Sequential sandwich immunoassay for simultaneous detection in trace samples using single-channel surface plasmon resonance.

To analyze multiple analytes in trace samples, low-dosage and high efficiency are crucial in many common cases. Herein, we developed a facile method using a single-channel surface plasmon resonance (SPR)-based biosensor for the simultaneous detection of gentamicin (GEN) and melamine (MEL) in milk and serum with only one sample injection. Based on a sandwich immunoassay, non-interfering antibodies against GEN from mouse (AbGEN) and against MEL from rabbit (AbMEL) were chosen to capture the analytes. Secondary antibodies against mouse (AbM) and rabbit (AbR) were used to bind with AbGEN and AbMEL to determine the concentrations of GEN and MEL on a single channel of an SPR sensor. All of the detection process could be done in 10 min with 50 µL of sample injection. According to the response shifts of AbM and AbR, two standard curves for GEN and MEL were obtained successively, with the limit of detection (LOD) values at 4.4 ng mL-1 and 1.3 ng mL-1, respectively. Moreover, the feasibility was determined by spiking milk and serum samples with GEN and MEL, with recoveries in the range of 81.6%-118.0%. Importantly, the analytes can be substituted by others for much more applications. This method is also expected to multiply the detection efficiency of multi-channel SPR biosensors with low-dosage samples in the future.

Drug-induced immune hemolytic anemia associated with anti-vancomycin complicated by a paraben antibody.

BACKGROUND: Drug-induced immune hemolytic anemia (DIIHA) is rare, but potentially life-threatening. A high index of clinical suspicion is required for diagnosis, since the number of medications known to induce DIIHA continues to expand. Additionally, in vitro antibody reactivity against reagent additives has been reported, which may complicate test interpretation. CASE REPORT: A 61-year-old group A, D+ woman with a history of negative antibody detection tests developed hemolytic anemia on Postoperative Day 7 after repeat incision and drainage of a chronically infected right knee prosthesis. She was treated with multiple antibiotics in the postoperative period, including three cephalosporins and vancomycin intravenously as well as vancomycin and gentamicin-containing intraarticular cement spacers. STUDY DESIGN AND METHODS: A workup for possible DIIHA was performed. Testing was performed using vancomycin and cephalosporin antibiotics. Initially, gentamicin injection solution was used for testing, followed by testing with its component ingredients. RESULTS: A vancomycin antibody was detected and anemia resolved after vancomycin was discontinued. Reactivity was seen when gentamicin injection solution was used for testing, raising the possibility of a gentamicin antibody as well. However, testing with purified gentamicin as well as methylparaben and propylparaben demonstrated a paraben antibody that reacted with the paraben-containing gentamicin solution. The patient also demonstrated an anti-N. Neither the paraben antibody nor the anti-N appeared to cause in vivo hemolysis. CONCLUSION: This is the second reported case of DIIHA associated with anti-vancomycin. It is the fourth report describing a paraben antibody.

Preparation of a Chicken scFv to Analyze Gentamicin Residue in Animal Derived Food Products.

Chicken is an ideal model for simplified recombinant antibody library generation. It has been rarely been reported to apply chicken single-chain variable fragments (scFvs) in immunoassays for the detection of antibiotic and chemical contaminants in animal food products. In this study, the scFvs (S-1 and S-5) were isolated from a phage display library derived from a hyperimmunized chicken. The checker board titration revealed that the optimum concentrations of S-1 and S-5 were 0.78 µg/mL and 0.44 µg/mL respectively, to obtain OD450 around 1.0 at 5 µg/mL of Gent-OVA coating concentration. Both S-1 and S-5 exhibited negligible cross reactivity with kanamycin and amikacin. The 50% inhibitory concentration (IC50) of S-1 and S-5 were 12.418 ng/mL and 14.674 ng/mL respectively. In the indirect competitive ELISA (ic-ELISA), the limits of detection for S-1 and S-5 were 0.147 ng/mL and 0.219 ng/mL respectively. The mean recovery for Gent ranged from 60.91% to 118.09% with no more than 10.35% relative standard deviation (RSD) between the intra-assay and the inter-assay. These results indicate the chicken scFv based ic-ELISA method is suitable for the detection of Gent residue in animal derived edible tissues and milk.

Sensitive immunochemical approaches for quantitative (FPIA) and qualitative (lateral flow tests) determination of gentamicin in milk.

