Purified Fab Fragments Research Articles

Biophysical characterization and structure of the Fab fragment from the NIST reference antibody, RM 8671

Monoclonal antibody pharmaceuticals are the fastest-growing class of therapeutics, with a wide range of clinical applications. To assure their safety, these protein drugs must demonstrate highly consistent purity and stability. Key to these objectives is higher order structure measurements validated by calibration to reference materials. We describe preparation, characterization, and crystal structure of the Fab fragment prepared from the NIST Reference Antibody RM 8671 (NISTmAb). NISTmAb is a humanized IgG1κ antibody, produced in murine cell culture and purified by standard biopharmaceutical production methods, developed at the National Institute of Standards and Technology (NIST) to serve as a reference material. The Fab fragment was derived from NISTmAb through papain cleavage followed by protein A based purification. The purified Fab fragment was characterized by SDS-PAGE, capillary gel electrophoresis, multi-angle light scattering, size exclusion chromatography, mass spectrometry, and x-ray crystallography. The crystal structure at 0.2 nm resolution includes four independent Fab molecules with complete light chains and heavy chains through Cys 223, enabling assessment of conformational variability and providing a well-characterized reference structure for research and engineering applications. This nonproprietary, publically available reference material of known higher-order structure can support metrology in biopharmaceutical applications, and it is a suitable platform for validation of molecular modeling studies.

Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli.

BackgroundDisulfide bonds are the most common structural, post-translational modification found in proteins. Antibodies contain up to 25 disulfide bonds depending on type, with scFv fragments containing two disulfides and Fab fragments containing five or six disulfide bonds. The production of antibody fragments that contain native disulfide bonds can be challenging, especially on a large scale. The protein needs to be targeted to prokaryotic periplasm or the eukaryotic endoplasmic reticulum. These compartments are specialised for disulfide bond formation, but both compartments have limitations.ResultsHere we show that the introduction into the cytoplasm of a catalyst of disulfide bond formation and a catalyst of disulfide bond isomerization allows the efficient formation of natively folded scFv and Fab antibody fragments in the cytoplasm of Escherichia coli with intact reducing pathways. Eleven scFv and eleven Fab fragments were screened and ten of each were obtained in yields of >5 mg/L from deep-well plates. Production of eight of the scFv and all ten of the Fab showed a strong dependence on the addition of the folding factors. Yields of purified scFv of up to 240 mg/L and yields of purified Fab fragments of up to 42 mg/L were obtained. Purified fragments showed circular dichroism spectra consistent with being natively folded and were biologically active.ConclusionsOur results show that the efficient production of soluble, biologically active scFv and Fab antibody fragments in the cytoplasm of E. coli is not only possible, but facile. The required components can be easily transferred between different E. coli strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0419-5) contains supplementary material, which is available to authorized users.

Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment

Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

Binding Affinity and Specificity of Neuromyelitis Optica Autoantibodies to Aquaporin-4 M1/M23 Isoforms and Orthogonal Arrays

Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for the neuroinflammatory disease neuromyelitis optica (NMO). We measured the binding of NMO autoantibodies to AQP4 in human astrocyte-derived U87MG cells expressing M1 and/or M23 AQP4, or M23 mutants that do not form orthogonal array of particles (OAPs). Binding affinity was quantified by two-color fluorescence ratio imaging of cells stained with NMO serum or a recombinant monoclonal NMO autoantibody (NMO-rAb), together with a C terminus anti-AQP4 antibody. NMO-rAb titrations showed binding with dissociation constants down to 44 ± 7 nm. Different NMO-rAbs and NMO patient sera showed a wide variation in NMO-IgG binding to M1 versus M23 AQP4. Differences in binding affinity rather than stoichiometry accounted for M1 versus M23 binding specificity, with consistently greater affinity of NMO-IgG binding to M23 than M1 AQP4. Binding and OAP measurements in cells expressing different M1:M23 ratios or AQP4 mutants indicated that the differential binding of NMO-IgG to M1 versus M23 was due to OAP assembly rather than to differences in the M1 versus M23 N termini. Purified Fab fragments of NMO-IgG showed similar patterns of AQP4 isoform binding, indicating that structural changes in the AQP4 epitope upon array assembly, and not bivalent cross-linking of whole IgG, result in the greater binding affinity to OAPs. Our study establishes a quantitative assay of NMO-IgG binding to AQP4 and indicates remarkable, OAP-dependent heterogeneity in NMO autoantibody binding specificity.

