Electron microscopy study of human myeloma immunoglobulin G1

Human immunoglobulin G1 Van was studied by negative staining, freeze drying and high resolution shadow casting. The Fab and Fc subunits of an intact IgG1 molecule were shown to possess limited mobility. It was found that about 70% of molecules in the IgG1 Van specimen are not flat but have a tripod-like shape.     

Cleavage of human serum immunoglobulin G by an immobilized pepsin preparation

In order to obtain an efficacious and safe immunoglobulin G (IgG) preparation for intravenous use, the digestion of IgG with an immobilized pepsin (EC 3.4.23.1) preparation was studied. Thus, pepsin was immobilized onto glutaraldehyde-activated AH-Sepharose 4B under acidic conditions. THe enzymatic properties, such as proteolytic activity, pH-activity profile and heat stability, of the immobilized pepsin preparation were examined. The immobilized pepsin retained more than 40% of its proteolytic activity toward N-acetyl-L-phenylalanyl-L-3,5-diiodo-tyrosine and more than 30% toward IgG, and also remarkable stability as compared with free pepsin. The immobilized pepsin thus prepared was efficiently used for the limited cleavage of IgG and the gel-filtration effect of the column made it easily possible to yield the F(ab')2-rich fraction for intravenous use.     

Physicochemical and biological properties of poly(ethylene glycol)-coupled immunoglobulin G

In order to develop a new intravenous immunoglobulin G (IgG), IgG was covalently coupled to poly(ethylene glycol) previously activated by cyanuric chloride. The poly(ethylene glycol) coupled IgG obtained was studied for physicochemical and biological properties such as molecular structure, size-exclusion chromatographic behaviour, surface activity, interfacial aggregability, heat aggregability inducing nonspecific complement activation, and antigen-binding activity. The poly(ethylene glycol) coupling to IgG increased the apparent Stokes' radius and the surface activity of IgG and stabilized IgG on heating and/or on exposure to interface, while no structural denaturation of IgG was observed. The suppressed nonspecific aggregability was interpreted mainly by difficulty in association between the modified IgG molecules. These results indicated the use of the poly(ethylene glycol)-coupled IgG as an intravenous preparation and also as an additive stabilizing intact IgG for intravenous use.     

A simple method of isolating monoclonal immunoglobulin M, possessing rheumatoid activity]

A simple procedure for obtaining highly purified preparations of native monoclonal (Waldenstr?m's disease) immunoglobulin M possessing a rheumatoid activity (IgM-RF) has been developed. The method is based on the use of affinity chromatography with a new readily available adsorbent (immunoglobulin G-porous glass) and 3 M LiCl in Tris-buffer pH 8.3-8.4 able to induce the dissociation of the IgM-RF-IgG complex. The IgM-RF preparation thus obtained was characterized in terms of amino acid composition (relative to conventional monoclonal IgM), carbohydrate composition and structure of oligosaccharide moieties of a complex type. It was shown that some dissociation conditions for the IgM-RF-IgG complex routinely used to isolate IgM-RF provoke irreversible denaturation of IgM-RF when applied to a preliminarily purified complex.     

Ordering of antibodies, that react with cardiac muscle fiber and interstitial connective tissue antigens, in different immunoglobulin classes

Sera of patients suffering from rheumatic diseases and myocarditis were examined on the sections of human and bovine myocardial tissue by indirect immunofluorescence with the use of pure IgG antibodies or monospecific sera against IgG, IgA and IgM. It was shown that antibodies reacting with different myofibers and interstitial connective tissue of the heart belong to the main immunoglobulin classes (IgG, IgA and IgM). There was a significant predominance of IgG antibodies as shown by the frequency of their detection and by the titer height. The predominance of antibodies to certain classes of immunoglobulins did not correlate with a specific disease entity. The frequency of detecting antibodies to a certain immunoglobulin class was in good agreement with the time of the disease onset. Moreover, the frequency of positive reactions due to IgG, IgA, and IgM antibodies correlated with the level of the appropriate immunoglobulins in the test sera.     

The fractional composition of immunoglobulin preparations studied by gel filtration on different carriers and by high-pressure liquid chromatography]

The fractional composition of immunoglobulin preparations produced by different manufacturing enterprises of this country has been studied by gel chromatography in columns packed with different carriers (Sephadex G-200 and ultragel AcA-34) and by high-performance liquid chromatography (HPLC). This study has revealed the nonstandard character of immunoglobulin preparations produced according to the same technological procedure (modified Cohn's method). The fractionation of immunoglobulins on different carriers with the use of different methods has yielded similar results confirmed by the statistical processing of the data. The results obtained in the study of the fractional composition of immunoglobulin preparations evidence that gel filtration with the use of ultragel and HPLC have greater resolving capacity in comparison with the method of gel filtration on traditionally used Sephadex G-200.     

