Immunoassay for bovine serum thymopoietin: discrimination from splenin by monoclonal antibodies

A polyclonal rabbit anti-bovine thymopoietin antiserum was used to develop a radioimmunoassay and sandwich enzyme-linked immunosorbent (ELISA) assay for the thymic hormone thymopoietin. Both assays showed slightly less sensitivity for the closely related splenic hormone splenin (SP) than thymopoietin (TP) and markedly less sensitivity for the human as compared with the bovine polypeptides. A number of murine monoclonal antibodies specific for bovine thymopoietin were generated; they were unreactive with bovine splenin and were also unreactive with human thymopoietin and splenin. A sandwich ELISA using these monoclonal anti-TP antibodies together with polyclonal rabbit anti-TP was specific for bovine thymopoietin and measured 300-500 ng/ml thymopoietin in bovine serum. Similar approaches are being pursued to develop an immunoassay for thymopoietin in human serum.     

Enzyme immunoassay using monoclonal antibodies to study conformational changes of human serum albumin

A method for studying conformational changes induced in the human albumin molecule, either in its purified form or in serum, is described. Plates were coated with albumin or human serum at varying pHs and were reacted with peroxidase-labeled anti-albumin monoclonal antibodies of different specificities. The data showed that albumin molecules were coated in conformations induced as a result of pH changes, allowing us to demonstrate that pH modifications involved the N-terminal portion of the albumin molecule whether in its purified form or in serum. This method should be applicable to the study of conformational modifications in other proteins as well.     

Site of conjugation of bovine serum albumin to corticosteroid hormones and specificity of antibodies

The influence of the site of attachment of bovine serum albumin (BSA) to corticosteroids on the specificity of the antisera obtained in rabbits was investigated. The steroids and positions studied were cortisol and 11-desoxycortisol at C-3, C-6α, C-6β and C-21 and 21-desoxycortisol and C-21-desoxycortisone at C-6α and C-6β. None of the antisera to cortisol showed high specificity. Similar cross reactions with antisera derived from cortisol coupled at C-6β, C-3 and C-21 to BSA were observed. 11-Desoxycortisol coupled at C-6α to BSA yielded the most specific antisera to this adrenal hormone. Cross reactions of antisera derived from coupling the protein to the extreme ends (C-3 and C-21) of 11-desoxycortisol were similar. The orientation of the conjugate at C-6 in 21-desoxycortisol and in 21-desoxycortisone did not influence the relative specificity of the antisera derived from the epimers. Highly specific antibodies were obtained against 21-desoxy-cortisone. Except tor 15% cross reaction with 17-hydroxyprogesterone, the antibodies to 21-desoxycortisol were relatively specific. It was concluded that the site on the steroid molecule to which BSA is attached influences the specificity of the antisera produced but there are also other factors operative.     

Immunoregulation of the anti-bovine serum albumin response by polyclonal and monoclonal antibodies

Monoclonal or polyclonal antibodies directed toward determinants on limited structures of bovine serum albumin (BSA) (P505-582) were shown to regulate the entire anti-bovine serum albumin (BSA) immune response when passively administered to mice 24 hr prior to immunization. Regulation was observed as suppression of the humoral IgG immune response toward all BSA determinants except those on fragment P505-582. By Day 21 suppression of humoral response was most pronounced toward determinants present on the carboxy terminal end of the molecule (N 307-582). These observations demonstrate that monoclonal antibodies directed against a single determinant on a protein molecule have the capacity to regulate the immune response to a multiplicity of determinants present on the same protein. The data lend support to concepts of antibody-induced regulation by induction of suppressor cells or idiotype recognition.     

Thermal features of the bovine serum albumin unfolding by polyethylene glycols.

Albumin showed very poor affinity for polyethylene glycol molecular weight (Mw) 1000 (30 M(-1)) and Mw 8000 (400 M(-1)) (PEG 1000 and PEG 8000). Polyethylene glycol of low Mw favours the ionization of the tyrosine (TYR) residues of albumin. Such variation might be a consequence of the change in dielectric constant at the domain of the protein by PEG binding. PEGs of high Mws stabilize the native compact state of human albumin showing negative preferential interaction with the protein. Interaction between PEGs and albumin is thermodynamically unfavourable, and becomes even more unfavourable for denatured proteins whose surface areas are larger than those of native ones leading to a stabilization of the unfolded state, which is manifested as a lowering of the thermal transition temperature. PEG 8000 perturbs the structure of the protein surface, partially modifying the layer of water and the microenvironment of the superficial aromatic residues (tryptophan, TRP and TYR) which is in agreement with the modifications of the UV spectrum of albumin by PEG 8000 and circular dichroism (CD) spectrum at high temperatures.     

