Heterogeneity in the reactivity of various groups of IgM antibodies with kaolin.

Kaolin strongly adsorbed rheumatoid factor (RF) and mono-nucleosis antibodies, while cold agglutinins and some antimicrobial IgM antibodies were poorly adsorbed. Maximum adsorption took place at a pH of about 7. The degree of adsorption also depended on the amount of kaolin. Rheumatoid factor could be eluted from kaolin at pH 11 after adsorption at pH 7. The reported heterogeneity with regard to reactivity with kaolin is suggested to be due to hydrophobic interactions.     

高岭土对Fe~(2+)的吸附及其对高岭土中Fe~(3+)生物还原的影响研究

利用异化铁还原微生物可将高岭土中不溶性的Fe3+还原成可溶性的Fe2+,但是此过程中产生的Fe2+能够被高岭土以及异化铁还原微生物吸附,从而影响高岭土中铁的异化还原。本文研究了pH、高岭土量、Fe2+浓度、温度4个因素对高岭土吸附Fe2+的影响;并采用Logistic方程拟合,研究Fe2+及温度对高岭土中Fe3+的生物还原特征。结果表明:pH、高岭土量、浓度、温度4个因素均会影响高岭土吸附Fe2+,当Fe2+吸附在高岭土和微生物菌体表面时,微生物的活性下降,同时高岭土表面Fe3+的生物可利用性也降低,Fe3+生物还原的最大速率减小。     

Effect of a Modified Kaolin Treatment on Serum Immunoglobulins

After kaolin treatment of fetal rabbit serum, 7S antibody titers were reduced more than 19S titers. This reduction was less when the kaolin treatment was performed at pH 9.0 than when it was performed at pH 7.3. A modification of the kaolin treatment of sera for use in the hemagglutination-inhibiting antibody titration, in which the hemagglutination reaction is performed at a neutral pH, is recommended. The advantage of the modified method is that adsorption of immunoglobulins to kaolin is minimized when serum is treated at a lower dilution with pH 9.0 kaolin, followed by reduction of the pH of the supernatant fluid to neutrality with a "serum adjusting diluent." When the serum was diluted with physiological saline before kaolin treatment, a great decrease in serum immunoglobulin concentrations occurred. This decrease was found to be less in the modified kaolin treatment than in the conventional pH 7.3 kaolin treatment.     

The effect on kaolin adsorption of serum on the virus neutralization and enzyme-linked immunosorbent assays of antibody to bovine herpesvirus 1

Two procedures have been used for measuring antibody titres to bovine herpes virus 1 (BHV1): the serum neutralization (SN) test and enzyme-linked immunosorbent assay (ELISA). One hundred and thirty-two sera selected for their low SN titres were tested both unadsorbed and after adsorption with kaolin to determine the effect of kaolin on the titres. With ELISA, the titres of unadsorbed and kaolin adsorbed were not significantly different but with the SN test many treated sera, originally with weak positive titres, became negative after kaolin adsorption. Thus, if the ELISA results are specific for BHV1 antibody then the SN test findings suggest that treatment of sera with kaolin, rather than removing a viral inhibitor, removes a substance from the serum which potentiates SN antibody. This in turn indicates that low SN titres (reciprocal of titre less than or equal to 4, for instance) are probably specific for BHV1 SN antibody whether or not they are abolished by kaolin treatment of the serum.     

Optical biosensors for real-time measurement of analytes in blood plasma

The preparation of assemblies consisting of multiple molecular layers of bovine serum albumin (BSA), monoclonal antibodies against horseradish peroxidase (anti-HRP), and monoclonal antibodies against methotrexate (anti-MTT), as well as interaction of the assemblies with human blood plasma were observed using a grating coupler and Young interferometer (YI). The assemblies could be arranged according to decreasing amounts of nonspecific deposits bound irreversibly to them from blood plasma as follows-an adsorbed antibody monolayer saturated with adsorbed BSA, antibody multilayers linked with polycations, antibodies covalently immobilized on a BSA layer densely crosslinked with glutaraldehyde (GA), slightly crosslinked BSA double layer, slightly crosslinked antibody double layers. The occurrence of human serum albumin (HSA), human fibrinogen (Fg), IgG, and IgM in the plasma deposits was studied by binding the respective antibodies. IgG, IgM, and Fg were detected in plasma deposits on the immobilized assemblies while the composition of a plasma deposit on the unmodified sensor surface reflected roughly the plasma composition containing mainly adsorbed HSA and Fg. A crosslinked anti-HRP double layer was immobilized on a waveguiding branch of YI and a similar anti-MTT double layer was immobilized on the other branch. The sensor response to blood plasma was fairly decreased owing to a compensation of the respective optical changes in the two branches, in which a similar non-specific adsorption took place. The addition of HRP or MTT to plasma induced specific responses of the corresponding branches.     

