytes with low detection rates were benzodiazepines and tetrahydrocannabinol. This study gives further support to the possibility of developing exhaled breath into a new matrix for drugs-of-abuse testing by extending the number of analytes that are documented to be detectable in breath.

Development of an ELISA procedure to study sorption of atrazine onto a sewage sludge-amended luvisol soil

Pesticides may contaminate ground and surface waters and one of the major factors governing this property is soil sorption. Sorption can be assessed by batch equilibrium technique which produces lots of extracts with high dissolved organic carbon concentration in which the pesticide concentration has to be determined. We developed an ELISA procedure to analyse atrazine based on polyclonal antibodies (C193) for which tracer structure and dilutions of immunochemical reagents were adapted to fit the purpose. After a 1000-fold dilution (or after an SPE clean-up procedure) extracts of a sewage-sludge amended luvisol (used as an example application of the methodology developed) could be reliably analysed. The Freundlich model is able to describe adsorption for this system (r2=0.977) delivering a distribution coefficient KF of 1.6±0.2 (mgkg−1) (mgL−1)−N and an isotherm nonlinearity factor N of 0.70±0.09.

Murine CENP-F Regulates Centrosomal Microtubule Nucleation and Interacts with Hook2 at the Centrosome

The microtubule (MT) network is essential in a broad spectrum of cellular functions. Many studies have linked CENP-F to MT-based activities as disruption of this protein leads to major changes in MT structure and function. Still, the basis of CENP-F regulation of the MT network remains elusive. Here, our studies reveal a novel and critical localization and role for CENP-F at the centrosome, the major MT organizing center (MTOC) of the cell. Using a yeast two-hybrid screen, we identify Hook2, a linker protein that is essential for regulation of the MT network at the centrosome, as a binding partner of CENP-F. With recently developed immunochemical reagents, we confirm this interaction and reveal the novel localization of CENP-F at the centrosome. Importantly, in this first report of CENP-F(-/-) cells, we demonstrate that ablation of CENP-F protein function eliminates MT repolymerization after standard nocodazole treatment. This inhibition of MT regrowth is centrosome specific because MT repolymerization is readily observed from the Golgi in CENP-F(-/-) cells. The centrosome-specific function of CENP-F in the regulation of MT growth is confirmed by expression of truncated CENP-F containing only the Hook2-binding domain. Furthermore, analysis of partially reconstituted MTOC asters in cells that escape complete repolymerization block shows that disruption of CENP-F function impacts MT nucleation and anchoring rather than promoting catastrophe. Our study reveals a major new localization and function of CENP-F at the centrosome that is likely to impact a broad array of MT-based actions in the cell.

Application of molecular and immuno-diagnostic tools for detection, surveillance and quarantine regulation of Karnal bunt (Tilletia indica) of wheat

Disease diagnostics and pathogen detection, a primary component of any crop management programme, is also helpful in monitoring sanitary and phyto-sanitary measures to seed quality. Molecular diagnostics based on immunological and DNA techniques can provide an efficient way for disease surveillance and disease forecasting. Karnal bunt (KB) of wheat has become a major disease of economic importance and an important issue in the international wheat trade. Due to its seed borne, soil borne and air borne nature, KB can be diagnosed in three pathogenic forms of pathogens viz. teliospores, sporidial and mycelial forms which occur in infectious or proliferative entities at different stages of the disease cycle. Suitable sampling and extraction procedures can improve the detection limits of modern molecular diagnostic methods used to test KB. For the extraction of teliospores from both infected and infested seeds, highly efficacious detergent washing techniques have been developed and extracted pathogenic entities were diagnosed by involving several species of specific primers and immuno-chemical reagents. Nucleic acid and antibody based techniques have been developed for the detection of KB pathogen in different forms. These techniques can also be successfully employed for differential diagnosis, disease surveillance of seed borne pathogens of quarantine importance and determination of teliospore load in wheat seeds. These tests can also be designed to assist in the trade of wheat, development of disease forecasting models and molecular and immuno-pathotyping of Tilletia indica isolates collected from different agro-climatic regions. Moreover, these formats could also be employed for molecular characterization of disease determinants and in comprehending their role in molecular basis of host-pathogen interaction. The present paper highlights the use of the principle of molecular diagnostics in exerting disease surveillance and quality assurances of seeds.

Human platelets circulating in mice: applications for interrogating platelet function and survival, the efficacy of antiplatelet therapeutics, and the molecular basis of platelet immunological disorders.

