Loss of Rubella Antibody from Immune Globulin Treated with Kaolin

Sera and immune globulin (IG) preparations are customarily treated with kaolin before titration of their rubella hemagglutination-inhibiting (HI) antibody in order to rid them of nonspecific inhibitors of hemagglutination. The treatment was shown in this investigation to have no adverse effect on the antibody level of the sera but was found to remove considerable amounts of gamma-globulin from IG preparations. Evidence of this removal was obtained by serological tests, by spectrophotometric determination of protein concentration and by disc electrophoresis. In contrast to kaolin, heparin-manganese chloride (MnCl(2)) treatment of IG preparations had essentially no effect on the level of antibody globulin by all the criteria used. Heparin-MnCl(2)-treated IG lots were in these respects similar, if not identical, to their untreated counterparts. Since nonspecific inhibitors associated with the beta-lipoprotein fraction of serum are removed by the method employed to fractionate the IG samples, it seems unnecessary to treat the latter in any way for the HI test. No difficulty was encountered in this investigation with several untreated IG lots.     

人巨细胞病毒IgG 抗体标准品研究进展

人巨细胞病毒(CMV)是威胁人类健康的最重要病原之一。高CMV抗体效价的免疫球蛋白G(IgG)制剂,为临床医生在预防和治疗用药上提供了一个有价值的选择。而CMV免疫球蛋白标准品对于制品的CMV抗体效价测定以及高效价血浆的筛选都至关重要。该标准品对于器官移植/输血安全测试,以及临床诊断都是不可或缺。本综述提供了一种人巨细胞病毒IgG标准品制剂方法以及目前研究进展的概述。此外,本文还关注应用于不同领域的不同CMV IgG抗体效价单位。故本文为人巨细胞病毒免疫球蛋白的开发,人巨细胞病毒IgG抗体诊断试剂的标准化,以及为其质量控制提供参考。     

Production of an diagnostic erythrocyte agent from Pseudomonas aeruginosa exotoxin A

The sensitization of formolized sheep red blood cells with exotoxin A by means of chromium chloride or glutaraldehyde is more effective with respect to their sensitivity in the passive hemagglutination test than loading by means of amidol, tannin and rivanol. The use of chromium chloride decreases the consumption of exotoxin A 2, 8, 16 and 16 times in comparison with the use of amidol, tannin, rivanol or glutaraldehyde respectively. The high specificity of erythrocyte diagnosticum obtained from exotoxin A by means of chromium chloride is indicated in the study of hyperimmune sera to 22 different antigens of enteric bacteria and staphylococci in the passive hemagglutination test and to 10 different enterobacterial and staphylococcal antigens in the antibody neutralization test.     

Design of stable immunoglobulin erythrocyte diagnostica. 1. Erythocyte fixation and their sensitization by specific immunoglobulins]

The work presents the results of developing the method of fixation of erythrocyte constituting the cellular base of immunoglobulin erythrocytic diagnostic preparations and the sensitization of erythrocytes with immunoglobulin preparations of various specificity. Based on Ingraham's method, modified method of erythrocyte stabilization has been developed; it consists in the treatment of 50% cell suspension with 4% formaldehyde solution in the presence of 0.5% sucrose (erythrocyte suspension and formaldehyde solution being in the ratio 1 : 2.5). An economic and highly productive technique of sensitizing erythrocytes with immunoglobulin preparations has been developed. The essence of this technique lies in the interaction between 6% suspension of erythrocytes treated with formalin and tannin and the equal volume of sensitin taken in a working dose. The work also presents the method of synthesizing the bifunctional compound fluoro-borate bis-daizonium complex (obtained from benzidine) and discusses the comparative possibilities of the methods of developing immunoglobulin erythrocytic diagnostic preparations by sensitization of tannin-treated erythrocytes and by chemical conjugation.     

Erythrocytic diagnostic agents for determining antibodies to Proteus O antigens

Highly sensitive and specific erythrocyte diagnostic agents (ED) for the determination of antibodies to Proteus O-antigens have been obtained by the sensitization of formolated sheep red blood cells (SPBC) with activated lipopolysaccharides (LPS) without the use of mediators. The tannin treatment of formolated SRBC and/or the increase of temperature from 45 degrees C to 100 degrees C in the process of the preparation of ED have been found to produce no increase in effectiveness. Antibody ED permitting the detection of Proteus O- and H-antigens has been obtained by the sensitization of formolated chick red blood cells with immunoglobulin preparations to Proteus hydroxylamine antigens, carried out with the use of amidol. The experiments have shown the possibility of using this antibody ED for the determination of O-antibodies in the antigen neutralization test with nonactivated LPS used as an agglutinating agent. The passive hemagglutination test with antibody ED has proved to be a more sensitive method for the detection of O-antibodies than the antigen neutralization test with antigenic ED. The determination of Proteus etiology in the passive hemagglutination test with the use of antigenic ED has been shown to be highly effective in the examination of patients with chronic osteomyelitis at the stage of exacerbation.     

