Normal Γ-globulin Research Articles

Prevalence of monoclonal proteins in patients with isolated hypogammaglobulinemia on serum protein electrophoresis

Isolated hypogammaglobulinemia (IH) is an electrophoretic pattern that can be encountered on serum protein electrophoresis (SPEP) and is defined as a decreased but morphologically normal γ-globulin fraction with normal α- and β-globulin fractions. SPEP is mainly used to detect monoclonal proteins which are usually observed as additional peaks in the electropherogram. However, they may also be more discretely present in a significant proportion of patients presenting with IH. Therefore, we aimed to evaluate i) via both retrospective and prospective analysis to what extent paraproteins as identified by immunofixation are present in patients demonstrating IH on SPEP and ii) whether other parameters may predict their presence in IH-patients. For this purpose, we first reviewed historic SPEP- and immunofixation results in our tertiary hospital and determined paraprotein prevalence in this retrospective cohort. This analysis showed immunofixation was requested in only 519/3938 (13.2%) historic IH-results with 52/519 (10%) patients demonstrating paraproteins. Next, various laboratory parameters were compared between paraprotein-positive and -negative patients and subjected to logistic regression models but regrettably, no parameter could be retained as promising predictor of paraproteins. Lastly, to confirm paraprotein prevalence seen in the historical query, we conducted a six-month prospective analysis during which immunofixation was requested more frequently in IH-cases during routine diagnostics and which showed paraproteins to be present in 20/83 (24.1%) of IH-patients. Hence, as up to 24% of patients with IH may harbour paraproteins, one should consider performing follow-up analyses (e.g. immunofixation, urine electrophoresis and/or free light chain analysis) for all IH-cases identified via SPEP.

Pediatric Clostridium difficile: a phantom menace or clinical reality?

Clostridium difficile is the leading cause of nosocomial gastrointestinal illness in adult patients in hospitals. Even though C. difficile disease in adults has been well studied, research on pediatric C. difficile disease is still in its infancy. For many years it has been believed that C. difficile was a disease that affected only adults and was not a problem for children. This erroneous belief arose from the observation that neonates acquire C. difficile quickly (within 48 hours of birth) but show no intestinal symptoms (1–4). More recent evidence has been documented in case reports of pediatric C. difficile and outbreaks of C. difficile disease in pediatric populations (5– 7). Pediatric C. difficile disease has also been associated with the occurrence of severe complications and high mortality rates (8–10). The cascade of events in the pathogenesis of C. difficile disease is similar in children and adults. Normal intestinal microflora are disrupted by antibiotic exposure, medications, or surgery. If the child is then exposed to C. difficile (or its spores), colonization may occur, with production of toxins A and B. These toxins act on enterocytes, causing an inflammatory response and morphologic changes that lead to diarrhea or colitis. Host factors (age, diet, immune response) play an important role in determining whether C. difficile develops into asymptomatic carriage or active disease. Treatment for pediatric C. difficile disease usually relies on metronidazole or vancomycin, but clinical guidelines have not been defined for the pediatric population (11). As in adults, recurrent C. difficile disease that does not respond to conventional therapy develops in a proportion of children treated with antibiotic therapy.

Brain neuropeptide Y in the control of adrenocorticotropic hormone secretion in the dog

An immunoneutralization technique with specific antibodies was used to explore the role of neuropeptide Y (NPY) in the adrenocorticotropic hormone (ACTH) release after hypoglycemic stress in the dog. Dogs received injections of rabbit antihuman NPY γ-globulin (anti-NPY) or normal γ-globulin (NGG) into the third cerebral ventricle, which was followed by i.v. injection of insulin. Hypoglycemia of a 40% fall in systemic glucose levels occurred in anti-NPY-treated dogs as well as NGG-treated animals. An intraventricular administration of anti-NPY significantly inhibited the ACTH and cortisol release to hypoglycemia, but had no effect on the pancreatic polypeptide (PP) response. These findings suggest involvement by endogenous NPY in the ACTH secretion induced by hypoglycemia.

Distribution of Actinobacillus actinomycetemcomitans in localized juvenile periodontitis plaque: an electron immunocytochemical study.

