Showing papers on "Bovine serum albumin published in 1971"

The carrier effect in the secondary response to hapten-protein conjugates. I. Measurement of the effect with transferred cells and objections to the local environment hypothesis

Abstract: A carrier effect is obtained typically when a hapten-protein conjugate is injected into an animal which has previously been primed with the same hapten conjugated to another carrier protein. Under these circumstances the anti-hapten secondary response is usually less than that which would have been obtained had the animal been injected with a conjugate prepared with the same carrier as that originally used for priming. Attempts have been made to account for the phenomenon in terms of the local environment hypothesis, which assumes that the receptor on immunologically competent cells recognises the hapten jointly with the area on the complete antigen which surrounds it. Alternatively the phenomenon can be accounted for by the hypothesis of cooperation, which assumes that the antigen is recognised by two receptors, one directed to the hapten and the other to a determinant on the carrier protein. Methods are described which enable carrier effects to be studied quantitatively in mice. They involve a cell transfer system in which cell suspensions prepared from large numbers of donors are distributed among irradiated syngeneic recipients. In these recipients the transferred cells can be made to perform a secondary response by appropriate antigenic stimulation. The response is monitored by binding tests in which the capacity of serum to bind highly radioactive haptens or proteins is measured. The haptens employed in this system are NIP (4-hydroxy-5-iodo-3-nitro-phenacetyl-) and DNP (2,4-dinitrophenyl-) and the proteins comprise chicken γ-globulin, bovine serum albumin, human serum albumin, ovalbumin, bovine γ-globulin, keyhole limpet hemocyanin and mouse γ-globulin. A carrier effect was regularly obtained when the proteins were tested against one another as carriers, for priming and for the secondary response. The effect could best be measured by comparing the relative potencies in the secondary response of the homologous conjugate (i. e. one with the carrier which had originally been used for priming) with heterologous conjugates (i. e. ones with new carriers). In this way the intrinsic potency of the individual protein could also be measured and allowance made for it in calculating the magnitude of the carrier effect. An average carrier effect of one thousand-fold relative potency was obtained. Priming by NIP-ovalbumin or NIP-chicken γ-globulin with secondary stimulation by NIP-bovine serum albumin (or the corresponding DNP conjugates) could be identified as the combination best suited to further study. Support for the cooperation hypothesis, particularly for that version of the hypothesis which postulates that recognition of carrier determinants allows an antigen-concentrating mechanism to operate, could be found in the parallel slopes of the dose-response curves obtained with homologous and heterologous conjugates. On the other hand the local environment hypothesis failed to pass either of the tests to which it was subjected. One, the weaker, was to compare haptens with and without spacer groups inserted between themselves and the carrier protein, in the expectation that spacers might reduce the local environment contribution: no difference could in fact be detected. The other, the stronger, was to attempt to inhibit the response with an excess of carrier protein even though the anti-hapten antibody had no detectable affinity for the carrier: such inhibition could regularly be obtained.

Radioimmunoassay of the f prostaglandins.

Abstract: Antibodies to prostaglandin F2α (PGF2α) were produced in rabbits immunized with a conjugate of PGF2α covalently linked to bovine serum albumin (BSA) by reaction with carbodiimide reagent. A radioimmunoassay was developed using dextran-coated charcoal to separate the free from antibody-bound PGF2α-3H. The sensitivity of the method was about 100 pg. Butanol was used for extraction of plasma and the various classes of prostaglandins were separated on a small silicic acid column. Recovery of PGF2α-3H through the extraction and separation procedures was 75–85%. Equilibration of PGF1α, PGE2, PGE1 PGA2 and PGB1, with antiserum and PGF2α-3H showed a progressive loss of ability to displace the labeled prostaglandin from the antibodies related to the degree of differences in molecular configuration from the hapten used in the immunization. The method should be sufficiently sensitive, precise, and simple to allow its use in the routine estimation of the F prostaglandins from biological samples.

Model experiments on the molecular mechanism of action of 6-hydroxydopamine.