Use of electrophoresis in polyacrylomide gel (SDS_PAGE) for evaluating IgG structure--the main component of human intravenous immunoglobulin

For the assessment of the structure of IgG particles in the immunoglobulin preparation for intravenous use (Bioglobulin) electrophoresis in polyacrylamide gel (SDS-PAGE) was applied in place of filtration on Sephadex G-200 gel. The method serves for determination of the molecular weight of polypeptide chains under conditions of reduction. The method is rapid, sensitive and of high resolution. Six batches of the preparation were examined finding that IgG monomers and dimers accounted for over 90%, with IgG aggregates about 3% and degradation products about 1%. The obtained values comply with the recommendation of the WHO and requirements in the latest edition of Europea Pharmacopaes.     

In vitro galactosylation of human IgG at 1 kg scale using recombinant galactosyltransferase

The Fc effector functions of immunoglobulin G (IgG) antibodies are in part determined by structural features of carbohydrates linked to each of the paired gamma heavy chains in the antibody constant domain (C(H)2). One glycoform that has been shown to be advantageous is G2, where both arms of complex bi-antennary N-glycans terminate in galactose. In vitro treatment with glycosyltransferases can remodel heterogeneous IgG glycoforms, enabling preparation of IgG molecules with homogeneous glycan chains. Here we describe optimization of conditions for use of a soluble recombinant galactosyltransferase in vitro to remodel glycans of human serum IgG, and we demonstrate a scaled-up reaction in which >98% of neutral glycans attached to 1 kg IgG are converted to the G2 glycoform. Removal of glycosylation reagents from the product is achieved in one step by affinity chromatography on immobilized Protein A.     

Characterization and measurement of normal platelet-associated immunoglobulin G by fluorospectrophotometry

Platelet-associated immunoglobulin G of normal healthy subjects was measured and its binding characteristics studied by use of a newly developed assay which is rapid, quantitative, sensitive and inexpensive. The assay is based on fluorospectrophotometry. Fluorescein isothiocyanate was used as a fluorescent marker to label IgG. Measurement of platelet-associated IgG by this method showed that normal platelets have at least two types of binding sites for IgG of normal healthy subjects. High- and low-affinity binding sites numbering 410 ± 200 and 1800 ± 500, respectively, were identified. Specificity of binding was shown by competition between fluorescein isothiocyanate-labeled IgG and nonlabeled IgG. The effect of pH and temperature and the kinetics of binding were also investigated.     

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.     

Production of immunoadsorbents based on cellulose carbonates and their application to the isolation of specific immunoglobulin light chains

The purification of rabbit immunoglobulin molecules expressing kappa (κ) light chains, utilizing the allotypic specificity b4, has been achieved in stages involving isolation of specific antibody, preparation of a solid phase immunoadsorbent of coupled antibody, and subsequent isolation of b4 (κ) IgG. Cellulose trans-2.3-carbonate is shown to be an effective matrix enabling chemical coupling of antibodies and antigens to the support at neutral pH thus preservng immunological activity. The trans-2,3-carbonate derived from microcrystalline cellulose is more effective as a matrix than the trans-2,3-carbonate derived from macroporous cellulose for the chemical coupling of rabbit a1a3/b4 IgG antigen and binding of specific anti-b4 antibody. The microcrystalline celulose carbonate is also more efficient for the coupling of rabbit anti-b4 antibody and the subsequent binding and elution of rabbit b4 (κ) IgG, thus separating immunoglobulin, expressing kappa light chain, from that expressing lambda light chain. The purification technique has potential application in other allotypic systems and antibody- antigen populations.     

F(ab')2 reagents are not required if goat, rather than rabbit, antibodies are used to detect human surface immunoglobulin.

The present report provides evidence that whole goat anti-human immunoglobulin, unlike similar reagents produced in the rabbit, binds both to the same number of and the same individual cells as the F(ab')2 fragments of rabbit or goat anti-human immunoglobulin. These results suggest that goat IgG has a lower affinity for the Fc receptors of human lymphocytes and monocytes than rabbit IgG. Because of this property, whole goat antibodies against human immunoglobulin can be used as simple, convenient relatively inexpensive reagents for the routine detection of immunoglobulin on cell surfaces by immunofluorescence microscopy. The preparation of F(ab')2 fragments of anti-immunoglobulin, which are necessary when rabbit antibodies are used, does not appear to -e required if goat antibodies can be empolyed. This observation has multiple practival applications in cellular immunology.     

Formalin inactivation of vesicular stomatitis virus impairs T-cell- but not T-help-independent B-cell responses.

