Separation of antigens by immunological specificity: Use of disulphide-linked antibodies as immunosorbents

1. γ-Globulin concentrates of antisera prepared against ovalbumin and human serum albumin were thiolated and cross-linked to form insoluble polymers. 2. These immunosorbents were of low solubility and of high capacity for homologous antigen. 3. The high specificity of these immunosorbents was demonstrated by fractionation of various binary mixtures of fluorescent ovalbumin, ¹³¹I-labelled human serum albumin, lysozyme and ribonuclease.     

Separation of antigens by immunological specificity. Studies on the desorption of homologous antigen from disulphide-linked antibody immunosorbents

1. Glycine-hydrochloric acid buffer, pH2.2, desorbed (131)I-labelled human serum albumin (100%), lysozyme (100%), ovalbumin (90%), fluorescent ovalbumin (50-60%) and fluorescent human gamma-globulin (20%) from their respective homologous disulphide-linked antibody immunosorbents; reasons are suggested for the low recoveries of the fluorescently labelled proteins. 2. Approx. 40% of the recovered (131)I-labelled human serum albumin and fluorescent ovalbumin was desorbed above pH6.0, but lysozyme was not eluted until the pH was 3.0 or below. 3. In all cases where high recoveries of antigen were obtained, the immunosorbents could be regenerated and recycled at least four times with full retention of specificity and minimal diminution of capacity. 4. The desorbed antigens were unchanged when compared with the original antigens by quantitative precipitin, specificradioactivity, fluorescent and enzymic analyses and by cellulose acetate electrophoresis. 5. Desorption of antigen with a variety of reagents was investigated. These reagents were less satisfactory than glycine-hydrochloric acid.     

Factors affecting production of contrasensitizing antisera in mice.

Using four different protein antigens, two different strains of mice, and various immunization protocols, we have studied production in mice of immunological enhancement antibodies that specifically suppress induction of delayed hypersensitivity. Primary assay of these antibodies was in vivo, because no in vitro test used detected them dependably. Any antigen priming that favored initiation of humoral antibody responses prepared mice to make these contrasensitizing antibodies vigorously following appropriate boosting. The method of boosting usually was more important than that of priming, high titers regularly developing only when primed mice were boosted with much antigen in a short time and were bled a few days later. The presence or absence of delayed hypersensitivity was immaterial. CAF₁ mice made these antibodies better than CF-1 mice, and antigen effectiveness correlated with propensity to induce humoral antibody formation in mice, decreasing from ovalbumin through human serum albumin and bovine serum albumin to methylated human serum albumin. In certain antigenmouse combinations (e.g., ovalbumin in CAF₁ mice) immunosuppressive antibody production was vigorous and prolonged; in others (e.g., bovine serum albumin in CF-1 mice) it was moderate and brief. From our results one can predict what conditions should induce formation of strongly enhancing/contrasensitizing antisera, and speculate that these conditions also should elicit strong, active immunologic tolerance for averting induction of delayed hypersensitivity.     

Uptake and degdradation of formaldehyde-treated 125I-labeled human serum albumin in rat liver cells in vivo and in vitro

The uptake of formaldehyde-treated ¹²⁵I-labelled human serum albumin in rat hepatocytes and nonparenchymal liver cells was measured in vivo and in vitro. Isolated liver cells were prepared by treating the perfused liver with collagenase. Purified hepatocytes and nonparenchymal cells were obtained by differential centrifugation. Human serum albumin was found to be taken up exclusively or almost exclusively by nonparenchymal cells in vitro and in vivo (after intravenous injection). The maximal rate of human serum albumin-uptake in vitro was comparable to that in vivo. Nonparenchymal cells degraded human serum albumin in vitro as indicated by release of trichloroacetic acid-soluble radioactivity. Degradation started about 20–30 min after addition of human serum albumin to cells and rate of degradation was proportional to rate of uptake. Human serum albumin-degradation could be studied without interference of concurrent uptake by separating cells that had been preincubated with human serum albumin from the medium and then reincubating them with human serum albumin-free medium. The lag phase before human serum albumin-degradation starts and the inhibitory effect of chloroquine on degradation indicate that human serum albumin is degraded in lysosomes. The data obtained show that enzymatically prepared nonparenchymal liver cells retain their endocytic activity in vitro. Denatured human serum albumin should be useful both as a marker for rat liver macrophages and for the study of intracellular proteolysis in these cells.     