Mice immunized with measles virus develop antibodies to a cell surface receptor for binding virus.

Mice were immunized with measles virus to determine whether an auto-anti-idiotypic antireceptor response could be generated as a probe for measles virus receptors. Mice initially responded to viral antigens (days 11 to 18) and subsequently developed antibodies to a putative measles virus receptor (peak at day 30 to 35) by three criteria: the sera (1) agglutinated erythrocytes which virus agglutinates, (2) reacted with Vero cells, and (3) inhibited virus attachment to Vero cells. Additionally, select sera inhibited virus infection of Vero cells. The cell-reactive activity was identified as immunoglobulin G antibody and was neutralized by sera reacting with virus (idiotype). The application of this anti-idiotypic antibody to identify measles virus-binding sites on Vero cells was revealed by the ability of sera to immunoprecipitate 20- and 30.5-kilodalton proteins from metabolically labeled ([35S]methionine) Vero cells.     

Induction of Immune Response by Synthetic Fragments of the Bovine Prion Protein and Their Analogues in Mice of Various Lines

Antibodies to the bovine prion protein were produced by immunizing mice of three lines with five synthetic fragments of the protein and six of their analogues. The analogues contained amino acid substitutions that, according to theoretical calculation, should lead to an increase in the immunogenic activity of peptides. All the peptides except for one induced the formation of antibodies. All the sera containing the antipeptide antibodies were tested by an immunohistochemical method. The sera effectively bound to brain preparations from an animal with spongiform encephalopathy were identified; it was shown that they do not interact with preparations of normal brain. Therefore, it was shown that the immunization of mice with synthetic fragments of a prion protein allows one to obtain specific antibodies suitable for the study and diagnostics of prion diseases.     

The improvement of immunobiological preparations against leptospirosis. An experimental study of a new concentrated purified vaccine against icterohemorrhagic leptospirosis for human immunization

The findings of the study of immunological structure of the population in regions endemic for leptospirosis indicate that the immune status of humans makes it impossible to obtain titrated blood sera for the preparation of antileptospirosis immunoglobulin. The data obtained in the study of the immunobiological properties of a new concentrated vaccine against icterohemorrhagic leptospirosis show the possibility of using this vaccine for the immunization of donors with the aim of obtaining blood sera to be used as raw material for the production of immunobiological preparations.     

The development by cytomegalovirus-infected cells of binding affinity for normal human immunoglobulin.

After infection with human cytomegalovirus (CMV), cells develop an affinity for normal human immunoglobulin G (IgG). This was demonstrated using 125iodine-labeled purified IgG. It was further demonstrated that the immunoglobulin molecule binds to CMV-infected cells via its Fc portion, and competition for binding to infected cells occurred between purified preparations of human IgG and the Fc fragment of human IgG. Whole sera from individuals with or without a high titer of anti-CMV antibody were labeled with 125iodine and it was demonstrated that serum from individuals with no anti-CMV antibody had an affinity for CMV-infected cells which probably reflected binding of IgG via its Fc fragment. The possible significance of these results in immunologic studies of human CMV is considered.     

Induction of immune response by synthetic fragments of the bovine prion protein and their analogues in mice of various lines

The antibodies to the bovine prion protein were produced by immunizing mice of three lines with five synthetic fragments of the protein and their six analogues. The analogues contained the amino acid substitutions that, according to theoretical calculation, should lead to an increase in the immunogenic activity of peptides. All the peptides, except for one, induced the formation of antibodies. All the sera containing the antipeptide antibodies were tested by an immunohistochemical method. The sera that were effectively bound to the brain preparations from the bovine with spongiform encephalopathy were identified; it was shown that they do not interact with the preparations of normal brain. Therefore, it was shown that the immunization of mice with the synthetic fragments of a prion protein helps obtain specific antibodies suitable for the study and diagnostics of prion diseases.     

Inhibition of the immune response by membrane-bound antibody.

