Search PubMed⌕ Search

Biomedical subjects

G W Welling

Publications and source records attributed to G W Welling.

At least 55 records · Page 3Linked to original sources
PubMed1990

Synthetic antibody fragment as ligand in immunoaffinity chromatography.

The possibility that a fragment of an antibody molecule may interact with a protein antigen was tested by studying the binding properties of a thirteen-residue synthetic peptide with an amino acid sequence similar to part of a hypervariable segment of a monoclonal antibody directed against lysozyme. Affinity adsorbents were prepared with this peptide and with non-related peptides as ligand. Non-specific interactions could be abolished by washing the column with 0.05 M sodium thiocyanate in 20 mM tris-HCl (pH 7.4). Lysozyme was only bound to the antilysozyme adsorbent and could be eluted with 1 M sodium thiocyanate. The results show that immunoaffinity chromatography with synthetic peptide ligands which mimic the antigen-binding site may be a useful tool in the selective purification of proteins.

Amino Acid Sequence↗
PubMed1990

Reactivity of human sera with overlapping synthetic peptides of herpes simplex virus type 1 glycoprotein D.

Thirty eight human sera, seropositive for herpes simplex virus (HSV) and 56 human sera, seronegative for HSV by immunofluorescence and by ELISA, were investigated for reactivity with a series of overlapping synthetic peptides of HSV type 1 glycoprotein D (gD-1). Thirty four out of the 38 human sera positive for HSV reacted with peptides located between residues 300 and 369; the HSV-negative sera reacted with six of the gD-1 peptides, but with none of the peptides within residues 300 to 369.

Amino Acid Sequence↗
PubMed1990

Virus neutralizing activity induced by synthetic peptides of glycoprotein D of herpes simplex virus type 1, selected by their reactivity with hyperimmune sera from mice.

Mice were immunized with synthetic peptides covering the first 56 amino acids of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) and a fusion protein, produced in Escherichia coli, containing the first 55 amino acid residues of gD. It was found that mice immunized with peptides composed of amino acid residues 1 to 13, 18 to 30. 22 to 38 and 38 to 56 of gD were not significantly protected against a lethal challenge with HSV-1. Immunization with peptide 9-21 and the gD fusion protein resulted in significant protection. Antisera, from mice immunized with HSV-1, were investigated for reactivity with a series of 57 overlapping gD peptides covering the entire amino acid sequence, except for the membrane-spanning region. All antisera reacted with peptides 9-21, 10-24, 151-165, 216-232, 282-301 and with peptide 340-354 located in the anchoring region of gD, and 15 other peptides were recognized by at least one antiserum. Twelve peptides (10-24, 151-165, 216-232, 244-267, 260-274, 270-284, 260-284, 282-301, 300-314, 340-354, 348-362 and 355-369) reacted most frequently with the hyperimmune sera from mice and were selected for further study. These were conjugated to bovine serum albumin and used to immunize rabbits. Only antisera against peptide 10-24, which covers the same epitope as peptide 9-21, neutralized HSV-1 in vitro.

Animals↗
PubMed1989

Immunological properties of multiple repeats of a linear epitope of herpes simplex virus type 1 glycoprotein D.

Several peptides containing the amino acid sequence 9-21 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were synthesized and investigated for reactivity with monoclonal antibody LP14 in a competition enzyme-linked immunosorbent assay (ELISA). Peptides containing two or four repeats of sequence 9-21 reacted at least one order of magnitude better with LP14 than with the monomeric form of sequence 9-21. Dimers in which one of the repeats of one or more essential residues were absent did not show this increased reactivity. Antisera obtained from rabbits immunized with a peptide containing two repeats of sequence 9-21 coupled to bovine serum albumin showed high antipeptide antibody titers with this peptide and were able to neutralize virus infectivity in vitro. Sera obtained from rabbits immunized with the free dimer could not neutralize virus infectivity.

Amino Acid Sequence↗
PubMed1989

The influence of different adjuvants on the immune response to a synthetic peptide comprising amino acid residues 9-21 of herpes simplex virus type 1 glycoprotein D.