Activation of delta5-3-ketosteroid isomerase of bovine adrenal microsomes by serum albumins.

The Δ⁵-3-ketosteroid isomerase (EC 5.3.3.1) of bovine adrenal microsomes is activated as much as 10- to 20-fold by micromolar concentrations of bovine serum albumin. Comparable activations are observed with the serum albumins of 10 other mammalian species, but are not seen with ovalbumin or conalbumin. Evidence that the activation is attributable to the serum albumins, rather than to a small, firmly-bound ligand, is based on: (1) Failure to remove the stimulatory activity from the albumin by chloroform extraction, dialysis, or gel filtration; (2) Destruction of the activity by heating or by trypsin digestion; (3) Precipitation of the stimulatory activity of bovine serum albumin by specific antibody. Bovine serum albumin induces small decreases in the Michaelis constant for Δ⁵-androstene-3,17-dione, but most of the activational effect reflects an increase in the maximum velocity. Low concentrations of Triton X-100, which are without effect on the isomerase activity, prevent the activation by bovine serum albumin.     

Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation

The nature of cholesterol-binding proteins acting upon human spermatozoa during in vitro capacitation was determined by measuring the efflux of [³H]cholesterol and of [³H]cholesteryl sulfate from labeled spermatozoa. Efflux of [³H]sterols was stimulated when the labeled gametes were incubated in Ham's F-10 medium supplemented with female serum or follicular fluid. Upon centrifugation of capacitated spermatozoa and application of the supernatant to density-gradient ultracentrifugation for lipoprotein analysis, both [³H]cholesterol and [³H]cholesteryl sulfate were found to be carried by very-low-density lipoproteins (VLDL), low-density lipoproteins (VLDL), high-density lipoproteins (HDL), as well as the albumin fraction (d > 1.21) in serum. When the capacitation medium was supplemented with follicular fluid, the [³H]sterols were bound to HDL's and to the albumin fraction; when the latter fraction was analysed by molecular sieve chromatography, 60–70% of the radioactivity eluted in fractions with a mean molecular weight corresponding to that of human serum albumin. Sperm cholesterol efflux was also stimulated when serum or follicular fluid was added to a simplified medium (50 mM Tris-HCl, 0.56% NaCl, pH 7.8); efflux of [³H]cholesterol from labeled gametes progressed in a time-dependent manner, but was low in the absence of serum components. The [³H]cholesterol/cholesterol ratios were higher in the albumin and HDL fractions, indicating some degree of specificity of these sterol acceptors. It was observed that follicular fluid albumin has a [³H]sterol binding capacity that is 2—3-fold higher than that of serum albumin. Commercial human serum albumin also promoted sperm cholesterol efflux. These results provide new information concerning those components of follicular fluid which may play a role in human sperm capacitation and provide further support for the concept that loss of cholesterol from the sperm plasma membrane is an important component of the capacitation process.     

The antibody–antigen interaction at an aqueous–solid interface: A study by means of polarized infrared ATR spectroscopy

The determination of structural changes in antibodies due to their specific interaction with antigenic proteins is an important problem in understanding immunological responses. The method of polarized ATR infrared spectroscopy applied to protein films adsorbed on an appropriate solid surface can give information about the conformation of the polypeptide chains, as well as their orientation with respect to the surface. The adsorption of anti-rabbit serum albumin onto monomolecular films of rabbit serum albumin, bovine serum albumin, and ovalbumin, and of anti-ovalbumin onto films of rabbit serum albumin and ovalbumin at a Ge-aqueous interface have been studied by this technique. The intensity of the amide I absorption indicates that the strengths of binding of these three albumin proteins with anti-rabbit serum albumin is, under appropriate conditions, in the order rabbit > bovine ? ovalbumin; with anti-ovalbumin, it is ovalbumin ? rabbit. Since the frequencies of the amide I band appear near 1655 cm^?1 for all the proteins and protein complexes studied, the major contributions to their conformation comes from α-helix and random-coil structures. The average orientation of the transition moments of the amide I and A bands has been shown to be about 75° with respect to the surface normal. This indicates that the polypeptides chains are on the average approximately parallel to the surface for all the systems studied. Consequently, the effect of the specific antibody-antigen interaction on the conformation and orientation of the former seems negligible in these films.     

