Activation of the human classical complement pathway by a mouse monoclonal hybrid IgG1-2a monovalent anti-TNP antibody bound to TNP-conjugated cells

A mouse hybridoma selected and cloned for anti-TNP specificity produced three distinct monoclonal antibody species that were separated on protein A-Sepharose by stepwise acid elution. The IgG1 kappa product of the parental myeloma was eluted at pH 6.0. An IgG2a kappa bivalent anti-TNP antibody was eluted at pH 4.5, whereas elution at pH 5.0 yielded a hybrid IgG1-2a kappa monovalent anti-TNP antibody. The IgG2a molecules agglutinated TNP-conjugated sheep erythrocytes (TNP-ES) and lysed TNP-ES in the presence of normal human serum (NHS). Hybrid IgG1-2a antibody was also capable of lysing the cells in NHS, although it did not agglutinate TNP-ES. A threshold in monovalent antibody input was necessary for the lysis of TNP-ES, indicating a requirement for a minimal density of bound monovalent IgG to trigger complement activation. Lysis occurred in NHS-VBS++ but not in NHS-MgEGTA, and it was associated with a dose-dependent consumption of C1, C4, and C2 hemolytic activities. Quantitation of the antibody bound to TNP-ES when using radiolabeled rabbit anti-mouse Fab antibody demonstrated that for similar inputs, 5.4 times as much bivalent as monovalent antibody bound to TNP-ES. When similar amounts of antibody were effectively bound to TNP-ES, monovalent hybrid IgG1-2a was five times less efficient than bivalent IgG2a to yield 50% cell lysis in the presence of NHS. These results indicate that neither bivalent binding nor the presence of two identical heavy chains are necessary requirements for antibody-dependent activation of the classical complement pathway.     

Use of Horseradish-Peroxidase Labelled Antibodies in ELISA for Plant Virus Detection

Detection of plant viruses by ELISA using horseradish peroxidase for antibody labelling (ELISA-peroxidase) has been standardized by evaluating variants of the procedure, regarding composition and concentration of buffers and additives. Immunoglobulins (IgG) are isolated from antisera by precipitation with ammonium-sulphate and by purification with DEAE-52 (Whatman) cellulose. IgG are conjugated with horseradish peroxidase by a modified oxidation-periodate method. In ELISA-peroxidase 0.05 M carbonate-bicarbonate coating buffer pH 9.6 has been substituted by 0.01 M carbonate buffer pH 9.2. Extraction buffer is used with 0.5% bovine serum albumin (BSA), without polyvinylpyrrolidone (PVP). Samples are diluted in, phosphate buffered saline (PBS) pH 7.2 with 0.05% Tween 20 and 0.5% BSA. IgG are conjugated with horseradish peroxidase, diluted in 0.1 M Tris-HCl, pH 7.4 with 0.05% Tween 20 and 1% BSA. The substrate is incubated in the darkness for 20 min at room temperature. ELISA-peroxidase proved to be equivalent in sensitivity and specificity with ELISA using alkaline phosphatase for antibody labelling. Its advantage is a lower cost of chemicals used in the test.     

Inhibition of immune precipitation by complement

Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.     

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.     

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%–60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.     

Isolation and characterization of immunoglobulin of the Indian major carp, rohu [Labeo rohita (Ham.)

Information on the structure and character of immunoglobulin of fishes is essential in health management. A study was carried out to characterize the serum immunoglobulin (IgM) of the Indian major carp, rohu Labeo rohita (Ham.). Rohu (500g) were immunised with bovine serum albumin (BSA) and the anti-BSA antibody was purified employing BSA-CL agarose affinity column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified Ig in a 3% gel under non-reduced conditions revealed a single protein having a molecular weight of 850kDa. Analysis of the purified serum in 10% SDS-PAGE under reduced conditions revealed that the immunoglobulin contained heavy and light chains with molecular weights of 85 and 23kDa, respectively. A polyclonal mouse anti-rohu IgM was prepared and used in an immunodot test which showed a specific reaction of the crude rohu anti-BSA antiserum and the purified anti-BSA IgM with BSA. Results indicate that the immunoglobulin of L. rohita is tetrameric IgM, similar to that of other fishes.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.     

Immunosuppressive properties of a peptic fragment of BSA.

