Expression and purification of a single-chain variable fragment antibody derived from a polyol-responsive monoclonal antibody

A previously described polyol-responsive monoclonal antibody (PR-mAb) was converted to a single-chain variable fragment (scFv). This antibody, PR-mAb NT73, reacts with the beta' subunit of Escherichia coli RNA polymerase and has been used for the immunoaffinity purification of polymerase. mRNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were used as the template for cDNA synthesis. The sequences were joined by the addition of a "linker" sequence and then cloned into several expression vectors. A variety of expression plasmids and E. coli hosts were used to determine the optimal expression system. Expression was highest with the pET22b(+) vector and the Rosetta(DE3)pLysS host strain, which produced approximately 60 mg purified His-tagged scFv per liter of culture (3.3 g wet weight cells). Although the production of soluble scFv was preferred, overproduced scFv formed inclusion bodies under every expression condition. Therefore, inclusion bodies had to be isolated, washed, solubilized, and refolded. The FoldIt protein refolding kit and enzyme-linked immunosorbent assay were sequentially used to determine the optimal refolding conditions that would produce active His-tagged scFv. Immobilized metal affinity chromatography was used for the final purification of the refolded active scFv. The polyol-responsiveness of the scFv was determined by an ELISA-elution assay. Although the scFv loses considerable affinity for its antigen, it maintains similar polyol-responsiveness as the parent monoclonal antibody, PR-mAb NT73.     

Immunofluorescent staining of mammalian nuclei and chromosomes with a monoclonal antibody to triplex DNA

Triplex DNA is an unusual conformation of DNA formed when two pyrimidine nucleotide strands share a common purine strand. A monoclonal antibody, demonstrated by numerous criteria to be specific for triplex DNA, was used to investigate the presence and distribution of this unique DNA configuration in nuclei and chromosomes of mouse LM cells and human lymphocytes. Indirect immunofluorescence microscopy revealed that constitutive heterochromatin in acetic-methanol fixed mouse nuclei was usually, but not always immunofluorescent, suggesting possible cell cycle related variations in the amount of triplex DNA or its accessibility in this condensed chromatin. In fixed mouse and human chromosomes, there was a positive correlation between immunofluorescent staining patterns, Hoechst 33258 banding, and G- and/or C-banding patterns. Unfixed, isolated mouse chromosomes also reacted positively with the antibody, particularly when they were gently decondensed by exposure to low ionic conditions at neutral pH. This result indicates that fixation is not mandatory for antibody staining, suggesting that some mammalian chromosomal DNA may be naturally organized in a triplex configuration. However, there is a possibility that fixation may facilitate the formation of additional triplex DNA complexes in potential sequences or expose previously inaccessible triplex DNA. The precise correspondence between the immunofluorescent patterns produced by anti-triplex DNA antibodies and G- and C-bands known to represent regions of chromatin condensation, suggests a potential role of triplex DNA in chromosome structure and regional chromatin condensation.     

Role of endogenous ANP on endocrine function investigated with a monoclonal antibody

Substantial volume expansion in conscious rats induces a strong natriuresis, cyclic GMP excretion, increase in cyclic GMP in plasma and kidney tissue, decrease in plasma renin activity and plasma aldosterone concentration. These effects are directly related to an increase in plasma levels of atrial natriuretic peptides. The renal response and the changes in plasma and kidney cyclic GMP, plasma renin activity and aldosterone could be totally blocked by simultaneous administration of monoclonal antibodies directed against ANP. From this study it seems to be clear that the rise in cyclic GMP and the inhibition of the renin-aldosterone system is not a direct effect of volume expansion but is specifically mediated by the released ANP. The great importance of ANP in acute volume expansion made us wonder about the role of ANP in chronic volume expansion and under basal conditions without volume loading. Chronic volume loading was induced pharmacologically by the sodium retaining vasodilatator minoxidil. Under both chronic volume expansion and basal conditions the neutralization of the circulation ANP by antibody administration leads to reduced plasma cyclic GMP levels. No alterations in urinary sodium excretion, plasma renin activity and plasma aldosterone concentration could be observed: In conclusion, the monoclonal antibody directed against ANP is a useful tool for the investigation of the physiological role of endogenous ANP.     