The use of an indirect electron immunocytochemical labelling procedure for labelling Actinobacillus actinomy cetemcomitans (serotype b) in ultrathin sections of pure bacterial cultures and in localized juvenile periodontitis‐derived plaque is described. Optimal labelling was obtained after fixation with 2.5% glutaraldehyde, embedding in glycol methacrylate and labelling with highly diluted absorbed rabbit anti‐A. actinomycetemcomtians γ‐globulin followed by incubation with diluted goat anti‐rabbit γ‐globulin conjugated with horseradish peroxidase. and subsequent histochemical visualization of the peroxidase. These labelling conditions were used to determine that no cross reactions existed with commonly found plaque bacteria. Two of 5 labelled plaque specimens were positive for this A. actinamycetemcomitans serotype. In one specimen large numbers of labelled bacteria were located only in the most apical portion of the plaque layer. In another specimen labelled cells were scattered throughout the plaque. Control experiments using normal rabbit γ‐globulin were all negative.

Natural Killing of Measles-Infected Cells by Human Lymphocytes

Abstract HeLa cells persistently infected with measles virus (M-HeLa) show markedly increased susceptibility to natural killing (NK) by normal human blood lymphocytes compared to uninfected HeLa cells. The killing is mediated by the same population of non-B, non-T lymphocytes with avid Fc receptors for IgG that mediate other forms of NK and antibody-dependent cellular cytotoxicity (ADCC). There is no evidence that this NK is mediated by antibody; specifically, the killing regenerates after being abolished by extensive treatment of the effector cells with trypsin or pronase, and it cannot be inhibited by large amounts of F(ab′)2 fragments of anti-measles antibody or by a large excess of normal γ-globulins. In addition, NK of M-HeLa is strongly correlated (r = 0.79, p < 0.001) with NK of an unrelated cell line (K-562) that does not contain measles virus and is a standard target for NK. The strong correlation between NK of M-HeLa and K-562 is confirmed by cold-target inhibition studies showing cross-competition between the two cell lines. Killing cannot be enhanced by addition of supernatants from lymphocytes cultured with M-HeLa to fresh lymphocytes cultured with M-HeLa, HeLa, K-562, or other cell lines, making it unlikely that interferon or other soluble factors have a role in this system. These studies suggest that persistent infection of HeLa cells with measles virus results in the modification of the HeLa cell membrane and that this modification is similar to some structure on K-562 that is important for NK. In addition, these studies raise the possibility that NK could play a role in host defenses against measles virus infection.

EFFICACY OF PROPHYLACTIC GAMMA-GLOBULIN IN PREVENTING NON-A, NON-B POST-TRANSFUSION HEPATITIS

Of 279 cardiac-surgery patients receiving a mean of twelve transfusions, 47 had significantly increased transaminase concentrations 14 to 180 days postoperatively and 10 were icteric. Preoperatively, each patient randomly received high-titre HbsAb γ-globulin, normal γ-globulin, or placebo and was followed at intervals for 9 months. Only 3 patients had serological evidence of hepatitis-B infection. 3 additional patients had serological evidence of cytomegalovirus infection, while none had evidence of hepatitis-A or Epstein-Barr infection. Less icteric hepatitis occurred in patients receiving the γ-globulin preparations (p=0·003), and the overall frequency of hepatitis was significantly reduced when compared with recipients of placebo. The protective effects of the two γ-globulin preparations were not significantly different. Most post-transfusion hepatitis today is neither viral hepatitis type B nor type A, and its severity and transmission are reduced by pre-transfusion γ-globulin.

Immunochemical studies of l-amino acid oxidase

Differences were found between l-amino acid oxidase ( l-amino acid:O 2 oxidoreductase (deaminating), EC 1.4.3.2) and its warm-inactivated and frozen-inactivated forms by complement fixation and immunoelectrophoresis. The enzyme inactivated by freezing had more alteration than that treated at 38°. These immunologically detected differences disappeared upon reactivation. Isozyme A also differed from the total mixture of isozymes. The antibody to the native does not inhibit enzymic activity but does protect against warm inactivation. Antibody also shifted the equilibrium between active and inactive forms towards the active form and accelerated reactivation of both warm and frozen forms. Normal γ-globulin also shifts the above equilibrium toward the active form.

