A simple and rapid chromatographic strip test for detection of antibody to porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a major economic problem for swine industries worldwide despite several disease-reduction strategies such as age-segregated early weaning and all-in-all-out pig movement. Routine diagnosis of PRRSV is carried out by the combined use of an antibody-detecting enzyme-linked immunosorbent assay (ELISA), immunofluorescence, reverse transcription-polymerase chain reaction, and virus isolation. These assays require specialized laboratory equipment in addition to multistep sample handling and sample preparation. The objective of this study was to evaluate a simple pen-side assay (BioSign PRRSV) for rapid detection of PRRSV antibody based on a lateral flow chromatographic strip immunoassay system. This assay uses Escherichia coli-expressed viral nucleocapsid protein antigen for detecting antibodies against PRRSV in swine sera. In this report, the authors describe the evaluation of this assay using sera from both clinical samples and experimentally infected piglets. The results were compared with those of a standard, commercially available antibody ELISA (HerdChek PRRS ELISA) and an indirect immunofluorescence assay using the same serum samples. The BioSign PRRSV assay was capable of detecting antibodies in sera known to contain antibodies to PRRSV, resulting in 93.2% sensitivity for samples from experimentally infected pigs and 98.7% sensitivity for clinical serum samples. For sera that did not contain antibodies to PRRSV, the specificity was found to be 98.5% and 99.2% for clinical and experimental serum samples, respectively.

2.8 A resolution crystal structure of human TRAIL, a cytokine with selective antitumor activity.

TRAIL is a newly identified cytokine belonging to the large tumor necrosis factor (TNF) family. TRAIL is a novel molecule inducing apoptosis in a wide variety of tumor cells but not in normal cells. To help in elucidating its biological roles and designing mutants with improved therapeutic potential, we have determined the crystal structure of human TRAIL. The structure reveals that a unique frame insertion of 12-16 amino acids adopts a salient loop structure penetrating into the receptor-binding site. The loop drastically alters the common receptor-binding surface of the TNF family most likely for the specific recognition of cognate partners. A structure-based mutagenesis study demonstrates a critical role of the insertion loop in the cytotoxic activity of TRAIL.

Determination of the limited trypsinolysis pathways of tumor necrosis factor-alpha and its mutant by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) is employed to directly analyze the limited trypsinolysis products of wild-type tumor necrosis factor-alpha (wtTNF-alpha) and its mutant, M3S. To determine the charge numbers of peaks of relatively small peptides in the ESI mass spectrum of a digest, a series of sodium-adduct ion peaks of each peptide are generated by adding a small quantity of NaCl to the digest before taking the spectrum. From the monitoring of the composition of proteolytic mixture as the incubation time is lengthened, it has been learned that the proteolysis of wtTNF-alpha by trypsin occurs sequentially: Arg2, Arg6, Arg32, Arg31, and Arg44, and that M3S is strongly resistant to the proteolysis. Since the cleavage sequence of wtTNF-alpha and the mutation-induced resistance of M3S are consistent with the structural features of the proteins, we can suggest a mutant more resistant to proteolysis than M3S, which has an additional point mutation, Ala35Leu or Ala35Ile.

High-level production of human growth hormone in Escherichia coli by a simple recombinant process.

Procedures have been devised for producing in Escherichia coli high yields of purified recombinant human growth hormone (hGH), by utilizing N-terminal pentapeptide sequence of human tumor necrosis factor-alpha, histidine tag and enterokinase cleavage site as a fusion partner. The fusion protein was produced as a soluble protein at the beginning of gene expression, but progressively became insoluble in Escherichia coli cytoplasm. The insoluble protein was solubilized by simple alkaline pH shift and purified to near homogeneity by Ni(2+)-chelated affinity chromatography. Following specific enterokinase cleavage, the recombinant hGH was purified by one-step anion exchange chromatography. The ease and speed of this recombinant process, as well as the high productivity, makes it adaptable to the large-scale production of hGH. Moreover, the highly efficient fusion partner could be applied to the production of other therapeutically important proteins.

A novel tumor necrosis factor-alpha mutant with significantly enhanced cytotoxicity and receptor binding affinity.

A novel tumor necrosis factor-alpha mutant (mutant M3), in which Ser and Tyr at positions 52 and 56 were substituted by Ile and Phe, respectively, along with deletion of 7 N-terminal amino acids, was prepared and its biological activities were investigated. The mutant exhibited a 14- to 24-fold increase in the cytotoxicity relative to the wild-type TNF on various cancer cell lines. The binding affinity of the mutant to TNF-R55 and TNF-R75 receptors was over 10-fold higher than that of the wild-type. TNF-alpha and the mutant show similar CD spectra in the far-UV region, indicating that the overall structure was not influenced by the mutations. The production of highly potent TNF-alpha mutant utilizing increase of hydrophobicity in the region 52-56 may provide a structural basis for a design of optimized TNF-alpha as a therapeutic purpose.

High resolution crystal structure of a human tumor necrosis factor-alpha mutant with low systemic toxicity.

A human tumor necrosis factor-alpha (TNF-alpha) mutant (M3S) with low systemic toxicity in vivo was designed, and its structures in two different crystal packings were determined crystallographically at 1.8 and 2.15-A resolution, respectively, to explain altered biological activities of the mutant. M3S contains four changes: a hydrophilic substitution of L29S, two hydrophobic substitutions of S52I and Y56F, and a deletion of the N-terminal seven amino acids that is disordered in the structure of wild-type TNF-alpha. Compared with wild-type TNF-alpha, it exhibits 11- and 71-fold lower binding affinities for the human TNF-R55 and TNF-R75 receptors, respectively, and in vitro cytotoxic effect and in vivo systemic toxicity of M3S are 20 and 10 times lower, respectively. However, in a transplanted solid tumor mouse model, M3S suppresses tumor growth more efficiently than wild-type TNF-alpha. M3S is highly resistant to proteolysis by trypsin, and it exhibits increased thermal stability and a prolonged half-life in vivo. The L29S mutation causes substantial restructuring of the loop containing residues 29-36 into a rigid segment as a consequence of induced formation of intra- and intersubunit interactions, explaining the altered receptor binding affinity and thermal stability. A mass spectrometric analysis identified major proteolytic cleavage sites located on this loop, and thus the increased resistance of M3S to the proteolysis is consistent with the increased rigidity of the loop. The S52I and Y56F mutations do not induce a noticeable conformational change. The side chain of Phe56 projects into a hydrophobic cavity, while Ile52 is exposed to the bulk solvent. Ile52 should be involved in hydrophobic interactions with the receptors, since a mutant containing the same mutations as in M3S except for the L29S mutation exhibits an increased receptor binding affinity. The low systemic toxicity of M3S appears to be the effect of the reduced and selective binding affinities for the TNF receptors, and the superior tumor-suppression of M3S appears to be the effect of its weak but longer antitumoral activity in vivo compared with wild-type TNF-alpha. It is also expected that the 1.8-A resolution structure will serve as an accurate model for explaining the structure-function relationship of wild-type TNF-alpha and many TNF-alpha mutants reported previously and for the design of new TNF-alpha mutants.