Biomedical subjects

J E Butler

Publications and source records attributed to J E Butler.

At least 109 records · Page 6Linked to original sources

PubMed1988

Production of streptavidin in a synthetic medium.

A simple, inexpensive procedure for producing streptavidin has been described. The biotin-binding protein was produced by growing Streptomyces avidinii in a synthetic liquid culture medium containing L-asparagine as the sole nitrogen source. With this procedure, extraneous proteinaceous substances inherently present in culture media prepared with yeast extract or with peptones were not present to interfere with isolation and purification of streptavidin. When harvested after 7-8 days of incubation, the culture fluid was relatively free of contaminating cell breakdown products. Maximal production of streptavidin (100-120 mg/l) was obtained in 8-10 day cultures. For some applications, the culture fluid can be used directly as a source of streptavidin. Under the same conditions used to grow S. avidinii, 11 other actinomycete strains and 134 eumycetes were found to lack the capacity to produce detectable amounts of an extracellular biotin-binding protein.

Bacterial Proteins↗

PubMed1988

Isolation and characterization of highly purified streptavidin obtained in a two-step purification procedure from Streptomyces avidinii grown in a synthetic medium.

A method is described for isolation of streptavidin from cultures of Streptomyces avidinii grown in a synthetic culture medium for 6-10 days. Streptavidin is precipitated directly from culture supernatant fluid using 80% ammonium sulfate, and the precipitate is dialyzed against water and centrifuged at 40,000 X g for 60 min. The absorbency coefficient at 280 nm of purified streptavidin was estimated to be 31.7142 +/- 0.1806 for a 1% solution. The protein appeared to be greater than 90% homogeneous by gel permeation chromatography and polyacrylamide gel electrophoresis. No biotin-binding molecules less than 70 kDa in size were detected at any step during the purification of streptavidin. Streptavidin was able to maintain a stable crosslink between two biotinylated molecules in a solid-phase assay. Streptavidin purified by this method was stable in 50% glycerol/water at -20 degrees C for more than 1 year. Lyophilization or iodination did not produce apparent damage to the protein.

Bacterial Proteins↗

PubMed1988

The immunochemistry of sandwich-ELISAs. IV. The antigen capture capacity of antibody covalently attached to bromoacetyl surface-functionalized polystyrene.

The antigen capture capacity of antibodies covalently immobilized on injection-molded polystyrene beads was evaluated. Bromoacetyl groups on the bead surfaces rendered them reactive to protein nucleophilic groups. The bromoacetyl surface exhibited up to a ten-fold greater capacity for protein compared to unmodified polystyrene, with no detectable dissociation such as occurs with simple adsorption. Biotinylated anti-fluorescein was immobilized on this surface both through direct covalent attachment and indirectly via streptavidin, which was first covalently attached to the bead. Comparisons of the resulting biological activity, normalized to the amount of anti-fluorescein on the bead, were made between the attachment methods and simple passive adsorption. The presence of the streptavidin spacer on the bromoacetyl surfaces improved the antigen capture capacity of antifluorescein, for fluoresyl-albumin by 45% compared to direct covalent linkage of the antibody to modified polystyrene and by 160% relative to antibody adsorbed on unmodified polystyrene.

Acetates↗

PubMed1988

Noninvasive measurement of glucose uptake by two populations of murine embryos.

Glucose uptake was measured by a noninvasive fluorescence technique on a total of 165 morula- and blastocyst-stage murine embryos in two different culture media. Eighty-four embryos were tested in M2 medium, and the remaining 81 embryos were tested in M16. Embryos assayed in M2 took up significantly less glucose over the 4-h assay period than did embryos assayed in M16. The lower uptake of glucose by embryos in M2 corresponded with a decrease in the quality of embryos cultured overnight in M2 as judged by morphological criteria. Embryos that were judged to be degenerate or had gross abnormalities took up significantly less glucose than did normal embryos. Glucose uptake in both populations of embryos covered a wide range of values and was normally distributed. A significant effect between mothers was noted in glucose uptake for embryos assayed in both M16 and M2 media. The possible uses of noninvasive measures of glucose uptake as a test of embryo viability or for optimizing culture conditions are discussed.