Competition of normal gamma-globulins with cytophilic antibody for corneal cells.

Sections of cornea from normal rabbits when incubated with rabbit antiserum to <i>β</i>-galactosidase (BG<sub>Z</sub>) and washed were shown to bind BG<sub>Z</sub>. This passive sensitization is presumably mediated by a ‘cytophilic’ antibody present in the hyperimmune serum. Incubation of the corneal discs with unfractionated normal rabbit serum or its <i>γ</i><sub>2</sub>-IgG fractions, significantly reduced the ability of the discs to bind cytophilic antibody. This inhibition was produced only by normal <i>γ</i><sub>2</sub>-IgG and not by <i>γ</i><sub>1</sub>-IgG which indicates the presence of specific receptors on cell surfaces for binding cytophilic antibody. Preemption of these receptors and thereby inhibition of passive sensitization of cornea by normal <i>γ</i><sub>2</sub>-globulin is presented as a possible means to reduce the manifestations of allergic responses in the eye.

Ratio of the Fab fragments I and II from goat antibodies normal γ-globulins

Ratio of the Fab fragments I and II from goat antibodies normal γ-globulins

The Search for Antibodies with Molecular Uniformity

Publisher Summary This chapter discusses the physical, chemical, and biological aspects of γM antibodies in depth. The chapter describes the physical characteristics, chemical composition, and subunit structure of the typical circular γM pentomers and the relationship of these molecules to low molecular weight γM-like proteins. The characteristics of the interaction of γM antibodies with antigens—for example, the nature and number of antigen combining sites—and the interaction of γM antibodies with the complement system are compared to the corresponding properties of γG antibodies. The biosynthesis and metabolism of γM antibodies and their peculiar role in the immune response are also discussed in the chapter. The chapter presents a brief summary related to the hallmarks of immunoglobulin heterogeneity and the criteria that must be used to judge the restricted heterogeneity or molecular uniformity of an antibody preparation. The chapter discusses human antibodies to certain selected antigens that appear to possess less heterogeneity than normal γ-globulin.

Reactions of intracellular crystals of foot-and-mouth disease virus with ferritin-tagged antibody.

Summary Crystals of intracellular foot-and-mouth disease virus, type A1, were reacted with ferritin conjugated to an A1 antibody, an O2 antibody, and normal γ-globulin as well as unconjugated ferritin. Specific tagging was demonstrated with both the full and ‘empty’ capsid crystals with type A1 virus and anti-A1 globulin. Anti-O2 ferritin-conjugated globulin, normal ferritin-conjugated γ-globulin and unconjugated ferritin did not attach to the A1 virus and showed only minimal reaction with tissue fragments and debris. Immunodiffusion in agar gel was used to demonstrate the specificity of the reagents.

The Synthesis and Secretion of γ-Globulin by Lymph Node Cells: V. THE DISSOCIATION OF THE MICROSOME-ANTIBODY COMPLEX WITH ENZYMATICALLY AND CHEMICALLY ALTERED γ-GLOBULINS

Microsomes derived from guinea pigs immunized with dinitrophenyl-protein conjugates possess firmly associated antibodies specific for the dinitrophenyl determinant. The fact that these antibodies can be specifically displaced by incubation with an excess of soluble, normal γ-globulins provides a method for studying the site (sites) on γ-globulin involved in the microsome-antibody complex. After digestion of γ-globulin or of the Fc fragment, several peptide fractions were separated by molecular sieve chromatography. All of these were found to be capable of solubilizing antibody from the microsomes. The peptides in one of the active fractions appear to be quite small as judged by their absorption into the matrix of Sephadex G-25 and G-15. The effect of mild periodate oxidation of γ-globulin on its ability to dissociate antibody from the microsomes was also examined. After oxidation, the ability of γ-globulin to displace antibody was increased 5-fold. In contrast, the reduction of a single disulfide bond located at the center of the heavy chains of γ-globulin resulted in a 5-fold decrease in the capacity of such γ-globulin to dissociate antibody. Direct reduction of the microsome-antibody complex also resulted in a loss of microsomal antibody. These findings and the fact that the reactive disulfide is adjacent to a threonine residue bearing one of the carbohydrate chains of γ-globulin are discussed in relation to the nature of the antibody-binding site on microsomes.