Nature of double-stranded DNA binding activity in seropositive rheumatoid arthritis: formation of low avidity DNA/rheumatoid factor/IgG/low density lipoprotein complexes

Sera from majority of patients with seropositive rheumatoid arthritis, which generally lacked detectable anti-double stranded DNA in Farr, Crithidia luciliae, and microcomplement fixation assays, exhibited high levels of dsDNA binding in the presence of 3.5% polyethylene glycol when using intrinsically labeled 3H-PM2 DNA as antigen. Except for SLE, such increased dsDNA binding was absent in normal and a variety of other disease sera, including those from patients with seronegative rheumatoid arthritis. In contrast to the situation in SLE, in which dsDNA binding is mediated by specific anti-DNA antibody, the increased dsDNA binding activity in seropositive rheumatoid arthritis was shown to be dependent upon complex low avidity interactions involving DNA, IgG, IgM rheumatoid factor, and low density lipoproteins. Analysis of the composition of the polyethylene glycol serum precipitates by 2-dimensional gel diffusion, immunoelectrophoresis, and sodium dodecyl sulfate polyacrylamide gel electrophoresis failed to reveal the presence of additional DNA-binding proteins unique to seropositive rheumatoid arthritis. The only feature distinguishing high DNA binding sera from those with low DNA binding activity was an increased amount of polyethylene glycol-insoluble IgG in the former, presumably reflecting IgG/IgG and/or IgG/IgM complexes. The significance of these unusual DNA/low density lipoprotein/IgG/rheumatoid factor complexes with respect to the diagnostic specificity and pathophysiology of the DNA/anti-DNA system is discussed.     

Interaction between collagen and adenosine 5'-triphosphate.

A quantitative study was made of the adsorption of adenosine 5′-triphosphate (ATP) on collagen by following the change in the absorbance at 258 nm of ATP in the soaking solution. The amount of ATP adsorbed decreased exponentially with the increase of pH up to pH 8 and fell off more rapidly at higher pH values. At a given pH, when the concentration of ATP was increased, the amount of ATP adsorbed increased following the pattern of a Langmuir isotherm. The adsorption was independent of the cation present. The adsorption of adenosine 5′-diphosphate was essentially the same as that for ATP. For tendons deposited with calcium phosphate, the amount of ATP adsorbed decreased compared to natural tendons. The adsorption of ATP on collagen fibers inhibited the contraction caused by calcium chloride, calcium bromide, and lithium bromide. In solution, ATP had very little effect on the denaturation of acid-soluble collagen caused by calcium chloride.     

THE ADSORPTION OF GELATIN BY COLLODION MEMBRANES

An experimental study has been made of the adsorption of gelatin from solution at 37°C. by collodion membranes. In the case of membranes of high permeability, very high concentrations of gelatin were required to produce maximum adsorption, and the maximum amounts adsorbed were independent of the pH values of the solutions over the range 3.8 to 4.8. With membranes of low permeability, maximum adsorption was reached at lower gelatin concentrations, and the maximum amounts adsorbed varied with the pH, being lower on either side of the isoelectric point, over the range 3.8 to 6.6. The addition of salt in such experiments raised the maximum amount adsorbed to a value equal to that obtained with solutions at the isoelectric point in the absence of salt. These experiments can be explained by, and seem to lend support to, the theory proposed by Loeb and further developed by Kunitz concerning the effects of pH and salt on the size of gelatin particles in solution.     

Adsorption, desorption, and activity of glucose oxidase on selected clay species.

The adsorption of the enzyme glucose oxidase (EC 1.1.3.4) to clays followed the pattern described for other proteins as being pH dependent. Maximum adsorption occurred at or below the isoelectric point of the enzyme. The amount of enzyme adsorbed to clay was influenced by the type of clay used, and also the saturating cations. Initially adsorbed enzyme showed low specific activities, and as amounts of enzyme adsorbed approached maximum stauration of clay, specific activities increased approaching that determined for free enzyme. The adsorption of glucose oxidase involved a temperature-independent cation-exchange mechanism, and enzyme adsorbed to surfaces of clay could be desorbed in active form by elevation of pH of suspending solution. This was followed by a slower temperature-dependent fixation, probably by hydrogen bonding, which resulted in protein being irreversibly adsorbed to clay surfaces. It is proposed that on adsorption of glucose oxidase to clay surfaces unravelling of the protein structure occurred, which allowed penetration of protein into the interlamellar spaces of montmorillonite. This proposal was based on the observed expansion of montmorillonite to 23 A, and the decreases in amount of a second-protein lysozyme adsorbed with extended incubation times of glucose oxidase - clay complexes at pH 4.5.     