聚乙二醇修饰牛血清白蛋白的反应与分析

采用N,N′-羰基二咪唑活化法活化单甲氧基聚乙二醇5000分子一端的羟基,对活化后的单甲氧基聚乙二醇分子进行了元素分析。用该活化产物对牛血清白蛋白的赖氨酸侧链氨基进行化学修饰。应用毛细管电泳对聚乙二醇修饰后的产物进行了分析,并与高效液相色谱分析结果作了对照研究,表明毛细管电泳对修饰后的牛血清白蛋白有更好的分析效果。     

Fractionation of bovine serum albumin and monoclonal antibody alemtuzumab using carrier phase ultrafiltration

Protein transmission and hence selectivity of separation can be significantly affected by solution pH and ionic strength in protein fractionation using ultrafiltration. Using parameter scanning ultrafiltration, the transmission of bovine serum albumin (BSA) and monoclonal antibody alemtuzumab (Campath-1H) through 300 kDa polyethersulfone (PES) ultrafiltration membranes were studied over a range of pH and salt concentrations, with focus on the likely conditions for achieving "reverse selectivity," i.e., obtaining purified alemtuzumab (approximately 155 kDa) in the permeate. Experimental results demonstrate that the reverse selectivity could be obtained by manipulating the operating conditions such as the solution pH, ionic strength, permeate flux, and system hydrodynamics. With a two-stage batch ultrafiltration process under suitable conditions, the monoclonal antibody alemtuzumab with a purity of > 98% was obtained in the permeate from a feed solution initially containing 0.50 g/l each of BSA and alemtuzumab. Further purity can be expected by selecting more suitable membranes and optimizing operating conditions.     

Binding of anandamide to bovine serum albumin

The endocannabinoid anandamide is of lipid nature and may thus bind to albumin in the vascular system, as do fatty acids. The knowledge of the free water-phase concentration of anandamide is essential for the investigations of its transfer from the binding protein to cellular membranes, because a water-phase shuttle of monomers mediates such transfers. We have used our method based upon the use of albumin-filled red cell ghosts as a dispersed biological "reference binder" to measure the water-phase concentrations of anandamide. These concentrations were measured in buffer (pH 7.3) in equilibrium with anandamide bound to BSA inside resealed human red cell membranes at low molar ratios below one. Data were obtained at 0 degrees C, 10 degrees C, 23 degrees C, and 37 degrees C. The equilibrium dissociation constant (Kd) increases with temperature from 6.87 +/- 0.53 nM at 0 degrees C to 54.92 +/- 1.91 nM at 37 degrees C. Regression analyses of the data suggest that BSA has one high-affinity binding site for anandamide at all four temperatures. The free energy of anandamide binding (DeltaG0) is calculated to -43.05 kJ mol-1 with a large enthalpy (DeltaH0) contribution of -42.09 kJ mol-1. Anandamide has vasodilator activity, and the binding to albumin may mediate its transport in aqueous compartments.     

Binding of isofraxidin to bovine serum albumin

The binding of isofraxidin to bovine serum albumin (BSA) was studied under physiological conditions with BSA concentration of 1.5 x 10(-6) mol x L(-1) and drug concentration in the range of 1.67 x 10(-6) mol x L(-1) to 2.0 x 10(-5) mol x L(-1). Fluorescence quenching spectra in combination with uv absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and CD spectroscopy was used to determine the drug-binding mode, binding constant, and the protein structure changes in the presence of isofraxidin in aqueous solution. The linearity of Scatchard plot indicates that isofraxidin binds to a single class of binding sites on BSA and the values given for the binding constants agree very closely with those obtained by the modified Stern-Volmer equation. The thermodynamic parameters, enthalpy change (DeltaH) and entropy change (DeltaS), were calculated to be -17.63 kJ x mol(-1) and 51.38 J x mol(-1) x K(-1) according to the van't Hoff equation, which indicated that hydrophobic interaction played a main role in the binding of isofraxidin to BSA.     

