Detection of biomolecules in the near-infrared spectral region via a fiber-optic immunosensor.

The design, development, and application of a fluorescent fiber-optic immunosensor (FFOI) procedure for the detection of antibody/antigen binding within the near-infrared (NIR) spectral region is reported. The technique was developed through the combined use of fiber-optics, semiconductor laser excitation, fluorescence detection, NIR dye, and immunochemical techniques. The antibody is immobilized on the FFOI's sensing tip and utilized as a recognition component for trace amounts of specific antigen. The FFOI is constructed to utilize antibody sandwich technique. Three individual immunoassays are reported. The first two assays utilize the FFOI and NN382, a commercial NIR dye, for the detection of human immunoglobulin G (IgG). In these assays, goat anti-human IgG antibody (GAHG) is immobilized on the sensitive terminal of the FFOI followed by the exposure of the antibody-coated terminal to human IgG. The probe is then introduced to GAHG labeled with NN382, generating a signal. The third assay utilizes the FFOI for the detection of trace amounts of Legionella pneumophila serogroup 1 (LPS1). In this assay, rabbit anti-LPS1 antibody is immobilized on the sensitive terminal of the FFOI followed by exposure to LPS1. The antigen-coated probe is then treated with monoclonal anti-LPS1 antibody followed by incubation with GAHG labeled with NN382. The assays are optimized to detect the corresponding antigen via the NIR-FFOI. Typical measurements are performed in 10-15 min. A 780-nm semiconductor laser provides the excitation of the immune complex and the resulting emission is detected by a 820-nm silicon photodiode detector. The intensity of the resulting fluorescence is directly proportional to the concentration of the antigen. Solutions of IgG and LPS1 with concentrations as low as 10(-11) M and 0.5 ng/ml, respectively, have been detected with a minimum interference.

Identification of some uncommon monounsaturated fatty acids of bacteria.

Location of the double-bond position of monounsaturated fatty acids of various bacteria was accomplished with combined gas chromatography-mass spectrometry analysis of dimethyl disulfide (DMDS) derivatives. The monoenoic fatty acids from whole cells were converted to methyl esters and then to DMDS adducts and analyzed by capillary gas chromatography-mass spectrometry. The mass spectra of DMDS adducts gave an easily recognizable molecular ion and two major diagnostic ions attributable to fragmentation between the two CH3S groups located at the original site of unsaturation. Twenty-one relatively novel monoenoic fatty acids were identified among the bacteria studied. All Flavobacterium species contained i17:1 omega 8c, Bacillus alvei contained i16:1 omega 11c and i17:1 omega 12c, and Psychrobacter immobilis contained 12:1 omega 9c. Resolution of cis and trans isomers with capillary gas chromatography and subsequent mass spectrometry permitted positive identification of 16:1 omega 7c and 16:1 omega 7t in Arcobacter (Campylobacter) cryaerophila and 16:1 omega 9c and 16:1 omega 9t in Aerococcus viridans.

Disposable reversed-phase chromatography columns for improved detection of carboxylic acids in body fluids by electron-capture gas-liquid chromatography.

Disposable reversed-phase chromatography columns were tested for their effectiveness in removing unreacted trichloroethanol (TCE) from derivatized samples for gas-liquid chromatography analysis. Derivatized acidic chloroform extracts of saponified whole cells of Mycobacterium species, spent culture media, and derivatized acidic chloroform extracts of serum and cerebrospinal fluids from patients with tuberculous meningitis were tested. Samples were added to preconditioned reversed-phase chromatography columns, and various solvents and solvent mixtures were tested to determine maximum recovery of the TCE derivatives. With this procedure, we were able to quickly remove the TCE reagent and efficiently recover TCE-derivatized carboxylic acids. Use of these columns improved the reagent cleanup procedure, simplified the derivatization step, permitted increased detection of trace components, such as tuberculostearic acid, in body fluids, and improved the selectivity of the procedure for detection of carboxylic acids.

