Purification Of Antigen Research Articles

The generation of monoclonal antibodies against human peroxisome proliferator-activated receptors (PPARs).

Monoclonal antibodies (Mabs) are valuable reagents for the purification, characterization and immunolocalization of proteins. In this study, we raised Mabs against human peroxisome proliferator-activated receptors (PPARs) using baculovirus particles displaying surface glycoprotein gp64-fusion proteins as the immunizing agent. In this system, to display fusion proteins on the viral surface, the amino terminal sequences of human PPARd and PPARg2 are inserted in-frame between the signal sequence and the mature domain of the gp64 nucleotide sequence.Mabs were raised by immunization with whole virus without a purification of the target antigens. The Mabs generated by this novel method were shown to recognize not only the gp64-PPARs fusion protein, but also mature, expressed proteins by a wide variety of techniques, including immunohistochemistry, immunoblotting, and electrophoretic mobility shift assays (EMSAs). Transfection of the transfer vector containing a nucleotide sequence encoding less than 30 amino acids along with linearized baculovirus DNA allows for the production of a high affinity antibody against the corresponding mature form. This method is of potential utility in that it allows the production of valuable antibodies without the requirement of a protein purification step.

Purification of Anisakis simplex antigen by affinity chromatography.

In order to improve the specificity and sensitivity of the techniques for the diagnosis of human anisakidosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG-specific antibodies were isolated by means of protein A-Sepharose CL-4B bead columns. IgG anti-Anisakis simplex, anti-Ascaris suum and anti-T. canis were coupled to CNBr-activated Sepharose 4B. For the purification of the larval Anisakis simplex antigens, it was loaded into the anti-A. simplex column and bound antigens were eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex-specific proteins were loaded into the anti-Ascaris suum and anti- T. canis columns. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis and immunoblotting were carried out. Likewise, immunoaffinity columns were prepared using specific IgG from patients with Anisakis simplex sensitization, previously diagnosed by fluoro-enzymo-immunoassay. The protein patterns of antigen after purification by the human columns were similar to those obtained using the rabbit columns.

Antibody response in salmonids against the 70 kDa serine protease of Aeromonas salmonicida studied by a monoclonal antibody-based ELISA

An antigen-capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies (mAb) was set up and evaluated for selective detection of salmonid antibody responses to the antigen P1, which is a weakly immunogenic exoprotease of typical Aeromonas salmonicida. This new assay permits a specific determination of anti-protease-antibodies, without antigen purification. Serum antibodies induced by the strongly immunogenic lipopolysaccharide could reliably be discriminated from anti-P1-antibodies. Antibody titres of 45 experimental antisera recorded by cELISA were moderately correlated with titres determined by routinely used indirect ELISA (iELISA) by detecting partially different antibody populations ( r=0.753). Substitutions of immunoreactants and confirmatory immunoblotting strongly suggest that the mAb-based assay selectively recognises antibodies directed to epitopes of native protease. A conjugate of inhibited protease and cationised bovine serum albumin (cBSA) was found to engender a significant anti-protease-response in three salmonid species ( P<0.05), whereas the unconjugated antigen and Apoject 1-Fural ® were proved to be ineffective. Recorded specific antibody titres were as high as 1:381,400, indicating a considerable enhanced immunogenicity of cBSA-conjugated P1 and high assay sensitivity. The established cELISA offers a promising approach to further improvement of monitoring fish humoral immune response to surface accessible epitopes of the immunosuppressive exoprotease, P1, and to scrutinise its protective significance.

Purification, characterization, and immunogenicity of a disulfide cross-linked Plasmodium vivax vaccine candidate antigen, merozoite surface protein 1, expressed in Escherichia coli.

The Plasmodium vivax merozoite surface protein 1 (MSP-1) 42-kDa fragment (PvMSP-1 p42) is a promising vaccine candidate antigen against the blood stage of the malarial parasite. We have developed a process for the production of this vaccine target, keeping in mind its use in human volunteers. A novel strain, Origami(DE3), of Escherichia coli with mutations in the glutathione and thioredoxin reductase genes yielded 60% more soluble PvMSP-1 p42 than the conventional E. coli BL21(DE3) strain. Recombinant PvMSP-1 p42 was purified to > or = 99% purity with a rapid two-step protocol designed for easy scaling up. The final product had a low endotoxin content and was stable in its lyophilized form. PvMSP-1 p42 was found to have the predicted primary and tertiary structures and consisted of a single conformer containing one free cysteine, as predicted. The product was recognized by conformational monoclonal antibodies against P. vivax MSP-1. Immunogenicity studies of PvMSP-1 p42 were carried out with two strains of mice and the adjuvants Montanide ISA51 and Montanide ISA720. Both formulations were found to induce high levels of immunoglobulin G1 (IgG1), IgG2b, and IgG2a antibodies along with low levels of IgG3. Lymphocytes from animals in all the PvMSP-1 p42-immunized groups showed proliferative responses upon stimulation with PvMSP-1 p42; the cytokines interleukin 2 (IL-2), gamma interferon, IL-4, and IL-10 were detected in the culture supernatants. These results indicate that PvMSP-1 p42 in combination with both of the adjuvants elicited cellular and humoral responses in mice.