Analysis of Variance↗

PubMed1987

A sandwich ELISA for properdin in clinical specimens.

We have developed a symmetrical sandwich ELISA for measuring human properdin (P) in serum by using the globulin fraction from a commercial antiserum as the capture antibody adsorbed on the plastic. The detecting reagent was a glutaraldehyde conjugate of this Ig fraction with alkaline phosphatase. Two types of inhibition were observed in this study. First, inhibition was observed when greater than 2.5 micrograms/ml of the globulin fraction was used to coat the plates. A second type of inhibition was observed for serum dilutions less than 1/400; it was independent of the concentration of capture Ab and did not occur when purified P was assayed. The data generated with this assay could be fitted in log-log mode by a quadratic equation. The coefficient of the linear term in this equation was found to be the same for serum and purified P, within the limits of experimental error. The results for different samples run on the same plate were expressed in terms of the relative concentration of each sample required to produce an OD405 = 0.2. A sample of pooled normal human serum was run on each plate as a reference; it was assigned a titer of 100 ELISA units/ml (EU/ml). The titers of the unknown samples were expressed in terms of EU/ml by reference to this standard. For purified P, the assay could readily detect 10 ng/ml. By comparing purified P with our reference serum pool, we found that 1 EU equals 0.57 microgram. Day-to-day variation for a group of nine normal sera showed a mean difference of -0.85 EU/ml, SD 5.85 EU/ml. The mean titer for these normal sera was 78.9 EU/ml, SD 15.7 EU/ml. In three recovery experiments in which purified P was mixed with pooled normal serum, the recoveries ranged from 96 to 117%. We conclude that the sandwich ELISA constitutes an adequate immunochemical assay for human P in serum specimens.

Antibody Specificity↗

PubMed1987

The heterogeneity of bovine IgG2--III. The ion-exchange heterogeneity of IgG2a is the result of VH-region variation.

Bovine IgG2a from an animal homozygous at the A-locus could be fractionated on DEAE columns into subpopulations; studies using analytical size columns showed that the number of subpopulations was dependent on the ionic strength of equilibration buffer. Subpopulations of IgG2a could be purified by this method which did not differ in their carbohydrate content but had mean Ips consistent with their elution behavior. The spectrotype of their H-chains but not their L-chains, paralleled the spectrotype of the corresponding intact IgG2a subpopulations. When papain digests of each subpopulation were fractionated by chromatofocusing, their Fc fragments eluted homogeneously with a pI ca 6.0 while their Fabs eluted heterogeneously with pIs ranging from 8 to 5. The distribution of carbohydrate among such fractions corresponded to the distribution of the Fcs. The behavior of the IgG2a Fabs on chromatofocusing paralleled the elution behavior of the parent IgG2a subpopulations from DEAE-Sephadex and the isoelectric behavior of their H-chains. The antibody activity of Fab fragments, separated by chromatofocusing, are consistent with the concept of idiotypic variation. The differential antibody activity of each intact IgG2a subpopulation for seven different antigens suggests that the ion-exchange behavior of IgG2a, in which the influence of allotypes and sub-isotypes has been excluded and for which charge-related L-chain heterogeneity is minimal, must reside in the VH-regions of the different IgG2a subpopulations. Investigators are cautioned against assigning ruminant subclass designations on the basis of subpopulations isolated by ion-exchange chromatography.

Animals↗

PubMed1987

The heterogeneity of bovine IgG2--IV. Structural differences between IgG2a molecules of the A1 and A2 allotypes.

