A simple saliva-based test for detecting antibodies to human immunodeficiency virus.

This study was performed to determine the feasibility of using saliva as a diagnostic medium for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 under nonlaboratory conditions and to evaluate the performance characteristics of such a test. We developed for this purpose a self-contained kit (Saliva. Strip [ST]), which combines the collection and processing, as well as the analysis, of the specimen. The kit's performance was evaluated in a blinded study. Saliva collection was facilitated with a specially designed device that contains a sample adequacy indicator, and immunochromatography test strips were used for the analysis. A total of 1,336 matched serum and saliva specimens (684 reactive and 652 nonreactive specimens) were tested. We tested sera using an enzyme immunoassay (EIA) and a rapid strip test. Sera reactive in one of the assays were also analyzed by Western blotting. Sensitivity and specificity were 99.4 and 99.4%, respectively, for ST, 100 and 99.1%, respectively, for EIA, and 99.7 and 100%, respectively, for the serum strip test. The saliva test performed well when HIV-2-positive sera or a low-titer performance panel (HIV-1) of serum or plasma specimens were diluted (1:2,000) in nonreactive saliva. Because the methodology we present here uses a noninvasively obtained medium, the methodology may be suitable for use in the field where laboratory support and personnel are limited, such as community outreach programs, doctors' offices, surveillance studies, and community hospitals.

A simple whole-blood test for detecting antibodies to human immunodeficiency virus.

We developed an immunochromatographic whole-blood test (WBT) which detects antibodies to human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) from fingerstick blood. The sensitivity and specificity of the WBT were 99.41% (1,018 confirmed positive patients) and 99.89% (941 uninfected patients), respectively (enzyme immunoassay [EIA] on serum or plasma as a reference). WBT performance was comparable to those of licensed EIAs and Western blotting, using 18 HIV-2 sera, 23 HIV-1 seroconversion panels, and a low-titer performance panel (in lieu of whole blood).

A field test for the detection of antibodies to human immunodeficiency virus types 1 and 2 in serum or plasma.

In response to the need for simple and rapid tests for infectious diseases, we have devised a test for antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 which resembles many contemporary strip-style pregnancy tests in format and ease of use. The test was evaluated with 2,928 serum specimens (1,541 reactive and 1,387 nonreactive) collected and tested at a Mexico City hospital clinic and was compared with a laboratory assay (Abbott) performed simultaneously. The sensitivity and specificity of the test using these serum specimens were 99.68 and 99.71%, respectively (before the code of the blinded study was broken). This compares with 100% sensitivity and 97.55% specificity with the laboratory assay (specificity upon reassay after the code was broken, 99.21%). In a survey of HIV-2 specimens, reactive (positive) specimens were detected in 51 of 51 cases. The test was examined with 21 commercially available (HIV-1) seroconversion panels. The performance of the test was comparable to that of a group of Food and Drug Administration-approved (antibody-based) HIV tests.

PIP: Simple and rapid methods of detecting infectious disease are needed. The authors therefore developed a test for antibodies to HIV-1 and HIV-2 which resembles many contemporary strip-style pregnancy tests in both format and ease of use. The test was evaluated with 1541 reactive and 1387 nonreactive serum specimens collected and tested at a Mexico City hospital clinic. A laboratory assay was performed simultaneously. The sensitivity and specificity of the test using these serum specimens were 99.68% and 99.71%, respectively. The laboratory assay was 100% sensitive and 97.55% specific. All reactive HIV-2 specimens were detected. The test was examined with 21 commercially available HIV-1 seroconversion panels. The performance of the authors' test was comparable to that of a group of Food and Drug Administration approved HIV tests.

Simplified testing for antibodies to human immunodeficiency virus.

Test strips for the detection of antibodies to human immunodeficiency virus type 1 were investigated using specimens from risk groups in Thailand (141 reactive; 445 nonreactive) in a local Thai laboratory. The diagnostic sensitivity and specificity were both 100%. Using a set of seroconversion panels, the sensitivity of the test strips was within the range of sensitivities obtained with enzyme immunoassays. The test was developed for performance at decentralized settings under nonlaboratory conditions.

PIP: During December 1992-January 1993, the Thai Ministry of Public Health collected blood samples from populations at high risk of HIV infection (prostitutes and men attending sexually transmitted disease clinic). All these samples were evaluated for HIV infection by enzyme immunoassay and, if positive, confirmed by the Western blot. There were 141 HIV-1 reactive samples. In a blind study, laboratory personnel of the Venereal Diseases and Control Center in Chonburi, Thailand, later analyzed 586 serum samples with test strips (Sero-Strip HIV; Saliva Diagnostics Systems; Singapore). The strips have a synthetic peptide representing a highly conserved region of viral transmembrane glycoprotein, gp41. This peptide captures HIV-1 antibodies. Three investigators read the test strips independently. There were no discrepant readings among them. The test strips were also evaluated under nonlaboratory conditions of decentralized settings: exposure for 21 weeks at 45 degrees Celsius, exposure for 20 weeks at 22 degrees Celsius, and 98% relative humidity. The results of the test strips correlated completely with those of the conventional immunoassays. Thus, there were neither false positive nor false negative reactions. In conclusion, these test strips can be used reliably in non-air-conditioned tropical settings.

