Effect of Polyclonal, Monoclonal, and Recombinant (Single-Chain Variable Fragment) Antibodies on In Vitro Morphology, Growth, and Metabolism of the Phytopathogenic Mollicute Spiroplasma citri

Antibodies are known to affect the morphology, growth, and metabolism of mollicutes and thus may serve as candidate molecules for a plantibody-based control strategy for plant-pathogenic spiroplasmas and phytoplasmas. Recombinant single-chain variable fragment (scFv) antibodies are easy to engineer and express in plants, but their inhibitory effects on mollicutes have never been evaluated and compared with those of polyclonal and monoclonal antibodies. We describe the morphology, growth, and glucose metabolism of Spiroplasma citri in the presence of polyclonal, monoclonal, and recombinant antibodies directed against the immunodominant membrane protein spiralin. We showed that the scFv antibodies had no effect on S. citri glucose metabolism but were as efficient as polyclonal antibodies in inhibiting S. citri growth in liquid medium. Inhibition of motility was also observed.     

Regulation of carbon metabolism in the mollicutes and its relation to virulence

The mollicutes are cell wall-less bacteria that live in close association with their eukaryotic hosts. Their genomes are strongly reduced and so are their metabolic capabilities. A survey of the available genome sequences reveals that the mollicutes are capable of utilizing sugars as source of carbon and energy via glycolysis. The pentose phosphate pathway is incomplete in these bacteria, and genes encoding enzymes of the tricarboxylic acid cycle are absent from the genomes. Sugars are transported by the phosphotransferase system. As in related bacteria, the phosphotransferase system does also seem to play a regulatory role in the mollicutes as can be concluded from the functionality of the regulatory HPr kinase/phosphorylase. In Mycoplasma pneumoniae, the activity of HPr kinase is triggered in the presence of glycerol. This carbon source may be important for the mollicutes since it is available in epithelial tissues and its metabolism results in the formation of hydrogen peroxide, the major virulence factor of several mollicutes. In plant-pathogenic mollicutes such as Spiroplasma citri, the regulation of carbon metabolism is crucial in the adaptation to life in plant tissues or the insect vectors. Thus, carbon metabolism seems to be intimately linked to pathogenicity in the mollicutes.     

Monoclonal antibody to calmodulin: development, characterization, and comparison with polyclonal anti-calmodulin antibodies

Specific anti-calmodulin rabbit polyclonal and murine monoclonal antibodies have been produced with a thyroglobulin-linked peptide corresponding to amino acids 128-148 of bovine brain calmodulin. The monoclonal antibody is IgG-1 with kappa light chains. Both sets of antibodies recognize native vertebrate calmodulin, with the polyclonal antibody exhibiting an approximately fourfold higher sensitivity than the monoclonal antibody in a radioimmunoassay. The affinity of both polyclonal and monoclonal antibodies is approximately 2.5-fold higher for Ca(2+)-free calmodulin than for Ca(2+)-calmodulin. Other selected members of the calmodulin family (S100, troponin, and parvalbumin) do not exhibit significant cross-reactivity with the monoclonal antibody. Troponin and S100 beta displace some 125I-calmodulin from the polyclonal antibody, but require at least 900-fold excess concentration. The monoclonal antibody recognizes intact vertebrate calmodulin in solution and also on solid-phase. In addition, plant calmodulin and some forms of post-translationally modified calmodulin (phosphorylated or glycated) bind the monoclonal antibody. The affinity of the monoclonal antibody is approximately 5 x 10(8) liters/mol determined by displacement of 125I-calmodulin. On dot blotting the sensitivity for vertebrate calmodulin is 50 pg. The epitope for the monoclonal antibody is in the carboxyl terminal region (residues 107-148) of calmodulin. This highly specific anti-calmodulin monoclonal antibody should be a useful reagent in elucidating the mechanism by which calmodulin regulates intracellular metabolism.     