The immunogenic properties of a peptic fragment of BSA were investigated. BSA was subjected to limited proteolysis by pepsin and the resulting fragments were separated on DEAE cellulose. The fragment under consideration, Fraction Ia (m.w. 8000 to 10,000), did not precipitate with anti-BSA serum but did inhibi, the binding of specific antibody to labeled BSA, indicating the presence of determinants found on the native antigen. BDF1 mice immunized with Fraction Ia in A1 (OH)3 gel or in complete Freund's adjuvant produced no significant antibody response as measured by passive cutaneous anaphylited a (PCA) or by a modified Farr assay. The fragment elicited a PCA reaction in mouse skin sensitized with anti-BSA serum. Treatment of mice with single doses of Fraction Ia at various time intervals before immunization with BSA resulted in significant suppression of the formation of anti-BSA antibody. The conditions of suppresion of the IgE response by the peptic fragment were studied in greater detail. Evidence is presented that such suppression can be attributed to the presence of specific T suppressor cells in our system.     

Conjugation of protein antigen to microparticulate β-glucan from Saccharomyces cerevisiae: a new adjuvant for intradermal and oral immunizations

Immunostimulatory glucose polymers known as β-glucans have been studied for many years. Our laboratory has prepared and characterized a novel microparticulate β-glucan (MG) from the budding yeast Saccharomyces cerevisiae. Because MG particles are rapidly phagocytized by murine peritoneal macrophages and induce the expression of B7 costimulatory molecules, we hypothesized that MG could serve as a vaccine adjuvant to enhance specific immune responses. Here, we describe a procedure for conjugating the test vaccine antigen bovine serum albumin (BSA) to MG via water-soluble carbodiimide linkage. Conjugates with up to 0.4 mg of BSA/mg MG were prepared. MG/BSA conjugates were still actively phagocytized by mouse peritoneal macrophages. When used to immunize mice by the intradermal route, these conjugates enhanced the primary IgG antibody response to BSA in a manner comparable to the prototypic complete Freund’s adjuvant. Although primary oral immunization with MG/BSA caused no increase in serum anti-BSA antibody titers, booster immunization elicited a significant anti-BSA antibody response. These results suggest that protein antigens can be conjugated to MG via a carbodiimide linkage and that these conjugates provide an adjuvant effect for stimulating the antibody response to the protein antigens.     

Rapid stimulation of large specific antibody responses with conjugates of antigen and anti-IgD antibody

Injection of mice with goat anti-mouse IgD antibody stimulates a large IgG1 anti-goat IgG antibody response, as well as polyclonal IgG1 production. To determine if this phenomenon could be used to induce large antibody responses to other Ag, covalent conjugates were produced between BSA or other Ag and H delta a/1, a mAb specific for IgD of the a allotype, and between BSA and AF3.33, a mAb specific for IgD of the b allotype. Injection of H delta a/1-BSA into BALB/c mice, which express Ig of the a allotype, or into (BALB/c x CB20)F1 mice (a x b allotype heterozygotes) induced IgG1 anti-BSA antibody responses that peaked 8 to 9 days after injection, and were more than 1000 times larger than those induced by injection of BSA alone, and 100 times larger than those induced by injecting unconjugated BSA plus H delta a/1. H delta a/1-BSA was no more immunogenic than unconjugated BSA when injected into CB20 mice, which express Ig of the b allotype, while AF3.33-BSA greatly enhanced anti-BSA antibody production in CB20, but not in BALB/c mice. Mice serially immunized with three different Ag conjugated to H delta a/1 made large antibody responses to all three Ag, provided that the mouse strain used did not recognize allotypic determinants on H delta a/1 as foreign and produce a neutralizing antibody response. Intravenous and s.c. routes of inoculation produced responses of similar magnitude and relatively low variability; responses to footpad or intramuscular inoculation were more variable, and i.p. inoculation induced smaller responses. Injection of BALB/c mice i.v. with 100 micrograms of H delta a/1-BSA induced an IgG1 anti-BSA response of 5.6 mg/ml, which was approximately 70% of the total IgG1 response. Anti-BSA responses to 30 micrograms of conjugate or less were much smaller, but could be considerably enhanced by adding unconjugated H delta a/1 to the inoculum. This system will be useful for the rapid stimulation of large antibody responses to biologically important Ag, and for investigating mechanisms of Ag processing and B and T cell activation.     

