Final Protein Concentration Research Articles

Refolding and aggregation of bovine carbonic anhydrase B: quasi-elastic light scattering analysis

Bovine carbonic anhydrase B (CAB) is chosen as the model protein to study the phenomenon of protein aggregation, which often occurs during the refolding process. Refolding of CAB from 5 M GuHCl has been observed by quasi-elastic light scattering (QLS), which confirms the formation of a molten globular protein structure as reported previously [Semisotnov, G. V., Rodionova, N. A., Kutyshenko, V. P., Ebert, B., Blanck, J., & Ptitsyn, O. B. (1987) FEBS Lett. 224, 9-13]. QLS analysis reveals the formation of multimeric species prior to precipitation. Activity and cross-linking studies have confirmed the presence of inactive multimeric protein species. The dimer formation has been determined to be the initiating step in the aggregation of CAB during refolding. Activity studies have indicated that the first intermediate observed in the refolding pathway of CAB aggregates to form the inactive dimer. The rate of formation of the dimer has a stoichiometric dependence on the final protein concentration. The dimer formation rate is a function of the final guanidine hydrochloride (GuHCl) concentration to the inverse 6.7 power, which correlates well with the binding of GuHCl to the native protein in 0.60-0.80 M GuHCl. These rate dependencies require the refolding of CAB to be performed at high GuHCl concentrations (1 M GuHCl) and low protein concentrations (less than 1 mg/mL) to avoid the formation of aggregates. Alternatively, refolding can be performed by allowing the first intermediate to form the second intermediate prior to further dilution or dialysis. The aggregation of a hydrophobic first intermediate species is likely to be common to the refolding of other molten globular proteins.

The role of mRNA and protein stability in gene expression.

How important is the stability of gene products in the process of gene expression? We use a dual-compartment mathematical model to demonstrate the effects that changing the rates of synthesis and degradation of hypothetical mRNAs and proteins would have on the final concentration of protein. The model predicts that the concentration of protein at steady state equals the product of the rate constants for synthesis of mRNA and protein (ks1 and ks2) divided by the product of the rate constants for degradation (kd1 and kd2) and that the rate at which protein concentration changes depends on the rate constants for degradation of both the mRNA and the protein. This permits great flexibility in controlling induction kinetics for particular gene products, since their synthesis, translation, and degradation may be regulated coordinately to permit induction to be stable or transient or to amplify the final yield of protein. We suggest single exons may encode structural features that cause both mRNAs and proteins to be labile, thereby ensuring that modal stabilities of highly regulated macromolecules are similar.

Influence of copolymer composition on protein adsorption and structural rearrangements at the polymer surface

The influence of copolymer composition on protein adsorption and subsequent structural rearrangements of the adsorbed protein has been studied by (1) investigating the ability of adsorbed proteins to be displaced by proteins in solution and (2) by determining the ability of an immobilized antibody to recognize an antigen—enzyme conjugate. Surfactant-free polystyrene copolymeric latexes were used to study the effect of specific comonomers [acrylic acid (AA), methacrylic acid (MAA), 2-hydroxyethyl acrylate (HEA), and acrylamide (A)] on protein adsorption. Although the classical surface techniques of microelectrophoresis and surfactant titration could not distinguish between the different polystyrene copolymers, the extent and nature of protein adsorption were influenced substantially by the copolymer composition. Adsorption was essentially irreversible with respect to dilution but adsorbed proteins could be displaced by proteins in solution. The order of effectiveness was fibrinogen > immunoglobulin > albumin. the ability of adsorbed proteins to be displaced from surfaces followed the order PS/PHEA > PS/PAA > PS/PMAA > PS (easiest to most difficult displacement). The retention of activity of an immobilized monoclonal antibody (Phe 1.9) at the surface followed the order PS/PHEA > PS/PAA = PS/PMAA > PS (greatest to least active). Protein adsorbed to polystyrene under low protein concentrations was less readily displaced than protein adsorbed at saturating protein concentrations. Similarly, the activity of the monoclonal antibody Phe 1.9 was dependent on the final adsorbed protein concentration at the surface of polystyrene homopolymer. In contrast, the activity of Phe 1.9 immobilized on copolymer containing HEA, AA, or MAA varied little if any with the final protein concentration at the surface. The retention of activity of covalently immobilized Phe 1.9 on poly(styrene—co-chloromethylstyrene) copolymers followed the order PS/PCS/PA > PS/PCS/PHEA(3) > PC/PCS/PHEA(1) > PS/PCS/PAA > PS/PCS/PMAA = PS/PCS (greatest to least activity). Thus, the inclusion of “modifier monomers” that are hydrophilic or are capable of hydrogen bonding with proteins appears to moderate structural rearrangements of an immobilized protein reflected by both the ease of displacement from the surface and the retention of biological activity.