Chemical characterization of clinical isolates which are similar to CDC group DF-3 bacteria.

Six clinical isolates, taken from blood or wounds, that had biochemical characteristics most similar to Centers for Disease Control group DF-3 bacteria were examined for cellular fatty acid composition and isoprenoid quinone content to evaluate their chemical relatedness to known bacterial species and groups. The fatty acids were liberated from whole cells by base hydrolysis, methylated, and analyzed by capillary gas-liquid chromatography. The isoprenoid quinones were extracted from lyophilized whole cells and analyzed by reverse-phase high-performance liquid chromatography. All six strains, which were designated group DF-3-like, possessed a distinct fatty acid profile that was characterized by large amounts (greater than 20%) of 13-methyltetradecanoate (i-C15:0) and 12-methyltetradecanoate (a-C15:0), moderate amounts of saturated branched-chain 13-carbon acids (i-C13:0 and a-C13:0) and hexadecanoate (n-C16:0), and small to moderate amounts of both branched- and straight-chain hydroxy acids (i-3-OH-C15:0, 3-OH-C16:0, i-3-OH-C17:0, and 2-OH-C17:0). This fatty acid profile was unique compared with the profiles of group DF-3 and other bacteria we have previously tested and is useful for the rapid identification of group DF-3-like isolates. The isoprenoid quinone content of four group DF-3-like strains was similar, with ubiquinone-9 (Q-9) and Q-10 as their major quinones, while the other two group DF-3-like strains contained Q-7 as their major quinones, with smaller amounts of Q-8 and Q-9.

Cellular fatty acid composition of Lautropia mirabilis.

Ten strains of Lautropia mirabilis (ATCC 51599(T) and nine phenotypically similar clinical isolates) were examined for cellular fatty acid (CFA) composition to evaluate their chemical relatedness to known bacterial species and groups. The CFAs were liberated from whole cells by base hydrolysis, methylated, and analyzed by gas-liquid chromatography. CFA profiles were generated by using a commericial software package (MIDI, Newark, Del.). All strains tested had an identical CFA profile characterized by major amounts of 16:1omega7c (41%) and 16:0 (44%); smaller amounts (1 to 4%) of 3-OH-10:0, 12:0, 14:0, 15:0, and 18:1 omega7c; trace amounts (<1%) of 10:0, 18:2 and 18:0; and no cyclopropane acids. This profile was similar to the CFA profiles of Acidovorax delafieldii, Comamonas terrigena, and strains of an unclassified Centers for Disease Control group designated weak oxidizer group 1. CFA analysis, when supplemented by phenotypic characterization, is useful for the identification of L. mirabilis isolates.

Biochemical characteristics and fatty acid composition of Gilardi rod group 1 bacteria.

Fifteen strains of eugonic, nonoxidative, gram-negative rods isolated primarily from human wounds of the extremities and blood formed a distinct group which was designated Gilardi rod group 1. The phenotypic characteristics of Gilardi rod group 1 were most similar to those of CDC group M-5, with the major difference that nitrite reduction was observed with CDC group M-5. All 15 strains of Gilardi rod group 1 possessed a distinct fatty acid profile which was characterized by large amounts (> 15%) of cis-vaccenic (18:1 omega 7c), palmitic (16:0), myristic (14:0), and lactobacillic (19:0 cyc11,12) acids and moderate amounts (3 to 5%) of lauric (12:0), 3-hydroxylauric (3-OH-12:0), and palmitoleic (16:1 omega 7c) acids. This fatty acid profile is unique compared with the profiles of CDC group M-5 and other bacteria we have tested and is useful for the rapid identification of Gilardi rod group 1 isolates.

Cellular fatty acid compositions of "Achromobacter groups B and E".

