Search PubMed⌕ Search

Biomedical subjects

J Pillot

Publications and source records attributed to J Pillot.

At least 127 records · Page 7Linked to original sources
PubMed1985

Demonstration of a firm association between hepatitis B surface antigen proteins bearing polymerized human albumin binding sites and core-specific determinants in serum hepatitis B viral particles.

Hepatitis B viral particles (HB-VP) were purified from sera of chronic hepatitis B surface antigen (HBsAg) positive carriers by consecutive isopycnic and rate-zonal sedimentation in sucrose gradients. Their immunological properties [HBsAg, hepatitis B core antigen (HBcAg) and hepatitis B e-antigen (HBeAg) activities] were examined by a radioimmunoassay based upon the classical "sandwich principle". A double antibody specificity radioimmunoassay (DAS-RIA) was then developed to determine whether envelope proteins (HBsAg) with binding activity for polymerized human serum albumin (pHSA-BA) were associated with core-specific antigenicities (HBc/HBeAg). An e-antigen activity cosedimenting with intact HB-VP (negative for HBcAg reactivity) was detected in association with HBsAg and receptors for pHSA. The presence of HBcAg-specific determinant(s) on HBeAg molecules was also indicated by DAS-RIA. So, we postulated that such hepatitis B virion (HBV) specific molecules are involved in immune complexes with anti-HBc as antibodies in sera of patients with chronic HBV infection. To define the significance of these molecular forms in HB-VP morphogenesis, we studied the effects of a mild treatment with a chaotropic salt, NaSCN, on HB-VP-rich fractions (DNA polymerase positive). A small mol. wt HBeAg derived from HB-VP by dissociating treatment was detected. We found that core-specific determinants (HBe/HBcAg) were bound to large surface proteins (HBsAg) with pHSA-BA and therefore probably contained the pre-S sequence. The selective release from HB-VP of such molecular forms, which could be a product of the major S-region transcript, suggests that they may be components of complete virions.

Binding Sites↗
PubMed1985

HBc and HBe antigenicity and DNA-binding activity of major core protein P22 in hepatitis B virus core particles isolated from the cytoplasm of human liver cells.

Highly purified hepatitis B virus core particles were obtained in large amounts from the cytoplasm of infected human liver cells. This DNA polymerase-negative core preparation had only hepatitis B core antigen-specific antigenicity and showed a surprising stability. Two forms of a single protein of 22,000 molecular weight, P22, were resolved electrophoretically; the slower moving species, P22a, appeared to be a reduced form of the protein, and the faster moving species, P22b, could have represented a conformational isomer containing an intramolecular disulfide bond(s). The immunological properties and DNA-binding activity of the reduced form, P22a, were examined following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by transfer onto nitrocellulose membranes (Western blotting). We found that the hepatitis B virus C gene protein shared the antigenic site responsible for both hepatitis B core and e antigen reactivity. We also demonstrated that the core protein(s) bound specifically the genomic hepatitis B virus DNA in comparison with a plasmid DNA (pBR322). This last observation was further substantiated by a radioimmunological method. P22a was also found to be phosphorylated in vitro by the endogenous protein kinase activity, copurified with the hepatitis B core antigen particles. These findings suggest that P22 is a multifunctional protein which is incorporated into core particles within the cytoplasm of the host cell before DNA encapsidation. A critical role of this protein in hepatitis B virus assembly is suggested.

Cytoplasm↗
PubMed1984

Prenatal diagnosis of congenital rubella.

In an attempt at prenatal diagnosis of fetal infection after primary rubella infection before 18 weeks of pregnancy fetal blood was taken by direct puncture under ultrasound guidance at 20-26 weeks of pregnancy from eighteen patients. Total IgM was assayed by radial immunodiffusion and rubella-specific IgM by IgM capture immunoassay. Rubella-specific IgM was detected in twelve of the eighteen fetuses. The parents decided whether to continue or terminate the pregnancy on the basis of the time of onset of maternal rubella. All six pregnancies in which rubella occurred before 12 weeks of gestation were terminated. Of the six with rubella after 12 weeks two were terminated. Among the six fetuses negative for rubella-specific IgM, five had no rubella-specific IgM at birth and no persistent IgG but one was found to be infected at birth; this false-negative diagnosis was due to sampling too early during pregnancy.