Superfused pituitary cell cultures: Effects of culture conditions on apparent responsiveness to LHRH stimulation administered as short duration pulses

We wished to study estrous cycle related differences in LH and FSH responsiveness to pulsatile LHRH. Such studies are very difficult to perform in vivo under controlled conditions; therefore, an in vitro superfused anterior pituitary cell culture system was evaluated for its capacity to support differences in estrous stage associated LHRH responsiveness. Three vital culture system parameters were evaluated; these parameters were (1) culture medium composition, (2) duration allowed for cell attachment to microcarrier beads and (3) superfusion flow rate utilized during pulsatile LHRH stimulation. It was found that a culture system which utilized 10% Nu Serum in DMEM (final protein concentration of 1.8 mg/ml; final serum concentration of 2.5%), an attachment time of 48 hrs and a flow rate of 0.125 ml/min most successfully maximized LH responsiveness at the lowest serum concentration. These studies indicated that although one may be able to observe LHRH responsiveness under a wide range of culture conditions, responsiveness may nonetheless be maximized by judicious adjustment of culture conditions.

Artificial pulmonary surfactant inhibited by proteins.

With a pulsating bubble surfactometer we assessed the ability of various agents, fibrinogen, human serum, albumin, and a 55,000-dalton serum protein, to interfere with the surface activity of Surfactant TA. From a highest final protein concentration of 4 mg/ml the potential inhibitors were diluted down to 0.125 mg/ml in six steps, and each concentration was evaluated together with two final phospholipid concentrations, 6.25 and 1.25 mg/ml, of the surfactant preparation. The strongest inhibiting action was exerted by fibrinogen, followed by human serum and the 55,000-dalton serum protein; the weakest inhibitor was albumin. Bilirubin, when added in an amount of 1.73 mg/100 ml dissolved in human serum, significantly (P less than 0.001) augmented the inhibition over that exerted by human serum alone. Adsorption rate, as reflected in the mean value of surface tension 2 and 10 s after creation of a bubble, not pulsating, was seriously affected by each of the protein-containing inhibitors in concentrations exceeding 1 mg/ml. Surface tension was raised significantly when the pulsating bubble was at maximal and minimal size. The effect was dose dependent. At maximal size it showed no tendency to disappear during the 10-min recording, but at minimal bubble size the inhibition gradually diminished. We conclude that proteins present in the airways may seriously interfere with the activity of Surfactant TA.

Effect of rearing density on thyroid and interrenal gland activity and plasma and hepatic metabolite levels in rainbow trout, Salmo gairdneri Richardson

There were significant inverse correlations between rearing density of rainbow trout, Salmo gairdneri Richardson, and final body weight, plasma L‐thyroxine (T4), trüodo‐L‐tryronine (T3), cortisol and protein concentrations, plasma T4/T3 ratios and thyroid epithelial cell height. In addition, hepatosomatic indices and plasma free fatty acid concentrations were higher in fish reared at low (134 g 1−1) density compared with groups reared at medium (210g1−1) or high density (299g 1−1), and the post‐feeding (3.5‐4h) elevation in plasma glucose and triglyceride levels evident in trout maintained at low rearing density was not found in those fish reared at higher densities. There were no significant effects of rearing density on hematocrit, carcass composition, hepatic glycogen and lipid levels and interregnal nucleus size.

Stability and self-assembly of the S-layer protein of the cell wall of Bacillus stearothermophilus.