Strains of "Achromobacter groups B and E" were examined for cellular fatty acid (CFA) composition to evaluate their chemical relatedness to known bacterial species and groups. The CFAs were liberated from whole cells by base hydrolysis, methylated, and analyzed by capillary gas-liquid chromatography. The CFA profiles of the two groups were identical and were distinct from CFA profiles of all other bacteria we have previously tested. These data provide support for results from whole-cell protein pattern analysis and DNA-DNA and rRNA-DNA hybridization studies, which show that "Achromobacter groups B and E" are biotypes of a single new genus and species.

Detection of metabolites by frequency-pulsed electron capture gas-liquid chromatography in serum and cerebrospinal fluid of a patient with Nocardia infection.

Serum (SR) and cerebrospinal fluid (CSF) from a patient suspected of having tuberculous meningitis were submitted to our laboratory for analysis by frequency-pulsed electron capture gas-liquid chromatography (FPEC GLC). The samples were tested for the presence of carboxylic acids, alcohols, hydroxy acids, and amines by methods described previously (C. C. Alley, J. B. Brooks, and D. S. Kellogg, Jr., J. Clin. Microbiol. 9:97-102, 1977; J. B. Brooks, C. C. Alley, and J. A. Liddle, Anal. Chem. 46:1930-1934, 1974; J. B. Brooks, D. S. Kellogg, Jr., M. E. Shepherd, and C. C. Alley, J. Clin. Microbiol. 11:45-51, 1980; J. B. Brooks, D. S. Kellogg, Jr., M. E. Shepherd, and C. C. Alley, J. Clin. Microbiol. 11:52-58, 1980). The results were different from previous FPEC GLC profiles of SR and CSF from patients with known tuberculous meningitis. Both the SR and CSF contained several unidentified compounds that were not previously detected in tuberculous meningitis or any of our other studies of body fluids. Nocardia brasiliensis was later isolated from the patient. Detection of these metabolites by FPEC GLC could prove to be useful for rapid diagnosis of Nocardia disease, and their identification will provide a better understanding of metabolites produced by Nocardia sp. in vivo.

Selective procedures for detecting femtomole quantities of tuberculostearic acid in serum and cerebrospinal fluid by frequency-pulsed electron capture gas-liquid chromatography.

Conditions are described for the detection of tuberculostearic acid (10-methyloctadecanoate; C18 X CH3) in cerebrospinal fluid and serum of patients with tuberculous meningitis. C18 X CH3 was found in both the cerebrospinal fluid and serum of patients with tuberculous meningitis at concentrations of 25 to 50 fmol (10(-15) mol). The necessary specificity and sensitivity for detection of C18 X CH3 were obtained by extraction under acid conditions with organic solvent, specific functional group esterification with trichloroethanol, cleanup with disposable reverse-phase sorption chromatography columns, analysis on high-resolution polar and nonpolar capillary columns, and detection by a frequency-pulsed electron capture detector. Use of an IBM 9000 computer equipped with CAP software significantly aided comparison between known C18 X CH3 standards and C18 X CH3 in clinical specimens. Scale expansion and attenuation changes were the major contributions obtained by use of the computer. The data indicate that detection of C18 X CH3 by frequency-pulsed electron capture gas-liquid chromatography may be a valuable aid for early detection of tuberculous meningitis.

Physiological studies on the growth and utilization of sugars by Listeria species.

Experiments, relevant to growth in milk, were done to delineate the aerobic and anaerobic growth of Listeria species on selected sugars in several media. All species grew on glucose aerobically, forming lactic acid and (or) acetic acid. Anaerobically, only lactic acid was formed; cell yields were 80% of those obtained aerobically. When incubated aerobically, small amounts (1.5 microns/mL) of isovaleric acid, 2-hydroxyisovaleric acid, and trace amounts of isobutyric acid were formed. These products were characteristically formed by 26 strains representing all the species of Listeria. Added leucine stimulated isovaleric acid formation. Anaerobic fermentations of glucose could be followed by 60 to 80% cell lysis; less lysis occurred in air. Anaerobically, only hexoses and pentoses supported growth; aerobically, maltose and lactose supported growth of some strains, but sucrose did not support growth of any strain tested. Listeria grayi and Listeria murrayi utilized the galactose and glucose moieties of lactose for growth; Listeria monocytogenes and Listeria innocua used only the glucose moiety. Glucosamine and N-acetylglucosamine supported aerobic and anaerobic growth as well as glucose, and their presence stimulated the utilization of lactose by "lactose-negative" strains. Analyses of cultures grown at 5 degrees C in sterile milk treated with glucose oxidase supported the conclusion that the glucose of the milk was the major, if not the limiting, substrate that supported growth.