Purification of aromatic L-amino acid decarboxylase from bovine brain with a monoclonal antibody.

Aromatic L-amino acid decarboxylase was purified from bovine brain for the first time by affinity chromatography using a monoclonal antibody to the enzyme, and it was compared with the decarboxylase purified from bovine adrenal medulla by the same procedure. The monoclonal antibody was produced from a hybridoma established for the enzyme highly purified from bovine adrenal medulla. The Mr values of brain and adrenal-medulla enzyme were both estimated to be approx. 100,000 by gel-permeation chromatography. SDS/polyacrylamide-gel electrophoresis revealed a single band with an apparent Mr of 50,000. Western immunoblot analysis showed that the antibody recognized each enzyme. With regard to substrate specificity, pH-dependence and effect of pyridoxal 5'-phosphate as a cofactor, both enzymes were similar.     

A rapid and sensitive ELISA to quantify an HBsAg specific monoclonal antibody and a plant-derived antibody during their downstream purification process.

An enzyme-linked immunosorbent assay to quantify the mAb CB.Hep-1 during downstream purification process was standardized and validated. This assay is characterized by a short time of incubation at high temperature, allowing the detection of this antibody with high specificity and sensitivity. Detection of antigen-antibody reaction was achieved using a horseradish peroxidase conjugated anti-mouse IgG whose enzyme activity was revealed with o-phenylenediamine substrate. The immunoassay is linear in a range between 3.12 and 50 ng/mL, with a recovery of 98.55-107.62%. According to results, it is possible to estimate the mAb CB.Hep-1 concentration with high precision and reproducibility. The intra- and interassay coefficient of variation ranged from 0.25 to 8.64% and 1.84 to 9.43%, respectively. Significant differences were not observed in the plant-derived antibody quantification by HRP-ELISA and PhoA-ELISA (n=18), demonstrating that plant endogenous peroxidases do not produce interferences in the quantification of this molecule. Therefore, both antibodies can be tested with the same immunoassay with high precision, specificity and accuracy during their respective purification processes without interference of the buffers and sample characteristics.     

Demonstration of an in vivo generated sub-picomolar affinity fully human monoclonal antibody to interleukin-8

The high specificity and affinity of monoclonal antibodies make them attractive as therapeutic agents. In general, the affinities of antibodies reported to be high affinity are in the high picomolar to low nanomolar range and have been affinity matured in vitro. It has been proposed that there is an in vivo affinity ceiling at 100 pM and that B cells producing antibodies with affinities for antigen above the estimated ceiling would have no selective advantage in antigen-induced affinity maturation during normal immune responses. Using a transgenic mouse producing fully human antibodies, we have routinely generated antibodies with sub-nanomolar affinities, have frequently rescued antibodies with less than 10 pM affinity, and now describe the existence of an in vivo generated anti-hIL-8 antibody with a sub-picomolar equilibrium dissociation constant. This confirms the prediction that antibodies with affinities beyond the proposed affinity ceiling can be generated in vivo. We also describe the technical challenges of determining such high affinities. To further understand the importance of affinity for therapy, we have constructed a mathematical model to predict the relationship between the affinity of an antibody and its in vivo potency using IL-8 as a model antigen.     

Rapid purification of the oxygenase component of toluene dioxygenase from a polyol-responsive monoclonal antibody.