Purification and properties of alternanase, a novel endo-alpha-1,3-alpha-1,6-D-glucanase.

A newly isolated soil bacterium strain NRRL B-21195, tentatively identified as a Bacillus species, was found to be a constitutive producer of a novel type of glycanase that hydrolyses in an endo-fashion the polysaccharide alternan, an alpha-1,3-alpha-1,6-D-glucan, referred to in the literature as B-1355 dextran (fraction S), synthesized from sucrose by alternansucrase of Leuconostoc mesenteroides. The glycanase, named alternanase, has been purified to homogeneity from a cell-free culture fluid of the bacillus grown in a liquid medium containing D-glucose, and has been characterized. The enzyme has a molecular mass of 110000 Da (SDS/PAGE) and an isoelectric point of approximately 4.0. Optimum activity occurs at pH 7 and at a temperature of 40 degrees C. The enzyme is stable up to 50 degrees C but loses activity rapidly at 60 degrees C. Its action is inhibited by EDTA and stimulated by Ca2+. The enzyme requires, for its action, D-glucan chains in which alpha-1,3-linkages alternate with alpha-1,6-linkages; i.e., it is specific for alternan. Monitoring of alternan hydrolysis by determination of liberated reducing sugars pointed to an unusually low extent of hydrolysis and a low specific activity of the enzyme. As shown in the accompanying paper [Côté, G. L. & Biely, P. (1994) Eur. J. Biochem. 226, 641-648] the reason for this finding is that the main hydrolytic products are non-reducing, novel types of cyclic oligosaccharides.

Two-dimensional gel analysis of serum apolipoprotein A-I isoforms: preliminary analysis suggests altered ratios in individuals with heart disease.

Apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, is quantified in a number of ways, typically by immunochemical methods. Commercial tests do not discriminate among isoforms of apo A-I. Two-dimensional electrophoresis, however, segregates and differentiates these isoforms, primarily due to charge variations among the various species. Stained two-dimensional gels can be scanned using high-resolution laser densitometry, and the isoforms can then be quantified using image analysis software. Human sera from coronary heart disease (CHD) patients (n = 36) and sex-matched and close-age-matched individuals (n = 36) with no known CHD were analyzed, to determine the relative abundance for each isoform within a given serum. In this preliminary study, we observed statistically significant differences between the two groups, suggesting altered post-translational processing from the proapo A-I and mature apo A-I isoforms to their adjacent isoforms for patients with histories of heart disease. In two instances, the P values were less than 0.005; in two others, P values were less than 0.001.

High-molecular-weight amylase activities from bacteria degrading starch-plastic films.

Amylases having unusually high molecular weights (M(r), >150,000) were found in culture supernatants of an environmentally derived microbial mixed culture selected for its ability to utilize starch-containing plastic films as sole carbon sources. The mixed culture produced amylases active at pHs 5.5 and 8.0.

Computerized quantitative analysis of coomassie-blue-stained serum proteins separated by two-dimensional electrophoresis.

Two-dimensional electrophoresis in combination with Coomassie Blue staining was refined for use as a quantitative method. Microcomputer software was developed for use with the IBM AT and compatible computers for analyzing the gels. To test the refined method to determine its usefulness in simultaneous measurements of 28 human serum proteins, we measured each protein relative to a single standard (bovine serum albumin) polymerized at different concentrations in a calibration scale, rather than using 28 individual standards. All samples were analyzed in triplicate. We evaluated calibration, linearity of response, recoveries, units, within-run CV, and between-run CV. The five isoforms of apolipoprotein A-I were analyzed in samples from 16 healthy donors and the isoform ratios determined. The method as presented here should prove useful for diagnosis of non-urgent disease states and for analysis for protein isoforms in relation to disease; it should also be applicable to assays of proteins in other fluids and tissues.

Biochemical characterization of a genetically altered calmodulin in Paramecium.

Recent evidence proposes that the calcium-binding protein, calmodulin, plays a crucial role in the regulation or modulation of the calcium-dependent potassium conductance in Paramecium tetraurelia (Hinrichsen, R.D., Burgess-Cassler, A., Soltvedt, B.C., Hennessey, T. and Kung, C. (1986) Science 323, 503-506). We purified the calmodulins from both the wild type and pantophobiac A (a mutant lacking the above-mentioned conductance and whose phenotypic defect is traceable to its calmodulin) by hydrophobic interaction and immunoaffinity chromatographies, and examined them biochemically. In this paper we address the preliminary characterization of the two calmodulins and discuss the consequences of the genetic alteration. The differences described here are in their electrophoretic mobilities in polyacrylamide gel electrophoresis and in their binding characteristics to monoclonal antibodies raised against calmodulin from wild-type paramecia. Also, we present data which indicate a difference in the stimulation of the calmodulin-dependent enzyme bovine brain phosphodiesterase under certain conditions.