Analyses of fermentation products of Listeria species by frequency-pulsed electron-capture gas-liquid chromatography.

Aerobic fermentation broths of eight Listeria monocytogenes strains, two or more strains of the remaining six Listeria species, and one strain of Jonesia denitrificans were examined by frequency-pulsed electron-capture gas-liquid chromatography for carboxylic acids, alcohols, amines, and hydroxy acids. All species produced acetic, isobutyric, butyric, isovaleric, phenylacetic, lactic, 2-hydroxybutyric, 2-hydroxyvaleric, and 2-hydroxyisocaproic acids. Propionic acid was not formed, and traces of isocaproic acid were observed. Of the alcohol and amine derivatives observed, only acetylmethylcarbinol, butylamine, and putrecine were identified. Recognition of the products of glucose and amino acid metabolism serves to further characterize the members of the genus Listeria both taxonomically and physiologically.

Phenotypic characteristics, fatty acid composition, and isoprenoid quinone content of CDC group IIg bacteria.

Eleven strains of eugonic, nonoxidative, gram-negative rods isolated from clinical specimens formed a distinct group that was designated CDC group IIg. Five of the 11 isolates were from wounds. The phenotypic characteristics of CDC group IIg were most similar to those of Weeksella species, with the major difference being that CDC group IIg strains grew on MacConkey agar in 1 to 2 days, did not hydrolyze gelatin, and did not produce urease. All 11 strains of CDC group IIg possessed a distinct fatty acid profile that was characterized by large amounts (19 to 29%) of 18:1 omega 7c, 16:0, and 16:1 omega 7c, moderate amounts (6 to 10%) of 3-OH-14:0 and 14:0, and smaller amounts (1 to 2%) of 18:2, 18:0, and 3-OH-16:0. This fatty acid profile differs from those of Weeksella species by the absence of branched-chain fatty acids. CDC group IIg contains ubiquinone-8, as opposed to menaquinone-6 in Weeksella species. The isolates were susceptible to a variety of antimicrobial agents, including the aminoglycosides, tetracyclines, quinolones, sulfonamides, and polymyxin B.

Rapid diagnosis of tuberculous meningitis by frequency-pulsed electron-capture gas-liquid chromatography detection of carboxylic acids in cerebrospinal fluid.

The frequency-pulsed electron-capture gas-liquid chromatography technique described previously by Brooks et al. was modified and applied to the studies of coded and routine clinical specimens. Uncentrifuged cerebrospinal fluid (2 ml) was extracted under acidic conditions, derivatized, and analyzed by frequency-pulsed electron-capture gas-liquid chromatography on large-bore fused silica polar and nonpolar capillary columns. The frequency-pulsed electron-capture gas-liquid chromatography profile of carboxylic acids (C2 through C22) along with identification of tuberculostearic acid, established by retention time comparison of derivatized tuberculostearic acid and derivatized sample extract, strongly suggests the presence of Mycobacterium tuberculosis in patients with lymphocytic meningitis. Results from 41 coded cases and 75 clinical cases showed that the frequency-pulsed electron-capture gas-liquid chromatography test had a specificity of 91% and a sensitivity of 95%.

Isoprenoid quinones of "Afipia" spp.