The surface layer of the cell envelope of Bacillus stearothermophilus consists of a regular array of protein subunits. As shown by dodecyl sulfate polyacrylamide gel-electrophoresis and ultracentrifugation, the fully solubilized S-layer protein represents a homogeneous entity with a subunit molecular mass of 115 +/- 5 kDa. Solubilization of the protein may be accomplished at acid pH, or using high concentrations of urea or guanidine X HCl. It is accompanied by (partial) denaturation, thus interfering with the characterization of the protein in its unperturbed native state. Removal of the solubilizing agent by dialysis or dilution allows the S-layer to be reassembled into two-dimensional crystalline lattices identical to those observed in intact cells. To determine the kinetics of association, optimum conditions are found to be rapid mixing with 0.1 M sodium phosphate pH 7.0, 20 degrees C, final protein concentration greater than 10 micrograms/ml. If the time course of the self-assembly is monitored by light scattering, as well as by chemical cross-linking with glutardialdehyde, multiphasic kinetics with a rapid initial phase and slow consecutive processes of higher than second-order are observed. The rapid phase may be attributed to the formation of oligomeric precursors (Mr greater than 10(6) ). Concentration-dependent light scattering measurements give evidence for a "critical concentration" of association, suggesting that patches of 12-16 protein subunits fuse and recrystallize into the final (native) S-layer structure. Recrystallization tends to be complete.

Mutagenic activation of 3-amino-1,4-dimethyl-5 H-pyrido(4,3- b)indole(Trp-P-1) and 3-amino-1-methyl-5 H-pyrido(4,3- b)indole (Trp-P-2) by primary cultures of adult rat hepatocytes: Effect of Aroclor induction in vitro

The mutagenic activation of tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, was studied in a Salmonella TA98/hepatocyte mutagenesis assay. Adult rat hepatocytes in primary culture were either untreated or induced by the addition of Aroclor 1254 (2 μg/ml) 18–20 h before the mutagenesis test which was performed at day 1 and at day 2 after the isolation of hepatocytes. The mutagenic activation of Trp-P-1 and Trp-P-2 was studied as a function of the time of incubation and of the concentration of chemical. Trp-P-1 and Trp-P-2 incubated for 20 min in the presence of untreated hepatocytes and bacteria gave rise to a weak number of revertants which doubled the level of spontaneous mutants. Aroclor-induced hepatocytes became highly competent in mutagenic activation of tryptophan pyrolysis products and the induction ratio reached 4.9 and 7.1 for Trp-P-1 and Trp-P-2, respectively, after 60 min of incubation, on day 2 of the experiment. It should be noted that the induction ratio was higher on day 2 than on day 1. When conditions were standardized, i.e. Aroclor-induced hepatocytes on day 2, final concentration of cellular protein about 1 mg/ml, 20 min of incubation, the Salmonella/hepatocyte assay produced a linear concentration-dependent mutagenic response for Trp-P-1 and Trp-P-2. By comparing the results obtained with Aroclor-induced hepatocytes and Aroclor-induced liver S9 fraction in the Salmonella test, it could be estimated that hepatocytes were 3 times less active than the S9 fraction with regard to mutagenic activation of both Trp-P-1 and Trp-P-2.

LDL-induced cytotoxicity and its inhibition by anti-oxidant treatment in cultured human endothelial cells and fibroblast

Low density lipoproteins (LDL) isolated by ultracentrifugation induce cytotoxic changes in cultured human endothelial cells (EC) and fibroblasts if the ratio between LDL cholesterol and the final protein concentration of the culture medium exceeds 0.10–0.12 mmol/g protein. In order to investigate if reactive oxygen species could contribute to the cytotoxicity, LDL were prepared in the presence of the antioxidants butylated hydroxytoluene (BHT) or superoxide dismutase (SOD), while routinely prepared LDL from the same donors served as control (N-LDL). A radiochromium release assay was used to evaluate cellular injury. BHT treatment of the LDL fraction virtually abolished LDL-induced cytotoxicity in cultured human EC and fibroblasts. SOD-LDL offered partial protection against LDL cytotoxicity. A positive correlation between the cytotoxicity of the various fractions and their content of malondialdehyde (MDA) further supports our conclusion that lipid peroxides in the LDL fractions mediate the cytotoxic effect on cultured cells.

Effect of protein on the determination of total bile acids in serum.

We measured total serum bile acids on a fluorescence-light-scattering micro centrifugal analyzer by the direct enzymatic method with 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) and with resazurin as a fluorogenic electron acceptor. We found that serum protein has an inhibitory effect on the measurement of bile acids, but this effect was eliminated by adding bovine serum albumin to the reaction mixture in a final protein concentration (12.2 g/L) that was high compared with that contributed by a normal serum specimen. The assay is a sensitive method that reaches equilibrium in 5 min. The method is microscale (5 microL of sample, 150 microL of working reagent), is easy to perform, and is accurate (analytical recovery = 104.1%) and precise (CV = 11.1 and 5.7% on specimens with bile acid concentrations of 7.6 and 35.4 mumol/L, respectively). Normal values are 1-12 and less than 9 mumol/L on nonfasting and fasting individuals, respectively. Pure 3 alpha-hydroxysteroid dehydrogenase must be used: we found several enzyme preparations that gave falsely high values for bile acid.

Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and by catecholamines.

The role of hormones and other humoral factors in the regulation of myocardial hypertrophy has been difficult to evaluate. We asked whether myocardial cell hypertrophy could be demonstrated in cultures from the day-old rat ventricle and evaluated the effect of serum concentration and catecholamines on the growth process. Two single-cell preparations were used: serum-supplemented, bromodeoxyuridine-treated cultures and serum-free cultures with transferrin and insulin. Both preparations were characterized by myocardial cell predominance (about 75--80% of total cells) and constant cell numbers. Myocardial cell size was documented by photomicroscopy and quantified by volume (microscopic diameter of suspended cells), surface area (planimetry of attached cells), and total cell protein concentration (Lowry method and cell counts). Growth was also evaluated in pure nonmyocardial cell cultures. In cultures with 5% (vol/vol) serum, myocardial cell size increased 2- to 3-fold over 11 days in culture. Final volume, surface area, and protein concentration were about 3000 micrometer3/cell, 5000 micrometer2/cell, and 1500 pg/cell, respectively. Serum had a dose-related effect on myocardial cell hypertrophy; myocardial cell size increased about 4-fold when serum concentration was increased from 0% to 5% or 10%. Cells maintained in serum-free medium with transferrin and insulin (each 10 microgram/ml) did not hypertrophy, but did remain responsive to the growth-promoting activity of serum. Chronic exposure to isoproterenol or norepinephrine (1 microM) significantly stimulated myocardial cell hypertrophy. This stimulation was dose-related, was not blocked by equimolar propranolol, was not associated with a sustained chronotropic effect, and was more pronounced in the serum-free preparation. In pure cultures of nonproliferating (bromodeoxyuridine-treated) nonmyocardial cells, cell size also increased with time in culture, but variation in serum concentration and addition of norepinephrine had no significant effect on cell size. Myocardial cell hypertrophy occurs in culture and is regulated by variations in the culture medium, including serum, with its contained hormones and growth factors, and catecholamines. The culture preparation can be used to explore the regulation of myocardial cell hypertrophy by nonhemodynamic factors.

Growth stage changes in the elemental composition, protein, fiber, and in vitro digestibility of fababean plants

Abstract The changes with growth stage in dry matter, concentrations of N, P., K, Ca, Mg, S, Fe, Mn, Cu, Zn, Se, protein and fiber, and in vitro digestibility of fababean leaves, stems, pods and beans during three successive growing seasons have been studied. At final harvest the beans made the dominant contribution to the total plant dry weight (43.7%). In plant parts most elements showed an overall decrease in concentration between initial and final sampling periods. However K increased in pods, and Ca, S, Mn, Cu and Se increased in leaves. Within the growth period Mn and Se in leaves exhibited the most marked fluctuations. Fe, Mn, Cu and Se showed the most rapid concentration changes over short time periods. The major contribution by the beans to the final protein concentration of the harvested plants makes the fababeans a valuable protein source for livestock. Fiber concentration and in vitro digestibility showed an inverse relationship. Comparison of the data with NRC Dairy Cattle Requirements 1978 showed that as a livestock feed fababean forage would only require Mn supplementation.

Growth of Iron-Oxidizing Thiobacilli in the Presence of Chalcopyrite and Galena

Iron-oxidizing thiobacilli were adapted to grow on a chalcopyrite and a galena ore concentrate. When grown on the chalcopyrite concentrate, the bacteria exhibited a doubling time of 38.4 +/- 2.9 h, with a final cellular protein concentration of 185 mug/ml and solubilization of 10.3 g of copper per liter. When grown on the galena ore concentrate, the generation time was 39.6 +/- 2.7 h, with a final cellular protein concentration of 120 mug/ml. Galena was converted to lead salts soluble in 1 M ammonium acetate to a concentration of 20.2 g of lead per liter. X-ray diffraction and refractive-image analysis indicated that the smaller-sized particles were favored in this process. Galena was converted to anglesite, and soluble copper was liberated from chalcopyrite with the concurrent formation of jarosite.