Production of a humanized Fab fragment of a neutralizing antibody against rabies virus

This paper describes the production of a functional humanized Fab fragment of a neutralizing antibody against the rabies virus. It is a prototype of a therapeutic agent that could be an alternative to equine anti-rabies immunoglobulins and anti-rabies serum immunoglobulins obtained from the blood of vaccinated human donors. Variable fragments of the high-affinity light and heavy chains neutralizing an antibody against the rabies virus were cloned and sequenced. Mouse constant regions were replaced by human constant regions followed by expression of the humanized Fab fragments in a yeast expression system. The immunochemical properties of the Fab fragments were evaluated using ELISA, polyacrylamide gel electrophoresis, and the Western blot assay. The binding capability of the produced humanized Fab fragments surpasses that of the parental antibody. A high degree of humanization was confirmed using sera against human immunoglobulins. The yield of the humanized Fab fragments was 21 mg per liter of culture medium. The purified Fab fragment preparation did not contain traces of the isolated light and heavy chains of the humanized Fab fragments.

Construction of an agglutination tool: recombinant Fab fragments biotinylated in vitro

The pComb3H vector system is used for constructing and panning recombinant antibody libraries. It allows for expression of monovalent Fab fragments, either on the surface of M13 phage, or in the form of soluble proteins secreted into the periplasmic space of bacteria. We constructed a modified pComb3H vector containing cDNA encoding for a 23-amino acid fragment of the Escherichia coli biotin carboxy carrier protein (BCCP), which is an acceptor sequence for biotinylation. The vector was used to express the Fab fragment recognizing human glycophorin A. The purified Fab fragment containing this biotin acceptor sequence was effectively biotinylated in vitro using biotin ligase (BirA). The specificity and avidity of the biotinylated Fab fragments were similar to the previously produced, unmodified Fab fragments. An avidin-alkaline phosphatase conjugate was used to detect the recombinant Fab fragments, instead of secondary antibody. In addition, when biotinylated Fab fragments were mixed with avidin, red blood cells were directly agglutinated.

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody–antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1–20 mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS–PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.

Anti-Moesin Antibodies Derived from Patients with Aplastic Anemia Stimulate Monocytes To Secrete TNF-α through an ERK1/2-Dependent Pathway.

Although aplastic anemia (AA) is a T-cell mediated disease, antibodies (Abs) specific to proteins derived from hematopoietic cells are often detected in the serum of patients with AA. It is not clear whether these auto-Abs play any role in the pathophysiology of AA. A previous study demonstrated that Abs specific to moesin, a membrane-cytoskeleton linker protein in the cytoplasm, were detectable in approximately 40% of AA patients (Takamatsu H, et al. Blood, 2007) and that anti-moesin Abs derived from AA patients could bind immune cells such as T cells and monocytes and enhance TNF-α secretion from a monocytic leukemia cell line THP-1. To substantiate the ability of anti-moesin Abs to induce the expression of myelosuppressive cytokines through immune cells, the secretion of other cytokines from THP-1 cells in response to binding of anti-moesin Abs was analyzed. THP-1 cells were incubated in the presence of PMA and LPS with 10 μg/ml of anti-moesin Abs purified from AA patients' serum or the same amount of control IgG for 48 hr. Anti-moesin Abs enhanced the secretion of TNF-α and IL-1β 2 to 5 times more than the control Abs. However, anti-moesin Abs did not enhance secretion of IL-2, IL-6 and IL-10. When peripheral blood mononuclear cells (PBMCs) isolated from 10 healthy individuals were incubated in the presence of 5 μg/ml of anti-moesin Abs alone, the amount of TNF-α in the medium was 2 to 10 times more than those of control cultures and was almost comparable to that of the culture stimulated by 100 ng/ml of LPS. Next, in order to determine whether the cytokine secretion induced by anti-moesin Abs requires FcγR cross-linking of the Abs, Fab fragments were prepared from anti-moesin Abs of an AA patient with papain treatment and the effect of the Fab fragment on THP-1 cells was examined. Purified Fab fragments failed to induce TNF-α from THP-1 cells while they moderately inhibited TNF-α secretion induced by anti-moesin Abs. Finally, to determine which signaling pathway is activated by the treatment of THP-1 cells with anti-moesin Abs, the effects of specific inhibitors of kinases were examined, including extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), all of which are involved in the signal transduction required for TNF-α secretion. The inhibition of ERK 1/2 using PD98059 suppressed TNF-α secretion induced by anti-moesin Abs in a dose dependent manner. In contrast, the interruption of JNK or p38 kinase with specific inhibitors JNK I, (L) form or SB202190 significantly enhanced TNF-α secretion in response to anti-moesin Abs in association with the up-regulation of the ERK 1/2 activity. These results indicate that anti-moesin Abs may therefore be involved in the suppression of hematopoiesis in AA patients by stimulating pro-inflammatory cytokine secretion from monocytes and that the cross-linking of the Fc portion of anti-moesin Abs and the activation of the ERK1/2-dependent pathway thus play an essential role in the anti-moesin Abs-induced TNF-α secretion from monocytes.