Tolerance induction by the peroral administration of protein antigens

The possibility of inducing systemic tolerance in animals by feeding them with ovalbumin and human serum was studied on mice, rats and rabbits. Antibodies to ovalbumin, human serum albumin and immunoglobulins (IgG, IgA, IgM) were determined by the passive hemagglutination test in the sera of the test and control animals after the second immunization made through a parenteral route. Tolerance to all the antigens under study was obtained in mice and rats, while in rabbits such feeding was found to produce the priming effect. The degree of tolerance was the greater, the more was the dose of the antigen and the longer was the period of feeding. Different proteins showed varying tolerogenic activity; the same degree of tolerance in mice was obtained by feeding them with IgG in a dose of 0.3-0.5 mg and with ovalbumin or human serum albumin in a dose of 6-12 mg (per gram of body weight). Tolerance was determined on day 3 after the course of feeding was over; in 3 weeks tolerance essentially decreased, and in 1.5-2 months it was replaced by normal reactiveness. Tolerance induced by the oral administration of antigens proved to be immunologically specific.     

Secretion of proteins by the fertilized mouse ovum

Fertilized one-cell mouse ova were injected with messenger RNA (mRNA) for chicken ovalbumin or rat albumin. The ova were labeled with [³H]amino acids, and endogenous proteins as well as proteins in the culture medium were separated by two-dimensional electrophoresis. Following injection of either message, ova were able to synthesize and secrete the appropriate protein. However, when the message for globin was injected, globin appeared only among the endogenous ovum proteins but was not secreted into the culture medium. The location of the secreted proteins, albumin and ovalbumin, on two-dimensional gels suggests that ova process the proteins before secretion by removing the amino-terminal signal sequence from rat albumin and glycosylating ovalbumin. Rat albumin may be exported into the culture medium more rapidly than ovalbumin. In addition, at least one endogenous ovum protein was secreted at the one-cell stage. The studies establish the ability of the newly fertilized mouse ovum to synthesize and secrete proteins from injected as well as endogenous messages.     

Antibodies specific to isoniazid and isonicotinic acid.

Rabbit antisera to isoniazid (INH) and its major metabolite, isonicotinic acid (INA), were prepared by immunization with conjugates of these compounds with human serum albumin. The antisera were rendered hapten-specific by exhaustive absorption with the immunizing carrier. Purified anti-hapten antibodies were also isolated with appropriate immunosorbents. As demonstrated by inhibition of the quantitative precipitin curves and of precipitating immune complexes in immunodiffusion tests, the antibodies to the two haptens reacted with either INH or INA, and also with isonicotinamide (INC); these three related molecules share the isonicotinyl group. The relative effectiveness of inhibition by free hapten of precipitating immune complexes consisting of either anti-INH or anti-INA antibodies and the related hapten-protein conjugates was INH greater than INC greater than INA.     

A general method for isolation of individual polyribosomes and pure mRNAs by immunosorption technique

A highly specific procedure has been developed for the isolation of individual polyribosomes by immunosorbents containing immobilized antigen-antibody complexes. Polyribosomes synthesizing immunoglobulin IgGl and serum albumin were quantificated and isolated in the native state from MOPC 21A plasmacytoma and rat liver cells. For albumin polyribosomes, the presumptive messenger RNA was demonstrated; it contained three components: 20S, 16S and 9S.Abbreviations IgG murine immunoglobulin - RSA rat serum albumin - RGG rat gamma-globulin - EDTA ethylene diamine tetraacetate - SDS sodium dodecyl sulphate     

Development of a new thiol-reactive prosthetic group for site-specific labeling of biomolecules with radioactive iodine

In this report, we describe the radiosynthesis of a new thiol-targeting prosthetic group for efficient radioactive iodine labeling of biomolecules. Radioiodination using the precursor 3 was performed to obtain ¹²⁵I-labeled tetrazole 4b with high radiochemical yield (73%) and radiochemical purity. Using the radiolabeled 4b, a single free cysteine containing peptide and human serum albumin were labeled with ¹²⁵I in modest-to-good radiochemical yields (65–99%) under mildly reactive conditions. A biodistribution study of [¹²⁵I]7 in normal ICR mice exhibited lower thyroid uptake values than those of ¹²⁵I-labeled human serum albumin prepared via a traditional radiolabeling method. Thus, [¹²⁵I]7 could be employed as an effective radiotracer for molecular imaging and biodistribution studies. The results clearly demonstrate that 4b has the potential to be effectively implemented as a prosthetic group in the preparation of radiolabeled biomolecules.     