A monoclonal antibody designated 302 beta that is specific for the beta subunit of the oxygenase component (ISPTOL) of toluene dioxygenase from Pseudomonas putida F1 was used to prepare an immunoaffinity column. ISPTOL in cell extracts of Escherichia coli JM109(pDTG611) bound to the column, and an enzyme-linked immunosorbent elution-screening assay with different combinations of polyols and kosmotropic anions was used to determine the conditions necessary for recovery of active enzyme. Elution from an 8-ml antibody column with 50 mM 2-(N-morpholino)ethanesulfonate buffer (pH 6.8) containing 50% ethylene glycol, 1.0 M ammonium sulfate, 1.0 mM dithiothreitol, and 0.2 mM ferrous ammonium sulfate gave approximately 2 mg of ISPTOL with a specific activity that was more than 300 times the specific activity previously obtained.     

Studies of mammalian ornithine decarboxylase using a monoclonal antibody.

A monoclonal antibody of the immunoglobulin M class was produced against mouse kidney ornithine decarboxylase. Screening for the antibody was carried out using alpha-difluoromethyl[5-3H]ornithine-labelled ornithine decarboxylase. The antibody reacted with this antigen and with native ornithine decarboxylase. The antibody attached to Sepharose could be used to form an immunoaffinity column that retained mammalian ornithine decarboxylase. The active enzyme could then be eluted in a highly purified form by 1.0M-sodium thiocyanate. The monoclonal antibody could also be used to precipitate labelled ornithine decarboxylase from homogenates of kidneys from androgen-treated mice given [35S]methionine. Only one band, corresponding to Mr of about 55000, was observed. The extensive labelling of this band is consistent with the rapid turnover of ornithine decarboxylase protein, since this enzyme represents only about 1 part in 10000 of the cytosolic protein.     

Inactive proenzyme to tissue-type plasminogen activator from human melanoma cells, identified after affinity purification with a monoclonal antibody

The human 66 000 mol. wt. plasminogen activator (HPA66; tissue-type plasminogen activator) has been purified from melanoma cells by a one-step affinity method with a monoclonal antibody. HPA66 purified in this way consists mainly of a one-polypeptide chain form with small amounts (15%) of a form containing two polypeptide chains held together by one or more disulphide bridges. The one-chain form was converted to the two-chain form by catalytic amounts of plasmin. During the conversion, the enzyme activity of HPA66, as measured by an [125I]plasminogen conversion assay and with a chromogenic substrate, increased linearly with the percentage of the two-chain form. A linear regression analysis showed that all enzyme activity could be accounted for by the two-chain form, while the one-chain form had no measurable enzyme activity (detection limit approximately 5% of the activity of the two-chain form). Together with previous findings of inactive proenzymes to murine and human approximately 50 000 mol. wt. (urokinase-type) plasminogen activators, these findings indicate that plasminogen activators are generally formed from inactive one-chain proenzymes which are converted to active two-chain enzymes by limited proteolysis, thus demonstrating a third step in a cascade reaction leading to extracellular proteolysis.     

Very fast purification of ornithine decarboxylase with high yield from mouse kidney and generation of a monoclonal antibody

Based on methods for ornithine-decarboxylase purification published previously we developed an improved procedure for purification of the enzyme from the kidneys of testosterone-treated NMRI mice. Advantages of the new procedure are, that inactivation of the enzyme during purification is largely reduced by fast methods for purification and by the use of proteinase inhibitors. That way we got pure ornithine decarboxylase within 60 h with a yield of about 70%. A part of the highly purified ornithine decarboxylase was used for the generation of monoclonal antibodies.     