Fine Mapping of an Epitope Recognized by an Invasion-inhibitory Monoclonal Antibody on the Malaria Vaccine Candidate Apical Membrane Antigen 1

Antibodies that inhibit red blood cell invasion by the Plasmodium merozoite block the erythrocytic cycle responsible for clinical malaria. The invasion-inhibitory monoclonal antibody (mAb) 4G2 recognizes a conserved epitope in the ectodomain of the essential Plasmodium falciparum microneme protein and vaccine candidate, apical membrane antigen 1 (PfAMA1). Here we demonstrate that purified Fab fragments of 4G2 inhibit invasion markedly more efficiently than the intact mAb, suggesting that the invasion-inhibitory activity of this mAb is not due solely to steric effects and that the epitope lies within a functionally critical region of the molecule. We have taken advantage of a synthetic gene encoding a modified form of PfAMA1, and existing x-ray crystal structure data, to fully characterize this epitope. We first validate the gene by demonstrating that it fully complements the function of the authentic gene in P. falciparum. We then use it to identify a group of residues within the previously described domain II loop of PfAMA1 that are critical for recognition by mAb 4G2 and demonstrate that the epitope lies exclusively within this loop with no contributions from residues in other domains of the molecule. This is the first complete characterization of a conserved invasion-inhibitory epitope on PfAMA1. Our results will aid in the design of subunit vaccines designed to generate a broadly effective, focused anti-PfAMA1 protective immune response and may help elucidate the function of PfAMA1.

Selection of SARS-Coronavirus-specific B cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice

Antibodies to SARS-Coronavirus ( SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosen for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed.

Expression of Fab fragment of catalytic antibody 6D9 in an Escherichia coli in vitro coupled transcription/translation system

The heavy chain (Hc) and light chain (Lc) genes of the Fab fragment of a catalytic antibody 6D9 were simultaneously expressed in an Escherichia coli in vitro transcription/translation system without a reducing agent. The intermolecular disulfide bond between the Hc and Lc was found formed, suggesting a correct formation of the Fab fragment in the in vitro system. In enzyme-linked immunosorbent assay, the Fab fragment synthesized in vitro exhibited an antigen-binding activity. Addition of reduced glutathione, oxidized glutathione, protein disulfide-isomerase and molecular chaperones, GroEL and GroES, increased the solubility and the antigen-binding activity of the Fab fragment greatly. The in vitro synthesized Fab was purified by means of a hexa-histidine tag attached to the C-terminus of the Hc. Catalytic assay of the purified Fab fragment showed that the His-tagged Fab fragment synthesized in vitro had a catalytic activity comparable to that produced in vivo.

