Monoclonal Antibodies against Enterocytozoon bieneusi of Human Origin

Enterocytozoon bieneusi is clinically the most significant among the microsporidia infecting humans, causing chronic diarrhea, wasting, and cholangitis in individuals with human immunodeficiency virus/AIDS. The lack of immune reagents is largely due to the absence of methods for laboratory propagation of E. bieneusi. We recently described a procedure for the concentration and purification of spores from diarrheic stool of infected humans. Purified spores were used to immunize mice for production and screening of monoclonal antibodies (MAbs) against E. bieneusi. The eight immunoglobulin M MAbs generated and fully characterized did not cross-react with other human microsporidia or with other microorganisms normally present in stool. One of the MAbs, 2G4, reacted with E. bieneusi spores in stools from monkeys and humans, without background fluorescence, which makes it an ideal diagnostic reagent. It also recognizes intracellular stages of the parasite and will be suitable for determining tissue distribution of E. bieneusi in infected hosts. At least two immunodominant antigens of E. bieneusi of 33,000 and 35,000 Da exist, which were recognized by rabbit and mouse antisera. The availability of MAbs against E. bieneusi will simplify considerably the diagnosis of this infection in humans and will provide tools for epidemiologic investigations regarding the true prevalence of the infection in various human and mammalian populations and the environmental sources of infection.     

Monoclonal Rat Antibodies to Rat Carcinoembryonic Antigen

An IgM-class rat monoclonal antibody, designated 5B1, was produced against rat carcinoembryonic antigen. This antibody, which can be considered an autoantibody, formed a single precipitation line in a gel diffusion test with an extract of the CEA-positive rat colonic adenocarcinoma, RCA-1. This line gave a reaction of complete identity with a line formed by a rabbit antiserum specific for rat CEA. 5B1 also bound to extracts of the RCA-1 tumor in EIA, but did not bind at all to extracts of the CEA-negative rat colonic adenocarcinoma, RCA-2, nor to extracts of normal rat tissues including liver, kidney and intestine. Inhibition tests showed that the activity of the monoclonal antibody in EIA was abolished by extracts of RCA-1 tumor, but was hardly affected by extracts of the other tissues.     

Monoclonal Antibodies against Human Monocytes

Three monoclonal antibodies (63D3, 63D2, and 61D3) with reactivity against human monocytes have been studied. They reacted with most adherent mononuclear cells but not with pure populations of peripheral blood B and T lymphocytes. Two of these antibodies, 63D3 and 63D2, competed in fluorescence inhibition experiments, recognized a monocyte surface antigem of about 200,000 daltons, and werre idiotypically related. The third antibody, 61D3, did not compete with the others in fluorescence inhibition experiments, recognized a different surface molecule, and was idiotypically distinct from 63D3 and 63D2. Whereas 63D3 and 63D2 reacted very weakly with some granulucytes, 61D3 did not, suggesting that it is specific for monocytes alone.     

Production of Non-pathogenic Human Monoclonal Antibodies to Desmoglein 3 from Pemphigus Vulgaris Patient

Pemphigus vulgaris is a potentially fatal autoimmune mucocutaneous disease associated with production of IgG autoantibodies to desmoglein 3, a 130 kDa epidermal protein. To further characterize the epitope(s) of pemphigus vulgaris antigen we established two human-human hybridoma by fusion of the peripheral blood mononuclear cells with a human and mouse heterohybridoma. These hybridomas designated as MAb Dsg-3: 06 and MAb Dsg-3: 10 and stable in culture and demonstrated yield of monoclonal antibodies specific for pemphigus vulgaris. Immunofluorescence, immunoblot, ELISA assays demonstrated that both the monoclonal antibodies bind to the intercellular cement substance and to 130 kDa protein present in the skin and specifically binds to recombinant desmoglein 3 protein, but not to desmoglein 1 protein. The IgG subclass distribution study demonstrated that both the antibodies are of IgG1 subclass in nature. Both the antibodies were non-pathogenic as demonstrated in vitro by their inability to produce acantholysis in normal human skin in organ culture or in vivo by the induction of disease in neonatal BALB/c mice. The relevance and value of these monoclonal antibodies in the pathogenesis of pemphigus vulgaris is discussed.     

