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Biomedical subjects

R Winston

Publications and source records attributed to R Winston.

At least 73 records · Page 4Linked to original sources
PubMed1991

Caveats and suggestions for the ELISA.

This study identifies three categories of errors that may arise from faulty procedures utilized in the ELISA and offers suggestions for their recognition and avoidance. The first deals with the potential sources of 'background' values indicating that the practice of subtracting those values from the total OD measurements of the reactivity of a specific antigen-antibody system is not valid in all instances. A simple method of curve analysis has been devised for determining whether that correction is valid. The second consideration is that of stoichiometry of binding of second antibody to the test antibody-antigen complex which is shown to be restricted to a narrow range of concentrations and a short period of incubation of that reagent. The need and method to determine the conditions for stoichiometry for each ELISA protocol is demonstrated. Thirdly, the need for and properties of a suitable blocking agent to prevent binding of ambiguous reactants to the surfaces of the test wells are reiterated and the greater efficiency of BSA, compared with other frequently recommended blocking agents, is demonstrated.

Antigen-Antibody Reactions↗
PubMed1990

Nine cases of heterotopic pregnancies in 4 years of in vitro fertilization.

The incidence of simultaneous intrauterine and extrauterine pregnancy increased after wider use of ovulation induction and the advent of techniques of assisted reproduction. Nine cases of heterotopic pregnancies are reported after in vitro fertilization (IVF) and embryo transfer (ET) between September 1984 and November 1988. The incidence of heterotopic pregnancies in clinical IVF pregnancies was 9 of 312 (2.9%). Superovulation and IVF-ET appear to predispose to heterotopic pregnancy, particularly after tubal disease.

Adult↗
PubMed1989

Genetic analysis of DNA from single human oocytes: a model for preimplantation diagnosis of cystic fibrosis.

Gene sequences in human oocytes were studied to investigate the possibility of diagnosing inherited or sporadic genetic disease before implantation after in vitro fertilisation. By specific amplification the possibility of analysing the DNA from single human oocytes for a specific gene was shown, and genotypes for markers closely linked to cystic fibrosis and Duchenne muscular dystrophy were determined. Single oocytes were used to approximate the total amount of DNA present in a single cell taken for biopsy from a 4-16 cell blastocyst. With a new technique for specific DNA amplification, the polymerase chain reaction, these data can be obtained within several hours of cell isolation. Extreme care must be taken to avoid any contamination of the sample with DNA from other sources. With this technique genotyping for single gene disorders is feasible with an accuracy and on a time scale that would allow implantation of the zygote after in vitro fertilisation without freezing.

Base Sequence↗
PubMed1989

Public health leads IMS legislative agenda.

Transportation safety, tobacco restriction and other vital issues such as mandatory Medicare assignment are IMS priorities for the upcoming session of the Iowa Legislature. The author, chairman of the IMS Committee on Legislation, discusses the 1990 IMS legislative agenda.

Health Policy↗
PubMed1988

Protamine-reactive natural IgM antibodies in human sera. Characterization of the epitope demonstrates specificity of antigenic recognition; occurrence indicates obscurity of origin and function.

We have identified a set of natural IgM antibodies in human serum that are reactive with protamines, a class of low molecular weight basic nucleoproteins that are synthesized de novo in the postpubertal testis and are unique to sperm. Those antibodies were detected by ELISA in significant titer in all of 100 sera of normal adult males and females and in 26 of 28 sera of normal pediatrics aged 7 d to 2 yr. Commonality between the protamine-reactive IgM antibodies of pediatric and adult sera was established by the demonstration of similarity in antigen recognition and reaction kinetics. Therefore, the role of protamines as either immunogenic stimulus or antigenic target of that set of natural antibodies is not likely. The antigenic site recognized by the protein-reactive serum IgM antibodies was characterized by comparison with the pattern of antigen recognition by a monoclonal antibody to human sperm protamines (HPmAb). By the use of synthetic peptides simulating the amino acid sequences of various segments of human protamine 2 and of polyarginine, polylysine, and histones as test antigens, the principle characteristic of the antigenic site recognized by both HPmAb and the serum IgM antibodies was inferred to be that of clustered arginyl residues with an apparent minimum requirement of four arginyl residues, including a triplet, within a six residue piece; for both, the reaction was shown to be not dependent upon charge attraction. A series of immunoabsorption procedures indicated that the protamine-reactive serum IgM antibodies are a discrete set with a high order of specificity. A search of protein data bases revealed that the putative minimum epitope is present in four or five human autogenous proteins, all moieties of the immune system, and in a number of viral proteins. The possible implications of those findings are discussed in the light of early hypotheses concerning the origin and function of natural antibodies and the many recent reports of identification of natural antibodies in normal human sera. The set of natural antibodies identified in this study may be unique or may represent a class of antibodies present in the repertoire that, by virtue of the obscurity of their origin or function, have not been previously or extensively recognized.

