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ELISA PRINCIPLES IN FLOW INJECTION IMMUNOANALYSIS (FIIA) BASIC STUDIES WITH MOUSE IMMUNOGLOBULIN G
No full textThe effects of incubation time, IgG subclass affiliation and
immunoassay type on microtiter plate ELISA and FIIA were investigated.
The advantages and limitations of the sandwich and the competitive
assay for mouse IgG are discusse
A NEW APPROACHTO THE NEWBORN SCREENING FOR METABOLIC DISORDERS: USE OF FLOW-INJECTION ANALYSIS WITH IMMOBILIZED DEHYDROGENASES
No full textThree highly specific NADH-dependent dehydrogenases are studied
for application in clinical analysis, particularly in newborn
screening of three metabolic disorders:
1. Hydroxyisocaproate dehydrogenase for the selective determination
of ketoisocaproate in Maple Syrup Urine Disease,
2. L-phenylalanine dehydrogenase for the determination of
L-phenylalanine in hyperphenylalaninemias, and
3. galactose dehydrogenase for the detection of galactosemia.
After immobilization, each of the three enzymes can be applied as
enzyme reactors in a flow-injection analysis system. Calibration
curves reveal that the sensitivity of the three methods enables
the measurement of the three metabolites in the normal and the
high range. First applications of these methods with plasma samples
are given, a sample frequency of 60 - 100 samples per hour
can be reached in each case
FIA based on Enzymes and Antibodies - developments at the GBF
No full textA major feature of the GBFis its capacity to carry out bioprocess development based on microorganisms,
recombinant microorganisms and animalcells in the 300 | to 5000 | scale. As a result, the GBF proved a
fertile testing ground for the development of bioprocess analysis.
The first attempt to employ FIA methods in this context was reported by GBFresearchers in 1985 (1-3).
Two years later, a major program in biosensor development was launched which now not only comprised
bioprocess control but also analytical procedures for food and environmental samples.
Though concepts for disposable sensors were an integral part of this program from thestart, flow injectionbased
procedures received particular attention, since FIA based on enzymes and antibodies proved a
powerful tool for the development of those analytical protocols designed for semi-continuous operation
and/or the analysis of large numbers of samples. In addition, FIA offers several advantages compared with
other biosensor concepts; thus, the linear detection range of biosensors, which is often quite limited, can
be largely extended in FIA, through the method of "zone sampling", and if two analytical reaction sequences
of largely different pH optima have to be combined, FIA mightoften beleft as the only possibility. In the
following, FIA-related work at the GBF over the past 6 years in the areas of bioprocess control, downstream
processing, food and pesticide analysis is summarized
CONSTRUCTION OF NEW EXPRESSION VECTORS FOR MAMMALIAN CELLS USING THE IMMEDIATE EARLY ENHANCER OF THE HUMAN CYTOMEGALOVIRUS TO INCREASE EXPRESSION FROM HETEROLOGOUS ENHANCER/ PROMOTERS
No full textMammalian expression vectors were optimized by the addition of a
human immediate early (hCMVIE) enhancer DNA fragment in cis to
heterologous enhancer/promoters. The hCMVIE enhancer fragment
mediated a 3-5 fold increase in stable, but not in transient
expression from SV40 expression plasmids ina position dependent, but
orientation independent manner. Maximum expression in BHK cells was
obtained region by separating the hCMVIE enhancer 2 kb from a target
SV40 promoter/enhancer. The effect of the hCMVIE fragment is not
restricted to the SV40 system and is also observed with expression
plasmids harboring the MPSv retroviral LTR. An up to eightfold
increase in luciferase expression is obtained after stable
transfection of CMV/MPSV-expression constructs if compared to the
respective SV40 expression plasmid
STIMULATION OF N-GLYCOSYLATION OF HUMAN CHORIONIC GONADOTROPIN BY cAMP
No full textIt is known that cAMP exerts multiple effects on the biosynthesis of human chorionic
gonadotropin (hCG) at the levels of gene activation and mRNAstabilization. We have
investigated the influence of 8-bromo-cAMP on the N-glycosylation and processing of the
hCG-o-subunit in first trimester placenta. By means of pulse-chase experiments (30 min
