Prenatal fragile X detection using cytoplasmic and nuclear-specific monoclonal antibodies.

We have been carrying out studies aimed at improving prenatal detection of the fragile X chromosome/mutation. Our current protocol requires a turnaround time (TAT) of several days. In an attempt to reduce the TAT, we have turned to the use of monoclonal antibodies (mAbs). Monoclonal antibody 1A1 (provided by Dr. Mandel of INSERM) immunostaining was performed according to a modified three-step immunocytochemical procedure. We found that cytoplasmic staining intensities, using mAb 1A1/avidin biotinylated complex/diaminobenzidine, varied from light to heavy within each sample, with controls exhibiting a majority of heavily stained cells in both chorionic villus (CV) sample and amniotic fluid cultured cells. Using mAb 1A1 and a new nuclear-specific antibody, mAb 3F11, we found that CV cultured cells harboring the FMR1 full mutation could be distinguished from controls as early as 10 weeks of gestation in both male and female specimens. Western blot analysis showed that the antibodies have similar staining patterns but that mAb 3F11 has fewer background/nonspecific bands. Our results demonstrate that it is feasible to detect fragile X full mutations within one day after obtaining cells from CV specimens taken as early as 10 weeks of gestation.

Expression of the human cytomegalovirus 65K tegument phosphoprotein in insect cells by baculovirus vectors.

The gene encoding the 65K tegument phosphoprotein (pp65) of human cytomegalovirus (HCMV) was cloned into pAc373 to construct a recombinant baculovirus (Acpp65-3) expressing pp65 in insect Sf9 cells. A baculovirus that carried a fragment of the gene, corresponding to the first 442 amino acids of pp65, was also developed, using vector pVL941 (Acpp65-2). Recombinant proteins migrating in SDS-polyacrylamide gels with an M(r) of either 65K (Acpp65-3) or 56K (Acpp65-2) were detected in cytoplasmic and nuclear extracts of infected Sf9 cells. The 56K and 65K proteins were recognized in immunoblots by monoclonal antibodies (MAbs) 28-77 and 28-19, which are specific for pp65. The insect cell-expressed antigens were also analysed on Western blots using MAbs 4D11, 7D2, 8E3, 7B4 and 8E10, which recognize the HCMV antigen GP66 in immunoblots. The truncated pp65 antigen of Acpp65-2 was reactive with MAbs 4D11, 7D2, 8E10 and 7B4. The protein expressed by Acpp65-3 reacted only with MAb 4D11. The data proved that the epitopes recognized by MAbs 4D11, 7D2, 8E3 and 7B4 mapped in the region of pp65, comprising amino acids 1 to 442, and also that GP66 and pp65 represent the same HCMV antigen. Immunoblot analysis of human sera from individuals seropositive for HCMV showed that the recombinant pp65 products were as antigenic as the native 65K phosphoprotein produced in HCMV-infected human embryonic fibroblasts.

Production and characterization of monoclonal antibodies specific for a glycosylated polypeptide of human cytomegalovirus.

Nine hybrid cell lines producing antibodies specific for cytomegalovirus (CMV) antigen were obtained after fusion of P3/X63-Ag8 myeloma cells with spleen cells from BALB/c mice immunized with CMV complement-fixing antigen. By the immunoblot technique, five of nine antibodies (4D11, 7B4, 7D2, 8E3, and 8E10) were identified as being reactive to a CMV glycosylated polypeptide with molecular weight of 66,000 (GP66). Four other antibodies (1B8, 8E9, 4D2, and 7E2) appeared to be reactive with CMV antigen(s) only if the antigen was not denatured by sodium dodecyl sulfate. These remain unassigned until further studies are done. With the enzyme-linked immunosorbent assay (ELISA), competitive bindings were performed with a constant amount of horseradish peroxidase-conjugated antibody and various concentrations of unconjugated homologous and heterologous antibodies on CMV antigen-coated ELISA wells, and the antigenic determinant specific for each antibody was determined. The nine antibodies could be classified into six different groups, each group reacting with a different epitope or a different region with two or more antigenic determinants which are so close to each other that they cause binding inhibition. They are groups A (4D11), B (7B4, 8E10), C (7D2), D (4D2, 7E2, 8E9), E (8E3), and F (1B8). The extent of competition among antibodies within each group was the same. By using the two antibodies that reacted with different epitopes on GP66, a double-antibody sandwich ELISA method was developed. The method was sensitive enough to detect as little as 50% of the antigen present in one infected cell or 0.000245 U of CMV complement-fixing antigen per test well. Other strains of CMV (David, Kerr, Espilat, C-87, and five clinical isolates) gave positive results, whereas herpes simplex virus types 1 and 2, varicella-zoster virus and Epstein-Barr virus nuclear antigen preparations did not. By the indirect immunofluorescence assay, antibodies 4D11 and 8E3 were able to detect GP66 in the nucleus of CMV-infected F-5000 human embryonic fibroblasts as early as 2 h postinfection and were superior in this respect to the remaining seven antibodies tested. By the double-antibody sandwich ELISA, the presence of GP66 in CMV-infected cells was detected as early as 2 h postinfection.