Stepwise dissociation of subcomponents of C1, the first component of human complement, upon activation on an affinity sorbent

An affinity sorbent comprising macroporous glass coated with the polymer with the polymer with immobilized immunoglobulin IgG was used for the isolation from human serum of the first component of the complement and for its separation into subcomponents C1r, C1s and C1q by the one-step procedure. Serum C1 was quantitatively bound to the sorbent at 0 degrees C. The unbound part of the serum can be used as a R1 reagent for determining the hemolytic activity of C1. After activation of bound C1 by heating (30 degrees C, 40 min) the activated subcomponent C1r is eluted from the sorbent. Stepwise elution with EDTA at pH 7.4 or with EDTA + 1 M NaCl at pH 8.5 results in a selective and quantitative elution of the activated subcomponent C1s and subcomponent C1q. Stepwise elution of C1 subcomponents from the affinity sorbent after activation reflects the process of C1 breakdown following its activation on immune complexes.     

纳米多孔硅阻抗生物传感器的研究

构建了1种基于多孔硅材料,无需标记的纳米生物传感器,用于对牛血清白蛋白分子进行检测。通过对多孔硅进行表面处理,形成氧化膜,将抗体固定到多孔硅氧化层表面。在磷酸盐缓冲液中,通过电化学检测系统检测加入抗原后,传感器的阻抗值的变化。磷酸缓冲液（PBS）/抗体-氧化层/硅,构成电解液/绝缘层/半导体（electro-lyte-insulator-semiconductor,EIS）结构。传感器的线性检测范围为0.01～0.27mg/mL,检测限为0.01mg/mL。     

Purification of antibodies against N-homocysteinylated proteins by affinity chromatography on Nomega-homocysteinyl-aminohexyl-Agarose

Modification with homocysteine (Hcy)-thiolactone leads to the formation of N-Hcy-Lys-protein. Although N-Hcy-Lys-proteins are immunogenic, pure antibodies have not yet been obtained. Here we describe synthesis and application of Nomega-homocysteinyl-aminohexyl-Agarose for affinity purification of anti-N-Hcy-Lys-protein antibodies. Nomega-homocysteinyl-aminohexyl-Agarose was prepared by N-homocysteinylation of omega-aminohexyl-Agarose with Hcy-thiolactone. Immune serum was obtained from rabbits inoculated with N-Hcy-Lys-keyhole limpet hemocyanine and IgG fraction prepared by chromatography on protein A-Agarose. Anti-N-Hcy-Lys-protein IgG was adsorbed on Nomega-homocysteinyl-aminohexyl-Agarose column at pH 8.6 and eluted with a pH 2.3 buffer. Enzyme-linked immunosorbent assays demonstrate that the antibody recognizes specifically N-homocysteinylated variants of hemoglobin, albumin, transferrin, and antitrypsin.     

Affinity chromatography of antibodies directed against soybean lipoxygenase-1 and -2 and an enzyme-linked immunosorbent assay (ELISA) for antibodies and lipoxygenases

Crude immunoglobulin G (IgG) fractions of antisera directed against soybean lipoxygenase-1 and -2 were purified by being passed through an immunoadsorbent column containing lipoxygenase coupled to CNBr-activated Sepharose 4B. Bound immunoglobulin was desorbed with pulses of 2 M or 3 M ammonium thiocyanate or 0.1 M glycine-HCl buffer (pH 2.5). The total column recoveries of anti-lipoxygenase-1 IgG and anti-lipoxygenase-2 IgG were 45% and 58%, respectively. The affinity for lipoxygenase of immunospecific antibodies was determined in an enzyme-linked immunosorbent assay (ELISA). In a reaction with lipoxygenase-1, anti-lipoxygenase-1 IgG, which was eluted with glycine-HCl buffer (pH 2.5) with recovery of 24%, had a 6.5-times higher affinity than the whole IgG fraction of antiserum. The affinity of anti-lipoxygenase-2 IgG for lipoxygenase-2 increased 2.2-times after chromatography of IgG over an immunoadsorbent column using 2 M ammonium thiocyanate as eluent (recovery 21%).     

Isolation and molecular weight determination of two immunoglobulin heavy chains in the channel catfish, Ictalurus punctatus

Channel catfish (Ictalurus punctatus), a teleost fish, were immunized over a 4 month period with 4 intraperitoneal injections of bovine serum albumin (BSA) in Freund's adjuvant. The catfish anti-BSA antibody was purified by affinity chromatography and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). By elution of catfish anti-BSA antibody from BSA-affinity columns with 3.0 M KSCN and subsequent SDS-PAGE, two immunoglobulin heavy chains were demonstrated in the channel catfish. The molecular weights and the relative percentages found of the two immunoglobulin heavy chains were 72,000 (94%) and 56,000 (6%). The molecular weight of the single light chain found was 23,000. Using the 72,000 mol. wt heavy chain and 23,000 mol. wt light chain and including a molecular weight of 15,000 for the J-chain, the molecular weight of the predominant channel catfish tetrameric IgM immunoglobulin molecule was calculated to be 775,000. Using the 56,000 low mol. wt heavy chain, the molecular weight of a second subclass of the channel catfish tetrameric IgM molecule was calculated to be 647,000. After Sephadex G-200 gel filtration, anti-BSA antibody activity was found only in the 14S globulin fraction by indirect hemagglutination testing.     

