An antigen localized at the centriolar region has been identified by indirect immunofluorescence studies in African green monkey kidney, human, hamster, rat, and mouse cells. The antigen consists of two polypeptides of 14,000 and 17,000 daltons. A related antigen is also present at the basal body region in ciliated cells from chicken, cat, mouse, pig, steer, and rabbit trachea and from rabbit fimbria. Immunoelectron microscopy shows that the immunoreactive antigen is indeed located in the region around the basal bodies of ciliated cat tracheal cells. Thus, we have found an antigen that is common to a variety of cell types from many different animal sources and is specifically associated with both centrioles and basal bodies. The possible role of the antigen in differentiation is discussed.Immunofluorescent staining at the centriolar region ofcells has been observed in several different systems by using both immune (1) and preimmune sera (2). The sera that give staining at the centriolar region also have been shown to stain the basal body regions of ciliated cells (1, 2). The centriolar staining by the above sera was observed as 0.2-to 0.9-,um granules, which appear in pairs near the interphase nuclei in most of the cells. Kasamatsu et al. (3,4) have shown similar staining at centriolar and basal body regions by using both preimmune and immune sera. However, they demonstrated the centriolar staining as a single 2-ntm structure at the centriolar region of TC7 cells at frequencies of only 15-20%. Thus, the sera in our studies seem to detect an antigen different from that described by others. The appearance of this antigen is stimulated up to a frequency of about 80% by simian virus 40 (SV40) infection (3). A wild-type SV40 small-tumor antigen gene function is required for the stimulation of this host antigen, and protein synthesis up to 5 hr after infection is essential (4). Two polypeptides with apparent molecular sizes of 14,000 and 17,000 daltons are detected on NaDodSOJpolyacrylamide gels (4). These two polypeptides were shown to be the antigens which give rise to the specific immunostaining because the immunoglobulin fraction that adsorbed to the TC7 14,000-to 17,000-dalton polypeptides also stained the centriolar and the basal body regions (4). We shall refer to the two immunoreactive polypeptides together as the centriolar antigen.Centrioles and basal bodies of ciliated cells are considered to be related based on structural similarities (for review, see refs. 5-7). A cultured cell normally possesses a pair ofcentrioles, whereas a differentiated ciliated epithelial cell may have several hundred basal bodies. During the differentiation to a multiciliated cell, the formation of cilia is preceded by the formation of the appropriate number of basal bodies, which then become arranged in rows beneath the cell surface. Each basal body gives rise to a single cilium. In cultured 3T3 cells, it has been shown that one centriole of the pair generates a primary cilium at a particular phase ofthe cell cycle (G1) (...
We have developed a murine monoclonal antibody (mAb) specific for the delta chain of hemoglobin (Hb) A2 that does not cross-react with alpha, beta, or gamma chains. The mAb reacted with Hb P-Nilotic (beta delta hybrid), but not with Hb Lepore-Boston (delta beta hybrid), indicating an epitope consisting of positions 116 (Arg) and 117 (Asn) or 125 (Gln) and 126 (Met) of the delta chain. By using this antibody, we have established a simple and rapid enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of Hb A2 in adult, cord, and fetal hemolysates. We analyzed 70 adult, 8 newborn, and 19 fetal hemolysates from normal subjects and those with various hemoglobinopathies. The mean percentage of Hb A2 was 2.5 for normal adults, 5.4 for beta thalassemic (beta thal) heterozygotes, and less than 0.1% in beta thal fetal samples. We were able to distinguish and characterize certain phenotypes of beta thal patients such as beta thal heterozygotes, beta 0 thal homozygotes, and C beta 0 thal, and C beta + thal double heterozygotes with the aid of this and other mAbs we have generated. This technique is a valuable addition to current methods for the diagnosis of beta thal based on quantification of Hb A2.
We have developed a monoclonal antibody-based enzyme immunoassay and a solid-phase radioimmunoassay for human myoglobin. Both assays are based on competition for the monoclonal antibody between the free myoglobin present in the standards or serum samples and the myoglobin coated to the wells of microtiter plates. Consequently, the absorbance at 630 nm and the radioactivity are inversely related to the concentrations of free myoglobin. The sensitivity of both assays was 10 micrograms/L with linearity up to 1,000 micrograms/L. There was no interference with other serum proteins, as judged from analysis of specimens with high concentrations of lactate dehydrogenase, creatine kinase, or hemoglobin. The average serum myoglobin concentration in 30 normal individuals was 67 micrograms/L. Five patients with cardiac arrhythmias had normal values (average, 63 micrograms/L) while four patients with myocardial infarction had abnormally high concentrations of myoglobin (300-1,000 + micrograms/L). In a typical case of myocardial infarction, serum myoglobin rose 21 hr earlier and peaked 12 hr earlier than creatine kinase and its cardiac isoenzyme. These rapid immunoassays appear to be useful for the early detection of increased serum myoglobin indicative of myocardial infarction.
We have generated a murine hybridoma that secretes a monoclonal antibody (mAb) that is highly specific for hemoglobin C (HbC) [alpha 2 beta 2 6(A3)Glu----Lys] and shows no cross reactivity with HbA, HbA2, HbF, HbS, HbE, or Hb O-Arab. Using this antibody, we developed a simple and rapid enzyme linked immunosorbent assay (ELISA) technique for the detection of HbC in both adult and cord blood. The assay can be carried out using either whole blood samples or hemolysates. With as little as 10 microliters/well of whole blood or 5 micrograms Hb/well of hemolysates, and, with dilutions of the antibody up to 10(-5), we were able to detect HbC unequivocally in cord blood samples. The ELISA procedure could detect HbC in proportions as low as 0.01%. This simple diagnostic test represents a technological advance in Hb identification and can easily be used for mass screening (96 samples in less than 45 min) to detect HbC. Furthermore, this assay, when employed in conjunction with an mAb specific for beta 6GLU of HbA, allows the discrimination between HbC homozygotes, heterozygotes, and normals.
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