Cells from the spleens of "normal" swine, which were pretreated with pronase to remove surface membrane-bound immunoglobulin, gave an enhanced hemolytic plaque-forming cell response to sheep red blood cells in vitro in comparison with untreated controls. The enhancement could be abrogated by preincubating pronase-treated spleeen cells in preparations containing antibody to sheep red blood cells. This effect was demonstrated by autologous sera, immune sera, and all three known classes of porcine serum immunoglobulins, including IgM, IgA, and IgG and could be removed by absorption with sheep red blood cells. Surface membrane-bound antibody exerted its effect by binding to the nonadherent cell population. The response of normal spleen cells was unaffected by antibody treatment. Pronase-treatment was not mitogenic, did not function as a polyclonal B cell activator, and did not selectively eliminate T or B cells. The results indicate that removal of antibody from the surface of lymphoid cells enhanced the humoral immune response invitro and confirm that membrane-bound antibody can inhibit response to antigen.     

Modified Hemagglutination-Inhibition Test for Rubella Employing Human Group O Erythrocytes

A hemagglutination-inhibition (HI) test for rubella is described which utilizes human group O, rather than 1-day-old chick, erythrocytes. The test was found to be as sensitive and reproducible for detection of rubella antibody as HI tests employing chick erythrocytes. Advantages to the use of human erythrocytes are (i) they are more available, (ii) it is unnecessary to absorb natural agglutinins from human test sera, and (iii) heparin-MnCl₂-treated sera do not agglutinate human erythrocytes, as is sometimes the case with chick erythrocytes. Factors influencing the reliability of the test are discussed.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

The production of anti-hapten antibody after immunization with trinitrophenylated (TNP) hamster erythrocytes (HRBC) or sheep erythrocytes (SRBC) was determined in high- and low-responder mouse strains against HRBC antigen. 1) Anti-TNP antibody was detected in sera of high-responder DDD and CF1 mice after primary immunization with TNP-HRBC, but not in those of low-responder C57BL/6 mice. 2) Anti-TNP antibody was detectable in sera of all the strains after primary immunization with TNP-SRBC. 3) Production of anti-TNP antibody was elicited after a booster injection of TNP-HRBC in low-responder C57BL/6 mice pre-sensitized with HRBC in Freund's complete adjuvant. These results suggest that functions of thymus-derived cells specific for HRBC antigen are deficient in low-responder mice.     

IMGT-Choreography for immunogenetics and immunoinformatics

IMGT, the international ImMunoGeneTics information system (http://imgt.cines.fr), was created in 1989 at Montpellier, France. IMGT is a high quality integrated knowledge resource specialized in immunoglobulins (IG), T cell receptors (TR), major histocompatibility complex (MHC) of human and other vertebrates, and related proteins of the immune system (RPI) which belong to the immunoglobulin superfamily (IgSF) and MHC superfamily (MhcSF). IMGT provides a common access to standardized data from genome, proteome, genetics and three-dimensional structures. The accuracy and the consistency of IMGT data are based on IMGT-ONTOLOGY, a semantic specification of terms to be used in immunogenetics and immunoinformatics. IMGT-ONTOLOGY has been formalized using XML Schema (IMGT-ML) for interoperability with other information systems. We are developing Web services to automatically query IMGT databases and tools. This is the first step towards IMGT-Choreography which will trigger and coordinate dynamic interactions between IMGT Web services to process complex significant biological and clinical requests. IMGT-Choreography will further increase the IMGT leadership in immunogenetics and immunoinformatics for medical research (repertoire analysis of the IG antibody sites and of the TR recognition sites in autoimmune and infectious diseases, AIDS, leukemias, lymphomas, myelomas), veterinary research (IG and TR repertoires in farm and wild life species), genome diversity and genome evolution studies of the adaptive immune responses, biotechnology related to antibody engineering (single chain Fragment variable (scFv), phage displays, combinatorial libraries, chimeric, humanized and human antibodies), diagnostics (detection and follow up of residual diseases) and therapeutical approaches (grafts, immunotherapy, vaccinology). IMGT is freely available at http://imgt.cines.fr.     

Brucellar antibody erythrocytic diagnostic agent made from monoclonal antibodies

Two preparations based on monoclonal antibodies to bacteria of the genus Brucella have been obtained. From the monoclonal preparations and globulins isolated from them erythrocyte diagnostica have been obtained with the use of amidol. Experiments on the cross indication of brucellae and other bacteria by means of these diagnostica and a similar preparation obtained from polyclonal serum have shown very high specificity of erythrocyte immunoreagents prepared from monoclonal antibodies.     