The major product of digestion of bovine IgG2a(A1) with immobilized pepsin is F(ab')2 while similar treatment of IgG2a(A2) yields Fab, Fc and other products. It is postulated that structural differences in the hinge region, including the absence of the most N-terminal disulfide bridge in IgG2a(A2) and a displacement of the primary pepsin cleavage site toward the Fd, can explain this effect. The immunodominant A1 allotope(s) appears to be located in the CH3 domain of IgG2a(A1) while the A2 allotopes are located elsewhere and are apparently affected by digestion. The allotypic bias of rabbit anti-IgG2a is also present in anti-IgG2a raised in goats. However, the A2 allotypic determinants of bovine IgG2a are recognized by goat precipitins although precipitins of this specificity are not detectable in rabbits immunized with IgG2a(A2). Rabbit anti-A2 antibodies are detectable using the ELISA in rabbits immunized with IgG2a(A2).

Animals↗

PubMed1987

The transport and metabolism of bovine IgM.

IgM is present in cows milk, is able to bind secretory component (SC), has a purported role as a secretory immunoglobulin in other species and has been identified with various antibody functions in cows milk. To determine the origin of cows milk IgM, we administered extrinsic 131I-IgM to lactating cows with cannulated bile and parotid ducts and studied the kinetics of its disappearance from serum and its appearance in milk, bile and parotid saliva for 60 hr post-injection. Pentameric IgM appeared to require a long equilibration time and disappeared from serum with a T1/2 of 40 hr. The transport of IgM into bile also appeared biphasic. Results showed that no 131I-IgM was transported intact into parotid saliva and that most radioactivity in milk and bile after 6 hr was in the form of low mol. wt, TCA-precipitable fragments rather than of the size of a pentamer. During the first 24 hr only 0.83% of the administered dose reached the milk in pentameric form, nevertheless, isotope dilution calculations indicated that nearly all milk IgM was derived from serum. During a 12 hr collection period, corresponding to one milking, greater than 200 mg of serum IgM is secreted in milk. During the first 24 hr, only 0.70% of the administered IgM reached the bile as a pentamer. It was calculated that 50% of the pentameric IgM in bile, 3 hr after administration, was serum-derived. Twenty-five per cent of the IgM appearing in bile and ca 10% of the IgM appearing milk, becomes associated with secretory component. A hypothesis to explain the degradation associated with this inefficient transport mechanism is presented.

Animals↗

PubMed1987

The isotypic, allotypic and idiotypic heterogeneity of bovine IgG2.

The antigenic heterogeneity of bovine IgG2 observed by single radial diffusion, when different cattle sera are tested with a panel of IgG2-specific reagents, was examined by a combination of biochemical and serological assays. Using an autologous anti-A1 allotypic reagent, the major antigenic heterogeneity detected by swine, rabbit and goat anti-IgG2 reagents was due to their propensity to recognize the AI allotope. All heterologous reagents and most monoclonals so far test are biased in their specificity toward this determinant. A second type of serological heterogeneity, recognized by only certain heterologous reagents, was their specificity for what we have called the IgG2b isotype of IgG2. This isotype is found in all cattle, has a restricted ion-exchange elution behaviour and does not bear a-locus allotypic determinants; molecules bearing the latter are now designated IgG2a. IgG2a elutes from anion-exchange columns in subpopulations over a wide range of ionic strength including fractions which contain IgG2b and IgG1 as well. Using IgG2a from an AI homozygous steer, these subpopulations were shown to result from idiotypic variation which appears to primarily reside in their VH-regions.

Animals↗

PubMed1987

The heterogeneity of bovine IgG2. II. The identification of IgG2b.

Goat and rabbit polyclonal reagents can be raised which recognize a new isotype of bovine antibodies. The polyclonal goat reagent was raised against a preparation enriched in the major IgG2 isotype (IgG2a) which contained the new isotype as a contaminant. The polyclonal rabbit reagent was prepared against a trypsin-derived Fc fraction of bovine IgG1 which contained the Fc of the new isotype as a contaminant. This new isotype is present in the sera of the cattle of all breeds tested regardless of their IgG2a allotype and is antigenically distinct from IgG2a, IgG1, IgA, IgM and IgE. The new isotype coelutes from DEAE anion exchangers with IgG1 and the more acidic populations of IgG2a. The isotype is tentatively designated IgG2b. The distribution of IgG2b antibody activity to E. coli K99 and phosphorylcholine among 15 cattle of different A allotypes is not correlated with the IgG2a or IgG1 antibody activity in these animals.

