Affinity targeting of Sendai virions to desialized human erythrocytes using hybrid antibody molecules.

F(ab') fragments obtained from anti-Sendai virus antibodies were chemically coupled to F(ab') fragments obtained from anti-human red blood cell antibodies (anti-hRBC-Ab). This led to the formation of hybrid antibody molecules (anti-SV-anti-hRBC(F(ab')2) each of whose F(ab') fragment possessed different binding specificity. The anti-SV(F(ab'] part of the hybrid molecule interacted specifically with Sendai virus particles, while the anti-hRBC(F(ab'] part interacted with the surface of hRBC. These hybrid antibodies were able to mediate binding and fusion of SV to hRBC, from which the virus receptors were removed by treatment with neuraminidase (desialized hRBC). Neither anti-SV-anti-SV(F(ab')2) nor anti-hRBC-anti-hRBC(F(ab')2) possessed the same ability. Thus, it is shown that soluble, hybrid antibody molecules can effectively mediate functional binding of Sendai virus to virus-receptor-depleted cells.

RNAase-sensitive DNA-dependent DNA polymerase from rat cells transformed by avian sarcoma virus.

An RNAase-sensitive DNA polymerase from rat cells transformed by avian sarcoma virus has been characterized. The enzyme requires RNA for its activity, as shown by its sensitivity to RNAase with endogenous as well as exogenous DNA templates. This sensitivity is maintained after its purification by sucrose gradients and ion exchange columns. A molecular weight of about 100 000 has been estimated. This DNA polymerase requires high salt concentration for its activity, is resistant to high concentrations of phosphonoacetic acid (400 micrograms/ml), is partially inhibited by 5 mM N-ethylmaleimide, and is completely inhibited by 0.3 mM parahydroxymercuribenzoate.

Use of chelating agents as terminators of alkaline phosphatase activity in enzyme-linked immunosorbent assay (ELISA) tests.

For a long time chelating agents have been known to be inhibitors of alkaline phosphatase activity. However, the use of chelating agents in stopping alkaline phosphatase activity in ELISA reactions is a novel application with several advantages over conventionally used acids or bases.

Synthesis of a compound soluble in organic solvents from deoxycytidine triphosphate in permeabilized normal human lymphocytes.

Normal human lymphocytes may be rendered permeable to deoxynucleoside triphosphates. When [3H]dCTP is furnished to permeabilized lymphocytes tow compounds are formed: DNA and a compound soluble in organic solvents. [3H]dCTP incorporation is higher in stimulated lymphocytes than in unstimulated cells. Some characteristics of the system are reported.

DNA polymerases of tumor virus: specific effect of ethidium bromide on the use of different synthetic templates.

The DNA polymerase enzymes from avian, murine, and feline RNA tumor viruses can be distinguished by their ability to read specific, synthetic primertemplates. The copying of templates containing adenylic and thymidylic acids by all these DNA polymerases is inhibited by ethidium bromide, though this compound affects the polymerases from mammalian tumor viruses much more than the enzyme from avian tumor viruses. Conversely, ethidium bromide stimulates the ability of the enzymes from avian tumor viruses to use primertemplates containing only guanylic and cytidylic acids, whereas the mammalian tumor virus enzymes are moderately inhibited.

Deoxyribonucleic acid polymerase of wild-type and phosphonoacetic acid-resistant mutant of herpes simplex virus.

The phosphonacetic acid (PAA)-susceptible deoxyribonucleic acid (DNA) polymerase of herpes simplex virus type 1 was partially purified and isolated in sucrose gradients and on double-strand DNA cellulose columns. The DNA polymerase isolated from cells infected with the PAA-resistant mutant had the same molecular weight as the wild-type enzyme (140,000 to 149,000) but was consistently more resistant to PAA.

Multiplication of Coxsackie B1 virus in synchronized HeLa cells.

A higher yield of Coxsackie B(1) virus was obtained when HeLa cells were infected late during S phase as compared to the amount produced by random cultures.

A new synthetic RNA-dependent DNA polymerase from human tissue culture cells (HeLa-fibroblast-synthetic oligonucleotides-template-purified enzymes).