Antiviral activity of arildone on deoxyribonucleic acid and ribonucleic acid viruses.

Arildone (3 micro/ml) reduced the replication of murine cytomegalovirus, Semliki Forest virus, vesicular stomatitis virus, and coxsackievirus A9 by 64, 68, 94, and 98%, respectively. When the plaque reduction method was used to evaluate the antiviral effect for the viruses, a concentration of 3 to 5 micrograms/ml yielded a 50% reduction in plaque numbers. The effect of arildone on virus replication was greatest when the drug was present from the time of inoculation. The effectiveness decreased as the time interval from the inoculation of the virus to the addition of the drug increased. The removal of the drug from infected cells by washing readily reversed the effect, and viral replication resumed at a significant level. Infectivity of these viruses was not inactivated by the drug. Tissue culture cells used for viral growth and assay grew well in arildone (3 micrograms/ml), with cell yields that were comparable to those for cultures in the absence of drug. At 3 micrograms/ml there were minimal effects of the drug on the uptake of 3H-labeled amino acids and [3H]-thymidine into cells. Furthermore, incorporation of these precursors was not affected. However, there was a reduction in uptake of [3H]uridine into the acid-soluble pool and a concomitant reduction in incorporation into acid-insoluble counts.

Murine cytomegalovirus-induced protein synthesis.

Murine cytomegalovirus (MCMV)-induced protein synthesis in mouse embryo fibroblast (MEF) cells was studied using polyacrylamide gradient SDS gel electrophoresis and autoradiography. Synthesis of at least 14 virus induced proteins (VIPs) was consistently detected in a lytic cycle. They were designated VIPs 132, 118, 99, 98, 88, 81, 76, 74, 58, 56, 51, 38, 36 and 33 on the basis of their mol. wt. Judging from the pattern of the rate of protein synthesis, VIPs can be classified into three groups: group A VIPs were synthesized actively for a brief period of time and then their synthesis was no longer detectable. This group included two major VIPs, 98 and 88 and three minor VIPs, 58, 56 and 38. Group B VIPs 81, 74, 36 and 33 were similar to group A except that, following a brief period of active synthesis, a low level of synthesis continued during the entire lytic cycle. Group C VIPs 132, 118, 99, 76 and 51 were synthesized at low steady levels at all times after initiation and seemed to accumulate slowly. According to temporal sequences of initiation of VIP synthesis, these proteins can also be divided into three groups: immediate early, early and late VIPs. The synthesis of the immediate early VIPs 132, 98, 88, 81, 76, 74 and 38 was initiated immediately after virus infection. The early VIPs included 58, 56, 51, 36 and 33 and their synthesis was initiated from 1 to 3 h post-infection. VIPs 118, 99 and several minor VIPs were first synthesized during 12 to 13 h post-infection which corresponded to the time of initiation of virus DNA synthesis and they are classified as late VIPs. Cycloheximide reversal experiments indicated that the initiation of synthesis of early VIPs must be preceded by the synthesis of immediate early VIPs. In the presence of actinomycin D, the immediate early VIPs (0 to 1 h post-infection) were not synthesized indicating that immediate early VIPs are translated from virus mRNA synthesized after virus infection.

Complement-fixing antigen of human cytomegaloviruses.