Trypanosoma lewisi: Avidity and adsorbability of ablastin,the rat antibody inhibiting parasite reproduction

The relation of naturally acquired host IgG in the surface coat of bloodstream forms of Trypanosoma lewisi to ablastin was studied to determine whether, contrary to a long-held conclusion, the antibody is avid and adsorbable. It was found by immunofluorescence and agglutination tests with monospecific antisera to rat IgG that bloodstream forms collected from immunosuppressed hosts, in contrast to those from immunocompetent hosts, have little or no detectable surface IgO. Specificity of adsorption was also demonstrated in other immunofluorescence experiments in which bloodstream forms from immunosuppressed hosts adsorbed IgG from immune serum with ablastic activity only (previously adsorbed with trypanosomes from immunocompetent hosts to remove the trypanocidal antibodies), but did not adsorb IgG from normal rat serum. To determine whether this specific adsorption of IgG by the parasite could be correlated with a reduction in ablastic activity, immune sera were adsorbed with bloodstream forms from immunosuppressed hosts at packed cell/serum ratios of either 1.2 or 2.0, and the adsorbed sera were then tested for ablastic activity in vitro. With both cell/serum ratios, ablastic activity was reduced by 50%. In comparison, similar adsorptions of immune sera with trypanosomes from immunocompetent hosts resulted in reductions of ablastic activity of only about 9 and 27% with the low and high cell/serum ratios, respectively. It is concluded that the failure to effect significant adsorption of ablastin in earlier studies resulted from the use of ablastinsensitized trypanosomes from immunocompetent hosts.     

Analyses of Idiotypes on Anti-α-1, 6-Dextran Antibodies in Mice

When mice of strains C57BL/6, C3H/He, and BALB/c were immunized with native dextran B512, only a small amount of IgM antibody was produced, but a substantial amount of anti-dextran antibody of IgG class was produced after immunization with a conjugate of dextran T10 and keyhole limpet hemocyanin regardless of the mouse strain used. Isoelectric focusing (IEF) spectra revealed limited heterogeneity of anti-dextran antibody of IgG class with strict consistency in all individual sera from C57BL/6 mice, even after secondary immunization, whereas antibodies from C3H/He and BALB/c mice showed more heterogeneous IEF spectra with some individual variations. Rabbit anti-idiotypic (Id) antibodies were raised by immunization with a subfraction of anti-dextran antibody of IgG class from C57BL/6 mice, which showed major bands focused at around pH 7.7 upon IEF. It was found by using the anti-Id antibodies that virtually all anti-dextran antibody molecules of both IgG and IgM classes from C57BL/6 mice possessed common Id determinants which can be classified into two specificities, one specific for antibody from C57BL/6 mice and the other cross-reactive with antibodies from BALB/c and C3H/He mice. About 80% of the antibody molecules from BALB/c and less than 20% of those from C3H/He mice were positive for the interstrain cross-reactive Id. Both Id determinants seemed to be closely related to the antigen binding sites, or at least to reside in the vicinity of the antigen binding sites of anti-dextran antibody.     

Labelling of colloidal gold with protein A. A quantitative study

Colloidal gold complexes with protein A are extensively used in immunocytochemistry as secondary reagents for the localization of antigens. However detailed information on the process and extent of adsorption of protein A onto gold particles, the optimal condition of preparation and the stability of such complexes are lacking. The adsorption isotherm of 125I-protein A onto gold particles (11.2 nm in diameter) was studied quantitatively with gold sols buffered at pH 4-7. At low coverage of the particles, the isotherm was independent of pH. However in the presence of a large excess of protein A, the highest coverage was obtained with a gold sol buffered at pH 5.1, the isoelectric point of the protein. The association constant was decreased at high coverage of the particles. Maximum binding of the complex to immobilized IgG occurred with particles labelled with at least 9 molecules of protein A. The complex was stable under storage with up to 12 molecules adsorbed per particle. At high coverage (26 molecules per particle), a progressive loss of protein A was observed. The optimum condition for preparing the complex are reported.     