Monoclonal Antibodies to Human Choriocarcinoma

ABSTRACT: We have established two monoclonal antibodies (TM7-3 and TM3-8) that react to choriocarcinoma cells. Both of these monoclonal antibodies have shown a similar reactive pattern to human cell lines, normal and neoplastic trophoblast tissues, and other fetal and adult tissues. They have reacted to nine of the ten choriocarcinoma cell lines, as well as to Hela cells (a cervical carcinoma cell line). During a cellular radioimmunoassay, neither TM7-3 nor TM3-8 reacted to two T lymphoblastoid cell lines or three B lymphoblastoid cell lines. Immunofluorescence and immunoper-oxidase staining showed that both monoclonal antibodies reacted selectively to the cytotrophoblast-like tumor cells of a choriocarcinoma and a hydatidiform mole but not to syncytiotrophoblast-like tumor cells. TM7-3 and TM3-8 also reacted slightly to the normal cytotrophoblast of early human chorionic villi under the same conditions as they did to choriocarcinoma tissue, but not to syncytiotrophoblast. In various normal tissues, TM7-3 and TM3-8 bind only to a part of the urinary tubles of the kidney and to the ducts of the pancreas of both adult and fetus.     

Diversity of Antigen Recognition by Serum Antibodies in Experimental Bovine Tuberculosis

Tuberculosis in cattle remains a major zoonotic and economic problem in many countries. The standard diagnostic assay for bovine tuberculosis, the intradermal tuberculin test, has low accuracy. Therefore, alternative immunodiagnostic methods, such as serological assays, are needed for detection of infected animals. Development of an accurate serodiagnostic test requires a detailed understanding of the humoral immune responses during bovine tuberculosis and, in particular, identification of the key antigens of Mycobacterium bovis involved in antibody production. In this study, we characterized antibody responses in cattle experimentally infected with M. bovis. Sequential serum samples were collected every 3 to 4 weeks for up to 27 months postinfection. Circulating immunoglobulin G antibody levels were measured by an enzyme-linked immunosorbent assay using 12 highly purified recombinant proteins of M. bovis. Six proteins, ESAT-6, 14-kDa protein, MPT63, MPT70, MPT51, and MPT32, were identified as major seroreactive antigens in bovine tuberculosis. A remarkable animal-to-animal variation of antigen recognition by serum antibodies was observed. Kinetic analyses of the antibody production to individual antigens during infection revealed that the heterogeneous antigen recognition profile changed markedly in a given infected animal as disease progressed.     

Monoclonal Antibodies to Acetylcholine Receptor Secreted by Human X Human Hybridomas Derived from Lymphocytes of a Patient with Myasthenia Gravis

Peripheral blood lymphocytes of a patient with myasthenia gravis (MG) were fused to the non-secreting human lymphoblastoid line HuNSI to produce human x human hybridomas that secrete monoclonal antibodies (mAb) to acetylcholine receptor (AChR). Screening of hybridomas for antibody production involved an enzyme-linked immunosorbent (ELISA) assay with AChR from Torpedo californica (TAChR). 25 of 302 wells tested (8.3%) were positive for anti-AChR antibody production and have been stable in their secretion of mAb for eleven months. Nine lines have been studied in detail. All produced IgM mAb, and most had greater activity against membrane-bound TAChR, than against solubilized TAChR. For anti-AChR clones, the mAb concentration in culture supernatants ranged from 2 to 33 ug/ml. Saturation curves of binding to TAChR performed on 4 lines demonstrated dissociation constants (Kds) estimated to range from 0.1-1.0 nM. The patient whose lymphocytes were used in this study had a serum anti-AChR antibody concentration of 243nM against human AChR and 15nM against AChR from T. californica. The results demonstrate the feasibility of producing stable human x human hybridomas secreting mAb to the autoantigen from the peripheral blood of patients with organ-specific autoimmune diseases. The mAb produced here may prove to be useful in analyzing, and possibly treating, the autoimmune phenomena in MG.     

