Biological repair of thyroid cartilage defects by osteogenic protein-1 (bone morphogenetic protein-7) in dog.

The efficacy of human recombinant osteogenic protein-1 (OP-1; bone morphogenetic protein-7) in regeneration of dog larynx was examined by treating thyroid cartilage defects (1.5 cm2) in dogs with thyroid allografts covered with host perichondrium or fascia. Prior to implantation allografts were frozen, thawed and demineralized. The treatment groups were as follows: I--Allograft control implant (n = 3); II--Implants coated with 500 micrograms OP-1 (n = 4); III--Implants coated with 100 micrograms OP-1 (n = 3); IV--Implants coated with 500 micrograms OP-1 and covered with neck fascia (n = 3); and V--Implants extracted with 1 M NaCl and guanidine hydrochloride, and coated with 500 micrograms OP-1 (n = 4). Dogs were sacrificed four months following surgery. Each larynx was removed, carefully dissected and a three-dimensional reconstruction of the defect area was performed on serial sections. The results revealed that the implants of control dogs remained intact with no apparent reduction in size and new tissue formation. OP-1 enriched thyroid allografts, dose dependently induced bone, cartilage and ligament-like structures comprising up to 80% of the total regenerated defect area. Boundaries of the defects healed by formation of new bone when bone resided within the old thyroid cartilage layers. Old cartilage not containing bone within its layers healed by complete integration with newly formed cartilage. Both new bone and cartilage were embedded into layers of new ligament-like tissue which expressed specific morphologic and molecular markers. The three newly formed tissues were tightly connected into a "bone-cartilage-ligament continuum" of tissues, suggesting that OP-1 served as a multiple tissue morphogen in this specific microenvironment.

Lessons learned in preparing method 29 filters for compliance testing audits.

Companies conducting compliance testing are required to analyze audit samples at the time they collect and analyze the stack samples if audit samples are available. Eastern Research Group (ERG) provides technical support to the EPA's Emission Measurements Center's Stationary Source Audit Program (SSAP) for developing, preparing, and distributing performance evaluation samples and audit materials. These audit samples are requested via the regulatory Agency and include spiked audit materials for EPA Method 29-Metals Emissions from Stationary Sources, as well as other methods. To provide appropriate audit materials to federal, state, tribal, and local governments, as well as agencies performing environmental activities and conducting emission compliance tests, ERG has recently performed testing of blank filter materials and preparation of spiked filters for EPA Method 29. For sampling stationary sources using an EPA Method 29 sampling train, the use of filters without organic binders containing less than 1.3 microg/in.2 of each of the metals to be measured is required. Risk Assessment testing imposes even stricter requirements for clean filter background levels. Three vendor sources of quartz fiber filters were evaluated for background contamination to ensure that audit samples would be prepared using filters with the lowest metal background levels. A procedure was developed to test new filters, and a cleaning procedure was evaluated to see if a greater level of cleanliness could be achieved using an acid rinse with new filters. Background levels for filters supplied by different vendors and within lots of filters from the same vendor showed a wide variation, confirmed through contact with several analytical laboratories that frequently perform EPA Method 29 analyses. It has been necessary to repeat more than one compliance test because of suspect metals background contamination levels. An acid cleaning step produced improvement in contamination level, but the difference was not significant for most of the Method 29 target metals. As a result of our studies, we conclude: Filters for Method 29 testing should be purchased in lots as large as possible. Testing firms should pre-screen new boxes and/or new lots of filters used for Method 29 testing. Random analysis of three filters (top, middle, bottom of the box) from a new box of vendor filters before allowing them to be used in field tests is a prudent approach. A box of filters from a given vendor should be screened, and filters from this screened box should be used both for testing and as field blanks in each test scenario to provide the level of quality assurance required for stationary source testing.

Single-chain Fv radioimmunotargeting.