The ACVD task force on canine atopic dermatitis (IV): environmental allergens

Numerous environmental allergens have been incriminated in the pathogenesis of canine atopic dermatitis (AD). These include dust and storage mite antigens, house dust, pollens from grasses, trees and weeds, mould spores, epidermal antigens, insect antigens, and miscellaneous antigens such as kapok. In this paper, we review the literature concerning the allergens that have been reported to contribute to canine AD. We conclude that attempts to identify the relevant canine antigens in the past have been plagued by a lack of standardisation of extracts and techniques, and the presence of false-positive and -negative reactions in allergy tests. Until these problems are rectified, it is unlikely that we will be able to provide a list of major and minor antigens for dogs. Hence, we recommend that future studies should be aimed at determining the major patterns of reactivity and cross-reactivity to specific protein allergens within antigenic extracts using electrophoresis and immunoblotting techniques. Once this information becomes available, it may be possible to use a selection of genetically engineered, highly pure antigens for both diagnostic and therapeutic purposes in canine allergy investigations. The use of such antigens will allow standardisation of canine allergy testing and immunotherapy so that the reliability and efficacy of these procedures can be objectively assessed.

Rapid Purification of Recombinant Anthrax-Protective Antigen under Nondenaturing Conditions

Anthrax-protective antigen is the central moiety of the anthrax toxin complex that mediates the entry of the other two toxin components, lethal factor and edema factor into the cells. It is also the main immunogen of the cell-free vaccine against anthrax. However, in addition to PA, the vaccine contains trace amounts of other culture-derived proteins that contribute to the side effects of the vaccine like pain, edema, erythrema, etc. Thus there is a need to develop high-resolution purification methods to purify PA to homogeneity. In this study we have presented a purification strategy for rapid purification of recombinant protective antigen under nondenaturing conditions, which ensures that not only biological activity but also the conformational integrity of immunological epitopes is well-preserved. The protein was purified to homogeneity in a two-step purification procedure that takes just 6 h for completion. Three milligrams of recombinant protective antigen obtained from 1-liter culture was comparable to B. anthracis protective antigen in terms of functional and biological activity. Moreover, the immunogenicity elicited by the purified protein in mice was also studied. The studies reported here are part of continuing research that aims to provide a safe and efficacious alternative to the current vaccine against anthrax.

HHV-8 ELISA based on a one-step affinity capture of biotinylated K8.1 antigen

The immunogenic envelope antigen gp35-37 of human herpesvirus-8 (HHV-8) is encoded by orfK8.1. An ELISA is described using streptavidin capture of bacterially expressed and biotinylated recombinant K8.1 antigen. The antigen capture strategy provides a simple and reliable method, which does not require high yield production and purification of the recombinant antigen before the serological assay. The specificity and sensitivity of the K8.1 ELISA were validated by gp35-37 envelope antigen Western blot and anti-lytic membrane immunofluorescence assay using lytically induced HHV-8 infected BCBL-1 cells. Under the established ELISA conditions, eight of the 10 Kaposi's sarcoma patients and five of the 180 healthy blood donors had IgG antibodies to K8.1 envelope antigen.