The immuno-modulating properties of different adjuvant systems on the murine humoral and cellular immune response to a synthetic peptide comprising amino acid residues 9-21 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were investigated. For immunization, the peptide was conjugated to ovalbumin or bovine serum albumin by glutaraldehyde and the adjuvants used in this study were Freund's complete adjuvant (FCA), aluminium hydroxide, the Ribi adjuvant system (RAS) and two non-ionic block polymer surfactants, viz. L101 and 31R1, in oil in water emulsions. High anti-peptide antibody titers were obtained after immunization with FCA, aluminium hydroxide, RAS and L101. All adjuvants, except RAS, stimulated the induction of delayed type hypersensitivity obtained after immunization with peptide 9-21 coupled to ovalbumin and elicited by injection of purified HSV-1 virions in the footpad. Challenge with a lethal dose of HSV-1 showed that mice immunized with peptide 9-21 coupled to ovalbumin in combination with FCA, RAS and L101, respectively, were significantly protected. Although immunization with peptide 9-21 coupled to ovalbumin combined with aluminium hydroxide stimulated induction of delayed type hypersensitivity, no significant protective immunity against the challenge was generated.

Adjuvants, Immunologic↗
PubMed1989

Comparison of non-ionic detergents for extraction and ion-exchange high-performance liquid chromatography of Sendai virus integral membrane proteins.

The integral membrane proteins of Sendai virus haemagglutinin-neuraminidase (HN) and fusion protein (F) were extracted from purified virions with 2% of a non-ionic detergent, i.e., polyoxyethylene alkyl ethers varying by 8-14 hydrocarbon units in the alkyl chain and by 4-8 ethylene glycol units in the oxyethylene chain. Triton X-100 and octyl glucoside were included as reference detergents. The hydrophile-lipophile balance (HLB) and the critical micelle concentration (CMC) of the detergents were determined. A decrease in length of the oxyethylate by 8-5 ethylene glycol units and an increase in the alkylate by 8-12 hydrocarbon units resulted in higher yields of extracted proteins. The highest yields were obtained for C12E5 with an HLB of 11.7. Yields of extracted protein could be correlated with the HLB values of the polyoxyethylene alkyl ethers. The structural integrity of HN and F was not affected during extraction by either detergent as measured by their reactivity with monoclonal antibodies directed against native HN and F. Extracts were subjected to anion-exchange high-performance liquid chromatography (HPLC) on a Mono Q column in the presence of 0.1% of the detergent used for extraction. Eluate fractions were analysed by sodium dodecyl polyacrylamide gel electrophoresis and recoveries of HN and F protein were determined by size-exclusion HPLC. The immunological activity of HN and F was tested in an enzyme-linked immunosorbent assay. The highest recoveries of HN and F (80%) were obtained with C10E5 in the elution buffer. HN and F were partially purified and the immunological activity was well preserved.

Animals↗
PubMed1989

Comparison of ion-exchange high-performance liquid chromatography columns for purification of Sendai virus integral membrane proteins.

The recovery and separation of the integral membrane proteins, the haemagglutinin-neuraminidase (HN) and the fusion protein (F), from a Sendai virus detergent extract were compared on three different ion-exchange high-performance liquid chromatography (IE-HPLC) columns: Mono Q, TSK DEAE-NPR and Zorbax BioSeries SAX. The detergent, either 1-O-n-octyl-beta-glucopyranoside (octylglucoside) or decyl polyethylene glycol-300 (decyl PEG-300), used for extraction of HN and F proteins from the virions, was also present in the elution buffers at a concentration of 0.1%. Recovery of HN and F proteins was primarily dependent on the detergent present in the eluent, resulting in yields of HN varying from 18 to 28 and 56 to 67%, when octylglucoside and decyl PEG-300, respectively, were used. The highest yield for HN protein was obtained by separation on either a Mono Q or a TSK DEAE-NPR column with decyl PEG-300 as the additive. Yields of F protein were lower, and the highest recovery of 46% was found in the presence of decyl PEG-300 by separation on the Mono Q column. Analysis of the fractions by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by size-exclusion HPLC indicated that the HN protein eluted in the presence of decyl PEG-300 from the Mono Q and the TSK DEAE-NPR columns was obtained in pure form, while the F protein was slightly contaminated with HN. Analysis of the fractions with monoclonal antibodies directed against conformational epitopes of HN and F proteins indicated that after IE-HPLC the conformation of the proteins is largely retained.