Engineered human IgG antibodies with longer serum half-lives in primates

The neonatal Fc receptor (FcRn) plays an important role in regulating the serum half-lives of IgG antibodies. A correlation has been established between the pH-dependent binding affinity of IgG antibodies to FcRn and their serum half-lives in mice. In this study, molecular modeling was used to identify Fc positions near the FcRn binding site in a human IgG antibody that, when mutated, might alter the binding affinity of IgG to FcRn. Following mutagenesis, several IgG2 mutants with increased binding affinity to human FcRn at pH 6.0 were identified at Fc positions 250 and 428. These mutants do not bind to human FcRn at pH 7.5. A pharmacokinetics study of two mutant IgG2 antibodies with increased FcRn binding affinity indicated that they had serum half-lives in rhesus monkeys approximately 2-fold longer than the wild-type antibody.     

Separation of immunoglobulin G from human serum by pseudobioaffinity chromatography using immobilized l-histidine in hollow fibre membranes

l-Histidine, intended as a pseudobiospecific ligand, was immobilized on poly(ethylenevinyl alcohol) hollow fibre membranes after their activation with epichlorohydrin or butanediol diglycidyl ether. The affinity membranes obtained allowed the one-step separation of immunoglobulin G (IgG) from untreated human serum. Elution was possible under mild conditions with discontinuous pH or salt gradients. IgM was also adsorbed to a certain extent and partially separated from IgG by pH gradient elution. The bound IgG fractions showed pI values between 8 and 9.5 and contained IgG₁ and IgG₃. The dissociation constants for IgG on the bisoxirane- and epichlorohydrin-activated membranes coupled with histidine were determined by equilibrium binding analysis to be 2.5·10⁻⁵ and 2.0·10⁻⁵ M, respectively. The maximum binding capacity of the affinity hollow fibre membranes was 80 and 70 mg of IgG per gram of support, respectively. With a cartridge of surface area 1 m² (about 19 g of fibres), during a 60-min run, theoretically up to 1.5 g of IgG can be removed from human serum. The histidine affinity membranes are very stable owing to the simple nature of the ligand and the coupling via an ether linkage. Reproducible results were obtained over more than 1 year even with untreated human serum being used regularly.     

A novel magnetic adsorbent for immunoglobulin-g purification in a magnetically stabilized fluidized bed

A novel magnetic poly(ethylene glycol dimethacrylate-N-methacryloly-L-histidinemethylester) [m-poly(EGDMA-(MAH)] support was prepared for purification of immunoglobulin G (IgG) in a magnetically stabilized fluidized bed by suspension polymerization. Elemental analysis of the magnetic beads for nitrogen was estimated as 70 micromol MAH/g polymer. Magnetic poly(EGDMA-MAH) beads were used in the separation of immunoglobulin-G (IgG) from aqueous solutions and/or human plasma in a magnetically stabilized fluidized bed system. IgG adsorption capacity of the beads decreased with an increase in the flow rate. The maximum IgG adsorption was observed at pH 6.0 for MES buffer. IgG adsorption onto the m-poly(EGDMA) was negligible. Higher adsorption values (up to 262 mg/g) were obtained in which the m-poly(EGDMA-MAH) sorbents were used from aqueous solutions. Higher amounts of IgG were adsorbed from human plasma (up to 320 mg/g) with a purity of 87%. IgG molecules could be repeatedly adsorbed and desorbed with these sorbents without noticeable loss in their IgG adsorption capacity.     

Effect of cold exposure on the metabolism of immunoglobulins in rabbits.

The effect of low environmental temperature on the metabolism of IgG and IgM was examined in unimmunized rabbits. The half-lives of both IgG and IgM were less in animals kept at 4 degrees C for 6 weeks than in animals kept at 22 degrees C. Serum concentration of IgM and GG were unaltered by cold exposure but intravascular pool sizes tended to increase as a consequence of an expanded serum volume. Fractional turnover rates of both IgM and IgG were greater in cold-exposed animals. At both 4 degrees C and 22 degrees C, the fractional catabolic rate of IgM was independent of its serum concentration whereas that of IgG was correlated directly with its serum concentration. Absolute turnover of both IgM and IgG was accelerated by cold exposure. It is suggested that increased synthesis of immunoglobulin could account for the higher levels of antibody reportedly found in cold-exposed rabbits.     