The isoprenoid quinone contents of seven strains of "Afipia felis," the type strains of "A. clevelandensis" and "A. broomeae," and reference strains of three unnamed "Afipia" genospecies were determined by reverse-phase high-performance liquid chromatography. The quinone profiles of all "Afipia" strains were essentially identical, with ubiquinone 10 as the major component. The identity of ubiquinone 10 was confirmed by mass spectrometry.

Cellular fatty acid composition and phenotypic and cultural characterization of CDC fermentative coryneform groups 3 and 5.

Seventy strains of fermentative, asporogenous, gram-positive coccobacilli or short rods form two closely related groups which have been designated CDC fermentative coryneform groups 3 (32 strains, xylose fermenters) and 5 (38 strains, xylose nonfermenters). The two taxa are otherwise similar to each other phenotypically and culturally and by a distinctive Staphylococcus-like odor and by cellular fatty acid (CFA) composition. CDC group 3 and CDC group 5 strains have been isolated from clinical sources (blood, abscesses, and wounds but not urine or respiratory specimens) in Canada and the United States and among referrals from Belgium, Sweden, and Spain. Coryneform CDC group 3 strains were phenotypically similar to CDC coryneform group A-3 but were distinguishable by their inability to reduce nitrate and by their lack of motility. Coryneform CDC group 5 isolates were phenotypically somewhat similar to Actinomyces viscosus and Rothia dentocariosa, except that none of this group reduced nitrate. Both CDC groups could be differentiated from these similar bacteria by the ability to decarboxylate lysine and ornithine. The CFA compositions of CDC group 3 and 5 strains were similar to each other, were distinctive from those of other coryneforms, and were of the branched-chain type. API CORYNE codes were consistent for both CDC group 3 and CDC group 5 bacteria, suggesting that this method could be useful as an identification method.

CDC group IIc: phenotypic characteristics, fatty acid composition, and isoprenoid quinone content.

Twenty strains of glucose-utilizing, small gram-negative slightly pleomorphic rods that grew well aerobically and that were isolated from clinical specimens formed a phenotypically similar group that was designated CDC group IIc. The phenotypic characteristics of CDC group IIc were most similar to those of CDC groups IIe and IIh, the major differences being that CDC group IIc produced acid from sucrose, hydrolyzed esculin, and usually reduced nitrate. The CDC group IIc strains were analyzed by gas-liquid chromatography for their cellular fatty acid compositions, and all contained relatively large amounts of isobranched hydroxy and nonhydroxy acids. High-performance liquid chromatography and mass spectrometry analysis of the quinone extract showed menaquinone-6 as the major component. Both the cellular fatty acid and isoprenoid quinone compositions were consistent with the profiles of CDC groups IIe and IIh. Thirty percent of the isolates were from human blood.

Legionella waltersii sp. nov. and an unnamed Legionella genomospecies isolated from water in Australia.

Two Legionella-like organisms were isolated from water samples obtained in Adelaide, Australia. One organisms was isolated from a drinking water distribution system, and the other was isolated from a cooling tower at a sewage treatment plant. Both strains required L-cysteine for growth and contained cellular branched-chain fatty acids and ubiquinones typical of the genus Legionella. These strains were serologically distinct from each other as determined by a slide agglutination test. STrain 2074-AUS-ET (T = type strain) was serologically distinct from all previously described Legionella species and serotypes. Strain 2055-AUS-E could not be differentiated biochemically or serologically from Legionella quinlivanii. Both strains were shown by DNA hybridization studies (Hydroxyapatite method) to be members of new Legionella species. Legionella waltersii sp. nov. is the name proposed for strain 2074-AUS-ET (= ATCC 51914T). L. waltersii was less than 10% related to other Legionella species. Strain 2055-AUS-E (= ATCC 51913) was informally named Legionella genomospecies 1, since it could not be phenotypically distinguished from L. quinlivanii. Legionella genomospecies 1 was closely related to L. quinlivanii strains (53 to 69% related with 4.5 to 6.5% divergence at 60 degrees C and 31 to 52% related at 75 degrees C).

