Highly sensitive optical chip immunoassays in human serum.

Over the past decade the ability of refractometric optical sensors to quantitatively measure a wide range of biomolecules has been demonstrated. These include proteins, nucleic acids, microorganisms, and in competitive formats small molecules such as drugs and pesticides. Furthermore, by using high refractive index nanoparticles to amplify the biomolecular binding signal, sensitivities approaching those of well established diagnostic assays have been achieved. However, to date it has not been possible to show rapid detection of analytes in complex bodily fluids such as serum, in a one-step procedure, due to the interference resulting from non-specific binding (NSB) to the sensor surface. We have carried out preliminary work on the control of interference due to NSB using an optical chip based on the Hartman interferometer. This interferometer configuration employs a reference sensing region that can be functionalized separately from the specific sensing region. Optical chips were stored dry after surface functionalization, and rehydrated in serum. The observed level of background drift in serum was reduced by an order of magnitude when an exposed reference was used, compared to a reference which was blind to the sample. An additional 70% reduction in signal drift in serum was achieved by controlling the surface chemistry of the optical chip using a biotin-poly(ethylene glycol) (PEG) blocking agent. This functionalization procedure was combined with a sandwich assay using gold nanoparticles to develop a one-step assay for human chorionic gonadotropin (hCG) in human serum with a detection limit of 0.1 ng/ml for a 35 min assay.

Chronic constipation management for institutionalized older adults.

Bran's effectiveness in reducing the need for bowel medication for intermediate care patients was examined at a VA medical center in New York. Twelve older men with chronic constipation and bowel medication use were matched and randomized to receive either bran treatment or usual care (controls). After baseline assessment, six patients were given gradually increasing daily doses of a bran mixture. Results over a 4-month period showed that these patients completely discontinued oral laxative use and had an 80% reduction in total bowel medication use without adversely affecting bowel frequency. The six men who received usual care (controls) experienced no changes.

Optical chip immunoassay for hCG in human whole blood.

We report on the development of an integrated optic chip sensor for performing rapid and sensitive immunoassays with human whole blood using human chorionic gonadotropin (hCG) as the model system. The optical chip is based on the Hartman interferometer, which uses a single planar lightbeam to address multiple interferometers, each comprising a signal/reference pair of sensing regions. The binding of antigen to specific capture antibodies on the signal sensing region causes a change in the refractive index of the surface layer, which is detectable by its effect on the evanescent field of the guided lightbeam. The reference-sensing region is coated with an irrelevant antibody, which optically cancels a large fraction of the non-specific adsorption that occurs on the specific-sensing region when the sensor is tested with clinical specimens. This work extends previous experiments with buffer and human serum to measurements in undiluted whole human blood. Optical chips were stored dry after surface functionalization, and rehydrated with blood. Colloidal gold nanoparticles conjugated to a second anti-hCG monoclonal antibody were used to provide signal amplification, thereby enhancing assay sensitivity, in a one-step procedure with the gold conjugate added to the test sample immediately prior to measurement. Background signals due to non-specific binding (NSB) in blood were found to be higher than those previously reported with human serum. In addition, a high level of background signal was found with the gold conjugate, which had not been observed in experiments with either buffer or serum. Nevertheless, hCG could be detected at 0.5 ng/ml within 10 min of sample application. The sensor response was linear over the concentration range 0.5-5 ng/ml hCG, as compared with the clinically-relevant range 0.3-1.5 ng/ml. Detection at higher concentrations was affected by scattering from large amounts of bound gold nanoparticles. However, initial binding rate measurements could be used to maintain assay quantitation.

Comparison of enzyme immunoassay and culture for diagnosis of chlamydial conjunctivitis and respiratory infections in infants.

The efficacy of Chlamydiazyme (Abbott Laboratories, North Chicago, Ill.) in detecting neonatal conjunctival and respiratory infections caused by Chlamydia trachomatis was determined by comparison of this enzyme immunoassay (EIA) with the method of isolation of chlamydiae in tissue culture. The sensitivity and specificity of Chlamydiazyme for detecting C. trachomatis in conjunctival specimens from infants with conjunctivitis were 98 and 94%, respectively. For nasopharyngeal infection in infants with conjunctivitis, the sensitivity and specificity were 87 and 92%, respectively. There were nine nasopharyngeal specimens that were Chlamydiazyme positive and culture negative. All of these specimens demonstrated the presence of typical fluorescing chlamydial elementary bodies when pellets of the original specimens were examined with a fluorescein-conjugated monoclonal antibody. When the EIA was performed on nasopharyngeal specimens from infants with suspected chlamydial pneumonia, 6 culture-positive and 10 culture-negative specimens were correctly identified.

Evaluation of chlamydiazyme for the detection of genital infections caused by Chlamydia trachomatis.