Ephrin B1 Is Expressed on Neuroepithelial Cells in Correlation with Neocortical Neurogenesis

To identify molecules involved in neurogenesis, we have raised monoclonal antibodies against embryonic day 12.5 mouse telencephalon. One antibody, monoclonal antibody 25H11, stains predominantly the ventricular zone of the anterior and lateral telencephalon. Purification of the 25H11 antigen, a 47 kDa integral membrane protein, from approximately 2500 mouse telencephali reveals its identity with ephrin B1. Ephrin B1 appears at the onset of neocortical neurogenesis, being first expressed in neuron-generating neuroepithelial cells and rapidly thereafter in virtually all neuroepithelial cells. Expression of ephrin B1 persists through the period of neocortical neurogenesis and is downregulated thereafter. Ephrin B1 is present on the ventricular as well as basolateral plasma membrane of neuroepithelial cells and exhibits an ventricular-high to pial-low gradient across the ventricular zone. Expression of ephrin B1 is also detected on radial glial cells, extending all the way to their pial endfeet, and on neurons in the mantle/intermediate zone but not in the cortical plate. Our results suggest that ephrin B1, presumably via ephrin-Eph receptor signaling, has a role in neurogenesis. Given the ventricular-to-pial gradient of ephrin B1 on the neuroepithelial cell surface and its known role in cell migration in other systems mediated by its repulsive properties, we propose that ephrin B1 may be involved in the migration of newborn neurons out from the ventricular zone toward the neocortex.

Fast selection of antibodies without antigen purification: adaptation of the protein fragment complementation assay to select antigen-antibody pairs

We have adapted the protein fragment complementation assay (PCA) to the screening and selection of antibodies in the single-chain Fv (scFv) format. In this assay, two interacting proteins (target and antibody) are genetically fused to the two halves of the dissected enzyme dihydrofolate reductase. Binding of the two partners reassembles this enzyme and reconstitutes its activity, thus allowing growth on minimal medium. We have optimized this system with regard to linker length and orientation, and can reach an efficiency for antigen/antibody interactions similar to that with fused leucine zippers. Using several model antibodies specific for peptides and proteins, we show that cognate interactions give rise to about seven orders of magnitude more colonies than non-specific interactions. When transforming mixtures of plasmids encoding different antigens and/or antibodies, all colonies tested contained plasmids encoding cognate pairs. We believe that this system will be very powerful as a routine system for generating antibodies, especially in functional genomics, since it does not require purification and immobilization of the antigen. The identification of an antibody specific for a cDNA or EST-encoded protein will require only cloning, transformation and plating of bacteria.

Purification of woodchuck hepatitis surface antigen using a monoclonal antibody raised against the antigen

Woodchuck hepatitis virus (WHV) is closely related to the human hepatitis B virus (HBV) and infection of woodchucks with WHV creates a useful model for studies of immunity, pathogenesis and therapy of HBV infection. To increase the usefulness of this model, monoclonal antibodies were raised to woodchuck hepatitis surface antigen (WHsAg) and one of these antibodies was used to purify the antigen by affinity chromatography from serum, a simpler and quicker method of purification than the current ultracentrifugation methods. The bands found by SDS-polyacrylamide gel electophoresis of WHsAg were the major 25 and 29 kilodalton (kDa) bands and a triplet of 45, 51 and 55 kDa which are thought to be the glycosylated and unglycosylated middle and large WHsAg. Both the antibody and the antigen are valuable reagents for the study of WHV infection.

Stirrer tank: an appropriate technology to immobilize the CB.Hep-1 monoclonal antibody for immunoaffinity purification

The CB.Hep-1 monoclonal antibody was coupled to CNBr-activated Sepharose CL 4B at three different immobilization scales for purification of recombinant hepatitis B surface antigen. Standard laboratory apparatus to obtain immunosorbents of 1 l (scale I) and 3 l (scale II) as well as a stirrer tank to prepare 6 l immunosorbents (scale III) were used. The binding capacity at scale III was 2- and 1.5-fold higher with respect to the scales II and I, while a reduction in the ligand leakage of 5- and 2-folds was observed. Immunosorbents from scale II showed a significantly reduced adsorption, and an increased ligand leakage. Differences in the coupling efficiency were not observed. Antigen purity eluted from the immunosorbents was always above 85%.

Purification of recombinant HBc antigen expressed in Escherichia coli and Pichia pastoris: comparison of size-exclusion chromatography and ultracentrifugation

Hepatitis B virus core protein (HBc) is an important serology marker of hepatitis B infection and patient follow-up. It is an M r 21 000 protein, which has the intrinsic capacity to self-assemble as a capsid-like particle. The hepatitis B core protein has been expressed in Escherichia coli and Pichia pastoris (three different constructions) in order to select a HBc recombinant antigen suitable for serodiagnosis requirements with a cost effective downstream strategy. The expression and purification of the different forms of recombinant HBc have been described. For the last step, ultracentrifugation and size-exclusion chromatography were compared. The morphology of these capsids was observed using an electron microscope. Our data shows that HBc antigen is produced in large quantities in E. coli but some contaminants remained which were associated with the E. coli HBc protein after ultracentrifugation or size-exclusion chromatography. The ultracentrifugation enables a higher purity of HBc antigen to be obtained than size-exclusion chromatography but the latter enables a higher recovery rate. P. pastoris enables the expression and extraction of a highly purified HBc antigen suitable for diagnostic purposes.

