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G Himmler

Publications and source records attributed to G Himmler.

27 records · Page 2Linked to original sources
PubMed1989

The complete nucleotide sequence of plum pox virus RNA.

The complete nucleotide sequence of the RNA of an aphid non-transmissible plum pox virus (PPV-NAT) isolate has been determined from five overlapping cDNA clones. cDNA prepared by primer extension was used to determine the 5' terminus. The assembled RNA is 9741 nucleotides in length, excluding a 3' terminal poly(A) sequence. One large open reading frame starts at nucleotide positions 36 to 38 and is terminated with an UAG codon at positions 9522 to 9524. The putative start codon is located at positions 147 to 149. The encoded polyprotein has a predicted Mr of 353.8K. Comparison of cistrons from tobacco vein mottling virus and tobacco etch virus with those predicted for PPV-NAT indicated a similar genome organization. A highly conserved sequence of 12 nucleotides was found in the 5' non-coding region of these three potyviruses. The potential polyadenylation signal from yeast (UAUGU) was found in the 3' non-coding region of PPV-NAT and several other members of the potyvirus group.

Amino Acid Sequence↗
PubMed1988

Early screening for anti-plum pox virus monoclonal antibodies with different epitope specificities by means of gold-labelled immunosorbent electron microscopy.

The technique of gold-labelled immunosorbent electron microscopy for the initial screening of monoclonal antibodies 10 days after cell fusion from 96-well culture plates is described. The technique is used to identify clones that secrete antibodies binding on the surface of the virion or to viral subunits, and compared to ELISA and Western blotting. High sensitivity was demonstrated.

Antibodies, Monoclonal↗
PubMed1987

Screening for monoclonal antibodies with covalently linked antigen.

Microtitre plates prepared for ELISA are treated for the maximum adsorption of gamma-globulins (at pH 9). The adsorption of other soluble proteins (e.g. antigens) is sometimes less effective and therefore requires a higher amount of protein for the primary coat of wells. In order to reduce the amount of pure antigen required for the screening of mAb-producing hybridomas, we improved the sensitivity of our screening ELISA technique by coupling the antigen covalently to the surface of the microtiter wells. The antigen (urokinase) was coupled by the glutaraldehyde or carbodiimide procedures respectively using "aminoplates" (Nissho Iwai, Japan). The glutaraldehyde method led to at least a five fold increase of the sensitivity compared to coventional adsorption. Reduced requirement of antigen for the assay is thus achieved by a simple procedure.

Animals↗
PubMed1987

Cross flow diafiltration of serum with basal medium suitable for growth of hybridomas, sterilization and protein reduction.

Fetal Calf Serum (FCS) was extensively extracted by cross flow diafiltration (Pellicon, Millipore) and sterilized using the basal growth medium (DMEM) for the extraction. Ultrafiltration membranes of 10(5) and 3 X 10(5) Dalton cut off were used respectively. The diafiltrates were used for hybridoma cultivation and the results of growth and mAb-production were compared with standard medium (DMEM + 5% FCS). Slightly reduced mAb-titers were achieved. These were, however, compensated by decreased concentration of contaminating protein and higher specific mAb/protein ratio as examined by SDS-PAGE and enzyme linked immuno electro transfer blot (EITB).

Animals↗
PubMed1985

Production kinetics of monoclonal antibodies.

Two typical patterns of kinetics of monoclonal antibody (mAb) production are observed. Hybridomas which produce high mAb-titers do not show any feed back inhibition upon secretion of mAb into the culture supernatant over long periods of cultivation. "Low producing hybridomas" generally display high initial rates of mAb production, however, after a few hours the mAb secretion is stopped at a certain low concentration level in the order of some 100 mg mAb per ml. To illustrate the case that the high initial production rates of mAb of low producing hybridomas could be maintained for a period of at least 24 hours, mAb titers comparable to those produced by high-mAb-titer producing hybridomas were achieved.

Animals↗
PubMed1985

A laboratory fermentor for agarose immobilized hybridomas to produce monoclonal antibodies.

Mass culture of immobilized cells in airlift-fermenters usually ends up with the beads accumulating in the foamy layer on the surface of the reactor fluid or, in stirred tankreactors, with partial destruction of the beads. We tried to use an airlift fermenter vessel for growing cells, immobilized in agarose beads. Instead of using the gas for driving, we mounted a slowly turning marine type impeller within the drought tube. Oxygen was supplied on occasional demands by the original sparger. This set up leads to sufficient operational characteristics of the reactor without accumulation of the beads in the foamy layer and without mechanical destruction. Different productivities of either immobilized cells or cells in free suspension culture are reported.

Animals↗
PubMed1985

Monoclonal ELISA for the determination of BNYV-virus.

Monoclonal antibodies have been developed against the BNYV-virus (beet-necrotic-yellow-vein) which causes the rhizomania disease in sugar beet (Beta vulgaris). The B103 hybridoma produced predominantly IgG2a at greater than 100 micrograms/ml. IgM-production could not, however, be ruled out. In a polyclonal-monoclonal indirect sandwich ELISA, the B103-antibody bound to BNYVV of Austrian, German, French and Bulgarian origin at a sensitivity of 1 ng/ml or less. Comparison of sugar beet samples in ELISA and immuno-electronmicroscopy showed good correlation, demonstrating the good performance of B103 antibody for broad application in BNYVV diagnosis.

Antibodies, Monoclonal↗
PubMedDate not supplied

A PCR membrane spot assay for the detection of plum pox virus RNA in bark of infected trees.

A procedure for sensitive detection of plum pox virus RNA in infected bark of trees is described. The method is based on the extraction of bark material with buffer containing proteinase K followed by partial purification of RNA using QUIAGEN anion exchange resin. The RNA is then reverse transcribed, the single stranded cDNA is amplified by the polymerase chain reaction using biotinylated deoxynucleotides as label. The amplified cDNA can subsequently be detected by spotting the reaction mixture onto a nitrocellulose membrane. After fixation and washing the incorporated label is detected enzymatically using streptavidin-alkaline phosphatase. It was shown that this non-radioactive detection system is more sensitive than ELISA and a DNA/RNA hybridization test using 32P-labelled probes. It is also possible to detect plum pox virus infection with this assay in trees in the non-vegetative period.

Base Sequence↗