Animals↗

PubMed1987

Roentgenologic diagnosis of stress fractures and stress reactions.

We examined 26 athletes with continued pain after strenuous exercise. We found stress fractures in 15 patients, all of whom had positive roentgenographic studies. Seven of these 15 also had bone scans, all of which were positive. Five patients had stress reactions, all of which showed positive results on bone scan and negative roentgenographic results. Five had occult stress reactions manifested by pain, with normal roentgenographic and scintigraphic results, and one had minor pain not requiring radiologic studies. Stress reaction is contrasted with stress fracture by demonstrating the role of bone imaging in the diagnostic continuum of stress reaction to stress fracture.

Adult↗

PubMed1987

Triple arthrodesis in young children: a salvage procedure after failed releases in severely affected feet.

A total of 19 triple arthrodeses were performed in 13 children aged 10 years or less for severe hindfoot deformities after failure of soft tissue releases. This was considered a salvage procedure and an alternative to talectomy. The average age at surgery was 8.4 years and average follow-up was 4 years. There were 68% excellent and good results, 16% fair, and 16% poor results. The nonunion rate was 7% of joints. Average shortening of the foot was 0.81 in but was not significant when compared with a group of clubfoot control patients. Except for one infection, fair and poor results were due to residual rather than recurrent deformity. Triple arthrodesis may be indicated in the young child for residual hindfoot deformity as an alternative to talectomy. The potential problems of increased nonunion, severe compromise of foot length, and recurrence of deformity were not observed in this series.

Age Factors↗

PubMed1987

Composition of sow milk during lactation.

The composition of sow colostrum and milk was quantitated in 25 sows at 14 time points throughout lactation. All animals belonged to the same experimental herd of German Landrace, farrowed within 4 d, and were of various lactation numbers and various litter sizes. In the first 6 h of lactation colostrum total solids (TS) and protein contents were higher, while fat and lactose contents were lower than in mature milk. Decreased total protein and whey protein contents and concomitantly increased fat and lactose content, with nearly unchanged TS levels, indicate transition from colostrum to mature milk. The high protein content of colostrum was largely due to immunoglobulin (Ig). During the first 6 h, IgG accounts for nearly all the protein in colostrum but plays a decreasing role in sow milk as lactation proceeds. After 2 wk, IgA levels begin to increase and at the end of lactation, IgA constitutes 40% of the total whey protein. No influences of lactation number and litter size on milk composition could be ascertained in this study.

Animals↗

PubMed1987

Production of ovine chimeras by inner cell mass transplantation.

Ovine chimeras were produced by micro-injection of isolated inner cell masses (ICM) into recipient blastocysts. Inner cell masses were isolated by immunosurgery. A total of 57 chimeric embryos was produced, 52 of which were transferred to recipient ewes. Thirty-seven live lambs were born, of which 15 were determined to be chimeric on the basis of blood type analysis. One lamb, although not a blood chimera, exhibited overt signs of chimerism. An additional six lambs were determined to have developed solely from the injected ICM. The rate of chimerism in live lambs was 43% (16/37) while the survival rate of injected ICM was 59% (22/37). The method presented allows the production of relatively large proportion of viable, chimeric embryos without the use of an intermediate recipient.

Animals↗

PubMed1987

Production of sheep-goat chimeras by inner cell mass transplantation.

Embryos were surgically flushed from goats and sheep on d 6 and 7, respectively, following the first day of estrus (d 0). After enzymatic removal of the zonae pellucidae, inner cell masses were isolated from caprine blastocysts by immunosurgery. The intact inner cell masses were injected into ovine blastocysts with the aid of a micromanipulator. Twenty-two manipulated blastocysts were surgically transferred into 12 ovine recipients. Nine ewes gave birth to a total of 13 young (59% embryo survival). Ten were classified by serum electrophoretic assays or karyotypes as lambs, one as a kid, and two as interspecific chimeras.