Abstract: 6-Hydroxydopamine is rapidly oxidized to its p -quinone and indoline derivatives at pH 7.4, as shown by ultraviolet spectroscopy. Incubation of bovine serum albumin (0.5 mM) with 3 H-6-hydroxydopamine resulted in covalent binding (nonextractable with alcoholic perchloric acid) of radioactivity to time protein. The amount of radioactivity bound was concentration-dependent. At pH 7.4 and 38°, saturation was reached at a concentration of 70 mM 3 H-6-hydroxydopamine. The radioactivity bound at this concentration corresponds to the equivalent of 11 moles of 3 H-6-hydroxydopamine per mole of albumin. If oxidation of 3 H-6-hydroxydopamine was prevented by Na 2 S 2 O 5 , the binding of radioactivity to bovine albumin was virtually completely abolished. Acetylation of albumin reduced the bound radioactivity to 19% of that of controls, whereas heat denaturation reduced it to only 75%, indicating that not denaturation as such, but the blockade of nucleophilic groups, is the main cause for the reduced binding of radioactivity. The results of the present experiments are compatible with the hypothesis put forward recently [H. Thoenen, J. P. Tranzer and G. Hausler, in "New Aspects of Storage and Release Mechanisms of Catecholamines" (H. J. Schumann and G. Kroneberg, eds.), p. 130. Springer-Verlag, Berlin, 1970] that the selective destruction of adrenergic nerve terminals by 6-hydroxydopamine results from the covalent binding of its oxidation products to nucleophilic groups of biological macromolecules. The reaction seems to be nonspecific, and the high gelectivity of the destructive effect results from the efficient uptake of 6-hydroxydopamine into the adrenergic nerve terminals.

pK change of imidazole groups in bovine serum albumin due to the conformational change at neutral pH.

Abstract: a b s t r a c t : The pH-dependent change in conformation o f bovine serum albumin, located between pH 6 and 9 (neutral transition), was studied by means o f optical rotation measurements at 313 nm and hydrogen ion titration experiments, both in the presence o f KC1 or CaClj. The optical rotatory dispersion measurements revealed that with CaCl2 the transition proceeds at lower pH values and within a smaller pH range than without calcium. From an analysis o f the titration curves, combined with the observed influence o f calcium ions on the neutral transition, it could be concluded that the transition causes a p K shift o f certain groups, probably imidazole groups, since as a consequence o f the pK shift protons are released in the neutral region. That these groups are indeed imidazole groups was further confirmed by measuring the apparent heat o f proton dissociation. The highest pK was found in the low pH conformation. This suggests that in this conformation several histidyl residues are involved in salt bridges. The effect o f calcium on the neutral transition indicates that the affinity o f albumin for protons decreases upon calcium binding. The relation between calcium and proton binding to albumin shows much resemblance with the Bohr effect o f hemoglobin, i.e., the relation between oxygen and proton binding.

Effect of free fatty acids on binding of drugs by bovine serum albumin, by human serum albumin and by rabbit serum