The effects of formalin on the infectivity and immunogenicity of vesicular stomatitis virus (VSV) serotype Indiana were investigated. We found that formalin inactivation of VSV prevents infection of Vero cells in a concentration- and time-dependent manner, as shown by fluorometric cell analysis and inhibition of plaque formation. Inactivated VSV failed to induce significant cytotoxic T-lymphocyte responses in vivo or after restimulation in vitro. In contrast, the early immunoglobulin M (IgM) response, which is T help independent in the VSV system, was unaltered, suggesting normal antigenicity for and induction of B cells. However, no switch to IgG occurred, demonstrating failure of induction of T help. If cross-reactive T help was provided by previous infection with a second serotype of VSV (New Jersey), the IgG response was almost completely restored, confirming that the absence of IgG was due to lack of T help. A formalin-treated preparation of glycoprotein of VSV led to a delayed but otherwise normal IgG response, whereas treatment of VSV with UV light or beta-propiolactone reduced IgG titers to the same extent as did formalin. These results suggest that loss of infectivity and the ensuing lack of amplification of viral antigens of formaldehyde-inactivated VSV is the major factor impairing induction of specific T-helper cell responses.     

The spontaneous release of a high-molecular-weight aggregate containing immunoglobulin G from the surface of Ehrlich ascites tumor cells

The spontaneous release of tumor cell antigens from the cell surface into the circulation has been proposed as a mechanism whereby tumors may escape the immune response of the host. In this study we have found that Ehrlich ascites tumor cells after removal from the host (mouse) spontaneously release significant amounts of cell surface components during incubation for 1 h in cold isotonic buffer. Immunodiffusion studies revealed that immunoglobulin G (IgG) and a complement component (C3) are included in this spontaneously released material. These surface-bound humoral immune components are apparently released in the form of a high-molecular-weight aggregate (cell coat particle) as shown by ultracentrifugation and ultrafiltration experiments. Precipitation of IgG from the cell coat particle preparation with antibodies directed against mouse IgG followed by detergent gel electrophoresis of the immune precipitate revealed five major bands in addition to the heavy and light chains of IgG. These results suggest that host IgG is tightly bound to several other components at the cell surface, perhaps in the form of immune complexes. IgG is localized on the tumor cell surface in a highly heterogeneous pattern with the appearance of patches and caps in some cells as shown by immuno-fluorescence analysis. The possibility that humoral immune components bind to the tumor cell surface and result in the shedding of high-molecular-weight aggregates of cell surface antigens into extracellular fluids is discussed.     

Membrane associated immunoglobulin in pig thymocytes

The polypeptides and glycoproteins in plasma membranes of thymocytes and mesenteric lymph node cells have almost identical patterns in polyacrylamide gels. Thymocyte plasma membranes contain IgG which comprises about 0.3% of the total membrane protein. IgM has not been detected. Previous studies with lymph node lymphocyte membranes have shown 0.6% immunoglobulin, containing both γ and μ determinants. Therefore, in their overall polypeptide composition and the possession of immunoglobulin, the membranes from the two types of cells are strikingly similar to each other.     

Bovine Immunoglobulin/Protein Isolate Binds Pro-Inflammatory Bacterial Compounds and Prevents Immune Activation in an Intestinal Co-Culture Model

Intestinal barrier dysfunction is associated with chronic gastrointestinal tract inflammation and diseases such as IBD and IBS. Serum-derived bovine immunoglobulin/protein isolate (SBI) is a specially formulated protein preparation (>90%) for oral administration. The composition of SBI is greater than 60% immunoglobulin including contributions from IgG, IgA, and IgM. Immunoglobulin within the lumen of the gut has been recognized to have anti-inflammatory properties and is involved in maintaining gut homeostasis. The binding of common intestinal antigens (LPS and Lipid A) and the ligand Pam3CSK4, by IgG, IgA, and IgM in SBI was shown using a modified ELISA technique. Each of these antigens stimulated IL-8 and TNF-α cytokine production by THP-1 monocytes. Immune exclusion occurred as SBI (≤50 mg/mL) bound free antigen in a dose dependent manner that inhibited cytokine production by THP-1 monocytes in response to 10 ng/mL LPS or 200 ng/mL Lipid A. Conversely, Pam3CSK4 stimulation of THP-1 monocytes was unaffected by SBI/antigen binding. A co-culture model of the intestinal epithelium consisted of a C2BBe1 monolayer separating an apical compartment from a basal compartment containing THP-1 monocytes. The C2BBe1 monolayer was permeabilized with dimethyl palmitoyl ammonio propanesulfonate (PPS) to simulate a damaged epithelial barrier. Results indicate that Pam3CSK4 was able to translocate across the PPS-damaged C2BBe1 monolayer. However, binding of Pam3CSK4 by immunoglobulins in SBI prevented Pam3CSK4 translocation across the damaged C2BBe1 barrier. These results demonstrated steric exclusion of antigen by SBI which prevented apical to basal translocation of antigen due to changes in the physical properties of Pam3CSK4, most likely as a result of immunoglobulin binding. This study demonstrates that immunoglobulins in SBI can reduce antigen-associated inflammation through immune and steric exclusion mechanisms and furthers the mechanistic understanding of how SBI might improve immune status and reduce inflammation in various intestinal disease states.     