pulse with [°SS]Met, 5-120 min chase) three intracellular precursors of the a-subunit of
secreted hCG were observed with apparent molecular weights of 11 kDa (non-glycosylated),
16.5 kDa (one N-glycosyl residue), and 19.5 kDa (two N-glycosyl residues)). HCG
secreted in vitro as well as purified from the urine of pregnant women contained a
sialylated a-subunit (20.6 kDa) which was digested by endo-@-N-glucosaminidase H (Endo H)
yielding a 16.5 kDa form which indicates that one carbohydrate residue was probably of
the hybride type. 8-bromo-cAMP caused the 16.6 kDa form to be converted faster into the
19.5 kDa precursor than in control cultures. In addition, the Endo sensitivity of the
a-subunit of secreted hCG was almost completely abolished in cultures treated with 0.5 mM
8-bromo-cAMP. This seems to indicate that cAMP influences the velocity as well as the
extent of N-glycosylation of the hCG-g-subunit. The specificity of this cAMP-action was
investigated by means of the N-glycosyl acceptor peptide (octanoyl-tripeptide, OTP) N-octanoyl-
asparagyl-tyrosyl-threonine amide. In JEG-3 cells N-glycosylation of OTP and its
secretion was significantly stimulated by 8-bromo-cAMP.In cells treated with 1 mM
8-bromo-cAMP a 5-22-fold higher amount of glycosylated OTP was secreted into the
culture media. Whereas in the control cultures more than 70% of N-glycosyl-OTP was
accumulated in the cells, about 60% was secreted in the presence of 1 mM 8-bromo-cAMP.
In placenta tissue similar results were obtained. These results show that the stimulation
of N-glycosylation by cAMP occurs in a general way and is not confined to the
synthesis of the hCG-a-subunit
CHARACTERIZATION OF TWO STRUCTURALLY RELATED PROTEOCHONDROITINSULFATES FROM A HUMAN B LYMPHOBLASTOID CELL LINE
No full textTwodifferent proteochondroitinsulfates were purified from culture supernatant andcellular lysate of the
human B-lymphoblastoid cell line LICR-LON-HMy2. The proteoglycansconsist of a comparatively small protein
core (supernatantproteoglycan: 21,5 kDa, cell surface proteoglycan: 30 kDa) to whichthree to four
chondroitinsulfate chains (CS-4-sulfate and CS-6-sulfate), each of 26 to 30 kDa are attached. The molecular
massof the mature proteoglycan wasestimated as approximately 130-150 kDa. Both proteochondroitinsulfates
could be distinguished by a different structure of the protein core which became apparentafter amino acid
analysis and comparative peptide mapping. In contrast to the cell surface proteoglycan, the protein core of the
supernatant proteoglycan contained additionally N-linked oligosaccharides
GLYCOSYLATION ENGINEERING: CHO MUTANTS FOR THE PRODUCTION OF GLYCOPROTEINS WITH TAILORED CARBOHYDRATES
No full textMost glycoproteins require their carbohydrates in order to be
synthesized efficiently and in a biologically active, stable form.
However it is clear that the actual structures of these carbohydrates
can vary widely; severly truncated forms will often confer the desired
characteristics. Minimizing carbohydrate heterogeneity may be
advantageous for many reasons, especially in the production of
recombinant glycocoproteins. To obtain glycoproteins with a limited
set of carbohydrate structures glycosylation mutants with mutations in
carbohydrate biosynthesis can be used. Chinese hamster ovary (CHO)
glycosylation mutants that are missing an enzyme activity (e.g. a
transferase or translocase) synthesize truncated carbohydrates with
predictable structures. For purification or tissue-targeting
purposes, it may be desirable to embellish carbohydrates with
particular sugar residues. Dominant CHO mutants or CHO cells
transfected with a cloned glycosyltransferase can be used for this
purpose. Most of the CHO mutants that synthesize altered
carbohydrates grow well in culture showing that a wide range of
carbohydrate structures are compatible with viability. These lines
can readily be used to engineer the carbohyrates of recombinant
glycoproteins for a multitude of purposes
INVESTIGATION OF RECOMBINANT ANTITHROMBIN III PRODUCED BY BHK CELLS DURING FERMENTATION PROCESSES
No full textEfficient recombinant protein production processes should optimize both productivity and
yield. The fermentation process should also preserve the integrity and quality of the desired
protein product.