Herein we describe a novel animal model for examining the survival and function of human platelets following their circulation in non-obese diabetic/severe combined immunodeficient mice. Resting human platelets in platelet-rich plasma are introduced into the retro-orbital plexus, where they are absorbed with high efficiency and circulate for up to 2 days, comprising 10-20% of total circulating platelets. During this period of time, the human platelets can be exposed to a number of biochemical and immunochemical reagents, including novel antithrombotic compounds, or human antiplatelet antibodies that have been implicated in platelet destruction, activation or clearance. Platelets can also be subjected to a variety of storage conditions before infusion, and their relative survival and function following storage and circulation compared. The ability to evaluate in living mice the in vivo function and survival of circulating human platelets may prove valuable for determining mechanisms of antibody-mediated platelet passivation, and aid in the development of novel antiplatelet therapeutics.

Development of methods to measure humoral immune responses against selected antigens in the common marmoset ( Callithrix jacchus) and the effect of pyridostigmine bromide administration

This methodological study was carried out in preparation for a major long term study, also reported in this volume, which was designed to investigate whether the combination of vaccines and pyridostigmine bromide (PB) could have been responsible for adverse signs and symptoms reported by a number of veterans of the 1990/1991 Gulf conflict. In this context, the marmoset has been used to model aspects of the human immune system. The purposes of this methodological study were to select appropriate immunochemical reagents to measure humoral responses induced in marmosets in response to selected health and hygiene and biological warfare vaccines and to initially assess the effects of PB on the responses recorded. Vaccines were administered at 1/5th of a human dose, and also investigated in combination with the nerve agent pretreatment compound PB. PB dosing was selected to induce an inhibition of erythrocyte acetylcholinesterase by 30%. In order to assess the functionality of the immune system, antibody responses to a neo-antigen (keyhole limpet haemocyanin—KLH), administered some 2 months following the completion of the vaccination schedule, were measured. The present study identified appropriate isotyping reporter reagents which cross-reacted with equivalent marmoset immunoglobulins. Robust antibody responses were identified against anthrax protective antigen (PA), whole cell pertussis vaccine and KLH, while weaker responses were measured against cholera and typhoid vaccines. The killed whole cell plague vaccine induced a response which was at the limit of detection of the assay. Coadministered PB had no discernable effect on immunological responses in this study.

Antibodies to a fragment of the Bothrops moojenil-amino acid oxidase cross-react with snake venom components unrelated to the parent protein

It is widely accepted that immunological cross-reactivity of snake venoms is mediated by antibodies that recognize venom components bearing either amino acid sequence homology or similar biological functions. However, here we demonstrate that polyspecific Bothrops antivenom is a source of cross-reactive antibodies that interact with venom proteins of distinctive primary structures and biological functions. The homoserine lactone derivative of the undecapeptide IQRWSLDKYAM (Ile 1-Hse 11), excised from the l-amino acid oxidase (LAAO) of the Bothrops moojeni venom, was the ligand of an affinity resin used to isolate specific anti-Ile 1-Hse 11 antibodies which were instrumental in revealing immunological cross-reactivity among unrelated venom proteins. We examined the extent of the cross-reactivity of these antibodies by probing electroblots of venoms from representative snakes of genera Bothrops, Lachesis, Crotalus and Micrurus, and by unambiguous structural characterization of the affinity-purified proteins of B. moojeni venom recovered from an agarose-anti-Ile 1-Hse 11 column. Our results indicate that all venoms tested had at least three reactive components toward anti-Ile 1-Hse 11 antibodies, among which we identified two serine proteases, one phospholipase A 2 homologue, and LAAO. We hypothesize that the cross-reactivity of the anti-Ile 1-Hse 11 antibodies to unrelated venom proteins derives from their mechanism of antigen recognition, whereby complementarity is achieved through reciprocal conformational adaptation of the reacting molecules. Also, we believe these findings have implications both in the development of improved antivenoms and the preparation of immunochemical reagents for diagnostic and scientific investigation purposes in the field of snake venoms.