Electron Microscope Studies of Anti-Antibodies

The presence of anti-antibodies in sera was first proposed by Najjar and Fisher in 1955. There have subsequently been numerous demonstrations of anti-antibodies and the subject was recently reviewed.According to Najjar's proposal there should be at least two distinguishable types of antibodies involved in the formation of immune complexes. The first antibody is specific for the antigen while the second, anti-antibody, is directed against the antibody portion of the immune complex.This report involves visualizing the anti-antibody portion of complexes by positive staining. It also demonstrates their removal from equivalence complex in acid media.Thin sections of equivalence complex were prepared before and after partial dissociation by exposure to pH 2. 8. The sections were stained by floating on uranyl acetate solutions. Fig. 1 illustrates a preparation of equivalence complex while Fig. 2 shows the remainder of a complex partially dissociated at low pH. Equivalence complex contains large areas of interacting anti-antibodies which are detected by the absence of ferritin molecules. Such areas are greatly reduced or removed from the complex in Fig. 2. Normal γ-globulin mixed with ferritin forms no precipitate.

Carbodiimide-Induced Bentonite-Antigen Complexes: Readily Prepared Immunoadsorbents

Summary γ-globulin and albumin are bound to bentonite by l-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The reaction proceeds promptly at room temperature in physiologic solution. It is suggested than an organometallic bond results between protein and bentonite. The bond is stable under most conditions, and the resulting materials function well as immunoadsorbents. Rabbit antibody, isolated from the adsorbent, contains a trace of albumin and less than 1 part/1000 antigen. At equivalence, 92 to 97% of the protein in preparations of isolated antibody is precipitated by soluble antigen. Protein is adsorbed both to immunologically specific and non-specific sites on the antigen-bentonite complex. About 0.17 mole antibody is quantitatively bound per mole adsorbent antigen. In excess antibody, the capacity of the γ-globulin adsorbent exceeds 0.54 mole antibody per mole adsorbent antigen. Most antibody, in the presence of other serum protein, is bound to specific sites on the adsorbent and is readily eluted at pH 2.5. Normal γ-globulin and other proteins are bound to nonspecific sites, and remain largely bound at the pH of elution. It is likely that a small amount of antibody is bound to nonspecific, adsorbent sites and remains bound during elution. Nevertheless, estimates of serum antibody with the adsorbents exceed estimates made by quantitative precipitation and more accurately reflect total serum antibody.

The genetic control of gamma-globulin heavy chains. Studies of the major heavy chain subgroup utilizing multiple genetic markers.

The genetic control of gammaG1-heavy chains was investigated by taking advantage of two recently described genetic antigens, Gm(z) and Gm(y), both produced by heteroimmunization of rabbits with myeloma proteins. These were studied in conjunction with known genetic markers, Gm(a) and Gm(f). The results indicated that among Caucasians there are two major allelic genes, Gm(za) and Gm(fy), coding for distinct varieties of gammaG1-heavy chains. Each of these contains a pair of genetic antigens which are located on different fragments of the chain and can be separated by enzymatic splitting with papain. The different areas of the heavy chains appear to be under the control of the same gene. In Mongoloid populations a grouping of three genetic antigens, Gm(f), (y), and (a), was found on isolated myeloma proteins and normal gamma-globulins indicating the presence of a Gm(fya) gene. The possible genetic events leading to the contrasting Caucasian and Mongoloid genes are discussed. In the gamma-globulin system the occurrence of multiple genetic antigens in different positions of the same heavy chains is the general rule. A better understanding of the relationships between the genes for the gammaG1-subgroup to those for the gammaG2- and gammaG3-subgroup has been obtained through the use of the multiple genetic markers. Strong evidence was obtained for intergenic crossover mechanisms to explain racial differences in the relationships of these genes as well as certain unusual gene complexes found through family studies. Further evidence was obtained for mapping the closely linked genes for the three subgroups in a specific order.