AT III (antithrombin III) is one of the most important serine protease inhibitors in plasma.
It is an @,-glycoprotein of M, 60,000 to 64,000 containing three or four polysaccharide chains
attached by N-glycosidic linkage to asparagine residues. The biological activity of AT III can be
enhanced 2-3 orders of magnitude by binding with the cofactor heparin (1). Affinity chromatography
of plasma-derived AT III on immobilized heparin resulted in two fractions of AT II
which differ from each other in their affinity to heparin; their difference in affinity to
heparin is due to different degrees of glycosylation (2). Concerns have been raised that if
BHK-derived AT III exhibit the same behavior on purification as its plasma-derived counterpart.
In the fermentation of AT III producing BHK (baby hamster kidney) cells, productivity was
monitored and the possible influence of serum-free cultivation conditions on protein quality in
termsof both integrity (against proteolytic activity) and affinity to heparin was investigated
Crystallization and Preliminary X-ray Studies of Lipase from Geotrichum candidum
No full textWehave succeeded in the production of new crystals of lipase from Geotrichum candidum which
were suitable for a refined X-ray analysis. First measurements proved the lattice constant and space
group to be similar to that reported by Hata et al. /1/. However, these crystals can be analyzed
without any cross-linking. This should enable a higher resolution for the structure determination of
the lipase from Geotrichum candidum.
Inhibition experiments with this lipase proved metal ions, iodine and p-chloromercuribenzoate
to effect the enzyme activity. Metal ions inactivated lipase at a 1000-fold excess and in the order
of Ag*>Hg’* >Co**>Zn**. In contrast, I, and p-chloromercuribenzoate modified lipase from
Geotrichum candidum in stoichiometric amounts. These modified as well as a deglycosylated lipase
could becrystallized. Such crystals ofslightly modified lipases offer the chance of analyzing further
details of the lipase structure, e.g. the influence of the glycosylation on the tertiary structure, and
they are an alternative approach to high quality crystals
PANCREATIC COLIPASE:STRUCTURAL AND IMMUNOLOGICAL STUDIES
No full textLipases hydrolyze triacylglycerols in the form of micellar aggregates or emulsions.
Adsorption of the enzyme to lipid particles is a key step in interfacial catalysis.
Pancreatic lipase is inhibited by bile salt and phospholipid which prevent enzyme
binding to interface.Inhibition is specifically reversed by colipase,a protein of
10.5 KD found with lipase in the pancreatic secretion.Human,porcine and equine
colipases have been sequenced.The amino acid sequence of the human and rat proteins
has been deduced from the nucleotide sequence of cloned cDNA.Comparison of the
primary structures reveals extensive homology.Colipase is secreted aS a precursor
form (procolipase) and activated by tryptic cleavage at one single bond (Arg,-Gly,).
Limited proteolysis increases the lipid binding capacity of colipase.Kinetic studies
of the activation of bile salt inhibited pancreatic lipase by colipase have provided
evidence that the cofactor forms a stoechiometric active complex with enzyme at
interface.Results of binding studies with model substrates are consistent with the
view that colipase binds to interface coated with bile salt and further anchors lipase
to its substrate.It has been postulated that colipase possesses two specific surface
domains for binding to lipid and lipase.Attempts to identify the lipid and lipase
binding sites were made using a physico-chemical approach.Spectroscopic studies have
shown that the region containing the three tyrosine residues is involved in the
binding to lipid-water interface.Chemical modification of the free carboxyl groups of
residues Glu), and/or Asp, which has no effect on lipid binding prevents interaction
with lipase.Polyclonal and monoclonal (MAb) antibodies have been raised against porcine
colipase and used for the characterization of the binding sites in spatial relationship
with antigenic regions.Results of studies carried out with eight MAb and fractions of
polyclonal antibodies separated on immobilized synthetic peptides bring evidence that
the tyrosine containing region and the N-terminal fragment are involved in lipid
binding and that Glu, is implicated in interaction with lipase