David Higginson Clarke

Introduction of an Immunochemical Label in a Cytidine Analogue

Abstract A new immunochemical label was synthesized and used as an artificial hapten to obtain a monoclonal antibody that specifically recognizes it. The new cytidine analogues, 5-methyl-4-N-{4-[6-(p-bromobenzoylamido)caproylamido]but-1−yl}-2′-deoxycytidine and 4-N-[∊-(p-bromobenzoylamido)caproylamido]cytidine, which could be useful for nonradioactive detection of DNA and RNA, were also synthesized.

Antigenic Activity of Three Chimeric Synthetic Peptides of the Transmembrane (Gp41) and the Envelope (Gp120) Glycoproteins of HIV‐1 Virus

Abstract The antigenicity of three chimeric synthetic peptides (Qm, Qm‐16, and Qm‐17) incorporating an immunodominant epitope of the gp41 transmembrane protein (587–617) and the different epitopes of the gp120 envelope protein (495–516), (301–335), (502–516) of human immunodeficiency virus (HIV‐1), separated by two glycine residues, was evaluated by UltramicroEnzyme‐linked immunosorbent assay (UMELISA) by using panels of anti‐HIV‐1 positive sera (n = 47). The specificity was evaluated with samples from healthy blood donors (n = 20) and anti‐HIV‐2 positive samples (n = 10). The results indicate that the chimeric peptide, Qm, was the most reactive one because it detected antibodies to virus efficiently. This may be related to peptide adsorption onto the solid surface, the C‐terminal region of HIV‐1 gp120 (495–516) combined with gp41 (587–617) in the chimera, and the epitope accessibility to the antibodies. This study showed the usefulness of the chimeric peptides as antigen to detect antibodies to HIV‐1 virus.

Antigenic Mixture of Synthetic Peptides for the Immunodiagnosis of HTLV I/II Infection

Abstract Monomeric and chimeric synthetic peptides were used as coating antigens in four different mixtures in a solid phase immunoassay to select an optimal combination for the detection of antibodies to human T‐cell lymphotropic virus (HTLV) in serum samples. The peptides, P‐13 (gp21 I), Q5 (gp21 II)‐GG‐(gp46 II), and Q (gp46 I)‐GG‐(p19 I), represent immunodominant sequences from transmembrane protein (gp21), envelope protein (gp46), and core protein (p19) of HTLV I/II viruses; they were the most antigenic and specific peptides in previous studies. The sequences of the chimeric peptides were separated by two glycine residues. An indirect UltramicroEnzyme‐linked immunosorbent assay (UMELISA) was used to evaluate the antigenicity of these peptide mixtures by using samples from anti‐HTLV I/II PRP205(M), (n = 20), HTLV I‐infected individuals from Cuba (n = 7), and HTLV I‐positive sera from Colombia and Chile (n = 9). The specificity was evaluated with healthy blood donor sera (n = 300), anti‐HIV 1‐positive samples (n = 10), and other seropositive samples to different infectious agents. The highest sensitivity and specificity was obtained with mixture 1, which could be very useful in the immunodiagnostic of HTLV infection.

Apolipoprotein E alleles in Cuban patients with mild cognitive impairment.

Background: Apolipoprotein E (ApoE) ∊4 genotype is the most clearly documented risk factor for Alzheimer’s disease (AD). Epidemiological studies demonstrate an accelerated rate of progression to dementia and AD in patients with mild cognitive impairment (MCI). We assessed the ApoE allele and genotypes frequencies in Cuban patients with MCI. Methods: We performed ApoE genotyping of 74 Cuban patients more than 65 years old. Cognitive assessments included the Mini-Mental State Examination (MMSE) and a cognitive battery for evaluating memory, attention, perception, and executive function. Results: Cognitive impairments were characterized by amnesia and executive deficits in patients with MCI. The Apo ∊4 allele frequency was 0.196 in patients with MCI, 10-fold higher than that in the controls. Patients carrying the ∊4 allele exhibited poorer performance in MMSE and tests assessing executive function and short-term memory than noncarriers. Conclusions: The patients exhibited amnestic MCI multiple domains. Cognitive performance was worse in patients who carried the ApoE ∊4 allele.

PCR of Immobilized DNA Molecules Isolated from Human Blood Cells by a Non‐Enzymatic Method

Abstract DNA molecules suitable for amplification by Polymerase Chain Reaction were obtained by immobilizing whole blood or isolated leukocytes and incubating the immobilized cells for one hour with the known non‐enzymatic solution described for preparing intact DNA molecules for PFGE. Cell immobilization was done in agarose gels and punches of 1.2 mm of diameter had the amount of DNA needed for amplifying chromosomal and mitochondrial sequences, many times. The approach was successfully used in preparing DNA molecules from multiple samples in flat‐bottom 96‐well ELISA plates. The procedure is simple and does not demand special conditions for sample transportation or conservation; thus, it should be useful to collect and process samples under field conditions in epidemiological studies.