Guidelines for the preparative fractionation of human serum proteins on gradient-eluted columns of concanavalin A-sepharose: elution positions of fourteen well-characterized proteins and evidence for concanavalin A-reactive albumin-IgA and -IgG complexes

Systematic studies on the fractionation of serum proteins on gradient-eluted columns of concanavalin A-Sepharose have been carried out to determine if the oligosaccharide residues were sufficiently different to permit a reasonable separation and to determine where in the chromatogram these proteins would be eluted. Human whole serum and ammonium sulfate fractions derived therefrom were used in conjunction with 2.1 x 75 cm columns of concanavalin A-Sepharose and a 4 x 400 ml gradient (Varigard) with 0.5 M methyl alpha-D-glucopyranoside as limit buffer. The elution positions and chromatographic limits of 14 well-characterized human serum proteins have been determined by double diffusion of aliquots of the effluent fractions (10X concentrated) in agarose gel against specific antibody and the general chromatographic distribution of the proteins by immunoelectrophoresis. Overall, the results demonstrate that the composition of the oligosaccharide side chain, like differences in molecular size, solubility, and charge density, is a useful parameter in the chromatographic separation of protein from serum. Although it is well-known that albumin is a nonglycoprotein, 1.0% of the protein was tightly bound by concanavalin A-Sepharose. Subsequent experiments showed that albumin binding was due to complex formation with IgA and IgG both of which possess the necessary complement of concanavalin A-reactive residues for strong binding. Sodium dodecylsulfate polyacrylamide gel electrophoresis of 2-mercaptoethanol-reduced albumin-IgA and -IgG complexes produced bands corresponding to the molecular weights of albumin and the heavy and light chains of IgA and IgG whereas unreduced samples were not dissociated. When these complexes were reacted with concanavalin A-Sepharose and treated with 2-mercaptoethanol, free albumin was eluted. The remaining adsorbed glycoprotein(s), IgA and IgG, could be eluted with methyl alpha-D-glucopyranoside. These results strongly suggest that these proteins and albumin are linked via a disulfide bond(s).     

Echinococcus multilocularis: distribution and persistence of specific host immunoglobulins on cysts membranes

Cyst vesicles were obtained from larval cyst masses of Echinococcus multilocularis grown in Medium 858 in vitro or isolated from the intraperitoneal or subcutaneous larval cyst mass of C57BL/6J mice infected 12 weeks previously. Antigenic determinants were present on the outermost section of the laminated layer and throughout the germinal layer of the cysts. Specific host antibodies of the IgG1, IgG2a, IgG2b, and IgM classes and complement component C3 but not host IgA or C-reactive protein were detected on the intact cysts by the indirect immunofluorescent technique. Specific antibodies were bound to the epitopes on the laminated layer but were not detected on the germinal layer. Host albumin, however, was found on the laminated layer, germinal layer, and within the intact cyst. IgG2a and IgG2b were high-affinity antibodies but IgG1 and IgM were low-affinity antibodies as they were eluted easily from the laminated layer with two washes of sodium phosphate buffer (0.01 M, pH 7.2) containing 0.15 M NaCl. The significance of bound antibody in complement activation and antibody-dependent cell-mediated cytotoxicity of the proliferative phase (cyst) of alveolar hydatid cyst is discussed.     