The availability of engineered antibody species has catalyzed new developments in radioimmunotargeting. This chapter summarized recent studies of single-chain Fv (sFv) proteins, which are minimal antibody binding sites engineered as single polypeptide chains. The single-chain Fv can be as small as 26 kDa monomers or may be engineered as larger fusion proteins designed to self-associate into dimeric or multimeric species. They typically exhibit rapid clearance that results in high targeting specificity within a matter of hours. We have compared different modes of administration to allow further manipulation of their biodistribution and targeting properties. Results of the present study comparing intravenous (i.v.) and intraperitoneal (i.p.) administration show comparable long-term retention in circulation, but the i.v. route showed an initially high peak blood level while i.p. injection did not. As with a single sFv dose, repeated bolus injections of sFv attained high target-to-background ratios, whereas continuous sFv infusion reached a steady state level of free sFv in blood and kidney that exceeded that in tumor xenografts. We observed improved localization of radioiodinated sFv in tumor xenografts if the radioiodine label resisted dehalogenation from the protein, which was accomplished, for example, through conjugation of a para-131I-benzoyl group to Iysyl epsilon-amino groups of the protein. Modification of the sFv by genetic incorporation of a cysteinyl peptide (to form sFv') provided a chelation site for radiometals that simplified incorporation of 99mTc with the opportunity for improved diagnostic imaging in cancer and other diseases. Therapeutic applications of sFv radioimmunotargeting could rely on sFv' complexed to 186Re or 188Re. Engineering sFv of sFv' with increased antigen-binding affinity and appropriately manipulating their mode of administration should promote sustained tumor retention conducive to clinically useful therapeutic indices.

Enhanced tumor specificity of 741F8-1 (sFv')2, an anti-c-erbB-2 single-chain Fv dimer, mediated by stable radioiodine conjugation.

UNLABELLED: The goal of this study was to determine if the stabilization of the radioiodine-protein bond by the N-succinimidyl p-iodobenzoate (PIB) method improved the degree and specificity of tumor localization of 125I-741F8-1 (sFv')2, an anti-c-erbB-2 sFv dimer, in an immunodeficient murine model. METHODS: Gamma camera images were acquired 21 hr after intravenous administration of 131I-741F8-1 (sFv')2 labeled by the p-iodobenzoate or chloramine T methods. The stability of the radioiodine-protein bond also was assessed in plasma samples after intravenous injection of 125I-741F8-1 (sFv')2 labeled by either the chloramine T or p-iodobenzoate methods. RESULTS: By 6 hr postinjection, 97% of the activity associated with the 125I-741F8-1 (sFv')2 labeled by the p-iodobenzoate method was protein bound compared with 61% after labeling with the chloramine-T method. These observations indicate that increasing the stability of the conjugation between the radioiodine and the sFv molecule can significantly increase the degree and specificity of tumor targeting. Significantly greater tumor retention (p < 0.005) and lower blood (p < 0.001), spleen (p < 0.001) and stomach (p < 0.005) retention were observed in biodistribution studies when the p-iodobenzoate conjugate was used. This resulted in superior tumor-to-organ ratios for all tissue samples studied. CONCLUSION: These observations may have clinical relevance for the use of radiolabeled sFv as imaging agents.

Targeting c-erbB-2 expressing tumors using single-chain Fv monomers and dimers.