Lymphocyte Subsets Defined by Monoclonal Antibodies in Human Pregnancy

ABSTRACT: Using monoclonal antibodies, indirect immunofluorescence, and flow cytometry, the proportions and absolute numbers of various lymphocyte subsets in peripheral blood have been measured in normal human pregnancy. Groups of ten women were studied at 12, 28, and 36 weeks of gestation and compared with 16 nonpregnant control women. The percentage of T cells (OKT3 +) was constant throughout pregnancy, and this was confirmed in three women studied serially prior to and throughout early pregnancy. A slight fall in the proportion of helper cells (OKT4 +) and rise in the proportion of suppressor cells (OKT8 +) was observed at 12 and 28 weeks, but these changes, and the resulting fall in helper/suppressor ratio, were not statistically significant. Absolute lymphocyte counts determined by white cell count and differential were lower during pregnancy. The absolute numbers of T cells, helper cells, suppressor cells, and Ia-bearing cells (mainly B cells) were significantly lower at 36 weeks' gestation. T cells and helper cells were significantly reduced in absolute number at 12 weeks' gestation. There was no change in the ratio of T cells to B cells at any stage of gestation. The lack of any significant change in the balance between helper and suppressor cells in peripheral blood suggests that these cells are not important in the immune adaptation to pregnancy.     

人-鼠杂交瘤制备人单克隆抗体技术的研究

本文采用小鼠骨髓瘤细胞系(Sp2/0)与破伤风类毒素(TT)免疫的人外周血淋巴细胞(PBL)进行融合,对人—鼠杂交瘤细胞系制备人单克隆抗体的技术条件进行了探讨。按免疫时间(d)及体外刺激与否分为4组。结果表明:(1)PWM+TT体外刺激可导致B细胞总数增加,且融合率高,容易建成稳定分泌人单抗的细胞株。(2)经TT免疫的人PBL,6d就有抗TT抗体(IgG)的前体细胞出现。15d及21d时,抗TT抗体前体细胞的频率更高。(3)体内免疫7d及14d并分别经体外刺激的两组共建立5株阳性株。经双扩碓定IgG2株,IgM3株。冻存10个月后复苏、传代及适当的克隆化后仍为阳性。上清液中人Ig的分泌量为0.42～1.15μg/ml。经染色体核型鉴定,碓认为人-鼠杂种细胞。     

A Study of HL-A Antigen Phenotype in Rheumatic Fever and Rheumatic Heart Disease Patients

The distribution of the HL–A antigens was studied in 76 patients who have had rheumatic fever or have rheumatic heart disease and in 177 disease-free Caucasian individuals. There was a decreased incidence of A–3 in the disease group as compared to the control. No significant increase in the frequency of any HL–A antigen was detectable in the disease group as compared to normal controls. The results were not affected by the exclusion of non Caucasians from the disease group. The parents of patients with rheumatic fever and heart disease showed a significant increase in shared antigens as compared with the parents of disease-free individuals. The total number of HL–A antigens detected on lymphocytes from rheumatic patients was significantly less than on those from normal individuals.     

Hybridoma Monoclonal Antibodies to Human Fetal Haemoglobin

Monoclonal antibodies against human fetal haemoglobin (HbF) were obtained by fusion of mouse myeloma cells with spleen lymphocytes from mice immunized against HbF from human umbilical cord blood erythrocytes. Initial antibody screening was by indirect immunofluorescence assays on smeared cord blood and normal adult blood erythrocytes. The specificity of these antibodies was further studied by combined immunoprecipitation assays and direct or inhibition solid-phase enzyme immunoassays. The combined pattern enabled recognition of three types of antigenic determinants: (i) common to both HbF and HbA, by both immunofluorescence and enzyme immunoassays; (ii) common to both HbF and HbA by enzyme immunoassays, present on fixed cord blood erythrocytes and adult thalassaemic erythrocytes by immunofluorescence, but not found on fixed normal adult erythrocytes; and (iii) private HbF by both immunofluorescence and enzyme immunoassays.     