Cloning and characterization of a single chain antibody to glucose oxidase from a murine hybridoma

Glucose oxidase (GOD) is an oxidoreductase catalyzing the reaction of glucose and oxygen to peroxide and gluconolacton (EC 1.1.3.4.). GOD is a widely used enzyme in biotechnology. Therefore the production of monoclonal antibodies and antibody fragments to GOD are of interest in bioanalytics and even tumor therapy. We describe here the generation of a panel of monoclonal antibodies to native and heat inactivated GOD. One of the hybridomas, E13BC8, was used for cloning of a single chain antibody (scFv). This scFv was expressed in Escherichia coli XL1-blue with the help of the vector system pOPE101. The scFv was isolated from the periplasmic fraction and detected by western blotting. It reacts specifically with soluble active GOD but does not recognize denatured GOD adsorbed to the solid phase. The same binding properties were also found for the monoclonal antibody E13BC8.     

Probable insensitivity of mollicutes to rifampin and characterization of spiroplasmal DNA-dependent RNA polymerase.

The effect of rifampin on five mollicutes (Spiroplasma citri, Spiroplasma melliferum, Spiroplasma apis, Acholeplasma laidlawii, and Mycoplasma mycoides) was compared with that on Escherichia coli. We found that, in contrast to wild-type E. coli, mollicutes were insensitive to rifampin. DNA-dependent RNA polymerases from S. melliferum and S. apis were purified to the stage where the enzymes were dependent on the addition of exogenous templates for activity. The enzymes were then tested for their sensitivity to rifampin. Spiroplasmal enzymes were at least 1,000 times less sensitive to rifampin than the corresponding E. coli enzyme. This result provides a molecular basis for the resistance of mollicutes to rifampin. The RNA polymerase of S. melliferum was further purified and its subunit composition was investigated. The RNA polymerase has one small and two large subunits. The structure of S. melliferum RNA polymerase therefore resembles that of the eubacterial enzymes in spite of its insensitivity to rifampin.     

Selection of Ceratitis capitata (Diptera: Tephritidae) Specific Recombinant Monoclonal Phage Display Antibodies for Prey Detection Analysis

Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators.     

Lipid composition and lipid metabolism of Spiroplasma citri.

In a horse serum-based medium containing a full complement of fatty acids, cells of Spiroplasma citri were seen to preferentially incorporate palmitic acid. In the same medium, which had a steryl ester-to-sterol ratio of 3.64, a steryl ester-to-sterol ratio of 0.23 was seen in the cells, cholesterol being preferentially incorporated over cholesteryl ester. Like most other mycoplasmas, S. citri was shown to be unable to synthesize fatty acids or esterify cholesterol. The neutral lipids of S. citri grown in a medium containing horse serum consisted of free cholesterol, cholesteryl ester, free fatty acids, triglycerides and diglycerides. All polar lipids were phospholipids, with no glycolipids detected. These phospholipids, which are characteristic of many mycoplasmas, are phosphatidyl glycerol, diphosphatidyl glycerol, and their lyso derivatives. Sphingomyelin was also incorporated when cells were grown on horse serum. A sterol requirement for the growth of S. citri was confirmed using a serum-free medium supplemented with bovine serum albumin, palmitic acid, and various concentrations of sterols dissolved in Tween 80. The addition of palmitic acid stimulated growth but was not essential for growth. S citri was shown to grow best on cholesterol and beta-sitosterol and was able to grow on stigmasterol and ergosterol to a lesser degree. No growth was obtained using mevalonate, deoxycholate, or taurodeoxycholate as an alternative to sterol. S. citri was also able to grow when palmitic acid was replaced with oleic acid, linoleic acid, or linolenic acid. Alterations in the lipid composition of the growth medium and hence in the lipid composition of S. citri induced changes in the characteristic helical morphology of the cells, concurrent with loss of cell viability. Culture, age, and pH were also factors in determining cell morphology and viability.     

Cloning, expression, purification, and characterization of a novel single-chain variable fragment antibody against the 2-nitrobenzaldehyde derivative of a furaltadone metabolite in Escherichia coli