A mutant Paramecium with a defective calcium-dependent potassium conductance has an altered calmodulin: a nonlethal selective alteration in calmodulin regulation.

The Paramecium mutant, pantophobiac A, has a defect that results in an in vivo loss of calcium-dependent potassium efflux channel activity. This defect is corrected fully by the microinjection of wild-type Paramecium calmodulin into pantophobiac A cells and is partially restored by calmodulins from other organisms, but it cannot be restored by microinjection of pantophobiac calmodulin. Overall, these results suggested that wild-type Paramecium calmodulin has unique features that allow it to restore fully a normal phenotype and that the defect in pantophobiac A might be an altered calmodulin molecule. Previous studies established the amino acid sequence of wild-type calmodulin and showed that Paramecium calmodulin has several differences from other calmodulins, including the presence of dimethyllysine at residue 13. To test directly the possibility that calmodulin from the pantophobiac mutant might be altered, we purified the mutant calmodulin and compared its properties to those of wild-type Paramecium calmodulin. We found one amino acid sequence difference between the two Paramecium calmodulins: a phenylalanine in the mutant protein, instead of a serine, at residue 101. This change is at a calcium-liganding residue in the third calcium-binding loop. These and previous studies demonstrate that comparatively subtle changes in the structure of calmodulin can result in quantitative alterations in in vivo activity, provide insight into the in vivo roles of calmodulin and the regulation of ion channels, and demonstrate that functional alterations of calmodulin are not necessarily lethal.

Restoration by calmodulin of a Ca2+-dependent K+ current missing in a mutant of Paramecium.

A combination of genetics, biochemistry, and biophysics was used to show that calmodulin is involved in the regulation of an ion channel. Calmodulin restored the Ca2+-dependent K+ current in pantophobiac, a mutant in Paramecium that lacks this current. The restoration of the current occurred within 2 hours after the injection of 1 picogram of wild-type calmodulin into the mutant. The current remained for approximately 30 hours before the mutant phenotype returned. The injection of calmodulin isolated from pantophobiac had no effect. These results imply that calmodulin is required for the function or regulation of the Ca2+-dependent K+ current in Paramecium.

Genetic analysis of mutants with a reduced Ca2+-dependent K+ current in Paramecium tetraurelia.

Two mutants of Paramecium tetraurelia with greatly reduced Ca2+-dependent K+ currents have been isolated and genetically analyzed. These mutants, designated pantophobiac, give much stronger behavioral responses to all stimuli than do wild-type cells. Under voltage clamp, the Ca2+-dependent K+ current is almost completely eliminated in these mutants, whereas the Ca2+ current is normal. The two mutants, pntA and pntB, are recessive and unlinked to each other. pntA is not allelic to several other ion-channel mutants of P. tetraurelia. The microinjection of a high-speed supernatant fraction of wild-type cytoplasm into either pantophobiac mutant caused a temporary restoration to the wild-type phenotype.

Permeabilization of Bacillus subtilis to chemotaxis methyltransferase II.

Treatment of Bacillus subtilis with 0.4% (vol/vol) toluene renders cells permeable not only to small molecules but also apparently to proteins as large as 30,000 daltons. Methyl-accepting chemotaxis proteins and two smaller polypeptides were methylated when B. subtilis methyltransferase II was added to permeabilized cells.

Functional homology of Bacillus subtilis methyltransferase II and Escherichia coli cheR protein.

A chemotaxis-related methyltransferase enzyme from Bacillus subtilis has been shown to methylate membrane-bound proteins in Escherichia coli in vitro. The methylated proteins are in the same molecular weight range as authentic E. coli methyl-accepting chemotaxis proteins. It was also shown that wild type E. coli cytoplasmic extract could methylate membrane proteins from B. subtilis in its methyl-accepting chemotaxis protein region. Cytoplasmic extracts from methyltransferase mutants of either species could methylate neither set of methyl-accepting proteins in vitro. The B. subtilis enzyme was incapable of methylating any of a group of soluble eucaryotic proteins. These data suggest functional homology between B subtilis methyltransferase II and E. coli cheR protein (chemotaxis methyltransferase) despite the evolutionary divergence between these two species.

Purification and characterization of Bacillus subtilis methyl-accepting chemotaxis protein methyltransferase II.

A Bacillus subtilis methyltransferase capable of methylating membrane-bound methyl-accepting chemotaxis proteins (MCPs) of a chemotaxis mutant was purified to homogeneity. MCPs are normally unmethylated in this strain. Results of gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the enzyme is a 30,000 molecular weight monomer. The enzyme transfers methyl groups from S-adenosylmethionine to glutamate residues of the substrates. The enzyme is activated by divalent cations and has a Km for S-adenosylmethionine of about 5 microM. It is competitively inhibited by S-adenosylhomocysteine, with a Ki of about 0.2 microM, and exhibits an in vitro assay pH optimum of 6.9. This methyltransferase is very different from another methyltransferase from B. subtilis, described previously (Ullah, A. H. J., and Ordal, G. W. (1981) Biochem. J. 199, 795-805).