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Escherichia coli producing recombinant antibodies
... To check the presence of antibodies in humans, as a protection measure, against a future infection, by an isolation identical to the isolation of E. coli, which we use as an antigen; we determined that, when facing a drop of blood from people at risk, with an aliquot of the antigen (known E. coli), we obtained agglutination reaction or positive reaction in 11 people; of the 12 that made up the study sample, which percentage equates to 91.7%, a result that coincides with Abram et al (1998) [17], who states that agglutination occurs when an antibody meets an antigen that is part of a cell or an insoluble particle and that serves for the identification of pathogens and their products. Also endorsing this research, there is the report by Plückthun (1994) [18] who affirm that glutination is positive when a suspension of the microorganism that we want to diagnose is used as antigen and confronts serum. In this case, the positive result (+), of the serum of the people at risk, indicates the existence of antibodies, because the immunoglobulins, against the antigen, reacted with agglutination. ...
... To check the presence of antibodies in humans, as a protection measure, against a future infection, by an isolation identical to the isolation of E. coli, which we use as an antigen; we determined that, when facing a drop of blood from people at risk, with an aliquot of the antigen (known E. coli), we obtained agglutination reaction or positive reaction in 11 people; of the 12 that made up the study sample, which percentage equates to 91.7%, a result that coincides with Abram et al (1998) [17], who states that agglutination occurs when an antibody meets an antigen that is part of a cell or an insoluble particle and that serves for the identification of pathogens and their products. Also endorsing this research, there is the report by Plückthun (1994) [18] who affirm that glutination is positive when a suspension of the microorganism that we want to diagnose is used as antigen and confronts serum. In this case, the positive result (+), of the serum of the people at risk, indicates the existence of antibodies, because the immunoglobulins, against the antigen, reacted with agglutination. ...
... To check the presence of antibodies in humans, as a protection measure, against a future infection, by an isolation identical to the isolation of E. coli, which we use as an antigen; we determined that, when facing a drop of blood from people at risk, with an aliquot of the antigen (known E. coli), we obtained agglutination reaction or positive reaction in 11 people; of the 12 that made up the study sample, which percentage equates to 91.7%, a result that coincides with Abram et al (1998) [17], who states that agglutination occurs when an antibody meets an antigen that is part of a cell or an insoluble particle and that serves for the identification of pathogens and their products. Also endorsing this research, there is the report by Plückthun (1994) [18] who affirm that glutination is positive when a suspension of the microorganism that we want to diagnose is used as antigen and confronts serum. In this case, the positive result (+), of the serum of the people at risk, indicates the existence of antibodies, because the immunoglobulins, against the antigen, reacted with agglutination. ...
... To check the presence of antibodies in humans, as a protection measure, against a future infection, by an isolation identical to the isolation of E. coli, which we use as an antigen; we determined that, when facing a drop of blood from people at risk, with an aliquot of the antigen (known E. coli), we obtained agglutination reaction or positive reaction in 11 people; of the 12 that made up the study sample, which percentage equates to 91.7%, a result that coincides with Abram et al (1998) [17], who states that agglutination occurs when an antibody meets an antigen that is part of a cell or an insoluble particle and that serves for the identification of pathogens and their products. Also endorsing this research, there is the report by Plückthun (1994) [18] who affirm that glutination is positive when a suspension of the microorganism that we want to diagnose is used as antigen and confronts serum. In this case, the positive result (+), of the serum of the people at risk, indicates the existence of antibodies, because the immunoglobulins, against the antigen, reacted with agglutination. ...
The objective of this experimental research was to determine the immunity detection capacity for Escherichia coli in people at risk in Kerbala Province, against a standardized Escherichia coli antigen under laboratory conditions. The antiserum was obtained by inoculation of the inactive antigen of bacterial suspension of E. coli to rabbits breed Iraq. The serum with antibodies was obtained 28 days after starting the antigen inoculation program, in the respective specimens. Verifying the presence of antibodies, by means of the agglutination reaction in the 10 dilutions (1:10 to 1: 5120), with the respective antigen. The confronted antigen, with the serum and blood of people, was obtained agglutination. Concluding that 91.7% of the people under study have generated antibodies to this isolation.
... The VH and VL domains can be linked by one of various flexible linkers (scFv), by a disulfide bond (dsFv) or by both (sc-dsFv) [22,23]. However, the insoluble inclusion body formation of scFvs expressed in Escherichia coli is a significant problem [24,25]; therefore, a variety of methods for refolding proteins have been developed [26,27]. In this study, we expressed the insoluble scFv derived from the premature antibody against myoglobin in E. coli. ...
Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the VH-VL sequence with a (Gly4Ser)3 linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10-4 M-1·s-1 and 6.29 × 10-3 s-1, respectively, with an affinity value exceeding 107 M-1 (kon/koff), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor.