Demonstration of different metal ion-induced calcineurin conformations using a monoclonal antibody

It has been suggested that calcineurin, a calmodulin-stimulated phosphatase, may exist in different metal ion-dependent conformational states (Pallen, C.J., and Wang, J. H. (1984) J. Biol. Chem. 259, 6134-6141). Evidence in favor of this hypothesis comes from studies involving a monoclonal antibody, VA1, which is specific for the small (beta) subunit of calcineurin. This antibody inhibits Ni2+-stimulated but not Mn2+-stimulated phosphatase activity against p-nitrophenyl phosphate and phosphorylase kinase. Inhibition is not due to competition of the antibody with substrate or to interference with metal ion binding to the enzyme. Complex formation between the antibody and calcineurin can be demonstrated either in the presence of Mn2+ or Ni2+ or in the absence of metal ion activators. These results indicate that the active conformational states of calcineurin are metal ion dependent, that the monoclonal antibody VA1 affects the Ni2+-induced conformational change of the enzyme, and that the beta subunit of calcineurin plays a critical role in the expression of Ni2+-stimulated phosphatase activity.     

A monoclonal antibody to bovine brain inositol monophosphatase. Immunoaffinity purification of the brain and kidney enzymes and evidence for their structural identity.

A monoclonal IgG2b(K) antibody, G-2A4, has been generated against bovine brain myo-inositol monophosphatase (EC 3.1.3.25). The identity of the antigen recognized by the antibody was established by using e.l.i.s.a. and Western blotting procedures, and by immunoprecipitation of enzyme activity from crude brain supernatant. In addition, the hydrolysis of Ins1P by crude brain extract was inhibited by up to 83% by the pure antibody. Under identical conditions, the hydrolysis of Ins(1,4)P2 was unaffected. An immunoadsorbent column containing monoclonal antibody G-2A4 covalently attached to CNBr-activated Sepharose 4B has been used for rapid purification of the brain enzyme. Elution conditions have been optimized to allow isolation of the enzyme in high yield (54%) with full retention of column-binding capacity. The enzyme was electrophoretically homogeneous, Mr 30,000 and of higher specific activity than that purified conventionally. Chromatography of the pure enzyme on high resolution ion-exchange columns revealed some charge heterogeneity, possibly indicative of some type of post-translational modification. The immunoadsorbent column has also been used to purify the bovine kidney cortex enzyme to homogeneity. Partial proteolytic fragmentation patterns of the brain and kidney enzymes using endoprotease glu-C were identical, suggesting that they are almost certainly products of the same gene.     

Identification of an endogenous peptide-ligand for the benzodiazepine receptor

An inhibitor of ³H-diazepam binding with characteristics that distinguish it from the other endogenous ligands reported for the benzodiazepine receptor was obtained from bovine brain. After isolation by gel filtration and ion exchange chromatography, the inhibitory factor was found to be a weakly charged molecule of approximately 3000 daltons. Although heat stable, the activity of the factor can be destroyed by treatment with papain. This factor, presumably peptide in nature, inhibited ³H-diazepam binding competitively and in a concentration dependent fashion.     

Demonstration of a surface antigen of Clostridium tyrobutyricum by use of immunoblotting with a monoclonal antibody

A monoclonal antibody, prepared against whole cells of Clostridium tyrobutyricum, recognized a surface antigen extracted by heat treatment or by hot phenol-water treatment. This antigen, after analysis by polyacrylamide gel electrophoresis and immunoblotting, has been shown to present a regularly-spaced ladder pattern similar to those shown by the lipopolysaccharide of many gram-negative bacteria. The proteinase K has been shown to have no effect on the recognition of this epitope by the monoclonal antibody. On the contrary, the inhibition of the antigen reactivity to the monoclonal antibody after a mild periodate oxidation suggests the involvement of a carbohydrate moiety in the epitope. Moreover, the SDS-PAGE analysis of phenol-water extracts has shown an additional compound, detected by silver staining but not recognized by the monoclonal antibody.     

Production of a monoclonal antibody to an attachment protein derived from human cementum.