A single monoclonal antibody as probe to detect the entire set of native and partially unfolded rhEPO glycoforms

Human erythropoietin (hEPO) is a highly heterogeneous glycosylated protein that requires well-characterised immunochemical reagents to evaluate the glycoform profile along its biotechnological production as a recombinant hormone. These reagents should be suitable for several assay conditions (like those used for immunoblotting analysis, liquid or solid-phase quantitative assays, immunoaffinity purification) with no glycoform selectivity. Five anti-recombinant hEPO monoclonal antibodies (mAbs) were characterised with the aim of selecting the appropriate reagent. These antibodies mapped two spatially distinct epitopes and neutralised the in vitro biological activity of the cytokine. All of them were able to bind to both, the partially denatured and the native form of the protein. Isoelectric focusing analysis followed by immunoblotting confirmed that all the mAbs, herein described, were able to bind to each glycoform, recognising amino acid sequences of the hEPO. Nevertheless, only mAb 2B2 preserved the ability to bind to soluble recombinant human erythropoietin (rhEPO) when it was coated to polystyrene plates or immobilised on CNBr-activated Sepharose matrix. Besides, mAb 2B2 was able to bind to the complete set of soluble rhEPO glycoforms, showing the same affinity for the glycosylated and deglycosylated cytokine. Thus, mAb 2B2 was useful as a capture antibody to develop a sandwich enzyme-linked immunosorbent assay (ELISA), performing a simple, specific and fast assay to quantify rhEPO with a detection limit of 7 ng ml −1. mAb 2B2 was also satisfactorily employed as affinity ligand to purify rhEPO. Our work led us to find a suitable and single reagent to perform a variety of immunochemical approaches, where the binding of each glycoform in the native or partially unfolded form of rhEPO is required.

MAbs to Bacillus anthracis capsular antigen for immunoprotection in anthrax and detection of antigenemia.

Bacillus anthracis is surrounded by an antiphagocytic polypeptide capsule composed of poly gamma-D-glutamic acid (gammaDPGA). gammaDPGA has been identified recently as a potential target for vaccine development. Studies of the role of gammaDPGA in disease have been hampered by the poor Ab response to this antigen and the lack of immunochemical reagents. As a consequence, neither the extent of gammaDPGA production during anthrax nor the protective activity of gammaDPGA Abs in inhalation anthrax are known. Here we report production of IgG Abs to gammaDPGA in mice following an immunization regimen using gammaDPGA in combination with agonist mAbs to CD40. mAbs were produced that are specific for gammaDPGA. Passive immunization with gammaDPGA mAbs protected >90% of mice in a pulmonary model of anthrax that was lethal in control mice (P < 0.0001). Use of gammaDPGA mAb in an antigen detection immunoassay found that the appearance of gammaDPGA in serum coincided with the emergence of bacteremia. These studies identify CD40 stimulation as a means for production of Ab and generation of mAbs against a weakly immunogenic antigen and demonstrate that the capsule is an effective target for immunoprotection and for antigen detection in the diagnosis of anthrax.

Phosphorylation of the Yeast Phospholipid Synthesis Regulatory Protein Opi1p by Protein Kinase A

The Opi1p transcription factor plays a negative regulatory role in the expression of UASINO-containing genes involved in phospholipid synthesis in the yeast Saccharomyces cerevisiae. The phosphorylation of Opi1p by protein kinase A (cAMP-dependent protein kinase) was examined in this work. Using a maltose-binding protein-Opi1p fusion protein as a substrate, protein kinase A activity was time- and dose-dependent and dependent on the concentrations of Opi1p and ATP. Protein kinase A phosphorylated Opi1p on multiple serine residues. The synthetic peptides SCRQKSQPSE and SQVRESLLNL containing the protein kinase A motif for Ser31 and Ser251, respectively, within Opi1p were substrates for protein kinase A. Phosphorylation of S31A and S251A mutant maltose-binding protein-Opi1p fusion proteins by protein kinase A was reduced when compared with the wild type protein, and phosphopeptides present in wild type Opi1p were absent from the S31A and S251A mutant proteins. In vivo labeling experiments showed that the extent of phosphorylation of the S31A and S251A mutant proteins was reduced when compared with the wild type protein. The physiological consequence of the phosphorylation of Opi1p at Ser31 and Ser251 was examined by measuring the effects of the S31A and S251A mutations on the expression of the UASINO-containing gene INO1. The beta-galactosidase activity driven by an INO1-CYC-lacZ reporter gene in opi1Delta mutant cells expressing the S31A and S251A mutant Opi1p proteins was elevated 42 and 35%, respectively, in the absence of inositol and 55 and 52%, respectively, in the presence of inositol when compared with cells expressing wild type Opi1p. These data supported the conclusion that phosphorylation of Opi1p at Ser31 and Ser251 mediated the stimulation of the negative regulatory function of Opi1p on the expression of the INO1 gene.