Trophoblastic beta-globulin in immunoglobulin preparations

The content of trophoblastic beta-globulin in 142 lots of commercial immunoglobulin preparations from 20 manufacturers, produced from placental, abortion and donor blood sera, has been studied. 83% of lots from abortion blood serum and 94% of lots from placental blood serum have been found to contain the admixture of this beta-globulin, its concentration in the lots from placental blood serum being significantly higher. The method for the detection of trophoblastic beta-globulin may be used for evaluating the quality of immunoglobulin preparations as it indicates the degree of their purification from placental proteins.     

Hypotensive properties of antibodies directed against an endogenous pressor peptide isolated from rat blood

Hypertensive factor (HF), a compound isolated from the erythrocytes of rats and tentatively identified as a peptide, has been shown to influence tissue calcium metabolism and induce prolonged blood pressure elevation. In the present study, we investigated the biological properties of antibodies directed against this peptide. Partially purified antibody preparations significantly decreased HF stimulation of lanthanum-resistant calcium uptake in rat aortic tissue in vitro. Infusion of the antibody preparation into spontaneously hypertensive (SH) or normotensive Sprague-Dawley rats resulted in a rapid decline in mean blood pressure of 54 and 34 Torr (1 Torr = 133.332 Pa), respectively. In contrast, infusion of the serum immunoglobulin preparations from controls (unimmunized and ovalbumin-immunized rabbits) had no significant effect on the blood pressure of SH or normotensive rats. The systolic blood pressure of SH rats was reduced for at least 72 h following a single injection of the antibody preparations, whereas the blood pressure of normotensive rats had returned to normal levels within 24 h following antibody injection. The results indicate that the anti-HF antibody preparation antagonizes the stimulation of calcium uptake by the peptide and acutely lowers blood pressure in SH and normotensive rats.     

Antibodies against synthetic fragments of the prion protein for the diagnosis of bovine spongiform encephalopathy

Seven peptides matching fragments of the prion protein and containing from 17 to 31 amino acid residues were synthesized to obtain antibodies for diagnostics of bovine spongiform encephalopathy. Rabbits were immunized with either free peptides or peptide-protein conjugates to result in sera with a high level of antipeptide antibodies. Immunohistochemical assay revealed sera against four free peptides and a protein-peptide conjugate, which effectively bind to the pathogenic isoform of the prion protein in brain tissue preparations from cattle afflicted with bovine spongiform encephalopathy and do not interact with normal brain preparations. The resulting antipeptide sera can be used in developing a diagnostic kit for bovine spongiform encephalopathy.     

A subclass of albumin recognized by inhibition of phosphatidylethanolamine-mediated agglutination of mouse erythrocytes

Agglutination of mouse erythrocytes by non-choline phospholipids is inhibited by a factor in mammalian sera. The inhibitor cochromatographed with albumin on dye-agarose conjugates, was retained by an anti-albumin affinity column, was neutralized by anti-albumin antibody and found in a serum fraction in which only albumin could be detected. A variety of commercial preparations of albumin (fraction V, crystalline) did not inhibit. However, they acquired potent inhibitory activity when treated with low molecular weight thiols. The inhibitory activity of serum was increased 8-fold by treatment with dithiothreitol. Other proteins were not activated in this way. Inhibitory activity increased with average free sulphydryl content of treated albumin, up to six thiol groups per molecule. Alkylation of these sulphydryl groups did not diminish inhibitory activity. Thiols also induced polymerization of albumin. Inhibitory albumin in serum was largely monomeric. We propose that the inhibitor is a type of serum albumin which is lost or inactivated during preparation of commercial albumin, and which shares a structural feature, necessary for inhibition, with thiol-reduced albumin and the ligand on mouse erythrocytes.     

Elastin V: Syntheses, Characterization, and Staining Properties of Amidol Black I

One of the monoazo dyes reported by us in an earlier study (Lillie et at. 1972), amidol brown NAP, yielded an oxidation product, amidol black I, which was an excellent elastin stain. The present study revises the synthesis of amidol black I to a more convenient form giving much larger yields. Briefly, diazotized 4-nitro-2-aminophenol is azo-coupled into 2, 4-diaminophenol (amidol) and the product is oxidized to a quinoneimine with nitric. The product stains elastin black from an acid alcohol bath according to a Taenzer-Unna (1890) orcein type technic. The stain may be combined with a Van Gibson collagen stain or with an oil red O fat stain. The synthesis is presented in sufficient detail to permit its repetition in a hospital pathology laboratory.