Norovirus Antigen Detection with a Combination of Monoclonal and Single-Chain Antibodies

The performance of a norovirus antigen detection assay was assessed using monoclonal antibody NV23 and single-chain antibody HJT-R3-A9 to identify both virus-like particles and virus-containing fecal samples. The detection of 25 different norovirus genotypes as recombinant virus-like particles or in clinical samples was dependent on virus or antigen concentration.     

灵敏的甲状腺微粒体抗体酶联免疫吸附分析的建立

甲状腺微粒体抗体（ＴＭＡｂ）或称甲状腺过氧化物酶抗体（ＴＰＯ－Ａｂ）已广泛用于人类甲状腺自身免疫性疾病的诊断，但粗提的人甲状腺微粒体制备物含有残留的甲状腺球蛋白（ＴＧ）和其它膜抗原，影响方法的灵敏度，我们采用亲和层析的方法，得到纯度较高的ＴＭ抗原，建立了灵敏的ＴＭＡｂ－ＥＬＩＳＡ。该方法的批内变异系数为３．２±０．０１％（ｎ＝８０），批间变异系数为８．３±０．０２％（ｎ＝８）；临床甲亢病人的阳性率为８０．０％（ｎ＝４６），与国内同类放免试剂盒相比，对Ｇｒａｖｅｓ病的阳性检出率提高２０％（ｎ＝２０），桥本氏甲状腺炎的阳性检出率提高１１．７％（ｎ＝２６）。     

Phenotypes of Human Large Granular Lymphocytes as Defined by Monoclonal Antibodies

Four monoclonal antibodies VEP8, VEP9, VIM-D5, VIB-C5 against antigens expressed on human mature myeloid cells (polymorphonuclear leukocytes [PMNL] and/or monocytes) as well as on immature cells in the bone marrow were tested for reactivity with cell preparations highly enriched for large granular lymphocytes (LGL). These cells are known to be the main effector cells responsible for natural killer (NK) cell activity in human peripheral blood. Using indirect membrane immunofluorescence (IMF), none of these antibodies showed any reactivity at all. In addition, LGL-enriched cell preparations were tested with the anti-lymphocyte monoclonal antibodies OKT6, anti-Leu1, anti-Leu2a, anti-Leu3a, and anti-human Lyt3, and also with OKM1 antibody. Significant reactivity was found with anti-Leu2a (59 ± 8 %), anti-Lyt3 (55 ± 4 %) and OKM1 (81 ± 11 %) antibodies, whereas T6, Leu1, and Leu3a antigens were less pronounced or missing on LGL. As a further approach, another monoclonal antibody, VEP13, which reacts with LGL, granulocytes but not monocytes and is therefore different in its specificity from OKM1 and OKT10, was used for identification of LGL. The coexpression of antigens as defined by the above-mentioned antibodies and OKT10 on VEP13⁺ cells was studied. Again, phenotypes similar to those observed on LGL enriched by Percoll^® gradient centrifugation were found: of VEP13⁺ cells 84 ± 6 % reacted with OKM1, 82 ± 5 % with OKT10, 52 ± 17 % with anti-human Lyt3, and 48 ± 14 % with anti-Leu2a, whereas VEP8, VEP9, VIM-D5, VIB-C5, T6, Leu1, Leu3a antigens were not expressed on VEP13⁺ cells. Taken together as an overall evaluation of phenotypic characteristics, our data indicate that LGL cannot be integrated into one of the known lymphocytic or myelomonocytic lineages. LGL show an intermediate phenotype depending possibly on varying differentiation or activation stages of haemopoietic cells. However, the possibility also exists that LGL belong to a separate, yet undefined cell lineage.