Single-chain Fv proteins containing a COOH-terminal cysteine (sFv') were constructed by using an antidigoxin 26.10 sFv and an anti-c-erbB-2 741F8 sFv. The fully active sFv' proteins were prepared by expression in Escherichia coli as insoluble inclusion bodies, followed by in vitro refolding using glutathione redox buffers and purification. The COOH-terminal cysteines of the refolded sFv' proteins were protected by a blocking group presumed to be the glutathionyl peptide, which was easily and selectively removed by gentle reduction. Air oxidation of the reduced sFv' monomers resulted in the efficient formation of disulfide-linked sFv' homodimers, designated (sFv')2, which were stable under oxidizing conditions and relatively slow to be disrupted under reducing conditions. The (26-10-1 sFv')-(741F8-1 sFv') heterodimer was prepared and possessed dual-antigen specificity; the active bispecific (sFv')2 dimerized under native conditions, apparently as a manifestation of self-association by the 741F8 sFv' subunit. Biodistribution and imaging studies that were performed on mice bearing human SK-OV-3 tumor xenografts that express the c-erbB-2 as a cell surface antigen were reviewed. Radioiodinated 741F8-2 (sFv')2 homodimer localized to the tumors with high specificity, as evidenced by excellent tumor:normal tissue ratios. Sagittal section autoradiography of whole animals 24 h after administration of antibody species revealed that 741F8 (sFv')2 produced a stronger tumor image than comparable doses of the 741F8 Fab, monomeric sFv', and the 26-10 (sFv')2 control without the high nonspecific background distribution of the 741F8 IgG.

Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide.

We describe a method to facilitate radioimaging with technetium-99m (99mTc) by genetic incorporation of a 99mTc chelation site in recombinant single-chain Fv (sFv) antibody proteins. This method relies on fusion of the sFv C terminus with a Gly4Cys peptide that specifically coordinates 99mTc. By using analogues of the 26-10 anti-digoxin sFv as our primary model, we find that addition of the chelate peptide, to form 26-10-1 sFv', does not alter the antigen-binding affinity of sFv. We have demonstrated nearly quantitative chelation of 0.5-50 mCi of 99mTc per mg of 26-10-1 sFv' (1 Ci = 37 GBq). These 99mTc-labeled sFv' complexes are highly stable to challenge with saline buffers, plasma, or diethylenetriaminepentaacetic acid. We find that the 99mTc-labeled 741F8-1 sFv', specific for the c-erbB-2 tumor-associated antigen, is effective in imaging human ovarian carcinoma in a scid mouse tumor xenograft model. This fusion chelate methodology should be applicable to diagnostic imaging with 99mTc and radioimmunotherapy with 186Re or 188Re, and its use could extend beyond the sFv' to other engineered antibodies, recombinant proteins, and synthetic peptides.

Optimization of in vivo tumor targeting in SCID mice with divalent forms of 741F8 anti-c-erbB-2 single-chain Fv: effects of dose escalation and repeated i.v. administration.

Single-chain Fv molecules in monovalent (sFv) and divalent [(sFv')2] forms exhibit highly specific tumor targeting in mice as a result of their small size and rapid systemic clearance. As a consequence, there is a rapid reversal of the sFv blood/tumor gradient, resulting in diminished retention of sFv species in tumors. In this report we investigate two distinct strategies, dose escalation and repetitive intravenous (i.v.) dosing, aiming to increase the absolute selective retention of radiolabeled anti-c-erbB-2 125I-741F8 (sFv')2 in c-erbB-2-overexpressing SK-OV-3 tumors in mice with severe combined immunodeficiency (SCID). A dose-escalation strategy was applied to single i.v. injections of 125I-741F8 (sFv')2. Doses from 50 micrograms to 1000 micrograms were administered without a significant decrease in tumor targeting or specificity. High doses resulted in large increases in the absolute retention of 125I-741F8 (sFv')2. For example, raising the administered dose from 50 micrograms to 1000 micrograms increased the tumor retention 24 h after injection from 0.46 microgram/g to 9.5 micrograms/g, and resulted in a net increase of greater than 9 micrograms/g. Over the same dose range, the liver retention rose from 0.06 microgram/g to 1 microgram/g, and resulted in a net increase of less than 1 microgram/g. The retention of 9.5 micrograms/g in tumor 24 h following the 1000-micrograms dose of (sFv')2 was comparable to that seen 24 h after a 50-micrograms dose of 125I-741F8 IgG, indicating that the use of large doses of (sFv')2 may partially offset their rapid clearance. When two doses were administered by i.v. injection 24 h apart, the specificity of delivery to tumor observed after the first dose was maintained following the second injection. Tumor retention of 125I-741F8 (sFv')2 was 0.32 microgram/g at 24 h and 0.22 micrograms/g at 48 h following a single injection of 20 micrograms, while 0.04 microgram/ml and 0.03 microgram/ml were retained in blood at the same assay times. After a second 20-micrograms injection at the 24-h assay time, tumor retention increased to 0.49 micrograms/g, and blood retention was 0.06 microgram/ml, at the 48-h point. These results suggest that multiple high-dose administrations of radiolabeled 741F8 (sFv')2 may lead to the selective tumor localization of therapeutic radiation doses.