Furaltadone is an illicit veterinary drug that shows toxic, carcinogenic, and mutagenic effects, as does its metabolite 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ)(1). Recombinant antibodies with desirable affinity and specificity that can replace polyclonal or monoclonal antibodies are important factors for effective AMOZ immunoassays. In the present study, a novel single-chain variable fragment (scFv) antibody against the 2-nitrobenzaldehyde derivative of AMOZ (NPAMOZ) was prepared and characterized. The scFv gene was cloned into the pET-22b(+) expression vector, and 6His-tagged scFv antibodies expressed as inclusion bodies in Escherichia coli BL21 (DE3), which were then purified by nickel nitrilotriacetic acid column chromatography. Characterization of the target protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and a novel indirect competitive chemiluminescence enzyme immunoassay (icCLEIA) showed that the scFv antibody was ～27kDa and exhibited HRP-anti-His-tag antibody-recognized activity. The final purity, yield and mg of this scFv antibody after ultrafiltration concentration were 97%, 20% and 29.1mg, respectively. The icCLEIA indicated that the antibody competitively combined with NPAMOZ, exhibiting an IC(50) value of 1.46±0.01 ng/ml (n=6). Cross-reactivity studies revealed that the antibody showed desirable specificity to NPAMOZ and little reactivity to analogs except the parent furaltadone. In summary, these findings suggested that the prepared recombinant scFv antibody can be used for future immunoassay screening for AMOZ.     

Antigenic relatedness between the spiralins of Spiroplasma citri and Spiroplasma melliferum.

Four spiralins were compared by rocket immunoelectrophoresis, quantitative immunoblotting techniques, and the spiroplasma deformation test with the use of antispiralin (polyclonal) monospecific antibodies. This investigation revealed that the spiralins of Spiroplasma citri and S. melliferum are antigenically related and that probably no more than two epitopes simultaneously saturable with antibodies are shared by the two proteins. One at least of these epitopes is accessible to antibodies on the spiroplasma cell surface.     

Growth and division of Spiroplasma citri: elongation of elementary helices.

The smallest viable cell of Spiroplasma citri is a two-turn helix (elementary helix). This elementary helix grows into longer parental cells, which then divide by constriction. The helical morphology is conserved during this process. The growth pattern of S. citri membranes has been investigated by different methods of membrane labeling. When labeling is done with specific antibodies, a diffuse growth of the membrane is observed. On the contrary, pulse-labeling of the membrane with tritiated amino acids reveals a polar growth of the organism. Finally, labeling of oxydo reduction sites with potassium tellurite also indicates a polarity in the organism. These results are discussed, and a scheme for spiroplasma growth is proposed.     

Expression in Spiroplasma citri of an epitope carried on the G fragment of the cytadhesin P1 gene from Mycoplasma pneumoniae.

We have previously described the use of the replicative form (RF) of Spiroplasma citri virus SpV1 as a vector for cloning and expressing foreign genes in S. citri, an organism which reads UGA as a tryptophan codon (C. Stamburski, J. Renaudin, and J.M. Bové, J. Bacteriol. 173:2225-2230, 1991). We now report cloning and expression in S. citri of the G fragment of cytadhesin P1 gene from Mycoplasma pneumoniae. The G fragment was inserted in the SpV1 RF downstream of a synthetic ribosome binding site and introduced into S. citri by electroporation. Northern (RNA) blot analyses showed that in S. citri, the G fragment was transcribed from an SpV1 RF promoter as a 1.2-kb mRNA. The translation product was detected by Western blotting (immunoblotting) with a rabbit antiserum raised against total proteins from M. pneumoniae (strain FH) and was proved to be P1 specific by using monoclonal antibodies specific for the G region of the P1 protein. The apparent molecular mass of the polypeptide (24.5 kDa) indicates that in S. citri, the G fragment was fully translated in spite of the seven UGA codons present in the reading frame.     

Immunological study of a neurofilament protein variant (S150) with polyclonal and monoclonal antibodies

Ten polyclonal neurofilament antibodies were tested for domain specificity with immunoblots of chymotrypsin digests of a neurofilament protein of 150 kDa (NF 150K). In contrast to most monoclonal antibodies previously reported, the five polyclonal antibodies which showed domain specificity reacted with the 40 kDa α-helical rod domain of the molecule. (With one exception, monoclonal antibodies reacted with the 200 kDa carboxy-terminal peripheral domain.) Of these ten polyclonal antibodies only two reacted with an isoelectric variant of NK 150 K (S150) isolated by Liem and collaborators (Wong, J., Hutchison, S.B. and Liem, R.K.H. (1984) J. Biol. Chem. 259, 10867–10874) from bovine brain. 13 monoclonal antibodies were also tested for reactivity with S150 protein. With one exception, none of these antibodies reacted with this variant, not even a monoclonal antibody which we have previously shown to react with a non-phosphorylated epitope located in the rod domain of NF 150K. We suggest that either there are modifications other than dephosphorylation in the S150 isoelectric variant or, alternatively, that it is not derived from NF 150K.     