Recent advances in idiotype vaccination for the treatment of non-Hodgkin's lymphoma

RECENT ADVANCES IN IDIOTYPE VACCINATION FOR THE TREATIVl]ENT OF NON-HODGKIN'S LYMPHOMA. Hendrik Veelken, Stanford University Medical Center. The hypervariable regions of the antigen receptors (idiotypes, ld) expressed by non-Hodgkin's lymphoma (NHL) cells constitute individual tumor-specific antigens and hence attractive target molecules for induction of specific anti-tumor immune responses. In two notable phase II trials, vaccination of follicular lymphoma patients in clinical remitdon with Id (produced fiom an individual lymphoma-derived hybridoma cell line) was associated with disappearance of minimal residual disease, and superior disease-flee and overall survival in patients who developed a measurable anti-idiotype immune response. However, the vast effort to produce an individual Id vaccine by hybridoma technology has hitherto limited large-seale clinical testing. Therefore, three recombinant approaches for ld vaccine production are currently being evaluated in early clinical trials: Transfection of a cell line with expres~on vectors encoding lymphoma-derived IgH and lgL chains, production of Id scFv fragments in tobacco plants by a hybrid TMV vector, and expression of functionally folded Id Fab fragments in the periplasma orE. coil The current experience with the bscterinl expression system indicates the potential to produce a vaccine within 3 weeks of obtaining a lymphoma biopsy, and yields of up to 20 nag highly purified Fab fragment in a 10 1 fermentation system under GMP criteria. In vitro stimulation experiments with recombinant Id Fab proved the existence of Id-specific CTL in the blood of NHL patients. Immunizations with a mixture ofld Fab and a lipid-based adjuvant i.d., combined with s.c. GMCSF, appear to be safe and well tolerated. Naked DNA and viral vectors Its tumor vaccines Gabriele Pecher Charit6 Humboldt-University Berl~ Dept. of Oncology/Hematology, AG Molecular G~meand Innmmotherapy, Hessische Str. 3-4, 10115 Berlin, Germany

Capture of human Fab fragments by expanded bed adsorption with a mixed mode adsorbent.

A novel group of mixed mode adsorbents has been developed for purification of monoclonal and polyclonal antibodies from a broad range of raw materials such as hybridoma cell culture, ascites fluid, animal sera, milk, whey and egg yolk. The aim of this study was to determine whether such mixed mode adsorbents were also useful for the recovery of recombinant proteins from microbial feedstocks. This paper describes the performance of one of these adsorbents for expanded bed capture of a human Fab fragment from recombinant E. Coli cell extracts. It is concluded that the mixed mode adsorbent binds the Fab fragment efficiently from crude extracts without any requirement for preconditioning the extract by for example de-salting or dilution. The capacity of the mixed mode adsorbent is approx. 12 mg Fab/ml matrix. The novel mixed mode adsorbent can be useful during production of highly purified Fab fragments as the first step in a purification scheme. In this respect the mixed mode adsorbent is advantageous over alternative commercially available ion-exchange materials which require pre-conditioning of cell extract for Fab' capture. Together with the concentration and clarification effect a significant enrichment of the Fab fragment is obtained in one single high yield operation.

Plasmas from Lymphocyte- and Macrophage-Tropic SIVmac-Infected Macaques Have Antibodies with a Broader Spectrum of Virus Neutralization Activity in Macrophage versus Lymphocyte Cultures

We examined plasma from macaques infected with three different phenotypes of SIVmacfor their ability to neutralize the infectivity of homologous and heterologous virus in lymphocyte (CEMx174 cells or normal rhesus macaque peripheral blood lymphocytes) or normal rhesus macaque macrophage (Mφ) cultures. Similar to previous findings, we observed that some plasmas failed to neutralize or poorly neutralized the infectivity of SIVmac239 and SIVmac251(<1:20 plasma dilution) in lymphocyte cultures. In contrast, when primary rhesus Mφ cultures were used as the indicator cells, the same plasmas neutralized both viruses at high dilutions (1:200 to 1:20,000). Neutralization of virus infectivity by the various plasmas was confirmed by SIV core antigen capture assays. We excluded the possibility that this differential neutralization in Mφ was related to the differences in the ability of the virus strain to replicate in these two cell types by demonstrating that the replication efficiency of SIVmac251 in CEMx174 cells, PBMC, and Mφ cultures was very similar. The role of Fc receptors on the Mφ surface in the clearance of the virus–antibody complexes was also excluded since similar neutralizing results were obtained using whole plasmas, purified IgG antibodies, and purified Fab fragments derived from the IgG fraction of these plasmas. The mechanism of virus neutralization in Mφ does not appear to involve blocking of virus entry into the cells since radiolabeled virus reacted with anti-SIV antibodies was taken up by rhesus Mφ as efficiently as virus reacted with normal antibody. DNA of the neutralized virus was identified in the Mφ cultures, but virus replication, as evidenced by accumulation of viral protein products, was not detectable so long as the antibodies were present in the medium. Removal of the antibodies resulted in a resumption of virus replication in the Mφ. These results indicate that virus infectivity can be efficiently neutralized by antibodies in Mφ cultures by a mechanism that is fundamentally different from that in lymphocyte cultures.

