Summary Sickle cell disease (SCD) is a significant healthcare burden worldwide, but most affected individuals reside in low-resource areas where access to diagnostic testing may be limited. We developed and validated a rapid, inexpensive, disposable diagnostic test, the HemoTypeSC™, based on novel monoclonal antibodies (MAbs) that differentiate normal adult haemoglobin (Hb A), sickle haemoglobin (Hb S) and haemoglobin C (Hb C). In competitive enzyme-linked immunosorbent assays, each MAb bound only its target with <0.1% cross-reactivity. With the HemoTypeSC™ test procedure, the sensitivity for each variant was <5.0 g/l. The accuracy of HemoTypeSC™ was evaluated on 100 whole blood samples from individuals with common relevant haemoglobin phenotypes, including normal (Hb AA, N=20), carrier or trait (Hb AS, N=22; Hb AC, N=20), SCD (Hb SS, N=22; Hb SC, N=13), and Hb C disease (Hb CC, N=3). The correct haemoglobin phenotype was identified in 100% of these samples. The accuracy of the test was not affected by Hb F (0-94.8% of total Hb) or Hb A2 (0-5.6% of total Hb). HemoTypeSC™ requires <1 μl of whole blood and no instruments or power sources. The total time-to-result is <20 min. HemoTypeSC™ may be a practical solution for point-of-care testing for SCD and carrier status in low-resource settings.
Direct sequencing and analysis of viral genomes are definitive methods for identifying various hepatitis C virus (HCV) genotypes. However, HCV genome sequencing methods are cumbersome and unsuitable for analyzing large numbers of clinical samples. We have developed a convenient, reliable, and reproducible RIBA strip immunoblot assay system for determining HCV serotype. Briefly, the assay consists of an immunoblot strip on which there are five lanes of immobilized serotype-specific HCV peptides from the nonstructural (NS-4) and core regions of the genomes of HCV types 1, 2, and 3. HCV serotype is deduced by determining the greatest intensity of reactivity to the NS-4 serotype-specific HCV peptide band in relation to the intensity of the human immunoglobulin G internal control bands on each strip. HCV core peptide reactivity is used only in the absence of NS-4 reactivity. We used this assay to successfully serotype a high percentage of sera from well-documented HCV-infected patients. Our serotyping results correlated 99% with the findings from the standard restriction fragment length polymorphism genotyping methods. Less than 5% of the serum samples were untypeable. For a selected group of alpha interferon-treated patients we observed that the nonresponders (76.2%) and a majority of the responders who relapsed (72.2%) had type 1 HCV infection. A small population (n ؍ 8) of complete responders was split 3:4:1 as type 1, type 2, and type 3, respectively. Our data indicate that this new serotyping assay has the potential to be a highly specific and reliable method for typing of HCV infection in patients.
Sera from 483 patients at high (group 1, n = 313) and lower (group 2, n = 170) risk for exposure to hepatitis C were tested for antibodies to hepatitis C using first-generation (c100-3) and second-generation enzyme-linked immunosorbent assays and four-antigen recombinant immunoblot assay. The second-generation enzyme-linked immunosorbent assay and nitrocellulose-based immunoblot assay differ from c100-3-based systems in the addition of expression products from the NS3/NS4 (c33c, c200) and putative nucleocapsid (c22-3) region of the hepatitis C genome. In group 1, the sensitivity of detection of hepatitis C antibodies was 45%, 55% and 46% by the first- and second-generation enzyme-linked immunosorbent assays and recombinant immunoblot assay, respectively. In group 2, antibodies were detected by each test system in 26%, 32% and 7% of patients, respectively. Most sera (99%) reactive with the first-generation enzyme-linked immunosorbent assay were reactive with the second-generation enzyme-linked immunosorbent assay (in group 1, 89% of these specimens demonstrated reactivity to at least one antigen with the immunoblot assay, compared with only 31% in group 2). An additional 12% (group 1) and 6% (group 2) of specimens demonstrated reactivity with the second-generation enzyme-linked immunosorbent assay only (of these, 75% [group 1] and 9% [group 2] demonstrated reactivity to at least one antigen with the immunoblot assay). Ninety-eight percent of specimens not reactive with both enzyme-linked immunosorbent assay test systems were also nonreactive by recombinant immunoblot assay.(ABSTRACT TRUNCATED AT 250 WORDS)
To develop a rapid antibody test for Sin Nombre hantavirus (SNV) infection for diagnosis of hantavirus pulmonary syndrome (HPS) in field settings where advanced instrumentation is not available, a strip immunoblot assay bearing four immobilized antigens of SNV and a recombinant nucleocapsid protein antigen of Seoul hantavirus (SEOV) was prepared. The SNV antigens included a full-length recombinant-expressed nucleocapsid (N) protein (rN), a recombinant-expressed G1 protein (residues 35 to 117), and synthetic peptides derived from N (residues 17 to 59) and G1 (residues 58 to 88). On the basis of the observed reactivities of hantavirus-infected patient and control sera, we determined that a positive assay requires reactivity with SNV or SEOV rN antigen and at least one other antigen. Isolated reactivity to either viral rN antigen is indeterminate, and any pattern of reactivity that does not include reactivity to an rN antigen is considered indeterminate but is unlikely to represent hantavirus infection. Fifty-eight of 59 samples from patients with acute SNV-associated HPS were positive according to these criteria, and one was initially indeterminate. Four of four samples from patients with HPS due to other hantaviruses were positive, as were most samples from patients with SEOV and Puumala virus infections. Of 192 control serum samples, 2 (1%) were positive and 2 were indeterminate. Acute SNV infection was distinguishable from remote SNV infection or infection with hantaviruses other than SNV by the presence of G1 peptide antigen reactivities in the former. The strip immunoblot assay shows promise for the detection of SNV antibodies early in the course of HPS.
DOLPHIN. Can. J. Chem. 53, 1554 (1975.The sulfonation of meso-tetraphenylporphyrin, in concentrated sulfuric acid followed by neutralization and dialysis, provides an efficient and convenient route to meso-tetra(4-sulfonatopheny1)porphyrin. The initial studies by Winkelman (1) on the localization of meso-tetra(4-sulfonatopheny1)-porphyrin in tumors have been followed by an increasing interest in this water soluble porphyrin. This interest has been stimulated by the ready availability (2) of meso-tetraphenylporphyrin (TPP) 1 and its conversion into mesotetra(4-sulfonatopheny1)porphyrin 2 by direct sulfonation.Two methods for this sulfonation have recently been reported. Both methods bring about the sulfonation in hot sulfuric acid and differ only in the isolation and purification of the product. The method developed by Fleischer et al. (3) invokes preparation of the tetraammonium salt which is purified by six precipitations from methanol-acetone but, in our hands, a good recovery of the product always results in its contamination with ammonium sulfate. To remove the inorganic salts one must sacrifice large amounts of the product at each stage with a resultant poor yield.The second method reported by Srivastava and Tsutsui (4) requires dilution of the reaction 'Author to whom correspondence should be addressed. mixture and addition of lime to the resulting large volume of aqueous solution in order to remove the sulfuric acid as calcium sulfate. A number of time-consuming and cumbersome filtrations and concentrations are then required to isolate the product, which is always contaminated with inorganic salts unless careful precautions are taken, which again result in a lowering of the yield.We wish to report here the preparation and simplified purification procedure which involves dialysis to remove inorganic contaminants and results in a 7574 overall yield from TPP. For personal use only.
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