Chlamydiazyme is a 4-h enzyme-linked immunoassay that detects an antigen of Chlamydia trachomatis directly in clinical specimens. This immunoassay was compared with cell culture for the diagnosis of chlamydial infections of the genital tract. The assay was evaluated at five clinics with a total of 1,277 cervical specimens of which 239 were culture positive. At three of these clinics where urethral samples were taken from males, 99 of 363 samples were culture positive. The sensitivity of the assay averaged 89.5% for detecting cervical infections and 78.8% for detecting male urethral infections. Specificity was 97.0% when samples from either males or females were tested. Some patients who were culture negative were infected with chlamydiae according to both Chlamydiazyme and a monoclonal antibody test that detected a chlamydial antigen distinct from the antigen detected by Chlamydiazyme. If the 15 females and 2 males who were positive by both immunoassays but culture negative were considered positive for chlamydial infection, the specificity of the assay was 98.4% in females and 97.7% in males. Chlamydiazyme is a simple and relatively rapid immunoassay that has sufficient sensitivity and specificity to supplant culture in the detection of genital chlamydial infections.

Expansion of the tetragonally arrayed cell wall protein layer during growth of Bacillus sphaericus.

The outermost layer of the cell wall of Bacillus sphaericus strain P-1 is a tetragonally arrayed structure (T-layer) which is assembled from a single polypeptide. No turnover of T-layer was detected during growth of cultures. In contrast, the turnover of peptidoglycan was between 20 and 25% per generation. The sites of deposition of new T-layer on the cell surface were identified by the indirect fluorescent antibody technique, which labeled old T-layer, and by the reverse technique, which labeled new T-layer. These experiments demonstrated that the major area of T-layer deposition was a band at the site of an incipient cell division. This band subsequently split and covered the new pole of each progeny cell. Little or no T-layer was inserted into existing poles. In addition, multiple bands of new T-layer, which probably accommodate cell elongation, were inserted along the lateral surface of the cell.

Incorporation of [14C]methionine by Yersinia enterocolitica after invasion of HeLa cells.

Invasion of HeLa cells by Yersinia enterocolitica can be measured by the incorporation of [(14)C]methionine in the presence of streptomycin and cycloheximide.

Adherence of Vibrio parahaemolyticus to human epithelial cell lines.

Light microscopy and a radioassay detected no significant difference in adherence of Kanagawa-positive and Kanagawa-negative strains of Vibrio parahaemolyticus to human epithelial cell lines.

Animal model studies of genital chlamydial infections. Immunity to re-infection with guinea-pig inclusion conjunctivitis agent in the urethra and eye of male guinea-pigs.

A previous report demonstrated that male guinea-pigs could be infected in the urethra with guinea-pig inclusion conjunctivitis (GPIC) agent and that the infection was transmitted during mating from infected males to females. In the experiments reported here, inoculation of male guinea-pigs in the urethra with GPIC organisms resulted in infection which subsided spontaneously in about 2 weeks. Males were demonstrated to be completely resistant to urethral challenge with 10(3)ID50 when tested 6 weeks after urethral infection. These guinea-pigs, immune to re-infection of the urethra, remained susceptible to infection of the eye, but this ocular infection was shorter in duration than that in previously uninfected control animals. Infection in the eye resulted in immunity to both ocular and urethral infection when animals were challenged 6 weeks after the ocular infection.

Neutralization of Chlamydia trachomatis in cell culture.

Neutralization of Chlamydia trachomatis was assayed by the decrease in inclusion-forming units in baby hamster kidney cells grown in culture. Five percent fresh guinea pig sera increased neutralization titers of rabbit antisera 100- to 1,000-fold but had no effect when normal rabbit sera were tested. Neutralization of a type A or B trachoma isolate was strain specific. Neutralization by human eye secretions and sera also was demonstrated when guinea pig sera were included in the test. All of the six human sera tested showed strain specificity against types A or B, in agreement with typing by the fluorescent antibody technique.

Specificity of the autolysin of Streptococcus (Diplococcus) pneumoniae.

A Streptococcus (Diplococcus) pneumoniae autolysin, partially purified from cellular autolysates, was optimally active at pH 7.0 and was stimulated by monovalent cations. Addition of autolysin to walls resulted in the appearance of only N-terminal l-alanine, whereas no glycosidase activity was observed. Walls which had been solubilized by autolysin were separated by gel filtration into a low-molecular-weight peptide containing amino acids in the same ratios found in intact walls and a high molecular fraction containing the amino acid-deficient peptidoglycan backbone. Thus, the major activity is an N-acetylmuramyl-l-alanine amidase. In addition, walls undergoing spontaneous lysis revealed no glycosidase activity but showed an increase in only N-terminal alanine. Autolysin, which was bound to walls in saline, was almost completely removed when walls were washed in distilled water, and all of the activity was recovered in the water wash fluid.