Two DNA polymerases that can copy synthetic RNA polymers are present in human tissue culture cells. These enzymes which have each been purified about 500-fold, are present in both HeLa cells, which are derived from a cervical carcinoma, and in WI-38 cells, a normal diploid strain originating from human embryonic lung tissue. These synthetic RNA-dependent DNA polymerases are identified by their ability to copy efficiently the ribo strand of synthetic oligonucleotide-homopolymer complexes, and differ in this respect from the known DNA-dependent DNA polymerases found in HeLa cells. The template requirements of these new DNA polymerases resemble that of the RNA-dependent DNA polymerases of the RNA tumor-viruses.

Multiple use of one-piece microtitration plates in ELISA (enzyme-linked immunosorbent assay) tests for the detection of cytomegalovirus and rubella virus antibodies.

Ninety-six-well, one-piece microtitration plates coated with rubella virus or cytomegalovirus (CMV) antigen can be used for multiple ELISA (enzyme-linked immunosorbent assay) testings. Only the number of test wells required per test need be used and the remaining unused test wells can be retained for subsequent assay. Consequently, as the one-piece microtitration plate is not a single-use, "all or none" element of the ELISA system, it is therefore as suitable for multiple ELISA testings as for one-time use. An alternate system of result interpretation for ELISA is introduced. Results are presented comparing the conventional optical density (OD) readings to values of the ratio: OD sample/OD low-positive sample.

Stimulation of DNA polymerase alpha by a nuclear DNA/protein complex.

A nuclear DNA complex containing DNA polymerase and SV40 T-antigen was isolated from nuclei of SV40-transformed mouse fibroblasts. DNA polymerase could be separated from the complex. The remaining DNA/T-antigen-containing complex stimulated DNA polymerase alpha activity about 10-fold. The complex contained 4 major proteins with molecular weights of 46, 54, 76, and 94 kilo-dalton (KD). The stimulation activity was retained by protein A-Sepharose loaded with specific IgG from SV40-tumor bearer serum, or from antisera against the 94 KD and 76 KD components and was partially inhibited in the presence of these antisera. The stimulation activity was completely abolished by treatment of the complex with trypsin or DNase I.

Interaction of ultraviolet-irradiated herpes simplex virus type 1 with BSC-1 cells.

Ultraviolet irradiation of herpes simplex virus (HSV) did not affect the transfer of uncoated virus DNA to the nuclei of infected cells but the synthesis of virus DNA was suppressed. The virus-specific DNA polymerase was synthesized in cells infected with the u.v.-irradiated HF strain of HSV. In cells infected with the u.v.-irradiated KOS strain, the virus DNA polymerase activity was hardly detectable. The two strains of HSV differ in the sensitivity of the virus DNA polymerase gene to u.v.-irradiation.

Herpes simplex virus DNA polymerase, thymidine kinase and deoxyribonuclease activities in cells infected with wild type, ultraviolet-irradiated and defective virus.

The DNA polymerase, thymidine kinase and deoxyribonuclease activities were studied in cells infected with wild type (wt), ultraviolet (UV)-irradiated and defective herpes simplex virus type 1. All three enzymatic activities were expressed in cells infected with wt virus. In cells infected with UV-irradiated virus, the thymidine kinase and deoxyribonuclease activities were inhibited and the DNA polymerase activity was markedly suppressed. In cells producing defective virus, there was thymidine kinase activity, but the viral deoxyribonuclease activity was considerably reduced. The DNA polymerase activity was fully expressed in cells producing defective virus at passage level 5, but at passage level 6, the activity of the viral DNA polymerase declined.

Homogeneous substrate-labeled fluorescent immunoassay for human serum albumin.

A homogeneous substrate-labeled fluorescent immunoassay for human serum albumin (HSA) has been developed, similar to previously described immunoassays for Immunoglobulin G and Immunoglobulin M. HSA was covalently linked to 6-(7-beta-galactosylcoumarin-3-carboxamide) hexylamine. The resulting conjugate had minimal fluorescence at 450 nm (with excitation at 400 nm). However, when the acetal linkage of the galactosyl moiety was hydrolyzed by beta-galactosidase, a substantial increase in the fluorescence was obtained. This increase was specifically inhibited by antibody to HSA. A competitive binding immunoassay was established by letting the conjugate compete with HSA in the serum for the limited number of antibody-binding sites. The level of fluorescence resulting from the addition of enzyme was proportional to the amount of HSA in the serum. Precision, analytical recovery and serum dilution studies were carried out on the assay. The immunoassay was compared to an albumin assay using the dye-binding method.