... Just as the V L to V H orientation appears to be a functionally important manifestation of antibody sequence diversity (Narayanan et al. 2009) in the natural immunoglobulin composite, it is, thus, not at all surprising that functional expression of the unnatural synthetically tethered V H /V L domains in the minimalist scFv format should be even more highly influenced by anomalous domain associations. Notwithstanding variability arising from linker length, the modular order of the V domains is indeed acknowledged to affect functional expression of discreet scFv entities (Desplancq et al. 1994;Plückthun 1994;Hamilton et al. 2001;Lu et al. 2004). Our comparison of the MG4 scFv and its domain-inverted VH-VL-oriented 'E2' clone confirmed significantly improved expression in the latter case. ...
Rabbit-derived recombinant antibodies have traditionally been viewed as intractable molecules due to the presence of a cysteine in position 80 of the VL domain that becomes rendered ‘aberrant’ when present in the ‘unpaired’ context of a single chain Fv (scFv) and chimeric Fab formats. This aberrant Cys80 can severely impinge on the achievable expression levels when rabbit recombinant antibodies are produced in prokaryote systems. The unpaired Cys residue also renders purification problematic. Consequently, researchers often disregard rabbit antibody libraries due to perceived limitations in accessible repertoire diversity. We have shown that by switching the orientation of the VH and VL domains in an aberrant-Cys-containing rabbit scFv isolated in a bona fide screening campaign, it was possible to substantially increase the expression and purification yields of this clone. Furthermore, by incorporating a novel rabbit C-kappa constant fusion domain, we were able to potentiate a further increase in expression level and purify this antibody to a high degree of homogeneity, hitherto impossible to achieve using the aberrant-Cys-containing wild-type scFv. Cumulatively, these findings demonstrate that facile re-formatting can help make the rabbit antibody repertoire, a very valuable resource, more accessible to researchers in the field.
... A reduction in protein yield occurs when any of the aforementioned steps are compromised in any way . Among the factors influencing expression are Ab gene structure, stability of mRNA, intrinsic properties of the expression vector, and the presence of inducible promoters (Plückthun, 1994;Skerra, 1994;Daugherty et al., 1999;Arbabi-Ghahroudi et al., 2005). Expression of functional protein in E. ...
... Furthermore, the hybridoma technology relies on animal immunization, which are strictly controlled under new regulations. Although the hybridoma technology immortalizes the antibody producing B-cell, the advent of recombinant DNA technology provides a mean to immortalize the antibody gene (Plückthun, A., 1994). The technological advances in the recombinant DNA technology on one hand can help to solve the immunogenicity problem while on the other hand it allows us to tailor the antibodies to a desired size and specificity. ...
Aachen, Techn. Hochsch., Diss., 2004 (Nicht für den Austausch).
Antibody fragments have distinct advantages over antibodies, and with both commercially viable fragments in the marketplace and numerous candidates in various stages of development, they look set to offer a credible therapeutic alternative to full-length mAbs.
IntroductionMaking Monoclonal AntibodiesOther Antibody Formats: Antibody FragmentsMedical Application Areas for MAbsFrom Initial Failure to Success:Getting the Target RightThe Market PerspectiveDrug Targeting: The Next Generation in Cancer TreatmentDeveloping a Manufacturing Process for MAbsRoutine Manufacture of MAbsGlycosylation and Other Post-translational ModificationsEmerging Issues in MAb ProductionThe Future of MAbs
Production of properly folded, functional recombinant antibodies in a prokaryotic system is governed by multiple factors like codon usage, plasmid copy number, upstream elements such as leader sequence, mRNA stability and presence of tightly controlled promoters. Here we present a strategy for enhanced production of the functional scFv in Escherichia coli by codon optimization. We have previously reported the generation of humanized scFv form of a potentially neutralizing mouse monoclonal antibody (5S) to the Hepatitis B surface antigen. However, the expression level of 5S-scFv in E. coli was fairly low which was possibly due to the presence of rare codons. In the native 5S-scFv gene, almost 58% of codons showed poor codon bias with varying degrees of rare occurrence in the E. coli genes. We therefore designed a synthetic gene encoding the 5S-scFv protein by using E. coli preferred codon usage. The codon-optimized scFv gene was further cloned into a T7 expression system with a C-terminus His-tag and expressed as a soluble protein mainly in the periplasm. The scFv was both purified by IMAC and detected on Western blot with this His-tag. Using the codon optimization strategy, we were able to achieve a more than 100-fold increased periplasmic expression of soluble scFv. Further, the purified scFv was stable and retained its antigen-binding affinity and epitope specificity. Interestingly, based on secondary structure prediction, we observed that the mRNA secondary structure, including that of the 5'-end, may not have a significant role in the increased expression of this optimized gene.
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