Adsorption of lecithin liposomes to acid clay

The interaction between lecithin liposomes and acid clay was investigated to clarify the mechanism for liposome adsorption to the clay. It was found that the multilamellar vesicular structure of the liposomes was broken as a result of primary adsorption. The acid clay particles aggregated and were eventually covered by the lecithin layer structure. In the case of kaolin, on the other hand, the liposomes were weakly adsorbed to the clay and maintained the vesicular structure. The amount of primary adsorption to the clay surface, which was estimated from the adsorption isotherm, was more for acid clay than for kaolin, and the total amount adsorbed to the acid clay was also more than to kaolin. This result can be explained by the much higher density of the negative charge on the acid clay surface than that for kaolin. The liposomes are therefore considered to be adsorbed to the acid clay mainly by the choline positive charge residing at the end of the lecithin molecule, although this is of no net charge as a whole.     

Analysis of a common inheritable idiotype in IgA-deficient sera using monoclonal antibodies

In these studies we describe the production of three mAb raised to an idiotype on an IgG anticasein antibody isolated from the serum of one IgA-deficient blood donor. These are IgM kappa and block the binding of casein Ag to anticasein antibody. Sera of unrelated IgA-deficient donors were tested for the presence of the idiotype; 15 of 56 IgA-deficient sera (25%) contain the anticasein idiotype, whereas 1 of 45 normal sera was positive. Anticasein antibodies as a whole were predominantly of the IgG1 and IgG3 subclass; idiotype-positive anticaseins are predominantly of the IgG1 subclass. For IgA-deficient donors, the relative amount of idiotype-positive anticasein antibody was correlated with the level of anticasein present in the serum. Studies were done to investigate the potential inheritance of the idiotype in families; in three of four families the idiotype was inherited in an apparent autosomal dominant pattern. Our data show that a common cross-reactive idiotype can be detected in the sera of IgA-deficient individuals and their family members. This suggests that V region markers may be conserved in this humoral immunodeficiency disease.     

IgG autoantibody activity in normal mouse serum is controlled by IgM

In the serum of normal BALB/c mice, IgG antibody reactivity to mouse actin and tubulin, DNA, and TNP groups was very low compared to that of the IgM. This activity was considerably increased when IgG was separated, by affinity chromatography on protein A-Sepharose, whereas no difference in the IgM activity was observed. Addition of IgM to IgG isolated from the same serum resulted in the inhibition of IgG binding to these Ag. Isolation of IgG antibodies on actin, TNP, and tubulin immunoadsorbents has indicated that at least part of the IgG antibodies is polyreactive. In order to understand this inhibition better, experiments with F(ab')2 fragments of IgG were performed. IgM inhibited the binding of F(ab')2 to the antigens in a dose-dependent manner and reacted with immobilized F(ab')2. IgM isolated on F(ab')2 immunoadsorbent, as compared to the initial IgM preparation, were less active toward the Ag but more inhibitory for IgG binding to the Ag. In some pathologic situations, IgM failed to inhibit some IgG antibody activities. The anti-DNA IgG activity from (NZB x NZW)F1 mice was not affected by autologous IgM. Similarly the anti-tubulin IgG from mice infected with Trypanosoma cruzi were less inhibited by IgM from autologous serum than antitubulin IgG from normal mice. These results are compatible with the existence in normal mice of an idiotypic-like network, regulating via an IgM population in the serum, the binding of IgG autoantibodies to self Ag. Modifications of this idiotype-anti-idiotype system might lead to the expression and/or expansion of autoreactive IgG-producing clones.     

Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces

A procedure for covalent immobilization of functional proteins on silica substrates was developed using thiol-terminal silanes and heterobifunctional cross-linkers. Using this procedure, a high density of functional antibodies was bound to glass cover slips and silica fibers. The amount of anti-IgG antibody immobilized was determined to be in the range of 0.66 to 0.96 ng/mm2 using radiolabeled antibody. The relative amount of IgG antigen bound by the immobilized antibody (0.37 to 0.55 mol antigen/mol antibody) was three to five times greater than other investigators have reported. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was further reduced by the addition of blocking agents so that nonspecific adsorption of protein antigens represented only 2-6% of the total antigen binding. With this low background, IgG antigen binding could be measured at levels as low as 150 fmol when an antigen concentration of 3 pmol/ml was applied. The process for antibody immobilization is straightforward, easy to perform, and adaptable for modifying mass quantities of biosensor components.