Animals↗

PubMed1987

The immunochemistry of solid-phase sandwich enzyme-linked immunosorbent assays.

Multivalent antigens (Ags) such as membrane proteins can be quantitated by using sandwich enzyme-linked immunosorbent assays (ELISAs), which typically show sensitivity from 0.1 to 50 ng/ml. The percentage of antigen that binds in the log-log linear region reflects the affinity of the capture antibody (CAb), and the range of linearity for assays conducted with a particular CAb is proportional to the antibody (Ab) concentration. The sandwich ELISA titration plot reflects the actual amount of Ag bound when asymmetrical configurations are used; steric hindrance that occurs with certain symmetrical configurations, especially when enzyme-Ab conjugates of greater than or equal to 10(6) daltons are used, can alter this relationship. Monoclonal CAbs bind less Ag than polyclonal CAbs. Immobilization of monoclonal CAbs by using a modified avidin-biotin system can result in greater antigen capture capacity (AgCC) than when the Abs are directly adsorbed on plastic. Adsorption of proteins on polystyrene is noncovalent and proportional to the amount added for up to 150 ng/200 microliter in a microtiter well. Adsorption can result in substantial loss of antigenic or antibody activity. Desorption is continuous at a low level and can negatively influence the results of an immunoassay. Data from microtiter sandwich ELISAs can be readily acquired and analyzed by using a computer-based analysis system (ELISANALYSIS) written for the IBM PC. This analytical system considers the immunochemical principles of sandwich ELISAs predicted theoretically and demonstrated empirically.

Adsorption↗

PubMed1987

Absolute and relative concentrations of immunoglobulins G, M, and A, and albumin in the lacteal secretion of sows of different lactation numbers.

The concentrations of immunoglobulin (Ig)G, IgM, IgA, and albumin in the lacteal secretion of 80 sows were measured not more than 14 times during lactation to determine whether the age of the sow, the anatomic location of the mammae, or the farrowing barn housing of the sow influenced Ig concentrations. The combined data indicated that there were lactation stage-dependent changes in the total Ig concentration, as well as changes in the relative importance of the 3 classes of Ig studied. Mean values of any one time point showed enormous variation in absolute values, with coefficients of variation ranging up to 70% The largest variations were observed between colostral samples obtained on the first day. The IgM showed the least variation on day 1. Such variations hindered attempts to statistically identify their source. Normalization of Ig concentrations in relation to albumin concentrations decreased some of the apparent variation. Normalized data accentuated the changeover from colostrum to milk which occurred after the first 3 days. There was also a 3-fold increase in IgA concentrations which occurred during the last 4 weeks of lactation. Data showed that older sows tended to have higher concentrations of all Ig. Lactation number-dependent increases in IgM and IgG were restricted to colostrum in lactation numbers higher than 4. Immunoglobulin A increased in a similar pattern and, in addition, increased in mature milk in sows with more than 2 lactations. The enormous variations observed seemed to be individual animal variations. This allowed only trends, rather than statistically significant differences, to be recognized.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗

PubMed1986

The immunochemistry of sandwich ELISAs. II. A novel system prevents the denaturation of capture antibodies.

The studies reported here describe a model system by which the effect of surface adsorption on the antigen capture capacity (AgCC) of monoclonal antibodies (MoAbs) can be quantitated. Three mouse anti-fluorescein (FLU) MoAbs were biotinylated and iodinated with [125I]Na. The AgCC/ng of these MoAbs was compared when they were adsorbed directly on plastic or immobilized via a Protein Avidin Biotin Capture (PABC) system. In this system, biotinylated MoAbs were allowed to adsorb on biotinylated carrier proteins through a "bridging" process using Streptavidin. This permits MoAbs to complex with antigen at some distance from the plastic surface. The AgCCs of the MoAbs immobilized using the PABC system were 5-400-fold higher than when they were directly adsorbed on plastic.

Antibodies, Monoclonal↗