Antibody Specificity↗
PubMed1984

A modified gel filtration technique producing an unusual exclusion volume of IgM: a simple way of preparing monoclonal IgM.

The vast majority of monoclonal IgM proteins is eluted just before the total volume of the column when filtered through G200 Sephadex or S200 Sephacryl gels equilibrated in a 0.005 M phosphate buffer but eluted with 0.05 M phosphate buffer containing 1.7 M NaCl. This unusual behaviour in low-ionic buffer is probably due to the poor solubility of IgM in diluted buffers. It allows a 1-step purification procedure under mild conditions and is suitable for both large and small scale preparations.

Acrylic Resins↗
PubMed1984

Immunochemical structure of the hepatitis B surface antigen vaccine--I. Treatment of immobilized HBsAg by dissociation agents with or without enzymatic digestion and identification of polypeptides by protein blotting.

Since the immunosorbent techniques and the cycles of isopycnic and rate zonal velocity ultracentrifugations were shown to be unsuitable for the purification of hepatitis B surface antigen (HBsAg) particles from human sera because HBsAg was still largely contaminated by serum proteins, we applied a drastic dissociating treatment of HBsAg stabilized by adsorption on silica gel which appeared essential to remove extraneous components initially present in the HBsAg particles. Only albumin and sometimes IgG were recovered with the purified antigen. The polypeptide composition of our purified HBsAg preparations was analyzed by SDS-PAGE with subsequent transfer to a nitrocellulose sheet by blotting, incubation with 125I-anti-HBs and exposure to X-ray film. Samples from HBsAg-positive sera containing the hepatitis B virus e antigen (HBeAg) displayed three proteins: P 24.5 and GP 28 as major components and GP 36 as a minor component. Dimers of these polypeptides were also immunologically detected. When a supplementary step of trypsin or pepsin digestion was included in our purification procedure after adsorption to silica and acid dissociation of HBsAg, proteolytic cleavage fragments of HBsAg with mol. wts lower than 10,000 were obtained on SDS-PAGE after reduction. This finding shows that arginine and lysine residues inaccessible to tryptic digestion in the intact HBsAg lipoprotein particle were exposed to enzymatic hydrolysis by our treatment. However, HBsAg kept the antigenic and immunogenic properties of the native antigen. Therefore such a HBsAg preparation appeared as a new candidate for the vaccination against HBV and a useful material for the analysis of the HBs antigenic structure.

Animals↗
PubMed1984

Specificity and sensitivity of the IgM capture immunoassay: studies of possible factors inducing false positive or false negative results.

The specificity and sensitivity of the IgM-capture immunoassay (IgM-CI) were evaluated for detection of rubella specific IgM and hepatitis B core (HBc) specific IgM. For rubella specific IgM, antibodies bound to the solid phase were detected by haemadsorption and for HBc specific IgM, by using HBc antigen (HBcAg) and radiolabelled IgG anti-HBc. Rheumatoid factor (RF) was found to interfere in the test for HBc specific IgM because IgM-RF bound to the solid phase reacted with aggregated radiolabelled HBc specific IgG. This false positive reaction did not occur when radiolabelled F(ab')2 was used instead of the whole IgG molecule. HBcAg purified from biological fluids might be coated with host IgG and under these conditions, HBcAg could react with RF. It was also demonstrated that high levels of IgG antibodies could interfere with IgG anti-mu coated-surface by means of non-immunological protein-protein interactions. In fact, IgG did not interfere in the rubella assay, whereas it did in the very sensitive anti-HBc test. To prevent this false-positive reaction, different dilution media were tested. Only the addition of non-specific IgG and fetal calf serum (FCS), to the dilution medium, seems to improve the specificity of the test. Furthermore, in order to decrease this non-specific IgG-IgG interaction and an occasional prozoning phenomenon, the dilution of serum to be tested was taken into account. Parameters considered to decrease sensitivity were also studied. RF, anti-F(ab')2 antibodies and non-specific IgM did not decrease significantly the sensitivity of the assay.