The effects of antibodies and complement in macrophage-mediated cytotoxicity on metacercariae of the lung fluke, Paragonimus westermani

Paragonimus westermani is a tissue migrating parasite in the early stage until arriving at lung, and most of the parasites spend their life spans there. Considerable immune responses including activation of macrophages are taken place during the residence of parasites in the host. However, concerning the immunologic defense mechanisms of the host against this parasite, only a few document is available so far. In this study, the cytotoxic effect of peritoneal macrophages under the presence of antibody and/or complement against metacercariae of P. westermani was investigated in vitro. Metacercariae were collected from the crayfish, Cambaroides similis and hatched out in Tyrode solution (pH 7.4). Plastic adherent cells from normal or infected rat (Wistar) peritoneal exudates were used as experimental macrophages. Polyclonal antibodies were obtained from infected rats and a cat. Cat IgG was fractioned with ion exchange chromatography. Fresh rabbit complement was used according to experimental scheme. Various combinations of peritoneal macrophages, normal or infected rat serum, complement and cat IgG were incubated at 36 degrees C in 5% CO2 incubator for 6, 14, 24 and 48 hours. The results obtained were as follows: 1. P. westermani infection activated peritoneal macrophages non-specifically and this activation induced increases of cell adherence and cytotoxicity on metacercariae. 2. In the presence of infected rat serum the antibody-dependent cell-mediated cytotoxicity of peritoneal macrophages on metacercariae was significantly increased and showed a peak at 6-hour incubation. But the cytotoxic effect was markedly reduced after inactivation of complement and heat-labile IgE antibody by the heating of infected serum at 56 degrees C for 30 minutes. 3. The highest cytotoxic effect (100%) of concomitant incubation with IgG and complement showed 24 hours after incubation, although cell adherence was relatively low at 6-hour incubation and 0% at 24-hour incubation. 4. Coordinative functions of complement with serum and IgG were effective in cell adherence and in cytotoxicity, but it is not clear the independent role of complement on the macrophage-mediated cytotoxicity in this study. With these results it is assumed that P. westermani infection can induce the non-specific activation of peritoneal macrophages, and serum antibodies including IgE antibody might enhance the cytotoxicity by macrophages.     

Chemokinetic accumulation of human neutrophils on immune complex-coated substrata: analysis at a boundary

The locomotory behavior of human blood neutrophil leukocytes was studied at a boundary between two surfaces with different chemokinetic properties. This was achieved by time-lapse cinematography of neutrophils moving on coverslips coated with BSA, then part-coated with immune complexes by adding anti-BSA IgG with a straight-line boundary between the BSA and the immune complexes. Cell locomotion was filmed in microscopic fields bisected by the boundary, and kinetic behavior was assessed by comparing speed (orthokinesis), turning behavior (klinokinesis), and the rate of diffusion of the cells on each side of the boundary, using a recently described mathematical analysis of kinesis. In the absence of serum or complement, the proportion of motile cells and their speed and rate of diffusion were greater on BSA than on antiBSA, but there was no consistent difference in turning behavior between cells on the two surfaces. The immune complexes were therefore negatively chemokinetic in comparison with BSA, and this resulted from a negative orthokinesis with little or no contribution from klinokinesis. As would be predicted theoretically, this resulted in gradual accumulation of cells on the immune complexes even in the absence of a chemotactic factor. In further studies, a parallel plate flow chamber was used to show that, under conditions of flow, neutrophils accumulated much more rapidly on a surface coated with BSA- anti-BSA than on BSA alone. Moreover, neutrophils on immune complex- coated surfaces lost their ability to form rosettes with IgG-coated erythrocytes. This suggests that neutrophils on immune complex-coated surfaces redistribute their Fc receptors (RFc gamma) to the under surface, and that the lowered speed of locomotion is due to tethering of neutrophils by substratum-bound IgG-Fc.     

One-step separation from lactose: recovery and purification of major cheese-whey proteins by hydroxyapatite--a flexible procedure suitable for small- and medium-scale preparations

The recovery of cheese-whey proteins and lactose represents an important task both in environmental and in food sciences. Optimization of whey processing requires the quantitative separation of whey proteins from lactose, lower costs, harmless environmental impact, flexibility in protein recovery, and adaptability of the process to type and amount of available whey. Here we present a method based on the use of self-made, low-price, and nontoxic hydroxyapatite for one-step separation of lactose (non adsorbed) from bovine whey proteins (adsorbed). Recovery of proteins can be performed with high flexibility. Total protein fraction can be eluted with 0.4 M phosphate at pH 7.0. In alternative, proteins can be recovered in pairs with 0.4 M phosphate but at different pH's. About 56% of the proteins, primarily alpha-lactalbulmin and IgG, were eluted at pH 5.0. The other major proteins, beta-lactoglobulin and BSA, were eluted at pH 6.0. Fractions eluted with the two first eluants at pH 5.0 and pH 6.0 were applied to a Superdex 75 column for final purification by gel filtration. This method provides flexibility in whey protein recovery and quantitative separation of proteins from lactose before ultrafiltration and nanofiltration.