The conformation of γ-globulin adsorbed on polystyrene latices determined by quasielastic light scattering

Measurements of the conformation of γ-globulin adsorbed on monodisperse polystyrene latices, and the subsequent rates of flocculation of the latices, have been carried out in situ using quasielastic light scattering. The molecular extension was found to depend on the protein solution concentration, with end-on adsorption indicated at high surface coverage. Application of the Deutch-Felderhof model for the translational friction coefficient of microscopic multisubunit assemblies established, through a comparison of calculated with experimental values of the molecular extension, the possibility of a flattening in the adsorbed conformation at low surface concentration. Changes in adsorbed conformation as a function of pH were also found. Although the initial rate of flocculation was too great to measure, the particle radius at a given time after mixing varied with the concentration exhibiting a maximum at approximately 0.05 mg · ml −1 final protein concentration.

A heated Biuret-Folin protein assay which gives equal absorbance with different proteins.

Abstract This protein assay requires heating the sample with biuret reagent for 100 min at 100°C before addition of Folin. Different proteins give almost identical optical densities on a nitrogen basis with this procedure. The absorbance per microgram-atom of protein nitrogin is linear at or below 0.400. A convenient final concentration of protein in this assay is about 0.2 μg-atom of protein nitrogen per ml. Most of the absorbance is due to amino acid combinations other than tyrosine and trytophan in the protein. The specificity appears similar to that of the bluret where few compounds of biological importance interfere significantly in the assay at normal cellular concentrations.

Cyclid 3':5'-nucleotide phosphodiesterase. Interconvertible multiple forms and their effects on enzyme activity and kinetics.

An extract of rat liver or human platelet displayed three cyclic 3':5'-nucleotide phosphodiesterase activity peaks (I, II, and III) in a continuous sucrose density gradient when assayed with millimolar adenosine 3':5'-monophosphate (cAMP) or guanosine 3':5'-monophosphate (cGMP). The three fractions obtained from each nucleotide were not superimposable. The molecular weights corresponding to the three activity peaks of cAMP phosphodiesterase in rat liver were approximately: I, 22,000; II, 75,000; and III, 140,000. In both tissues, fraction I was barely detectable when assayed with micromolar concentrations of either nucleotide, presumably because fraction I has low affinity for cAMP and cGMP. Any one of the three forms upon recentrifugation on the gradient generated the others, indicating that they were interconvertible. The multiple forms appear to represent different aggregated states of the enzyme. The ratio of the three forms of cAMP phosphodiesterase in the platelet was shifted by dibutyryl cAMP (B2cAMP) and by the enzyme concentration. B2cAMP enhanced the formation of fraction I. Low enzyme concentration favored the equilibrium towards fraction I, while high enzyme concentration favored fraction III. When phosphodiesterase activities in the extract of rat liver, human platelets, or bovine brain were examined as a function of enzyme concentration, rectilinear rates were observed with micromolar, but not with millimolar cAMP or cGMP. The specific activity with millimolar cAMP was higher with low than with high protein concentrations, suggesting that the dissociated form catalyzed the hydrolysis of cAMP faster than that of the associated form. In contrast, the specific activity with millimolar cGMP was lower with low than with high protein concentrations. Supplementing the reaction mixture with bovine serum albumin to a final constant protein concentration did not affect the activity, suggesting that the concentration of the enzyme rather than that of extraneous proteins affected the enzyme activity. A change in enzyme concentration affected the kinetic properties of phosphodiesterase. A low enzyme concentration of cAMP phosphodiesterase yielded a linear Lineweaver-Burk plot, and a Km of 1.2 X 10(-4) M (bovine), 3 X 10(-5) M (platelet), or 5 X 10(-4) M (liver), while a high enzyme concentration yielded a nonlinear plot, and apparent Km values of 1.4 X 10(-4) M and 2 X 10(-5) M (brain), 4 X 10(-5) M and 3 X 10(-6) M (platelet), or 4 X 10(-5) M and 3 X 10(-6) (liver). Since a low enzyme concentration favored fraction I, the dissociated form, whereas a high enzyme concentration favored fraction III, the associated form, these kinetic constants suggest that the dissociated form exhibits a high Km and the associated form exhibits a low Km. In contrast, a high enzyme concentration gave a linear kinetic plot for cGMP phosphodiesterase, while a low enzyme concentration gave a nonlinear plot...