Chromatography, Gel↗
PubMed1989

Inactivation of aztreonam by faecal supernatants of healthy volunteers as determined by HPLC.

The enzymatic inactivation of aztreonam to its biologically inactive open ring form, by faecal enzyme preparations from 12 volunteers, was investigated. The percentage inactivation was determined by a high-performance liquid chromatography assay at months 0, 6 and 24. Faecal enzyme preparations from five volunteers could inactivate aztreonam by more than 50% within 20 h at one or more of these sampling times. Monitoring of aztreonam inactivation over a shorter period of time (14 days) in three of the volunteers showed that aztreonam inactivation cannot be predicted.

Adult↗
PubMed1989

Effects of selective oral antimicrobial prophylaxis and systemic antibiotics on the fecal flora and fecal beta-aspartylglycine concentration in patients with acute leukemia.

The influence of selective oral antimicrobial prophylaxis (SOAP) and systemic antibiotic treatment (SAT) on the intestinal flora of fourteen granulocytopenic patients was investigated. For SOAP, patients received Cotrimoxazole, Polymyxin and Nystatin; Gentamicin, Ticarcillin and Cefuroxime were administered for SAT. Under SOAP, a complete elimination of Enterobacteriaceae and a marked reduction of yeasts and staphylococci was found. A reduction in the number of anaerobic species was also observed; however, the concentration of anaerobic bacteria remained constant. In contrast, additional systemic antibiotic treatment caused a significant reduction of aerobic as well as anaerobic bacteria, except for microorganisms pathogenic to neutropenic patients (i.e. Staphylococcus epidermidis and Candida sp.). The fecal concentration of beta-aspartylglycine was inversely correlated with the presence of anaerobic bacteria.

Acute Disease↗
PubMed1989

The 'normalization' of germ-free guineapigs with host-specific caecal microflora.

Hysterectomy-derived germ-free guineapigs were given colonization-resistant caecal flora from mice (mCRF) or microflora obtained from the caecum of an antibiotic-decontaminated conventional guineapig (gpCRF) and compared with guineapigs raised conventionally with the sow. Body weight and the following intestinal parameters were determined for the groups: colonization resistance (CR) to Escherichia coli, relative caecal weight (RCW), beta-aspartylglycine (faeces), volatile fatty acids (caecum) and bile acids (faeces). mCRF guineapigs showed values quite different from control animals for CR and RCW, indicating the unsuitability of mouse CRF for normalizing guineapigs. In gpCRF guineapigs CR and RCW values were comparable with controls, indicating the suitability of the guineapig flora for normalizing guineapigs. mCRF guineapigs housed with gpCRF guineapigs, showed an improvement in CR and RCW, yielding values found in control animals.

Animals↗
PubMed1989

Intestinal "normalization" of germ-free rabbits with rabbit caecal microflora: effect of dosing regimens.

Hysterectomy-derived germ-free (GF) rabbits were given strictly anaerobic microflora obtained from the caecum of an antibiotic-decontaminated conventional rabbit. One group was given the caecal flora diluted in doe's milk. The second group received caecal flora without doe's milk and administration of the flora was repeated when the animals were given pelleted diet. Body weight and intestinal parameters determined in the two groups of rabbits were compared with values in rabbits conventionally raised with the doe. In GF rabbits given caecal flora, the values for most intestinal parameters were similar to values recorded in conventional animals. However colonization resistance to Escherichia coli was limited in GF rabbits given caecal flora diluted in doe's milk and severely impaired in GF rabbits given caecal flora without doe's milk, although relative caecal weight was normal. It is concluded that enteric microfloras should be preferably incorporated in doe's milk when dosed to GF rabbits to obtain acceptable gastrointestinal "normalization".

Animals↗
PubMed1988

Purification of fusion proteins expressed by pEX3 and a truncated pEX3 derivative.

A derivative of the pEX3 expression vector was constructed that codes for the first 407 amino acids of the 1051 amino acids of the pEX3 fusion protein. The amount of truncated fusion protein (40 mg/g cells), obtained by expression in E. coli, was similar to that produced by the original pEX3 vector. The truncated fusion protein was purified more easily from E. coli contaminants than the original fusion protein by washing with 2 M urea and 0.5% Triton X-100.