Electrophoretic Analysis of Polypeptide Chains Isolated from Antibodies in the Serum of Immunized Rabbits

Summary The electrophoretic patterns of the sera from rabbits hyperimmunized with group A-variant and group C streptococcal vaccines exhibit a prominent narrow band in the γ-globulin region. This electrophoretically homogeneous γ-globulin was identified as antibody. L chains isolated from purified antibody preparations were shown to migrate as a narrow band on acid urea starch gel electrophoresis, whereas L chains from normal γ-globulin traveled as a diffuse smear. A limited number of L chain bands was also demonstrable using alkaline urea starch gel. All of the rabbits exhibiting this unusual antibody response were allotypically homozygous at both the a and b loci. Precipitin gel diffusion experiments suggest that the purified antibodies represent a limited population of the antigenic spectrum of normal rabbit γ-globulin. Thus, the immunologic and physicochemical properties of the γ-globulin isolated from these rabbits suggest a selective manufacture of an unusually homogeneous population of group specific carbohydrate antibodies.

On the heterogeneity of the light chains of rabbit antibodies

The ligh polypeptide chains froms several purified anti-hapten rabbit antibodies and normal γ-globulin were prepared and subjected to acrylamide gel electrophoresis. The number and distribution of the bands formed were very similar in all cases. Antibodies to the 2,4 dinitrophenyl hapten, either pooled or from individual, allotypically homozygous, rabbits, were affinity labeled with tritiated m-nitrobenzenediazonium fluoborate. The labeling was confined to the antibody active sites, and was found on both heavy and light chains. The labeled light chains were subjected to gel electrophoresis and the label was found to be essentially uniformly distributed within the bands. These results are consistent with the suggestion that any population of anti-hapten antibodies contains a number of different kinds of light chains which is substantially larger than the number of electrophoretic bands, and by inference that the total number of possible light chains in a normal γ-globulin population may be very large indeed.

Some physical-chemical properties of chicken γ-globulins and their pepsin and papain digestion products

Chicken γ-globulin has been purified and subjected to physical-chemical and to enzymatic degradation studies. The normal γ-globulins have a molecular weight near 206,000, have an isoelectric point near 5·2, and contain near 3·1 per cent hexose. Electrophoretic “fast” and “slow” components are produced with both papain and pepsin. The papain fast components retain some antigen combining activity. It is suggested that the normal chicken γ-globulins are more similar to the γA- than to the γG-globulins of mammalian species.

Structure studies on human serum γ-globulins and myeloma proteins: III. Oxidative sulfitolysis of myeloma globulins and reconstitution of the macromolecules

Individual specimens of myeloma globulins were cleaved by sulfite and cuprammonium ion, and the fragments were separated by gel filtration in acetic acid. Molecular weight determination indicated that both the H and L chains, after separation, recovery, and redissolution in a weakly alkaline solvent, appeared as dimers. When the cleaved globulins were treated with mercaptoethanol, the disulfide bonds were reformed. The reconstituted globulins appeared similar to the original native material in molecular weight and immunodiffusion tests. Slight differences were found in the optical rotatory dispersion behavior of the native and the reconstituted globulins. Individual specimens of the globulins differed among themselves and from normal γ-globulin with respect to the H L ratio, quantity of free N-terminal amino acids, and ease of reconstitution; but their chain conformations were similar. Significant differences were found in amino acid composition and peptide patterns between the H and L chains, and between individual L chains. The L chains showed similarity to Bence-Jones proteins in redissolving on boiling, conformation, immunodiffusion, and electrophoretic mobility in gels.

Univalence of Half-Molecules of Rabbit Antibody

Summary Dissociation of specifically purified rabbit antiovalbumin into half-molecules, followed by recombination at neutral pH, resulted in a 6 S product, 81% of which was precipitable by antigen. The sedimentation coefficient, precipitability, and shape of the precipitin curve indicated that most of the molecules in the product were bivalent. When the recombination was carried out in the presence of a large excess of dissociated normal γ-globulin, the product did not form specific precipitates with antigen, but specifically inhibited the reaction of ovalbumin with untreated anti-ovalbumin. The results are consistent with univalence of half-molecules of antibody and the previously demonstrated capacity of half-molecules of anti-ovalbumin to combine with halfmolecules of normal γ-globulin, while retaining the integrity of antibody combining sites. A procedure is described for specific purification of rabbit anti-ovalbumin and anti-bovine γ-globulin.