Glutaraldehyde fixed-antigen sensitized stable cells in indirect hemagglutination test for rapid diagnosis of hydatid disease, trichinosis and amoebiasis

Glutaraldehyde fixed and antigen sensitized sheep red cells (SRBC) refrigerated or lyophilized in phosphate buffered saline, pH 7·2 (PBS) or in PBS containing 3% normal rabbit serum (PBS-NRS) were stable for more than 6 months in IHA test against sera from patients with hydatid disease, trichinoisis and amoebiasis. The final concentration of protein in hydatid, trichinella and amoebic antigens used for sensitization of SRBC was 0·43, 0·75 and 1·2 mg/ml, respectively. The lyophilized cells in general showed a slightly higher sensitivity but within acceptable limits. By contrast the sensitized cells refrigerated in PBS consistently showed a uniform reactivity and IHA titres were reproducible even 6 months after sensitization of the cells. No demonstrable change was observed in the sensitivity of the test when the PBS-refrigerated cells sensitized 6 months before were exposed to room temperature for 4 days. The following are the main improvements on existing serological tests: (a) a more rapid and reproducible test for sero-epidemiological investigations in areas with minimum laboratory facilities, (b) sensitized cells could be shipped to various laboratories without losing the sensitivity of the test, (c) reduction in the cost of personnel and reagents where these tests are needed infrequently.

Purification, Properties, and Synthesis of Dihydrofolate Reductase from Wild Type and Methotrexate-resistant Hamster Cells

Abstract Dihydrofolate reductase from a methotrexate-resistant hamster cell culture was purified to apparent homogeneity by affinity chromatography. A similar procedure was used to prepare enzyme from wild type cells, although the final enzyme protein concentration was too small to be measured. The purified enzymes were not significantly different with regard to substrate specificity, Km values for folate and NADPH, sedimentation coefficient, electrophoretic mobilities at pH values 8.3 and 7.0, turnover number per methotrexate-binding site, and degree of inactivation by antiserum. The rate of radioactive leucine incorporation into the reductase in wild type and resistant cells was compared by specific immunoprecipitation. By this method resistant cells showed a 20-fold increase in the rate of enzyme synthesis. These results indicate that methotrexate-resistant cells accumulate large quantities of a very similar, if not identical, reductase, due at least in part to an increased rate of enzyme synthesis.

Utilization of cellulose from waste paper by Myrothecium verrucaria

AbstractExtensive screening studies on cellulolytic bacteria and fungi led to the selection of Myrothecium verrucaria as the organism producing the maximum rate of protein biosynthesis from ball‐milled newspaper. Studies in aerated stirred‐jar fermentors were carried out to determine the conditions for maximum protein synthesis rate and maximum final protein concentration. The optimum aeration rate was 250 to 374 mM of oxygen at 300 to 400 rpm stirring rate. The pH optimum was broad, from 3.9 to 6.5. Urea at 0.03% and yeast autolysate at 0.1% stimulated growth rate and protein production. The maximum rate of protein biosynthesis and the maximum protein yield were 0.3 g/liter/day and 1.42 g/liter, respectively, from medium G3 with 4% ball‐milled newspaper. The final product, obtained by evaporation of the total culture, was 33.7 g from one liter of medium which originally contained 40 g of ball‐milled newspaper and 11.3 g of other dissolved materials. The protein content of this final product was 3.3 g, calculated from total organic N × 6.25 or 1.42 g calculated from the biuret method. Both the synthesis rate and the final cell yield are below those obtainable by growing Fungi Imperfecti, yeasts or bacteria on soluble materials such as glucose.

Differences in Golonic Staining with Technical Variations in Fluorescent Antibody Conjugation.

SummarySeveral different parameters involved in preparing direct fluorescent antibody stains were studied using autologous and homologous ulcerative colitis and normal colonic tissues. Diffuse nonimmunospecific staining was observed when conjugates were labeled unevenly, had high fluorescein: protein (F:P) ratios or when final protein concentrations exceeded 1.5 mg/ml. Some apparently nonimmunospecific staining of the mucosal epithelial glands was observed, but there was no specific fluorescence at these sites. Using an evenly labeled stain with an F:P ratio of 3.70 × 10-3 and final protein concentrations of either 0.50 or 1.50 mg/ml, specific fluorescence of the cytoplasm of some mononuclear cells was observed in autologous and homologous ulcerative colitis tissues, but not in normal colonic specimens.