Abstract: Do plasma free fatty acids inhibit the binding of other ligands by serum albumin, and if so, at what serum FFA concentration does this effect become manifest? To investigate these questions bovine serum albumin (BSA) and human serum albumin (HSA) were prepared with either 7, 3.5 or l0.05 mol of palmitate or oleate per mol of protein. These preparations correspond to serum free fatty acids (FFA) concentrations of 4000, 2000 and l30 µEq/l. By the dialysis-equilibrium technique, the capacity of these albumin preparations to bind some or all of these ligands was measured: Salicylate, octanoate, bromsulfophthalein (BSP), phenylbutasone, sulfadiasine, thiopental, bishydroxycoumarin and diphenyihydantoin. Analysis of SCatchard plots shows that Salicylate, octanoate, BSP and phenylbutazone occupied three classes of binding site in BSA, whereas for sulfadiasine, bisbydroxycoumarin, thiopental and diphenylhydantoin only one type of binding site was detected. The binding of all eight ligands to BSA was inhibited by palmitate at a molar ratio of 7. The inhibition affected either the number of binding sites in a particular class available to the ligand, or the association constant for the interaction of the site with the ligand or both. Binding to salicylate, octanoate, sulfadiasine, thiopental and diphenylhydantoin was markedly reduced, with 50% or more reduction in number of clasa 1 sites available, whereas binding of BSP, phenylbutasone and bishydroxycoumarin was less inhibited, the number of available class 1 sites being preserved. The inhibitory effect of oleate at a molar ratio of 7 on the binding properties of BSA for salicylate, octanoate and diphenylhydantoin was closely similar to that of 7 mol of palmitate. Palmitate at a molar ratio of 3.5 inhibited the binding of salicylate and octanoate to BSA, but the effect was only a small fraction of that observed with 7 mol of palmitate; 3.5 mol of plamitate did not inhibit binding of diphenylhydantoin. The binding of salicylate, octanoate and diphenylhydantoin to HSA containing 7, 3.5 or l0.05 mol of FFA was qualitatively similar to that observed with the corresponding series of BSA preparations. The binding of salicylate, octanoate and diphenylhydantoin by rabbit sera with FFA concentrations ranging between 300 and 3000 and 3000 µEq/l was also measured; these sara were obtained by injecting the animals with graded doses of the lipolytic hormone adrenocorticotropin. Below the concentration 1700 µEq/l, FFA did not inhibit binding capacity of the serum detestably. concentrations above 2200 µEq/l were consistently associated with a 20 to 26% reduction in the binding of all three ligands. These experiments suggest that serum FFA in the concentration range of 2000 to 4000 µEq/l have a general inhibitory effect on the capacity of serum albumin to bind many ligands of widely different structure, but that at concentrations below 2000 µEq/l this effect is minor or absent.

Receptors on immunocompetent cells : ii. specificity and nature of receptors on dinitrophenylated guinea pig albumin-125i-binding lymphocytes of normal guinea pigs

Abstract: Nonimmunized guinea pigs possess rare lymphocytes which bind sufficient 2,4-dinitrophenyl-guinea pig albumin-125I (DNP-GPA) to their surface to be detected by short-term radioautography. The cells occur in the lymph nodes, spleen, peripheral blood, and bone marrow with a frequency of ∼40/100,000 lymphocytes, but are absent from the thymus. The receptors of these cells are largely specific for the haptenic group (ϵ-DNP-L-lysine) as shown by inhibition of DNP-GPA-125I binding with ϵ-DNP-L-lysine and with DNP bovine serum albumin (DNP-BSA). Furthermore, these cells specifically adsorb to agarose beads to which either DNP-GPA, DNP-BSA, or DNP-keyhole limpet hemocyanin (KLH) has been covalently linked. This hapten specific depletion of DNP-GPA-125I antigen-binding cells (ABC) correlates with a similar diminution in the capacity of adsorbed populations to transfer primary responsiveness to DNP-KLH to irradiated syngeneic recipients. Fluoresceinated anti-immunoglobulin binds to the surface of some guinea pig lymphocytes, and all DNP-GPA-125I ABC, as shown by a double-label technique. The great majority of DNP-GPA ABC and human γ-globulin ABC possess surface Ig molecules of the γ2 heavy chain class. Preincubation of cell suspensions with anti-γ2 antibody markedly diminishes the number of DNP-GPA-125I ABC which are detected, strongly suggesting that the receptors of these cells are immunoglobulin molecules, most of which possess γ2 heavy chains. The specificity characteristics of DNP-GPA-125I ABC are strikingly different from those of cells mediating a cellular immune response to DNP-GPA, indicating major differences in the specificity and nature of the receptors of these cell types.

Isolation of Mycoplasma arginini from commercial bovine sera and its implication in contaminated cell cultures.

Abstract: SummaryMycoplasmas were isolated from 73 of 379 (19%) commercial bovine serum lots examined, including 55 of 239 (23%) unprocessed fetal bovine and calf serum sublots received from 4 suppliers and 18 of 140 (13%) final lots obtained from 2 of 5 manufacturers. Of 31 mycoplasma strains examined for identity, 28 were identified as M. arginini and 3 strains were unrelated to 39 well established Mycoplasma species and serotypes and may represent new species. Mycoplasma arginini was isolated from 10 uninoculated cell cultures and one virus pool also. Data presented indicate that M. arginini is a common contaminant of commercial bovine sera and of cell cultures and that bovine serum is a major source of bovine mycoplasma contamination of cell cultures.Addendum. Since Acholeplasma laidlawii is a common cell culture contaminant (1) of bovine origin, attempts to isolate this agent from commercial bovine serum were continued. The culture procedure was modified as follows: the horse serum component was deleted from t...