The use of antibody fragments for crystallization and structure determinations.

We thank Andrew Prongay for his earlier contributions to the crystallization of the HIV p24 Fab complex, Jan McClure (Bristol Myers-Squibb Pharmaceutical Corporation, Seattle, WA) for providing the anti-p24 monoclonals, Lorna Ehrlich and Carol Carter (SUNY, Stony Brook, NY) for the recombinant HIV p24, and Tianwei Lin for discussions on antibody sequencing. We are grateful for an MRC and an NIH grant for MGR and for a research scholar award to LCK from the American Foundation for AIDS Research.

A human monoclonal antibody obtained from EBV‐transformed B cells with specificity for myosin

We describe the preparation of a stable human lymphoblastoid cell line obtained during ex vivo studies in which peripheral blood lymphocytes of a Glanzmann's thrombasthenia patient were transformed with Epstein-Barr virus. Somatic hybrids secreted an IgM monoclonal antibody (B7) that reacted with the myosin heavy chain of human platelets by immunoblotting. Flow cytometry showed that B7 barely recognized unstimulated intact platelets, but bound abundantly after permeabilization of fixed cells with Triton X-100. The reactivity of the antibody on thin sections of human myocardium and aorta was studied by immunohistochemistry. B7 specifically stained myosin of myocytes, but there was no labelling of aortic smooth muscle cells. The epitope was conserved in cardiac or skeletal myosin prepared from pig or rabbit. Measurement of the dissociation constant in a competitive ELISA showed that B7 bound with high affinity (10(-8) M). Purified Fab fragments retained their ability to bind to myosin, suggesting that B7 may be useful in the imaging of myocardial necrosis after myocardial infarction, myocarditis, cardiac drug toxicosis or graft rejection. This work also shows that EBV transformation of B cells may uncover naturally occurring autoantibodies which under normal circumstances are inhibited by the immune surveillance system.

Direct Production of the Fab Fragment Derived From the Sperm Immobilizing Antibody Using Polymerase Chain Reaction and cDNA Expression Vectors

Sperm immobilizing antibodies cause infertility mainly through complement dependent sperm immobilization. To analyze any effect of sperm immobilizing antibody on fertilization, we had already established cell lines that secrete IgM monoclonal antibody (MAb H6-3C4) and IgG monoclonal antibody (MAb EnBCMGS). The latter was a class-switched recombinant IgG antibody that shares the same variable region as MAb H6-3C4. The biological effects of the IgG antibody were also reported previously to eliminate sperm immobilizing or sperm agglutinating activities. However, the method of chemical digestion of IgG had some disadvantage to prepare the purified Fab fragment stably and in large quantities. This time we report a unique method to obtain the recombinant Fab fragments (Fab EnBCMGS) using polymerase chain reaction (PCR) and cDNA expression vectors. Two kinds of PCR primers were designed to make a truncated heavy chain (Fd) gene of MAb EnBCMGS. The amplified Fd gene and light chain gene were ligated into cDNA expression vectors and then transfected into mammalian cells. Expression of the Fd gene and light chain gene were confirmed by Northern blotting. Secretion of the recombinant Fab fragment from mammalian cells was also confirmed by Western blotting. The Fab fragment showed biological activity as is expected by FACS analysis. This method enables the stable production of genuine Fab fragments of IgG in mammalian cells without any chemical treatment that may be time consuming and affect the quality of the Fab fragments.