牛血清蛋白酶联免疫检测试剂盒的研制及验证

为研制酶联免疫试剂盒以检测病毒性疫苗中残余牛血清蛋白(BSP)含量,制备高效价高纯度的兔抗BSP多克隆抗体作为包被抗体和酶标抗体,建立了ELISA双抗体夹心法并组建试剂盒,通过标准剂量曲线可对样品中所含BSP、BSA及B-IgG进行定量,经验证该方法标准曲线线性范围内r≥0.98,对BSP的检测限量为3ng/ml;分别检测5、10、20ng/ml含量的BSP时,试验内(n=12)和试验间(n=3)测定的变异系数在3.71%到7.29%之间,回收率在93.4%~106.3%,未见该方法与人血清白蛋白、卵清蛋白以及疫苗复合保护剂之间有交叉反应。该法敏感度高,准确性、重复性和稳定性好,可用于疫苗牛血清残余蛋白的质量控制。     

人血清白蛋白与金属离子作用的荧光光谱研究

通过金属离子对人血清白蛋白内源荧光的猝灭,探讨了金属离子Cu²⁺,Mn²⁺,Ni²⁺,Co²⁺,Cr³⁺等与人血清白蛋白的结合;基于Förster无辐射能量转移机理,得出了人血清白蛋白第一类Cu²⁺结合部位与214位色氨酸残基间的距离。     

Phosphoinositide turnover in human platelets is stimulated by albumin.

The effect of human serum albumin (HSA) on the hydrolysis of phosphatidylinositides in human platelets labeled with myo(3H)inositol was studied. Incubation of platelets with HSA (4 gm/dl) for 10 seconds increased IP2, and IP3, by 169% and 217% respectively. 93% of IP3 accumulated within the first 10 seconds. This effect was also shared by bovine serum albumin, although no changes in IP3 levels occurred with ovalbumin. All albumin species used induced 45Ca+2 release from platelets irrespective of its effect on IP3 accumulation. These findings indicate that albumin may function in biological systems by inducing intracellular signaling.     

Study on the resonance Rayleigh scattering and resonance non‐linear scattering spectra of ion‐association complexes of [PdI4]2−‐protein systems and their analytical applications

In an acid medium solution, proteins such as bovine serum albumin, human serum albumin, ovalbumin, hemoglobin, lysozyme, γ‐globulin, α‐chymotrypsin and papain could react with [PdI₄]^2? by virtue of electrostatic attraction and hydrophobic force to form ion‐association complexes. As a result, the resonance Rayleigh scattering (RRS) and resonance nonlinear scattering such as second‐order scattering (SOS) and frequency doubling scattering (FDS) intensities were enhanced greatly and new scattering spectra appeared. The maximum scattering peaks of RRS, SOS and FDS were at 367, 720 and 370 nm, respectively. The enhanced RRS, SOS and FDS intensities were directly proportional to the concentrations of proteins. The detection limits for the different proteins were 2.4–11.8 ng/mL for RRS method, 9.5–47.9 ng/mL for SOS method and 4.6–18.5 ng/mL for FDS method. In this work, the influences of the interaction of [PdI₄]^2? with proteins on spectral characteristics of RRS, SOS and FDS were investigated and the optimum conditions were tested. Meanwhile, the effects of coexisting substances were tested and the results showed that the method exhibited a good selectivity. Based on the above research, a highly sensitive, simple and rapid method for the determination of trace amounts of proteins by resonance light scattering technique has been developed. It can be applied to the determination of proteins in tablet, human serum and urine samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of the calcium-dependent interaction of calmodulin with bovine serum albumin

An air-driven ultracentrifuge has been used to investigate the calcium-dependent association between calmodulin and bovine serum albumin. Procedures were described which allowed the interaction to be analyzed to yield the equilibrium constant. At low ionic strength (25 mm Tris-HCl, pH 7.5, pCa 6.68, 9°C) the equilibrium constant for the interaction was estimated to be 2.1 × 10⁴m⁻¹, while at high ionic strength (25 mm Tris-HCl, pH 7.5, 150 mm KCl, pCa 6.68, 9°C) the value was 4.5 × 10³m⁻¹. Under similar conditions, calmodulin was also found to interact with β-lactoglobulin A and gelatin, but no detectable association was observed with ovalbumin.     