Chemical and cultural characterization of CDC group WO-1, a weakly oxidative gram-negative group of organisms isolated from clinical sources.

Ninety-six strains of weakly oxidative gram-negative rods isolated primarily from clinical specimens form a distinct group that has been designated Centers for Disease Control (CDC) group WO-1 (WO stands for weak oxidizer). The phenotypic characteristics of CDC group WO-1 were most similar to those of Comamonas acidovorans, Pseudomonas mallei, and CDC pink coccoid group III. The WO-1 group can be differentiated from C. acidovorans by the oxidation of glucose (often weak and sometimes delayed), motility by means of one or two polar flagella, and, when positive, the complete reduction of nitrate and nitrite. Motility and usually the failure to produce arginine dihydrolase distinguish this group from P. mallei. The WO-1 strains differ from the pink coccoid group III by the absence of pink growth pigment, the lack of predominantly coccoid cellular morphology, and usually the inability to produce acid from xylose. The cellular fatty acid compositions of 29 group WO-1 strains were characterized by large amounts of C16:0 and C16:1w7c; smaller amounts of C18:1w7c, C14:0, C12:0, and 3-OH-C10:0; and trace to small amounts of C15:1w6 and C17:0 acids. The fatty acid profile of WO-1, compared with the profiles of other bacteria we have tested previously, was most similar to the profiles of two phenotypically different organisms, Comamonas terrigena (a nonoxidative, multipolar gram-negative rod) and Chromobacterium violaceum (a fermentative gram-negative rod). Ubiquinone-8 was the major quinone in the five WO-1 strains examined. Eighty-five percent of the WO-1 strains were isolated from human specimens. Thirty-three percent were from blood, and 10% were from cerebrospinal fluid.

Design and development of a fiber-optic immunosensor utilizing near-infrared fluorophores.

The design and application of a fluorescent fiber-optic immunosensor (FFOI) are reported. The FFOI is utilized for the detection of antibody/antigen binding within the near-infrared (NIR) spectral region. The technique is developed through the combined use of fiber-optic, semiconductor laser-excitation, fluorescence detection, NIR dye, and immunochemical techniques. The antibody is immobilized on the FFOI and utilized as a recognition component for trace amounts of specific antigen. The FFOI is constructed to utilize an antibody sandwich technique. The assay involves the immobilization of the capture antibody on the sensing tip of the FFOI followed by the exposure of the immobilized sensing tip to the antigen. The antigen-coated FFOI is then introduced to a second antibody previously labeled with the NIR dye. Typical measurements are performed in about 15 min. A semiconductor laser provides the excitation (780 nm) of the immune complex. The resulting emission is detected by a silicon photodiode detector (820 nm). The intensity of the resulting fluorescence is directly proportional to the concentration of the antigen. The sensitivity of the analysis reaches 10 ng/ml and the response time is 10-15 min.

Characterization of Centers for Disease Control group NO-1, a fastidious, nonoxidative, gram-negative organism associated with dog and cat bites.

Seventeen strains of fastidious, nonoxidative, gram-negative rods, isolated from human wounds resulting primarily from dog or cat bites, formed a distinct group, which was designated Centers for Disease Control (CDC) group nonoxidizer 1 (NO-1). The phenotypic characteristics of CDC group NO-1 were most similar to non-acid-producing Acinetobacter species, with the major difference being a negative reaction in the transformation assay test for Acinetobacter spp. The cellular fatty acid composition of CDC group NO-1 was different from those of Acinetobacter species and all other bacteria tested to date. The isolates were susceptible to a variety of antimicrobial agents including the aminoglycosides, beta-lactam antibiotics, tetracyclines, quinolones, and sulfonamides. Fifty percent of the isolates were resistant to trimethoprim. Ubiquinone-8 was present as the major isoprenoid quinone in CDC group NO-1.