Adolescent↗
PubMed1988

Detection of the major epitopes of human protamine P1 recognized by rabbit and mouse antibodies.

The characterization of the major antigenic determinants present in human protamine P1 has been carried out by the use of specific rabbit polyclonal and mouse monoclonal antisera raised against protamine P1. This basic protein, the full amino acid sequence of which has been determined here, has been cleaved by cyanogen bromide and/or by pepsin to generate a discrete number of peptides. These have been purified, characterized by partial amino acid sequencing and used for the determination of their antigenic reactivities with antisera to native protamine P1. Both rabbit polyclonal and mouse monoclonal antibodies were able to recognize the NH2-terminal CNBr peptide encompassing residues 1-36 to the same extent as the intact protamine. A minor epitope present on the COOH-terminal peptide 37-50 could be detected only with the polyclonal rabbit antisera. Attempts to further cleave the P1 molecule in order to isolate peptides shorter than fragments 1-36 whilst retaining full antigenic reactivities, were unsuccessful. This suggests that the epitopes in P1 are conformation-dependent and located for the most part on the amino-terminal half of the molecule, which comprises the characteristic central arginine cluster. The implication of these findings for the studies of the specificities of autoantibodies in sera from infertile and vasectomized individuals is discussed.

Amino Acid Sequence↗
PubMed1987

Phosphorylation state of protamines 1 and 2 in human spermatids and spermatozoa.

The basic nuclear proteins of a fraction of elongating spermatids from human testes and of a fraction of motile spermatozoa from the ejaculate, separated by ion-exchange chromatography, were compared. Analysis by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) showed that, in both fractions, four proteins of lower mobility were coeluted with protamine 1 by 23% guanidinium chloride (GuCl) while protamine 2 alone was eluted by 50% GuCl. Treatment with alkaline phosphatase identified those four proteins as phosphorylated protamines, and cyanogen bromide (CNBr) treatment of the dephosphorylated protamines distinguished them as variants of protamine 2 and not of protamine 1. Thus far, phosphorylated forms of protamine 1 have not been detected in either spermatids or spermatozoa. Those observations indicate that protamine 2 functions in the cycle of phosphorylation-dephosphorylation, which is essential to the process of sperm chromatin condensation, while the role of protamine 1 in human spermiogenesis is not yet defined. The presence of phosphorylated protamine in motile, presumably mature spermatozoa appears to be characteristic of human sperm but not of the sperm of other mammals and is probably the basis for the heterogeneity of chromatin condensation frequently observed in human spermatozoa.

Alkaline Phosphatase↗
PubMed1986

An ELISA for IgM titer of human serum.

An ELISA for the measurement of serum immunoglobulins (Ig) of specific isotype is described which is reproducible, more sensitive than currently used methods and simple to perform. Data are presented to support the need to include a reference serum of known Ig content on the same plate as the specimen sera in order to compensate for plate-to-plate variability due to ambient conditions. This method should prove useful for monitoring changes in levels of specific Ig classes in the progression of diseases with immune system involvement or during a therapeutic regimen.

Enzyme-Linked Immunosorbent Assay↗
PubMed1986

Reactivity of natural IgM antibodies with sperm head surface proteins. Use of an ELISA to characterize the reaction.

The data and system of analysis presented in this report have defined a subset of natural IgM antibodies in human sera that are reactive with specific members of a fraction of proteins extracted from human sperm heads. An ELISA has been developed in which, by use of a standardized reference serum, the relative values for total IgM and for sperm protein-reactive IgM may be derived for each serum. Those values, determined for 32 adult and 9 neonatal sera, indicate that the sperm protein-reactive IgM antibodies represent a reasonably constant proportion of the total serum IgM titer. Other assay data indicate that the reactivity between the antigenic sperm proteins and IgM antibodies is of high affinity. Although immunotransfers of this study have confirmed an earlier report (Rodman et al., 1985) that the immunoreactive elements of the sperm protein fraction include 7-9 separate bands or doublets, the data suggest that the reactivity is not a composite of multiple heterogeneous reactions but may be that of a few or a specific reaction, possibly involving homologous or cross-reactive antigenic determinants.

Antibodies↗