Use In Production Of Antibodies Research Articles

Multiscale 2D-SPR Biosensing for Cell Chips

Evaluating cell activity and functions in different-sized cell chambers requires multiscale sensing. We have been developing multiscale biosensing applied from 10 µm to 1 mm. We measured mouse IgG in micro wells using a high-resolution two-dimensional surface plasmon resonance (SPR) imaging affinity sensor. This sensor uses high refractive optics, a 1X to 7X microscopic lens, and a cooled CCD camera. The micro-well array was prepared with a PDMS film on gold sensor film. Protein A immobilized on sensor film was used for IgG recognition. SPR sensitivity was dramatically decreased with 10 and 8.5 µm microwells. To improve sensor sensitivity, we optimized the sensor’s measurement angle and exposure time, enabling mouse IgG to be detected in wells of 1 mm, 30 µm, and 10 µm using the same 2D-SPR imaging sensor and measurement protocol. These results show the feasibility of multiscale biosensing use in antibody production in a micro well or a cell chamber.

Cloning, expression and purification of recombinant cotton rat interferon-gamma.

The hispid cotton rat (Sigmodon hispidus) has proven to be an excellent small animal model; however, immunological studies have been limited due to a lack of available reagents. We report cloning of the cotton rat interferon-gamma (IFN-gamma) cDNA from concanavalin A-stimulated spleen cells using a combination of reverse transcription polymerase amplification reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) protocols. The open reading frame of 513 nucleotides encodes a 170 amino acid (aa) protein followed by a stop codon with a predicted molecular mass of 19548Da. Cotton rat IFN-gamma shares 63, 60, 43 and 43% identity with the hamster, gerbil, mouse and rat counterpart, respectively. IFN-gamma nucleotide sequence corresponding to aa 18-153 was expressed in Escherichia coli under tryptophan promoter control, either fused to a single initiating codon or fused to the thioredoxin coding sequence. Both expression products were found exclusively in bacterial inclusion bodies. Two purification schemes have been developed to purify the product fused to a single methionine. One of them is fast and leads to the recovery of a pure product suitable for use in antibody production. The second protocol, which includes chromatographic steps, allows the use of the purified product for in vitro demonstration of biological activity in a viral cytopathic reduction assay on cotton rat cells.

Structural and functional characterization of estrogen sulfotransferase isoforms: distinct catalytic and high affinity binding activities.

Estrogen sulfotransferase (EST) purified from the guinea pig (gp) adrenal gland consists of multiple charge isoforms with isoelectric points (pls) ranging from 6.5 to 5.2. Four individual isoforms were isolated for use in antibody production, as well as for tryptic fragmentation analysis. The multiple charge isoforms manifested a high degree of relatedness as evidence by complete immunocross-reactivity. This relatedness was further demonstrated by peptide mapping analysis, which revealed essentially identical elution profiles for three of the isoforms, whereas the fourth most acidic isoform, although similar to the other three isoforms, demonstrated some differences. To further explore the nature of the charge isoforms, native gpEST and EST expressed by CHO-K1 cells transfected with the gpEST cDNA were subjected to additional biochemical characterization. An interesting finding was that, in addition to EST activity, gpEST bound estradiol with a high affinity [dissociation constant (Kd) 10 nM]; furthermore, the binding activity was absolutely dependent on the cofactor, adenosine-3',5'-diphosphate (3',5'-ADP). An unexpected and novel finding was that only the most basic pl 6.5 isoform was catalytically active, while estradiol-binding activity was associated with a more acidic isoform (pl 5.5-5.2); however, it has not as yet been possible to definitively assign the binding activity to a specific isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of phosphoenolpyruvate carboxylase subunits and isoforms from Vicia faba L. by preparative gel electrophoresis and their detection by enzyme-linked immunosorbent assay.

A purification procedure which yields nearly homogenous subunits of stomatal phosphoenolpyruvate carboxylase from epidermal strips of Vicia faba L. is reported. Preparative gel electrophoresis was found to be the most suitable technique for subunit purification. Denatured subunits of the stomatal enzyme in the eluate were immunologically detected by enzyme-linked immunosorbent assay (ELISA) tests. The enzyme preparation meets all requirements for its use in antibody production.

Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis.

The sfp gene is required for cells of Bacillus subtilis to become producers of the lipopeptide antibiotic surfactin. sfp was isolated and its nucleotide sequence was determined. sfp was expressed in Escherichia coli and its putative product was purified for use in antibody production and in amino acid sequence analysis. The gene was plasmid-amplified in B. subtilis, where it conferred a Srf+ phenotype on sfp0 (surfactin non-producing) cells. Overproduction of Sfp in B. subtilis did not cause production of an increased amount of surfactin and resulted in the repression of a lacZ transcriptional fusion of the srfA operon, which encodes enzymes that catalyze surfactin synthesis. We propose that sfp represents an essential component of peptide synthesis systems and also plays a role, either directly or indirectly, in the regulation of surfactin biosynthesis gene expression.

A monoclonal antibody to the insect prothoracicotropic hormone

The prothoracicotropic hormone (PTTH) is an insect cerebral peptide that stimulates the prothoracic glands to produce the steroid hormone ecdysone thus initiating molting and metamorphosis. "Big" PTTH, one of several molecular forms of the neurohormone, was isolated from brains of the tobacco hornworm Manduca sexta, and fractionated by high-pressure liquid chromatography (HPLC) for use in antibody production. A murine polyclonal antiserum and a monoclonal antibody (MAb) have been generated using this highly purified preparation of big PTTH. Antisera and hybridoma supernatants were screened with an indirect, brain whole-mount immunocytological assay, and antibody specificity was confirmed by immunocytological, ELISA, and functional criteria. In brain whole-mount preparations, the MAb (A2H5) and antiserum specifically immunostained the lateral protocerebral neurosecretory cells (L-NSC III), the prothoracicotropes, which produce PTTH. This immunostaining was blocked by preadsorbing the antibodies with big PTTH. Analysis of the elution of HPLC-fractionated big PTTH with an in vitro bioassay for the neurohormone and an ELISA employing the A2H5 MAb resulted in peaks of activity that were superimposable. Finally, the antiserum and A2H5 MAb inhibited big PTTH activation of the prothoracic glands to synthesize ecdysone in the in vitro bioassay for the neurohormone. With these specific antibodies, the organization of the PTTH neuroendocrine axis has been defined. It is now evident that both of the peptidergic neurons that comprise the L-NSC III are prothoracicotropes, and that the corpora allata are the neurohemal organs for the release of big PTTH into the hemolymph. This study indicates that these specific antibodies will be useful in investigations of numerous aspects of the biology of this cerebral neuroendocrine axis.