Antibodies, Viral↗
PubMed1984

Analysis of the vaccinating group specificity a of HBsAg with monoclonal antibodies. Identification of continuous and discontinuous epitopes.

The group specificity a of HBsAg is responsible for the induction of protective antibodies. Several synthetic peptides for vaccination are in preparation. It is not known if the structure of the epitope(s) implied in specificity a is continuous or discontinuous. For this analysis, we have prepared six monoclonal anti-a antibodies (all belonging to the IgG1 subclass) which precipitated native HBsAg and agglutinated sensitized red blood cells. The antibody affinities ranged from 1.2 X 10(9) to 10 X 10(10) 1/M. These monoclonal antibodies were comparatively tested against native radioiodinated HBsAg and polypeptides obtained from HBsAg after reduction and alkylation. Two antibodies with low affinity continued to bind about 40% of polypeptides (compared to 94% retained by polyclonal antibodies). Three antibodies with higher affinity did not bind any polypeptide under the same conditions. When an inhibition technique was used, the two antibodies binding the polypeptides could be inhibited by polypeptides in their reaction with HBsAg. For the other antibodies, polypeptides did not inhibit the reaction with HBsAg. These results show that continuous and discontinuous epitopes are implied in specificity a of HBsAg. Therefore, for effective synthetic vaccines, mere synthesis of peptides corresponding to selected sequences of the primary structure of the protein may not be sufficient and a sophisticated mimicking of a conformational epitope might also be necessary.

Antibodies, Monoclonal↗
PubMed1983

Human myeloma light chains with increased molecular weight: high frequency among lambda chains.

The discovery of a human myeloma protein comprising a kappa L-chain with an increased mol. wt of 30,000) (Bouvet et. al., 1980) prompted investigations on the incidence of such heavier L-chains among other human myeloma proteins. In 105 samples examined, 34 were found to have L-chains heavier than normal (23,000-24,000), ranging from 25,000 up to 31,000, and five of lighter mol. wt (21,000-22,000). These mol. wt abnormalities were detected by electrophoresis in sodium dodecyl sulfate 10% polyacrylamide gels (SDS-PAGE) after reduction with 2-mercaptoethanol. The mol. wt of three of the heavier kappa or lambda chains was also estimated by filtration through a Sephadex G100 column and by sedimentation equilibrium. All three methods indicated a mol. wt increase of about 15-25% as compared with the usual mol. wt. The distribution of the high mol. wt chains among all L-chains examined was found to be 11 out of 62 kappa chains (17.7%) and 23 out of 43 lambda chains (53%) (P less than 0.001). A preferential association of such L-chains with H-chains producing multiple bands in SDS-PAGE (P less than 0.01) and an association between multiple L-chain and multiple H-chain band (P less than 0.05) were also observed. In contrast, no abnormal L-chain was found in immunoglobulins from normal subjects. Spontaneous degradation of the normal H-chains sometimes yielded fragments of 30,000 mol. wt. These fragments were easily distinguishable from abnormal L-chains. The nature of extra mol. wt in heavy L-chains was investigated for the presence of carbohydrate moiety. Four large and three normal size L-chains were examined for amino-sugar and sialic acid content. A small amount (one residue per molecule) of amino-sugar was detected only in two normal and two heavy L-chains, whereas sialic acid was only found in the heaviest (27,000-30,000) L-chains (Lh) and in small percentage (one or two residues per molecule). Total sugar estimation in one Lh chain indicated a proportion not exceeding three or four residues per L-chain (mol. wt 1,000) and this is insufficient to explain the 15-25% (3,600-6,000) mol. wt increase. It is therefore possible that, at least in some heavy myeloma L-chains, an additional peptide is expressed. Whatever the nature of the increase it would be of interest to elucidate whether this is a marker of malignant process or of an intermediate step of normal Ig synthesis.