The Role of the Aceullular Pertussis Vaccine and the Comeback of 'Pertussis Pete'?

Pertussis or whooping cough is an acute infectious disease of the respiratory tract caused principally byBordetella pertussisand less commonly byBordetella parapertussis(1). Until two decades ago, pertussis in adults was a medical curiosity (2-4), but with the purification of specificBordetellaspecies antigens, the development of reliable enzyme immunoassays allowing accurate serological diagnosis and better understanding of the duration of immunity from vaccination, it has been clearly demonstrated thatB pertussisis a common cause of prolonged cough in adults. Indeed, its incidence has been increasing gradually over the past decade in both adults and adolescents. Given the recognition of the importance of pertussis as a cause of prolonged cough in adults and the advent of the new acellular pertussis vaccines, it is timely to review current concepts of the pathogenesis of pertussis, its epidemiology in adults and the utility of the anticipated impact of the acellular vaccine.

Expression, purification, and characterization of active recombinant prostate-specific antigen in Pichia pastoris (yeast).

Prostate-specific antigen (PSA), a member of the kallikrein family of serine proteases, is a chymotrypsin-like glycoprotein produced by the prostate epithelium. Elevated serum PSA (> 4 ng/ml) is a tumor marker for prostatic cancer and benign prostatic hypertrophy; increasing serum PSA over time is indicative of metastatic disease. It has been suggested that PSA may contribute to tumor metastasis through degradation of extracellular matrix glycoproteins, as well as cleavage of IGF binding protein-3, a modulator of IGF-1. To elucidate the role of PSA in the development and progression of prostatic cancer, it is necessary to have a reliable, cost-effective source of enzymatically active protein. Previous efforts to express recombinant PSA (rPSA) produced inactive proPSA, or mixtures of active and inactive PSA requiring activation by removal of the propeptide. We describe the expression of active recombinant mature PSA in yeast. Stable chromosomal integration of a construct consisting of the yeast alpha-factor signal sequence preceding the mature PSA sequence resulted in secretion of rPSA. The rPSA was purified from the yeast cell culture supernatant to homogeneity by strong cation-exchange chromatography, and characterized by SDS-PAGE, Western analysis, electrospray mass spectrometry, N-glycanase digestion, N-terminal amino acid sequencing, and inactivation by a PSA-specific inhibitor. We report the production of active, mature rPSA in Pichia pastoris. Two forms of rPSA varying slightly in glycosylation were identified. The specific activity of the rPSA was equal to that of human seminal plasma PSA (0.56 micromol/min mg) as determined using a chromogenic substrate. Large-scale production of active rPSA will be useful in the exploration of PSA effects on tumor cell proliferation, migration and metastasis. In addition, a large supply of enzyme should facilitate the discovery of novel inhibitors for in vitro and in vivo evaluation, and may provide a reproducible source of rPSA for use as a standard in diagnostic testing.

Immunogeneity and structural organisation of some pLCR-encoded proteins of Yersinia pestis.

A novel method of cultivation of Yersinia pestis EV-76 and its isogenic strains KM-217 (pPst-;pCad+;pFra-) and KM-218 (pPst-;pCad-;pFra-) and careful extraction of Y. pestis proteins (YPPs) permitted isolation of >35 low Ca2+ response plasmid (pLCR)-encoded products, some of which are potentially new members of the LCR family. Immunisation with each YPP demonstrated that 25-, 54-, 72- and 87-kDa YPPs provided the highest level of protection in mice challenged with Y. pestis virulent strain 231. Their immunological relationship was established with monoclonal antibodies (MAbs) and revealed several common properties, including oligosaccharide binding with specificity for N-acetylglucosamine. Affinity chromatography with MAb to the 25-kDa YPP permitted purification of the relevant antigen and its precursor. Their existence in the form of a complicated protein molecule was shown.