In vitro and in vivo characterization of a human anti-c-erbB-2 single-chain Fv isolated from a filamentous phage antibody library.

BACKGROUND: Antibody-based reagents have failed to live up to their anticipated role as highly specific targeting agents for cancer therapy. Targeting with human single-chain Fv (sFv) molecules may overcome some of the limitations of murine IgG, but are difficult to produce with conventional hybridoma technology. Alternatively, phage display of antibody gene repertoires can be used to produce human sFv. OBJECTIVES: To isolate and characterize human single chain Fvs which bind to c-erbB-2, an oncogene product overexpressed by 30-50% of breast carcinomas and other adenocarcinomas. STUDY DESIGN: A non-immune human single-chain Fv phage antibody library was selected on human c-erbB extracellular domain and sFv characterized with respect to affinity, binding kinetics, and in vivo pharmacokinetics in tumor-bearing scid mice. RESULTS: A human single-chain Fv (C6.5) was isolated which binds specifically to c-erbB-2. C6.5 is entirely human in sequence, expresses at high level as native protein in E. coli, and is easily purified in high yield in two steps. C6.5 binds to immobilized c-erbB-2 extracellular domain with a Kd of 1.6 x 10(-8) M and to c-erbB-2 on SK-OV-3 cells with a Kd of 2.0 x 10(-8) M, an affinity that is similar to sFv produced against the same antigen from hybridomas. Biodistribution studies demonstrate 1.47% injected dose/g tumor 24 h after injection of 125I-C6.5 into scid mice bearing SK-OV-3 tumors. Tumor:normal organ ratios range from 8.9:1 for kidney to 283:1 for muscle. CONCLUSIONS: These results are the first in vivo biodistribution studies using an sFv isolated from a non-immune human repertoire and confirm the specificity of sFv produced in this manner. The use of phage display to produce C6.5 mutants with higher affinity and slower k(off) would permit rigorous evaluation of the role of antibody affinity and binding kinetics in tumor targeting, and could result in the production of a therapeutically useful targeting protein for radioimmunotherapy and other applications.

Engineering disulfide-linked single-chain Fv dimers [(sFv')2] with improved solution and targeting properties: anti-digoxin 26-10 (sFv')2 and anti-c-erbB-2 741F8 (sFv')2 made by protein folding and bonded through C-terminal cysteinyl peptides.