Cementum is the mineralized structure that covers the surface of the roots of teeth; it serves as the attachment site for collagen fibers of adjacent soft connective tissues. Very little is known about how cementum formation is regulated or how it affects other periodontal structures. We have raised a monoclonal antibody that may aid in studies to determine the biology and function of cementum. Mice were immunized with a 55-kDa attachment protein partially purified from human cementum and a monoclonal antibody, H166, was produced. Incubation of tissue sections with this antibody and fluorescein isothiocyanate-conjugated secondary antibody revealed that it immunostains cementum but not dentin, gingiva, or periodontal ligament. Alveolar bone did not bind the antibody, although a few paravascular cells were positive. Long bones, kidney, liver, skin, and several other tissues were negative. Protein fractions separated from cementum extracts by binding to immobilized H166 column contained 55-, 49-, 39-, 29- to 31-, and 23- to 26-kDa components that cross-reacted with the antibody in Western blots; these components were previously shown to be derived from a common precursor. We conclude that the antibody recognizes a group of proteins related to 55-kDa attachment protein in cementum. Our data show that the antibody could serve as a marker for cementum.     

Process and economic evaluation for monoclonal antibody purification using a membrane-only process

In recent years, the market for therapeutic monoclonal antibodies (mAb) has grown exponentially, and with this there has been a desire to reduce the costs associated with production and purification of these high-value biological products. A typical mAb purification process involves three adsorption/chromatography steps [protein A, ion exchange (IEX), and hydrophobic interaction (HIC)], along with ultrafiltration, nanofiltration, and microfiltration. With the development of membrane adsorption/chromatography as a viable alternative to traditional pack bed systems, the opportunity exists to complete the entire downstream purification process using only membrane operations. In this study, the process simulation tool SuperPro Designer was used to evaluate the application of recently developed ultra-high capacity electrospun nanofibrous adsorption membranes as a replacement for conventional chromatographic media in the downstream mAb production process. The simulation showed that nanofibrous adsorption membranes in place of the three packed bed chromatography steps reduced the required volume of protein A, IEX, and HIC adsorptive medium by 25, 80, and 80%, respectively. In addition, the membrane-only process reduced the downstream processing time by 50%, decreased the number of labor hours associated with the purification steps by 40%, generated 40% less aqueous waste, and reduced the overall downstream process operating expenses per unit product by 23%. There were also significant savings in facility construction costs and the price of fixed equipment required for separations. With these savings not only is the membrane-only process economically competitive with the traditional packed bed operations, but it offers the possibility of moving toward more disposable process.     

Preliminary studies on the purification of a monoclonal antibody by affinity precipitation with Eudragit S-100

A simple procedure for the purification of an IgG-type monoclonal antibody by affinity precipitation using Eudragit S-100 is presented. The ligand, a microbial lipase previously used as antigen, was coupled to the polymer at a concentration of 40 mg lipase/g Eudragit. This macroligand was reversibly precipitated by manipulating the pH at values higher and lower than 4.8. The effects of polymer concentration and dilution of hybridoma culture supernatant on the overall precipitation process were evaluated. The best purification factor was achieved with a polymer concentration of 0.1% (w/v) and a supernatant dilution of 1:3. The preliminary studies reported here enabled the purification of a monoclonal antibody in one step with an activity yield (by ELISA) of 50%-55% and a purification factor of ca 6.     

Hexokinase A from mammalian brain: comparative peptide mapping and immunological studies with monoclonal antibodies

Immunological reactivity of partially purified hexokinase A (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from brain of several vertebrate species has been compared by using enzyme-linked immunosorbent assay and seven monoclonal antibodies raised against the rat brain enzyme. The epitopes recognized by three of these antibodies have been rather widely conserved among the species examined (rat, mouse, guinea pig, rabbit, cat, dog, sheep, cow, pig, chicken), while this was not the case for the epitopes recognized by the other antibodies, which differed markedly in their distribution among these species. The domain structure of these enzymes has been examined by peptide mapping (after limited tryptic digestion) in conjunction with immunoblotting techniques employing monoclonal antibodies. The results indicate that the overall domain structure of these enzymes is similar to that previously described for rat brain hexokinase A, but that there are significant differences in the size of these domains in enzymes from different species.