Aging research in India

Research on aging in India has been well documented since ancient times. As way back as 3000–1500 BC, the Indian medical system of Ayurveda was used as a means for the prevention of the effects of aging and generation of disease in organs or the whole organism, respectively. In recent years, the focus has been demographic studies on different aspects of aging and has been in isolation. Molecular aspects of aging have been addressed only by a few groups of scientists which has focused on regulation of gene expression, DNA damage and repair, development of immunochemical reagents to detect oxidative DNA damage and assessing the levels of circulating antibodies to reactive oxygen species modified DNA (ROS-DNA), etc. This review aims to recapitulate various research studies on aging since 3000 BC to date.

Antibodies to delta sleep-inducing peptide in ultralow doses: study of the effect by enzyme immunoassay.

The effects of potentiated homeopathic preparations containing antibodies to delta sleep-inducing peptide in ultralow doses were studied by enzyme immunoassay. Experiments were performed with the following immunochemical reagents: antigens of delta sleep-inducing peptide conjugated to various macromolecular carriers and specific antigens. Antibodies to delta sleep-inducing peptide were synthesized in dilutions of C3, C6, C12, C50, and C200. Enzyme immunoassay showed that test preparations of antibodies in dilutions of 1:400-1:3200 produce the combined effect on immune complex formation. The proposed method holds much promise for identification of medicinal preparations in ultralow doses.

A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold

We report the design, construction and use of an antibody bacteriophage display library built on the scaffold of a single-chain variable fragment (scFv) previously proven to be functionally expressed in the reducing environment of both bacterial and plant cytoplasm and endowed with intrinsic high thermodynamic stability. Four amino acid residues of the third hypervariable loop (CDR3) of both VH and VL were combinatorially mutated, generating a repertoire of approximately 5 × 107 independent scFvs, cloned in a phagemid vector. The ability of the antibody phage library to yield specific binders was tested by biopanning against several antigens. Successful selection of fully active scFvs was obtained, confirming the notion that combinatorial mutagenesis of few amino acid residues centrally located in the antigen-binding site is sufficient to provide binding specificities against virtually any target. High yields of both soluble and phage antibodies were obtained in Escherichia coli. Maintenance of the cognate scFv antibody stability in the newly selected scFv fragments was demonstrated by guanidinium chloride denaturation/renaturation studies and by soluble antibody expression in the bacterial cytoplasm. The antibody library described here allows the isolation of new stable binding specificities, potentially exploitable as immunochemical reagents for intracellular applications.

Characterization of monoclonal antibodies recognizing immunoglobulin κ and λ chains in pigs by flow cytometry

The existence of two types of the immunoglobulin (Ig) light chain in pigs was documented>30 years ago and has been confirmed by the cloning of porcine light chain genes homologous to human and murine Ig kappa (Igκ) and Ig lambda (Igλ). However, immunochemical reagents defining these two light chain isotypes have not been characterized. Here, we show that rabbit antisera specific for human Igκ and Igλ and certain anti-porcine light chain monoclonal antibodies (mAb) are useful in distinguishing light chain isotypes by flow cytometry (FCM). Porcine B cell lines L23 and L35 stained positive only with anti-human Igλ antiserum and were negative when tested using anti-human Igκ antiserum. While mAbs K139.3E1, 1G6 and 27.7.1 also tested positive on these cell lines, mAb 27.2.1 did not. Therefore, FCM was used to examine the hypothesis that K139.3E1, 1G6 and 27.7.1 are Igλ-specific whereas mAb 27.2.1 recognizes the Igκ chain in pigs. Double staining of peripheral blood mononuclear cells (PBMC) with pairs of anti-light chain mAbs and using cocktails of anti-light chain mAbs and anti-human polyclonal antiserum, confirmed this hypothesis with the exception that mAb K139.3E1 appears to recognize only a subset of Igλ + B cells in most pigs. In summary, we identified two pan-specific anti-pig Igλ mAbs, one anti-λ mAb that recognizes a λ-light chain subset and one anti-pig Igκ mAb.

Identification of phage antibodies toward the Werner protein by selection on Western blots.

A procedure was established for selecting phage antibodies (phage-abs) from phage-displayed antibody repertoires by panning against proteins, separated by sodium dodecyl phosphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes (Western blots). This immobilization strategy is applicable for secondary rounds of panning in selections against semipurified proteins, and directs the selection toward antibodies suitable as immunochemical reagents in Western blots. In model experiments, enrichment factors as high as 1.9x10(5) were obtained in a single round of panning. Furthermore, we demonstrate the application of this approach by selection of phage-abs recognizing the human Werner protein, which is defective in a premature aging syndrome.