Production of monoclonal antibodies to dehydroepiandrosterone-sulphate after immunization of mouse with dehydroepiandrosterone-bovine serum albumin conjugate

Monoclonal antibodies with a much higher specificity for DHA-S than for DHA were obtained from a BALB/c mouse immunized with a non-sulphated DHA-7CMO-BSA antigen. An improved fusion technique using PEG containing 10% DMSO instead of PEG alone increased the number of positive hybridomas. One of the five monoclonal antibodies obtained, showed a high affinity for DHA-S (Ka = 10(10) M-1) and very low cross-reactions with androsterone (0.62%) and androsterone sulphate (0.83%) which made it potentially useful for direct quantitation of DHA-S in human serum.     

抗牛血清IgG单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清IgG免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,用含山羊血清的培养基培养细胞,上清用间接ELISA法筛选。获得4株能稳定分泌抗牛血清IgG的单克隆抗体杂交瘤细胞株,分别命名为1G5、2A8、3F5、4C5。其中2A8为IgG2a,其余3株为IgG1;腹水单抗的ELISA滴度均超过10-5;除3F5株单抗与山羊血清有交叉反应外,1G5、2A8、4C5株与人、马、猪、羊、兔、豚鼠等血清均不发生交叉反应;4株单抗与制备病毒性疫苗的基质液呈阴性反应;4株单抗识别分子量为160kD的牛血清IgG的两个不同抗原表位;4株单抗相对亲和力大小依次为4C5>2A8>1G5>3F5,相对敏感度依次为2A8>4C5>3F5>1G5;4株杂交瘤细胞株的染色体计数均大于90条,连续培养三个月以及冷冻保存半年后复苏,细胞生长良好。使用这些单抗建立的双抗体夹心法检测生物制品中的残留牛血清IgG。     

ATP binding to bovine serum albumin.

Specific binding of ATP to bovine serum albumin (BSA) is demonstrated employing ATP derivatives spin-labeled at either N6 or C8 of adenine ring or at the ribose moiety. Based on a 1:1 stoichiometry binding constants are in the 50-100 microM range. Binding is largely competitive with ATP or stearic acid. A small fraction of the labeled nucleotides could not be liberated by these ligands. Binding of AMP is in the millimolar range, only.     

Isolation and characterization of sets of anti-l-fucose and anti-bovine serum albumin antibodies directed against a glycoconjugate ofl-fucose and bovine serum albumin

A set of anti-carbohydrate antibodies and a set of anti-protein antibodies were isolated from the serum of rabbits immunized with a glycoconjugate ofl-fucose and bovine serum albumin. The sets were separated by affinity chromatography by a two-column method on adsorbents withl-fucose or bovine serum albumin ligands. Isoelectrofocusing results showed that the anti-carbohydrate antibodies consisted of 11 molecular species and the anti-bovine serum albumin antibodies consisted of seven molecular species. The anti-carbohydrate antibodies are all of the IgG type while the anti-protein antibodies contain three types of globulin molecules, IgA, IgG, and IgM. The former antibodies should be useful as markers for unique glycoproteins of diseased cells and the latter antibodies may be useful for investigating the mechanism of simultaneous synthesis of three types of immunoglobulins.     

Electrospinning of bovine serum albumin. Optimization and the use for production of biosensors

Electrospinning of the globular protein, bovine serum albumin (BSA), was optimized to obtain proteinous fibers suitable as biosensors. It was shown that the as-spun protein preserves its native form, whereas solubility of the cross-linked in the ambient conditions BSA nanofibers evidently decreases. Insoluble BSA fibers can be easily modified to be used as two-dimensional biosensors. Here, we show the micro pH sensor obtained from the BSA fiber stained with a fluorescein derivative (FITC).     

Binding of berberine to bovine serum albumin: spectroscopic approach

Fluorescence spectroscopy in combination with UV–vis absorption spectroscopy was employed to investigate the binding of an important traditional medicinal herb berberine to bovine serum albumin (BSA) under the physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of BSA by berberine is a result of the formation of berberine–BSA complex. Fluorescence quenching constants were determined using the Stern–Volmer equation and Scatchard equation to provide a measure of the binding affinity between berberine and BSA. The results of thermodynamic parameters ΔG, ΔH, ΔS at different temperatures indicate that the electrostatic interactions play a major role for berberine–BSA association. Site marker competitive experiments indicated that the binding of berberine to BSA primarily took place in site II. Furthermore, the Effect of supramolecules to berberine–BSA system, and the distance r between donor (BSA) and acceptor (berberine) was obtained according to fluorescence resonance energy transfer (FRET).