Response and specificity of antibodies for Shigella flexneri: demonstration of type-specific factors in immunoglobulin G fraction of antiserum

Considerable amounts of immunoglobulin G (IgG) antibody appeared in hyperimmune rabbit serum at a late period during a course of immunization with several injections of Shigella flexneri O antigens. High yields of IgG antibody possessing homogenous specificity could be fractionated from crude gamma-globulin solution on a diethylaminoethyl-Sephadex column with 0.02 m phosphate buffer (pH 6.6) containing 0.1 m NaCl. Specificities of IgG antibodies for six serotypes of S. flexneri were demonstrated to be high as compared with those of whole sera and their IgM antibodies. Type-specific factors for antigens I to VI were shown in each IgG fraction according to serotype employed. Further, in most sera, subtype-specific factors could be detected in the IgG fraction. These results suggest that it would be desirable to use IgG antibodies for the typing of S. flexneri.     

Optimization of a ligand immobilization and azide group endcapping concept via "Click-Chemistry" for the preparation of adsorbents for antibody purification

This report describes and compares different strategies to deactivate (endcap) epoxide groups and azide groups on bio-chromatographic support surfaces, before and after ligand attachment. Adsorbents possessing epoxide groups were deactivated using acidic hydrolysis or were endcapped with 2-mercaptoethanol or 2-ethanolamine. The influence of surface-bound 2-ethanolamine was demonstrated for the triazine-type affinity adsorbent B14-2LP-FractoAIMs-1, which was tested in combination with the weak anion exchange material 3-aminoquinuclidine-FractoAIMs-3 (AQ-FA3). Azide groups were modified with 2-propargylalcohol using Click-Chemistry. Besides the conventional one-pot Click reaction, an alternative approach was introduced. This optimized Click protocol was employed (i) for the preparation of the weak anion exchange material AdQ-triazole-Fractogel (AdQ-TRZ-FG) and (ii) for the endcapping of residual azide groups with 3-propargyl alcohol. Using the new Click reaction protocol the ligand immobilization rate was doubled from 250 to 500 μmol/g dry adsorbent. Furthermore, the modified support surface was proven to be inert towards the binding of immunoglobulin G (IgG) as well as feed impurities. A thorough evaluation of modified surfaces and adsorbents was performed with dynamic binding experiments using cell culture supernatant containing monoclonal human immunoglobulin G (h-IgG-1). Besides SDS-Page, a recently introduced Protein A-size exclusion HPLC method (PSEC-HPLC) was used to visualize the feed impurity composition and the IgG content of all collected sample fractions in simple PSEC-Plots. A surprising outcome of this study was the irreversible binding of IgG to azide modified surfaces. It was found that organic azide compounds, e.g. 1-azide-3-(2-propen-1-yloxy)-2-propanol (AGE-N3) promote antibody aggregation to a slightly higher extent than the inorganic sodium azide. The possibility that the Hofmeister Series of salt anions may be applicable to predict the properties of the corresponding organic compounds is discussed.     

Humoral immune response in animals after the oral administration of group C meningococcal whole-culture preparation

Experimental data giving grounds for the development of a new group C meningococcal whole-culture preparation for oral administration are presented. The study revealed that the use of the controlled cultivation of group C meningococci and a nutrient medium with definite chemical composition in combination with the "soft" method of the isolation of the whole-culture preparation ensured the preservation of polysaccharide, outer membrane protein and lipooligosaccharide in a native state, as well as retaining their full antigenic value, in the preparation. The oral immunization with the whole-culture preparation stimulated the multiple elevation of the level of hemagglutinating and IgG antibodies to these antigens and their prolonged preservation in the blood of immunized animals.     

Effect of urea on aggregation of immunoglobulins in water solutions

The methods of molecular light scattering and of gel filtration were used to study the degree of urea-induced desaggregation of immunoglobulin (IgG) molecules preaggregated by heating. Urea was shown to desaggregate considerably (70--80%) protein IgG aggregates. The data obtained suggest an important role of hydrophobic interactions in the immunoglobulin aggregation.