Production and characterization of single-chain antibody (scFv) against 3ABC non-structural protein in Escherichia coli for sero-diagnosis of Foot and Mouth Disease virus

Differentiation of Foot-and-Mouth Disease infected from vaccinated animals is essential for effective implementation of vaccination based control programme. Detection of antibodies against 3ABC non-structural protein of FMD virus by immunodiagnostic assays provides reliable indication of FMD infection. Sero-monitoring of FMD in the large country like India is a big task where thousands of serum samples are annually screened. Currently, monoclonal or polyclonal antibodies are widely used in these immunodiagnostic assays. Considering the large population of livestock in the country, an economical and replenishable alternative of these antibodies was required. In this study, specific short chain variable fragment (scFv) antibody against 3B region of 3ABC poly-protein was developed. High level of scFv expression in Escherichia coli system was obtained by careful optimization in four different strains. Two formats of enzyme immunoassays (sandwich and competitive ELISAs) were optimized using scFv with objective to differentiate FMD infected among the vaccinated population. The assays were statistically validated by testing 2150 serum samples. Diagnostic sensitivity/specificity of sandwich and competitive ELISAs were determined by ROC method as 92.2%/95.5% and 89.5%/93.5%, respectively. This study demonstrated that scFv is a suitable alternate for immunodiagnosis of FMD on large scale.     

Sugar import and phytopathogenicity of Spiroplasma citri: glucose and fructose play distinct roles

We have shown previously that the glucose PTS (phosphotransferase system) permease enzyme II of Spiroplasma citri is split into two distinct polypeptides, which are encoded by two separate genes, crr and ptsG. A S. citri mutant was obtained by disruption of ptsG through homologous recombination and was proved unable to import glucose. The ptsG mutant (GII3-glc1) was transmitted to periwinkle (Catharanthus roseus) plants through injection to the leaf-hopper vector. In contrast to the previously characterized fructose operon mutant GMT 553, which was found virtually nonpathogenic, the ptsG mutant GII3-glc1 induced severe symptoms similar to those induced by the wild-type strain GII-3. These results, indicating that fructose and glucose utilization were not equally involved in pathogenicity, were consistent with biochemical data showing that, in the presence of both sugars, S. citri used fructose preferentially. Proton nuclear magnetic resonance analyses of carbohydrates in plant extracts revealed the accumulation of soluble sugars, particularly glucose, in plants infected by S. citri GII-3 or GII3-glc1 but not in those infected by GMT 553. From these data, a hypothetical model was proposed to establish the relationship between fructose utilization by the spiroplasmas present in the phloem sieve tubes and glucose accumulation in the leaves of S. citri infected plants.     

Mitogen-like monoclonal anti-actin antibodies

Monoclonal antibodies (IgM kappa) have been produced to actin isolated electrophoretically from L cell extracts. These monoclonal anti-actin antibodies bind to intact L cells and modulate DNA synthesis and cell proliferation, much like affinity-purified polyclonal rabbit antibody to the same Mr 42,000 actin. In addition, monoclonal antibodies specific for actin from Entamoeba histolytica also bound to and modulated the growth of L cells. A monoclonal antibody directed against a neuroblastoma surface antigen did not produce stimulation of L cells, and the binding activity of anti-actin monoclonal antibody to L cells was removed by absorption with actin covalently coupled to Sepharose. These observations demonstrate the specificity of interaction between the anti-actin monoclonal antibodies and the surface of intact L cells. We conclude that a surface actin-like molecule on the L cell, when bound by specific monoclonal antibody, initiates a stimulatory signal which results in enhanced cellular metabolism.     