The titer of complement-fixing (CF) antigen of human cytomegalovirus (CMV) strain C87 was not reduced by 10 cycles of freezing and thawing or by storage at 4 C for two months, although the virus was labile at 37 C and 100 C. The rapid increase in titers of CF antigen and plaque-forming units was seen from days 3 through 6 after infection; however, CF antigen was detected 24 hr after infection. The molecular weight of CF antigen was estimated by gel filtration to be greater than 1.5 X 10(7) daltons. Electrophoresis of CF antigens from five strains of human CMV (AD169, C87, Espilant [Esp.], Davis, and Towne) in a gradient polyacrylamide gel slab in the presence of sodium dodecyl sulfate revealed two specific polypeptides with molecular weights of 140,000 and 66,000 daltons, respectively. The smaller polypeptide was glycosylated. Preparations of CF antigen from murine CMV and herpes simplex virus type 1 contained polypeptides of 140,000 and 145,000 daltons, respectively. However, the polypeptide with a molecular weight of 66,000 daltons was seen only in preparations of human CMV.

Analysis of structural polypeptides of purified human cytomegalovirus.

Human cytomegalovirus strain C87 was purified by the following procedures. (i) Extracellular virus was concentrated by centrifugation at 100,000 X g for 90 min and passed through a Bio-Rad Bio-Gel A-15m column. Most of the virus was recovered in the void volume. (ii) After two consecutive isopycnic potassium tartrate gradient centrifugations (20 to 50%), coinciding peaks of plaque titer, protein, and radioactivity were found at a density of from 1.20 to 1.21 g/cm3. To characterize the structural polypeptides of human cytomegalovirus and to establish relative purification criteria, virus was purified from two mixtures: (i) [35S]methionine-labeled extracellular virus mixed with an equal volume of unlabeled normal culture fluid; (ii) unlabeled extracellular virus mixed with an equal volume of [357a1methionine-labeled normal culture fluid. The extent of purification, as judged by the ratio of cellular to viral radioactivity, was 39-fold; i.e. about 2.5% of the protein in the purified virus preparation could be accounted for by host protein contamination. Electrophoresis of purified [35S]methionine-labeled virus on a polyacrylamide gel slab showed that there were at least 33 viral structural polypeptides (VPs), and their molecular weights ranged from 11,000 to 290,000. Autoradiograms obtained from electropherograms of purified [14C]glucosamine labeled virus showed six bands. Four of these were so broad that several VPs corresponded to each of the glycosylated bands. When heavy (two fractions close to 1.21 g/cm3) and light (two fractions close to 1.20 g/cm3) fractions of the PFU peak from the second potassium tartrate gradient were analyzed separately, the number of polypeptides observed was the same, but the relative amounts of some polypeptides differed. The major polypeptide, VP17, was found in greater amounts in the heavy fraction (35%) than in the light fraction (22%). The amount of DNA as a percentage of the weight of protein was 2% for the light fraction and 1% for the heavy fraction.

Analysis of structural proteins of purified murine cytomegalovirus.

Murine cytomegalovirus propagated in mouse embryo fibroblasts was purified by the following procedures. (i) Extracellular virus was concentrated by centrifugation at 100,000 x g for 90 min. (ii) The concentrated virus was passed through a Bio-Rad Bio-Gel A-15m column to eliminate contaminating materials smaller than 15 x 10(6) daltons. Most of the virus was recovered in the void volume of the column. (iii) Two consecutive centrifugations through 20 to 50% potassium tartrate gradients were performed. After the second tartrate gradient centrifugation, symmetrical, coinciding peaks of plaque titer, protein, and radioactivity were found at a density between 1.20 g/cm3 and 1.21 g/cm3. To establish purification criteria, virus was purified from two different mixtures: [35S]methionine-labeled extracellular virus, mixed with an equal volume of unlabeled normal culture fluid, and unlabeled extracellular virus mixed with an equal volume of [35S]methionine-labeled normal culture fluid. At the end of the procedure, the extent of purification, as judged by the ratio of cellular to viral radioactivity was at least 70-fold. Virus proteins were analyzed by electrophoresis on a 5 to 20% gradient polyacrylamide gel slab. After gel electrophoresis,, Coomassie brilliant blue staining profiles and autoradiograms of the purified virus preparations were compared. At least 33 virus structural protein bands were present. The molecular weights of these proteins ranged from 11,500 to 255,000. The sum of the molecular weights of the virus structural proteins was 2,462,000. Autoradiograms obtained from electrophoresis of purified [14C]glucosamine-labeled virus showed that at lease 6 of the 33 viral structural proteins were glycoproteins.