Carbohydrates↗
PubMed1983

Long term cultivation of functionally active normal human adult hepatocytes.

Monolayer cultures were obtained from human liver explants. The cells, which extend in all directions, are trypsinized then collected by differential centrifugation and incubated at 38 degrees 5. The oldest culture is 12 month-old. Albumin and alpha 1-antitrypsin are constantly detected and measured in the supernatant by radioimmunoassay. The liver specific protein is always characterized by immunofluorescent staining on the surfaces of the cultivated cells.

Albumins↗
PubMed1982

[Prolonged culture of hepatocytes from healthy adult human livers; immunologic characterization of the hepatocyte nature of cultivated cells].

Monolayer cultures were obtained from human liver explants. Some cells, which extend in all directions, are trypsinized then collected by differential centrifugation and incubated at 38 degrees 5. The oldest culture is 12 months old. Albumin and alpha 1-antitrypsin are constantly detected and measured in the supernatent by radioimmunoassay. The liver specific protein is always characterized by immunofluorescent staining on the surface of the cultivated cells.

Adult↗
PubMed1982

A critical study of the use of staphylococci containing protein A for separation of IgG and IgM antibodies.

This study was to determine the best conditions for using staphylococci bearing protein A to separate IgG from IgM. The validity of the technique was evaluated for detection of IgM with antimicrobial activity and for typing monoclonal IgM. The results indicate that separation of IgG and IgM is not entirely satisfactory in normal sera and worse in hyperglobulinemic sera. The detection and titration of IgM antimicrobial antibodies (rubella and hepatitis B core (HBc) specific IgM) was unreliable because IgG was only partially absorbed by staphylococcal cells, while a significant portion of IgM was bound. The use of higher concentrations of staphylococci did not improve the results because the more IgG was absorbed, the more IgM was also bound. It is shown that with anti-HBc specific IgM the risk of misinterpretation is very high with a sensitive radioimmunoassay technique allowing detection of trace amounts of nonabsorbed IgG. In contrast staphylococcal protein A proved useful in typing monoclonal IgM.

Animals↗
PubMed1982

An indirect immunofluorescence staining procedure for detection of human Fc gamma receptors on streptococci.

Fc gamma receptors on streptococci are usually revealed by hemagglutinating techniques (IgG coated red blood cells) or uptake of radiolabeled IgG. The results obtained with these methods are not always satisfactory. For this reason, we developed a technique involving indirect immunofluorescence staining. Bacterial smears were treated with human Fc gamma fragment and their binding to streptococcal Fc gamma receptors was revealed by a fluorescent F(ab')2 fragment of anti-human Fc gamma sheep antibodies purified on an IgG immunosorbent. These purified sheep F(ab')2 fragments did not contain any IgG nor Fc gamma as shown by SDS polyacrylamide gel electrophoresis. Under these conditions indirect immunofluorescence staining was a highly specific and sensitive method of detecting Fc gamma receptors on streptococci. Distribution of Fc gamma receptors was studied in 237 streptococcal strains of human origin belonging to groups A, B, C, D and G; these receptors were also looked for in 21 strains of alpha-hemolytic streptococci which did not possess the group carbohydrate and 12 strains of pneumococci. Fc gamma receptors were found only in group A, C and G streptococci, but all strains of these groups did not possess Fc gamma receptors.

Animals↗
PubMed1982

False reactions in radioimmunoassay for viral hepatitis B markers in patients suffering from coagulation disorders.

Simultaneous occurrence of hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) has been frequently described in hemophiliac patients. During our working procedures, this phenomenon was also observed. Interference of coagulation disorders with radioimmunoassay (RIA) used in the detection of viral hepatitis B markers was studied. It was shown that, in vivo or in vitro, correction of coagulation disorders led to the disappearance of false positive reactions and, thus, simultaneous presence of HBsAg and anti-HBs never occurred. Confirmatory tests which are usually recommended to control RIA were reviewed and it found that only a completed coagulation of serum by thrombin addition, before performing the test, gave specific results.

Antibodies, Viral↗