Ca-Mg-ATPase activity in brain coated microvesicles purified on immunosorbents

Coated microvesicle fractions isolated from ox forebrain cortex by the ultracentrifugation procedure of Pearse (1) and by the modified, less time consuming method of Keen et al (2) had comparable Ca²⁺+Mg²⁺ dependent ATPase activities (about 9 μmol/h per mg protein). The Na⁺+K⁺+Mg²⁺ dependent ATPase activity was 3.2 μmol/h per mg (±1.0, S.D., n=3) when microvesicles were prepared according to (1) and 1.5 μmol/h per mg (±1.0, S.D., n=3) when prepared according to (2).Oligomycin, ruthenium red, and trifluoperazine, inhibitors of Ca²⁺ transport in mitochondria and erythrocyte membranes had no effect on Ca²⁺+Mg²⁺ dependent ATPase from any of the preparations.As demonstrated both by ATPase assays and electron microscopy, coated microvesicles could be bound to immunosorbents prepared with poly-specific antibodies against a coated microvesicle fraction obtained by the method of Pearse (1). The binding could be inhibited by dissolved coat protein using partially purified clathrin. The fraction of coated vesicles eluted from the immunosorbent was purified relative to the starting material as judged by electron microscopy.The Ca²⁺+Mg²⁺ ATPase activity and calmodulin content was copurified with the coated microvesicles and the specific activity of Na⁺+K⁺+Mg²⁺ ATPase was decreased.Na⁺+K⁺+Mg²⁺ dependent ATPase activity in the coated microvesicle fraction could be ascribed to membranes with the appearance of microsomes. These membranes were also bound to the immunosorbents, but the binding was not influenced by clathrin. The capacity of the immunosorbents for these membranes was less than for the coated microvesicles, resulting in a decrease of Na⁺+K⁺+Mg²⁺ dependent ATPase activity in the eluted coated microvescile fraction.It was concluded that Ca²⁺+Mg²⁺ ATPase activity is not a contamination from plasma membrane vesicles or mitochondrial membranes but seems to be an integral part of the coated vesicle membrane.     

Photoaffinity labeling of uncoupler binding sites on mitochondrial membrane

³H 2-azido-4-nitrophenol, a photoactive uncoupler, has been synthesized, and its uncoupling action on oxidative phosphorylation and its binding to the mitochondrial membrane have been studied. The uncoupler bound covalently to the mitochondrial membrane on photoirradiation was 3–4 times that bound reversibly in the absence of light. When irradiation was carried out in the presence of serum albumin, covalent binding was significantly depressed. The pattern of loss of ATP-P_i' exchange activity with increasing amounts of the uncoupler suggests that serum albumin prevents the binding of the uncoupler to the functional sites as well. Polyacrylamide gel electrophoresis of photoaffinity labeled submitochondrial particles in the presence of sodium dodecyl sulfate revealed that a 9000 dalton peptide bound high levels of uncoupler. Other proteins in the molecular weight range of 20,000–40,000 and 55,000 were also labeled. Photolysis in the presence of serum albumin or ATP decreased the covalent binding of the uncoupler to all the proteins, but particularly to the 20,000 dalton component. Soluble ATPase and the mitochondrial proteolipid purified from labeled mitochondria showed the presence of label.Abbreviations NPA 2-azido-4-nitrophenol - DNP 2,4-dinitrophenol - DCCD N, N¹-dicyclohexylcarbodiimide - AE particles=bovine heart submitochondrial particles prepared by treatment with NH₄OH and EDTA at pH 8.8 - RCI respiratory control index - BSA bovine serum albumin     

Molecular events in the processing of avidin by antigen-presenting cells (APC)

The immune response of T lymphocytes to avidin was measured by proliferative assays, antibody production and delayed-type hypersensitivity. Mice ofH-2 ^k haplotypes were found to be low responders, whereas mice of other haplotypes, and particularly ofH-2 ^s , were high responders.Ir genes controlling this response were mapped to theI subregion ofH-2. Helper T cells were found to be responsible for the Ir phenotype of antibody production. These results indicate the feasibility of using the avidin-biotin complex as a tool for studying molecular mechanisms by which antigens underIr gene control are processed and presented to T lymphocytes.Abbreviations used in this paper Ir genes, immune-response genes - H-2 murine major histocompatibility complex - APC antigen-presenting cell - OA ovalbumin - BSA bovine serum albumin - DNP dinitrophenyl - DNP-OA DNP-ovalbumin - DNP-Av DNP-avidin - DNP-BSA DNP-bovine serum albumin - CFA complete Freund's adjuvant - PPD purified protein derivative - PBS phosphatebuffered saline - IP intraperitoneal - LNC lymph-node cells - DTH delayed-type hypersensitivity