Single-chain Fv fusions with C-terminal cysteinyl peptides (sFv') have been engineered using model sFv proteins based upon the 26-10 anti-digoxin IgG and 741F8 anti-c-erbB-2 IgG monoclonal antibodies. As part of the 741F8 sFv construction process, the PCR-amplified 741F8 VH gene was modified in an effort to correct possible primer-induced errors. Genetic replacement of the N-terminal beta-strand sequence of 741F8 VH with that from the FR1 of anti-c-erbB-2 520C9 VH resulted in a dramatic improvement of sFv folding yields. Folding in urea-glutathione redox buffers produced active sFv' with a protected C-terminal sulfhydryl, presumably as the mixed disulfide with glutathione. Disulfide-bonded (sFv')2 homodimers were made by disulfide interchange or oxidation after reductive elimination of the blocking group. Both 26-10 (sFv')2 and 741F8 (sFv')2 existed as stable dimers that were well behaved in solution, whereas 741F8 sFv and sFv' exhibited considerable self-association. The 741F8 sFv binds to the extracellular domain (ECD) of the c-erbB-2 oncogene protein, which is often overexpressed in breast cancer and other adenocarcinomas. The recombinant ECD was prepared to facilitate the analysis of 741F8 binding site properties; the cloned ECD gene, modified to encode a C-terminal Ser-Gly-His6 peptide, was transfected into Chinese hamster ovary cells using a vector that also expressed dihydrofolate reductase to facilitate methotrexate amplification. Optimized cell lines expressed ECD-His6 at high levels in a cell bioreactor; after isolation by immobilized metal affinity chromatography, final ECD yields were as high as 47 mg/l. An animal tumor model complemented physicochemical studies of 741F8 species and indicated increased tumor localization of the targeted 741F8 (sFv')2 over other monovalent 741F8 species.

Mammalian cell expression of single-chain Fv (sFv) antibody proteins and their C-terminal fusions with interleukin-2 and other effector domains.

The production of several single-chain Fv (sFv) antibody proteins was examined by three modes of mammalian cell expression. Our primary model was the 741F8 anti-c-erbB-2 sFv, assembled as either the VH-VL or VL-VH, and expressed alone, with C-terminal cysteine for dimerization, or as fusion proteins with carboxyl-terminal effector domains, including interleukin-2, the B domain of staphylococcal protein A, the S-peptide of ribonuclease S, or hexa-histidine metal chelate peptide. Constructs were expressed and secreted transiently in 293 cells and stably in CHO or Sp2/0 cell lines, the latter yielding up to 10 mg per liter. Single-chain constructs of MOPC 315 myeloma and 26-10 monoclonal antibodies were also expressed, as were hybrids comprising unrelated VH and VL regions. Our results suggest that mammalian expression is a practical and valuable complement to the bacterial expression of single-chain antibodies.

Tumor targeting in a murine tumor xenograft model with the (sFv')2 divalent form of anti-c-erbB-2 single-chain Fv.

This investigation has utilized novel forms of the single-chain Fv (sFv), wherein a cysteine-containing peptide has been fused to the sFv carboxyl terminus to facilitate disulfide bonding or specific cross-linking of this sFv' to make divalent (sFv')2. The 741F8 anti-c-erbB-2 monoclonal antibody was used as the basis for construction of 741F8 sFv, from which the sFv' and (sFv')2 derivatives were prepared. Recombinant c-erbB-2 extracellular domain (ECD) was prepared in CHO cells and the bivalency of 741F8 (sFv')2 demonstrated by its complex formation with ECD. The tumor binding properties of 125I-labeled anti-c-erbB-2 741F8 sFv, sFv', and (sFv)2 were compared with radiolabeled antidigoxin 26-10 sFv' and (sFv')2 controls. Following intravenous administration of radiolabeled species to severe combined immune-deficient (SCID) mice bearing SK-OV-3 tumors (which over-express c-erbB-2), blood and organ samples were obtained as a function of time over 24 h. Comparative analysis of biodistribution and tumor-to-organ ratios demonstrated the 741F8 sFv, sFv', and (sFv')2 had excellent specificity for tumors, which improved with time after injection. This contrasted with nonspecific interstitial pooling in tumors observed with the 26-10 sFv, sFv', and (sFv')2, which decreased with time after administration. Tumor localization was significantly better for disulfide or peptide crosslinked 741F8 (sFv')2 having Gly4Cys tails than for monovalent 741F8 sFv' or Fab. The superior properties of the 741F8 (sFv')2 in targeting SK-OV-3 tumors in SCID mice suggests the importance of further investigations of divalent sFv analogs for immunotargeting.