Cloning, Molecular↗
PubMed1988

Effect of detergents on the structure of integral membrane proteins of Sendai virus studied with size-exclusion high-performance liquid chromatography and monoclonal antibodies.

The integral membrane proteins of Sendai virus, the fusion protein F (Mr = 65,000) and the haemagglutinin-neuraminidase protein HN (Mr = 68,000), were used as a model protein mixture. They were subjected to size-exclusion high-performance liquid chromatography on Superose 6HR columns with eluents containing various additives in order to solubilize the proteins. The effect of the additives on the structure of the membrane proteins was investigated with conformation-dependent monoclonal antibodies, either directed against F or HN protein, and by determination of the haemagglutinating capacity of the HN protein. The results show that the structure of the HN protein is more easily disturbed by eluents than that of the F protein. When the elution conditions are mild, e.g., 0.1% octylglucoside, the structure of both proteins is conserved but no separation is obtained. Elution with a buffer containing 0.05% sarkosyl (dodecyl methylglycine sodium salt) did not affect the structure and resulted in pure F protein. Pretreatment of the Amberlite XAD-2-treated Sendai virus envelope extract with 4% sodium dodecyl sulphate (SDS) and elution with 0.1% SDS in 50 mM sodium phosphate (pH 6.5) altered the structure of the HN protein but resulted in purification of the tetramer and the dimer of the HN protein, and the monomer of the F protein.

Antibodies, Monoclonal↗
PubMed1988

The influence of pH and ionic strength on the coating of peptides of herpes simplex virus type 1 in an enzyme-linked immunosorbent assay.

Rabbits were immunized with synthetic peptides of herpes simplex virus type 1 glycoproteins, coupled to a carrier protein with glutaraldehyde. Antibodies directed against the peptides were determined in an enzyme-linked immunosorbent assay (ELISA). Either free peptides or peptides coupled with glutaraldehyde to another carrier protein than the one used for immunization were used as the coating antigen. When conjugated peptides were used as the coat, it was necessary in some instances to correct the antibody titers for a substantial amount of antibody activity against glutaraldehyde. When free peptides were used, optimal coating conditions with regard to pH and ionic strength had to be determined, since some peptides failed to coat under standard conditions, at pH 9.6. The results show that some peptides needed stringent pH conditions while others could be coated in a broad pH range. The addition of 0.6 M NaCl had a favorable effect on peptide coating.

Antigen-Antibody Reactions↗
PubMed1988

Immunological properties of an N-terminal fragment of herpes simplex virus type 1 glycoprotein D expressed in Escherichia coli.

The N-terminal fragment, comprising residues -5 to 55 of herpes simplex virus type 1 glycoprotein D was expressed as a beta-galactosidase fusion protein in Escherichia coli. This gD-fusion protein reacts with monoclonal antibody LP 14 directed against glycoprotein D of HSV. Antisera obtained after immunization of rabbits with purified gD-fusion protein react with HSV-1 gD in a Western blot and with N-terminal synthetic peptides of gD. In addition, these antisera are able to neutralize viral infectivity in vitro.

Amino Acid Sequence↗
PubMed1988

A comparative study of the effect of oral treatment with augmentin, amoxycillin and bacampicillin on the faecal flora in mice.

Oral treatment of groups of four mice with different daily dosages of three related antibiotics, amoxycillin, augmentin and bacampicillin, has indicated the influence of the amount of the dose that reaches the intestine in a biologically active form. Augmentin (amoxycillin plus clavulanic acid to protect it against enzymatic hydrolysis) appeared to have a suppressive effect on the indigenous colonization-resistance-associated microflora. Dose-effect curves of amoxycillin alone, showed the same shape but at a lower level. Bacampicillin treatment practically did not have an effect on the faecal flora. Only at doses of bacampicillin of well above 1.5 mg per day, an indication was seen of CR-flora disturbance. At a dose level of 2 mg and more per day, a low concentration of beta-aspartylglycine was found in the faeces. A normal undisturbed intestinal flora normally produces in mice sufficient enzyme to degrade completely this dipeptide released by the host organism into the intestinal tract.

Administration, Oral↗