Hemocytes of Schistosome-Resistant and -Susceptible Biomphalaria glabrata Recognize Different Antigens on the Surface of Schistosoma mansoni Sporocysts

A cytoadherence assay was used to determine whether antibodies to plasma and hemocyte components of Schistosoma mansoni-susceptible (M-line) and -resistant (10-R2, 13-16-R1) strains of Biomphalaria glabrata affected attachment of hemocytes to chemically fixed schistosome sporocysts differentially. Experiments used purified, intact IgG and purified Fab fragments of each antibody. Indirect fluorescent antibody tests confirmed that the intact purified IgG to plasma and hemocytes from the 3 strains of snails bound to sporocysts. There was no qualitative difference in fluorescence among any of the antibodies to snail components, although the various antibodies affected adherence of hemocytes to the parasite differentially. Adherence assays revealed that antibodies to plasma components inhibited binding of hemocytes from the snail strain to which the antibody was generated. Additionally, antibody to plasma from resistant strains of B. glabrata inhibited binding of hemocytes from the homologous and heterologous resistant strains but not hemocytes from susceptible snails. Antibody to hemocytes from M-line snails did not inhibit binding of hemocytes from any of the snail strains. Since results of assays using intact IgG correlated well with assays using their Fab fragment counterparts, it was concluded that hemocytes from the 3 snail strains utilize specific antigen-binding sites on sporocysts. Results of these assays also indicate that, with regard to the mechanism of hemocyte binding to sporocysts, M-line and 13-16-R1 snails are more dissimilar than M-line and 10-R2 or 10-R2 and 13-16-R1 B. glabrata.

DNA-hydrolyzing autoantibodies.

Catalysis by antibodies could be a frequent phenomenon if the immune system generates a sufficiently diverse number of antibody-active sites, some of which may possess catalytic activity. A catalytic antibody can be expected to do more damage than one that simply binds antigen. The best biochemical marker of systemic lupus erythematosus (SLE) is presence of autoantibodies to DNA. In the present article, we describe the DNA-hydrolyzing activity of DNA-binding autoantibodies purified from SLE patients. The substrates employed were supercoiled plasmid, radiolabeled plasmid fragments, and oligonucleotides. Hydrolysis of DNA by the antibodies was indicated by the appearance of fragments visualized by ethidium bromide staining of agarose gels or autoradiography of polyacrylamide gels. Changes in linear dichroism values were also indicative of DNA hydrolysis. The antibody activity was purified by protein A-sepharose chromatography, high-performance liquid chromatography gel filtration, and DNA-affinity chromatography. Scrupulous control studies were done to demonstrate that DNA-hydrolyzing activity really belongs to the antibodies. Purified Fab fragments showed hydrolyzing activity, whereas the Fc fragment was inactive. The specificity of DNA cleavage was investigated, and the rate parameters of hydrolysis by antibodies and conventional nucleases were compared.

Induction of a secondary body axis in Xenopus by antibodies to beta-catenin.

We have obtained evidence that a known intracellular component of the cadherin cell-cell adhesion machinery, beta-catenin, contributes to the development of the body axis in the frog Xenopus laevis. Vertebrate beta-catenin is homologous to the Drosophila segment polarity gene product armadillo, and to vertebrate plakoglobin (McCrea, P. D., C. W. Turck, and B. Gumbiner. 1991. Science (Wash. DC). 254: 1359-1361.). Beta-Catenin was found present in all Xenopus embryonic stages examined, and associated with C-cadherin, the major cadherin present in early Xenopus embryos. To test beta-catenin's function, affinity purified Fab fragments were injected into ventral blastomeres of developing four-cell Xenopus embryos. A dramatic phenotype, the duplication of the dorsoanterior embryonic axis, was observed. Furthermore, Fab injections were capable of rescuing dorsal features in UV-ventralized embryos. Similar phenotypes have been observed in misexpression studies of the Wnt and other gene products, suggesting that beta-catenin participates in a signaling pathway which specifies embryonic patterning.