Highly specific in vivo tumor targeting by monovalent and divalent forms of 741F8 anti-c-erbB-2 single-chain Fv.

The in vivo properties of monovalent and divalent single-chain Fv (sFv)-based molecules with the specificity of the anti-c-erbB-2 monoclonal antibody 741F8 were examined in scid mice bearing SK-OV-3 tumor xenografts. 741F8 sFv monomers exhibited rapid, biphasic clearance from blood, while a slightly slower clearance was observed with the divalent 741F8 (sFv')2 comprising a pair of 741F8 sFv' with a C-terminal Gly4Cys joined by a disulfide bond. Following i.v. injection, the 741F8 sFv monomer was selectively retained in c-erbB-2-overexpressing SK-OV-3 tumor, with excellent tumor:normal organ ratios uniformly exceeding 10:1 by 24 h. The specificity of this effect was demonstrated by the lack of retention of the anti-digoxin 26-10 sFv monomer, as evaluated by biodistribution studies, gamma camera imaging, and cryomacroautoradiography studies. The specificity index (741F8 sFv retention/26-10 sFv retention) of 741F8 monomer binding, measured by the percentage of injected dose per g of tissue, was 13.2:1 for tumor, and 0.8 to 2.1 for all tested normal organs, with specificity indices for tumor:organ ratios ranging from 7.0 (kidneys) to 16.7 (intestines). Comparing divalent 741F8 (sFv')2 with the 26-10 (sFv')2, similar patterns emerged, with specificity indices for retention in tumor of 16.9 for the Gly4Cys-linked (sFv')2. These data demonstrate that, following their i.v. administration, both monovalent and divalent forms of 741F8 sFv are specifically retained by SK-OV-3 tumors. This antigen-specific binding, in conjunction with the 26-10 sFv controls, precludes the possibility that passive diffusion and pooling in the tumor interstitium contributes significantly to long-term tumor localization. 741F8 (sFv')2 species with peptide spacers exhibited divalent binding and increased retention in tumors as compared with 741F8 sFv monomers. Since the blood retention of the (sFv')2 is slightly more prolonged than that of the monomer, it was necessary to demonstrate that the increased tumor localization of the peptide-linked (sFv')2 was due to its divalent nature. The significantly greater localization of the divalent bismalimidohexane-linked 741F8 (sFv')2 as compared with a monovalent 741F8 Fab fragment of approximately the same size suggests that the increased avidity of the (sFv')2 is a factor in its improved tumor retention. This is the first report of successful specific in vivo targeting of tumors by divalent forms of sFv molecules. The improved retention of specific divalent (sFv')2 by tumors may have important consequences for targeted diagnostic or therapeutic strategies.

Rapid infarct imaging with a technetium-99m-labeled antimyosin recombinant single-chain Fv: evaluation in a canine model of acute myocardial infarction.

Studies of monoclonal antibody-based imaging agents show that blood clearance is inversely proportional to molecular size, i.e., Fab or Fab' > F(ab')2 > IgG. Indium-111-antimyosin Fab-DTPA is a highly specific and sensitive marker for myocardial necrosis. An improvement on current antibody diagnostic imaging may result from the use of smaller labeled fragments. We report the first in vivo targeting of acute myocardial infarction with a novel recombinant single-chain Fv (sFv) antimyosin protein. The sFv (MW = 27,594) is approximately one-half the size of the Fab and is comprised of the heavy and light chain variable regions from the myosin-specific murine monoclonal antibody R11D10 which were joined by a 15-amino-acid linker and expressed as a fusion protein (sFv) in E. coli. The binding affinity of the sFv for cardiac myosin was similar to the affinity observed for the Fab fragment. Technetium-99m labeling of the sFv was accomplished by the attachment of a cleavable, ester-linked bifunctional chelator (RP-1). Comparative studies in mice showed 99mTc-sFv-RP-1 cleared significantly faster (p < 0.001) than 99mTc-Fab'-RP-1 and 111In-Fab-DTPA antimyosin fragments. Furthermore, measurement of 99mTc-sFv-RP-1 blood clearance in a canine model of acute myocardial infarction gave a mean T1/2 of 0.54 +/- 0.13 hr versus 2.80 +/- 0.57 and 2.58 +/- 0.64 hr for Fab-DTPA and Fab'-RP-1 (p < 0.05), respectively. Despite its comparatively rapid clearance, 99mTc sFv-RP-1 had similar uptake in the infarct compared to the Fab'-RP-1. In addition, infarct visualization was more rapid with the sFv. Thus, these data demonstrate antimyosin sFv possesses characteristics necessary for rapid imaging of myocardial necrosis.