Process optimization for enhanced production of diphtheria toxin by submerged cultivation

When stationary culture was replaced by submerged cultivation in a fermentor, a significant increase in the yield of diphtheria toxin in a short cultivation time (less than 48 h as against 7–8 d) was noted. It was found that under optimal conditions of temperature, vortex mixing and surface aeration, an alkaline pH favoured toxin release. Furthermore, to enhance the production volume, two-and three-step semicontinuous batch cultivations were carried out. The toxin produced was of good titre with an adequate antigenic purity. Under optimal conditions, the variation in the Limes of flocculation (Lf titre) was likely due to the quality of the production medium, which in turn depended on the quality of the raw materials used. The process was also optimized in a pilot-scale fermentor.

Process Optimization for Enhanced Production of Diphtheria Toxin by Submerged Cultivation.

When stationary culture was replaced by submerged cultivation in a fermentor, a significant increase in the yield of diphtheria toxin in a short cultivation time (less than 48 h as against 7-8 d) was noted. It was found that under optimal conditions of temperature, vortex mixing and surface aeration, an alkaline pH favoured toxin release. Furthermore, to enhance the production volume, two-and three-step semicontinuous batch cultivations were carried out. The toxin produced was of good titre with an adequate antigenic purity. Under optimal conditions, the variation in the Limes of flocculation (Lf titre) was likely due to the quality of the production medium, which in turn depended on the quality of the raw materials used. The process was also optimized in a pilot-scale fermentor.

Liquid-liquid extraction of a Schistosoma mansoni recombinant protein from an Escherichia coli extract in aqueous two-phase system

This work describes the partition of a Schistosoma mansoni tegumental antigen produced by a recombinant Escherichia coli strain using an aqueous two-phase system composed of polyethylene glycol (PEG) and potassium phosphate. The effects of the polymer molecular weight, tie line length and pH on antigen partitioning were investigated. The detection of the antigen in both phases were determined by ELISA. The system composed of PEG 3550 (19.7% w/w) and potassium phosphate (17.7% w/w) led to a yield of 59% and an antigen purification factor of 3 in the PEG-rich phase. It was observed that the antigen partition in ATPS was strongly affected by the pH value and tie line length. In addition, it was possible in a single step, to remove the cell debris, that precipitated at the interface of the system.

A quantitative flow cytometry assay for the preclinical testing and pharmacological monitoring of rabbit antilymphocyte globulins (rATG)

Rabbit antilymphocyte or antithymocyte globulins (rATG) are currently used as immunosuppressive agents in clinical organ and bone marrow transplantation, but because of differences in antigen source and purification processes between manufacturers, determination of the dose to be administered remains empirical. Dosage may be adjusted to peripheral blood T cell counts or limited by side effects such as neutropenia or thrombocytopenia. We report here a measurement by indirect immunofluorescence of the amount of antibodies from rATG that bind to different blood cell types. A concentration-related immunofluorescence signal was obtained with reasonable replicability, allowing comparison of different rATGs by reference to the same rATG batch as internal standard. Ratios of antilymphocyte to undesirable antibodies (directed against antigens expressed on neutrophils, platelets or red cells) could be calculated for each rATG preparation. Finally, the method is applicable to the measurement of free antibodies in sera from patients treated with rATG and to the analysis of the effects of in vivo absorption of rATG during its administration. Marked differences in antibody content per mg of rIgG account for the differences in rATG dosage that were empirically established for clinical applications.

Purification of Prostate-specific Antigen from Serum by Indirect Immunosorption and Elution with a Hapten

Preparative purification of macromolecules, e.g., proteins, has reached a high standard because of efficient separation materials that are available for this task. However, this is limited to samples containing substantial amounts of the desired protein. When proteins that are present only in minute amounts (e.g., in the μg/L range) must be isolated from a limited supply of complex biological material (e.g., human serum), a one-step method is recommended to obtain the protein in sufficient yield. When suitable antibodies are available, immunopurification often is the method of choice (1), but nonspecific binding of proteins to affinity matrices is a substantial problem because elution with acid or chaotropic agents very often washes off the impurities as well as the analyte (2). Thus, there is a need for methods that allow the specific elution of the desired proteins without eluting the impurities. Specific elution by competing with a low-molecular weight analog is appealing, but suitable analogs may not be available. We wanted to isolate prostate-specific antigen (PSA) from serum and analyze its structure by mass spectrometry. The concentration of PSA in serum from patients with prostate cancer (PCa) ranges from ∼3 to >3000 μg/L, ∼105–107 times less than that of other serum proteins, e.g., albumin. We have developed a general indirect immunosorption method that follows to a certain extent an immunoassay principle developed by Hashida et al. (3) and Ishikawa et al. (4) and makes use of a digoxigenylated anti-analyte antibody. Initially, the …