Antibodies to Testosterone-3-Bovine Serum Albumin, Applied to Assay of Serum 17β-ol Androgens

Abstract: Testosterone was conjugated to bovine serum albumin at carbon 3, and antibodies to this conjugate were developed in virgin female rabbits. This antiserum, diluted 1:3200, was used to measure testosterone. With tritiated testosterone and anti-rabbit γ-globulin as the second antibody, a doseresponse curve was obtained for doses in the range 0.1-10 ng. The antibody cross-reacts extensively with dihydrotestosterone, so that dihydrotestosterone is measured with testosterone in extracted but unchromatographed serum. Results for pooled serum from men, as measured repeatedly by radioimmunoassay and competitive binding assay for testosterone, agreed well. Seventeen normal men had serum 17β-ol androgen concentrations of 667 ng/100 ml (SD, ±248); seventeen normal, menstruating women had a mean concentration of 69 ng/100 ml (SD, ±18). Values for 17β-ol androgens in adolescent males agreed reasonably with testosterone values reported by other investigators. Values are presented for patients with various androgenic disorders. This procedure is easy; many samples can be processed at one time.

Studies on "nonspecific" binding.

Abstract: The binding of flourescein to bovine serum albumin was studied by equilibrium dialysis under various conditions. At pH 8.0 and 5°C there are three binding sites with association constants of 2.8 × 104 M−1. Specific labelling of the binding sites using fluorescein-isothiocyanate showed that two of the binding sites are immediately adjacent. The labelled peptides were isolated and the sequence around one of the binding regions was determined revealing the presence of three lysines in this eight amino acid sequence. The binding is mainly casused by hydrophobic interactions and to lesser extent by electrostatic forces. The electrostatic contribution is much less than expected compared with other substances which are bound to bovine serum albumin. This is probably related to the fact that fluorescein is a nonphysiological compound and is “nonspecifically” bound to bovine serum albumin. Studies on binding properties of various peptic fragments of bovine serum albumin were performed. They showed that even slight fragmentation caused a drastic decrease in the ability to bind fluorescein.

Deficient IgA Antibody Responses to Arsanilic Acid Bovine Serum Albumin (BSA) in Neonatally Thymectomized Rabbits

Abstract: The anti-hapten antibody response to intravenously injected arsanilic acid-bovine serum albumin was studied in neonatally thymectomized, 8-week-old rabbits using the Cla fixation and transfer test. This test allows quantitation of antibody in individual immunoglobulin classes. The IgG antibody response of thymectomized rabbits was not significantly different from the response of intact rabbits and the IgM antibody response of thymectomized rabbits was only slightly diminished. In contrast, the IgA antibody response of thymectomized rabbits was absent or strikingly diminished. This depressed IgA antibody response was also observed when animals were stimulated with a dose of antigen 10-fold greater than the original dose. It is proposed that for at least certain antigens, serum IgA antibody responses are more dependent on the thymus-derived cell system than are antibody responses in other immunoglobulin classes.

Microtiter tests for detecting antibody in bovine serum to parainfluenza 3 virus, infectious bovine rhinotracheitis virus, and bovine virus diarrhea virus.

Abstract: Microneutralization tests for detection of antibody in bovine serum to three bovine viruses are described. The Madin-Darby bovine kidney cell line was used with parainfluenza 3 virus (PI 3), whereas serially cultivated bovine embryonic kidney cells were used for infectious bovine rhinotracheitis virus and bovine virus diarrhea virus. Comparison of micro-hemagglutination-inhibition (HI) with micro-serum-neutralization (SN) tests for PI 3 showed the SN test to be more sensitive, more specific, and therefore more useful than the HI test for detecting antibody. Although the effect of trypsin-periodate treatment of serum was to reduce the HI titer of numerous sera by a twofold dilution, sufficient evidence could not be found to indicate that nonspecific HI inhibitors to PI 3 are present in bovine sera.