Biosynthetic antibody binding sites: development of a single-chain Fv model based on antidinitrophenol IgA myeloma MOPC 315.

The functional antigen binding region of antidinitrophenol mouse IgA myeloma MOPC 315 has been produced as a single-chain Fv (sFv) protein in E. coli. Recombinant 315 proteins included sFv alone, a bifunctional fusion protein with amino-terminal fragment B (FB) of staphylococcal protein A, and a two-chain 315 Fv fragment. Successful refolding of the 315 sFv required formation of disulfide bonds while the polypeptide was in a denatured state, as previously observed for the parent Fv fragment. Affinity-purified recombinant 315 proteins showed full recovery of specific activity, with values for Ka,app of 1.5 to 2.2 x 10(6) M-1, equivalent to the parent 315 Fv fragment. As observed for natural 315 Fv, the sFv region of active FB-sFv315 fusion protein was resistant to pepsin treatment, whereas inactive protein was readily degraded. These experiments will allow the application of protein engineering to the 315 single-chain Fv; such studies can advance structure-function studies of antibody combining sites and lead to an improved understanding of single-chain Fv proteins.

Rapid purification of a recombinant protein using tandem radial flow ion-exchange column chromatography.

Tandem radial flow anion- and cation-exchange columns were used to partially purify and concentrate a dilute recombinant protein that had been refolded in vitro after production as insoluble inclusion bodies in E. coli. The refolded sample was first passed through a Q-Sepharose Fast Flow column in order to remove the majority of E. coli contaminating proteins and endotoxins, then purified on an S-Sepharose Fast Flow column connected to the outlet of the Q-Sepharose column. This tandem arrangement enabled the rapid processing of multiple preparations of refolded material during production method development.

Polyclonal antibody that recognizes calcium-dependent determinants in Tetrahymena calmodulin.

We report here that a precipitating antibody prepared against Tetrahymena pyriformis calmodulin recognizes calcium-dependent determinants in the native protein. The ability of the antibody to precipitate 35S-labeled Tetrahymena calmodulin in direct radioimmunoassays was enhanced at least 3-fold in the presence of calcium. Competitive radioimmunoassay using homogeneous preparation of endogenously 35S-labeled Tetrahymena calmodulin and protein A-Sepharose-purified immunoglobulin G demonstrated that this antibody preparation is specific for protozoan calmodulin. Homogeneous vertebrate, invertebrate, and plant calmodulins, as well as rabbit skeletal muscle troponin C, did not show significant competition with the 35S-labeled Tetrahymena protein at concentrations 100-fold greater than that at which the homologous unlabeled Tetrahymena calmodulin produced 50% competition. A cyanogen bromide digest of Tetrahymena calmodulin also showed partial competition with the intact 35S-labeled protein, but only in the presence of calcium. The major antigenic determinants were localized to the carboxyl-terminal half of the molecule by immunoassay of limited trypsin fragments of Tetrahymena calmodulin. The antibody bound native calmodulin complexed to bovine brain phosphodiesterase (EC 3.1.4.17) but failed to recognize the Tetrahymena calmodulin carboxyl-terminal fragment (76-147) when complexed to the enzyme.