Rare,high-affinity mouse anti-PD-1 antibodies that function in checkpoint blockade,discovered using microfluidics and molecular genomics

Conventionally, mouse hybridomas or well-plate screening are used to identify therapeutic monoclonal antibody candidates. In this study, we present an alternative to hybridoma-based discovery that combines microfluidics, yeast single-chain variable fragment (scFv) display, and deep sequencing to rapidly interrogate and screen mouse antibody repertoires. We used our approach on six wild-type mice to identify 269 molecules that bind to programmed cell death protein 1 (PD-1), which were present at an average of 1 in 2,000 in the pre-sort scFv libraries. Two rounds of fluorescence-activated cell sorting (FACS) produced populations of PD-1-binding scFv with a mean enrichment of 800-fold, whereas most scFv present in the pre-sort mouse repertoires were de-enriched. Therefore, our work suggests that most of the antibodies present in the repertoires of immunized mice are not strong binders to PD-1. We observed clusters of related antibody sequences in each mouse following FACS, suggesting evolution of clonal lineages. In the pre-sort repertoires, these putative clonal lineages varied in both the complementary-determining region (CDR)3K and CDR3H, while the FACS-selected PD-1-binding subsets varied primarily in the CDR3H. PD-1 binders were generally not highly diverged from germline, showing 98% identity on average with germline V-genes. Some CDR3 sequences were discovered in more than one animal, even across different mouse strains, suggesting convergent evolution. We synthesized 17 of the anti-PD-1 binders as full-length monoclonal antibodies. All 17 full-length antibodies bound recombinant PD-1 with K_D < 500 nM (average = 62 nM). Fifteen of the 17 full-length antibodies specifically bound surface-expressed PD-1 in a FACS assay, and nine of the antibodies functioned as checkpoint inhibitors in a cellular assay. We conclude that our method is a viable alternative to hybridomas, with key advantages in comprehensiveness and turnaround time.     

Isoenzyme-directed selection and characterization of anti-creatine kinase single chain Fv antibodies from a human phage display library

Epitopes differing among isoenzymes of creatine kinase (CK) are apparently limited in number and poorly immunogenic in vivo. Especially for the BB-CK isoenzyme, very few monoclonal antibodies (mAb) are available. Here, we use in vitro selection with a synthetic human phage display antibody library and develop isoenzyme competition and peptide panning strategies to obtain human single chain Fv (scFv) antibodies against specific CK isoenzymes. We isolated and characterized seven scFv clones that recognize native as well as denatured cytosolic BB-CK in ELISA, immunoblot, immunofluorescence histochemistry and surface plasmon resonance (SPR) spectroscopy. To a variable but minor degree, they also react with cytosolic MM-CK, but not with mitochondrial CK isoenzymes. Epitope mapping revealed that the scFv antibodies recognize different BB-CK epitopes, including the N-terminus and the isoenzyme-specific box, a highly conserved sequence of unknown function for which no mAb were available so far. With a K(D) of 3.5-9.6 x 10(-7) M, the isolated scFv compare favorably with mouse mAb and may overcome certain of their limitations. Our results demonstrate the advantages of in vitro antibody selection for the generation of isoenzyme-specific antibodies.     

Species-specific PCR for identification of common contaminant mollicutes in cell culture.

Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5' end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5' end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.     

Production and characterization of a humanized single-chain antibody against human integrin alphav beta3 protein

Anti-angiogenesis therapy is an emerging strategy for cancer treatment. This therapy has many advantages over existing treatments, such as fewer side effects, fewer resistance problems, and a broader tumor type spectrum. Integrin αvβ3 is a heterodimeric transmembrane glycoprotein that has been demonstrated to play a key role in tumor angiogenesis and metastasis. We have used a phage antibody display to humanize a mouse monoclonal antibody (mAb E10) against human integrin αvβ3 with a predetermined CDR3 gene. Three human phage antibodies were developed. Analysis of the humanized phage antibodies by phage ELISA revealed that the antibodies retained high antigen-binding activity and detected the same epitope as the parent mAb E10. A humanized single chain Fv (scFv) antibody was expressed in Escherichia coli in a soluble form. Analysis of the purified scFv indicated that it has the same specificity and affinity as the original mAb. Cell viability assays and xenograft model results suggested that the humanized scFv possesses anti-tumor growth activity in vitro and in vivo. This successful production of a humanized scFv with the ability to inhibit αvβ3-mediated cancer cell growth may provide a novel candidate for integrin αvβ3-targeted therapy.     

Development of a novel mammalian display system for selection of antibodies against membrane proteins

Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 10⁹ variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (K_D) as low as 0.8 nm. We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule–positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.