Three kinds of immunoassays for the determination of gentamicin in milk samples were developed and validated. First, a fast and easily-performed fluorescence polarization immunoassay was used for characterization of the employed polyclonal antibody. The calculated Kaff were (1.9±0.4)×10(9)М(-1) and (6.0±0.2)×10(6)М(-1) for the high- and low-affinity fractions respectively. The assay was characterized with a good sensitivity, the limit of detection being 5µgkg(-1). Two different kinds of detection labels, i.e. colloidal gold (CG) and quantum dots (QDs), were evaluated for use in lateral-flow format with respect to rapid visual on-site testing. The cut-off levels for both qualitative formats were selected based on the maximum level for gentamicin in milk established by the European Commission, 100µgkg(-1), resulting in a 10µgkg(-1) cut-off considering sample dilution. The intra-laboratory validation was performed with sterilized milk samples artificially spiked with gentamicin at concentrations less than, equal to, and greater than the cut-off level. It was shown that milk products could be analyzed without any sample preparation, except for dilution with the buffer solution. The rates of false-positive and false-negative results were below 5% for both labels. The different developed immunoassays were tested towards gentamicin determination in artificially-spiked and naturally contaminated milk samples.

[Effects of autologous plasma and gentamicin on immunophenotype and viability of cytokine-induced killer cells].

OBJECTIVE: To investigate the effects of treatment with different volumes of autologous plasma and gentamicin on immunophenotype and viability of cytokine-induced killer (CIK) cells in vitro. METHODS: Flow cytometry (FCM) was used to analyze the effects of autologous plasma and gentamicin on different immunocyte subtypes of CIK cells. The cell count method and MTT assay were used to detect the cytotoxicity of such pretreated CIK cells on HeLa cells. RESULTS: No significance was found in the relative amount of immunocyte subtypes CD3(+), CD56(+), CD3(+)CD4(+), CD3(+)CD8(+), CD3(+)CD56(+), CD4(+)CD25(+) between autologous plasma and control groups. Compared with control group, CD3(+), CD56(+), CD3(+)CD8(+), CD3(+)CD56(+) cells of gentamicin group were significantly down-regulated by 1.4, 1.7, 1.4 and 2.5 folds (P<0.05), respectively. Furthermore, the cytotoxicity of CIK cells in gentamicin and control groups on HeLa cells were 43.0% and 90.0%, respectively, with a statistically significant difference (P<0.05). CONCLUSION: Autologous plasma has little effect on different subtypes of CIK cells. On the contrary, gentamicin can cause a decrease in the number of CD3(+), CD56(+), CD3(+)CD8(+), CD3(+)CD56(+) cells with some cytotoxicity.

Development of a dot-blot assay for screening monoclonal antibodies to low-molecular-mass drugs.

Dot-blot is a versatile and simple analysis to perform. We adapted this method as a simple identity test for monoclonal antibodies to a number of small compounds: three transplant drugs, an anticonvulsant, a steroid, an anticancer drug, and an antibiotic. Immunology-based identity tests using low-molecular-mass organic compounds have historically been a challenge to develop. We modified the traditional dot-blot assay to serve as an identity test for monoclonal antibodies to carbamazepine, sirolimus, tacrolimus, cyclosporine, cortisol, methotrexate, and gentamicin. The primary obstacle was the immobilization of these organic compounds on nitrocellulose as nitrocellulose is also soluble in most of the organic solvents in which the compounds are soluble. We evaluated different membranes, solvents, and chemical forms of these organic compounds to overcome this challenge. A number of incubation and washing solutions were also investigated. By varying the chemical form, concentration, and incubation conditions, a set of effective and reproducible identity tests were developed for these monoclonal antibodies.

Immunoassays for the rapid detection of gentamicin and micronomicin in swine muscle.

A monoclonal antibody (mAb)-based ELISA and strip test for gentamicin (GEN) and its analogue micronomicin (MIN), are reported in this study. The conjugate gentamicin-glutaraldehyde-bovine serum albumin (GEN-GDA-BSA) was used as an immunogen. The produced anti-GEN mAB exhibited high cross-reactivity with micronomicin (MIN; 131.2%) and slight or negligible crossreactivity with other aminoglycosides. Based on this mAB, an ELISA and a strip test for GEN and MIN were developed and evaluated. The ELISA showed a 50% inhibition concentration (IC50) of 0.75 ng/mL for GEN and 0.58 ng/mL for MIN. For GEN, the average recoveries at 25-200 microg/kg ranged from 73 to 91%, with intraday CVs of 9-16% and interday CVs of 8-15%. For MIN, the average recoveries ranged from 108 to 131%, with intraday CVs of 10-16% and interday CVs of 8-15%. In contrast, the strip test for GEN or MIN had a detection limit of 5 ng/mL in phosphate-buffered saline and 50 microg/kg in muscle (n=24), and the results could be judged within 10 min. The detection results of incurred samples analyzed by the strip test, ELISA, and HPLC indicated that the two immunoassays correlated well with the HPLC method and could be used as convenient tools for the rapid screening of GEN and MIN residues in swine muscle.

Positive patch test reactions to gentamicin show sensitization to aminoglycosides from topical therapies, bone cements, and from systemic medication.

BACKGROUND: A history of prolonged use of topical antimicrobials is common among patients with positive patch test reactions to gentamicin and to aminoglycosides. OBJECTIVES: The aim of this study was to show sources of gentamicin sensitization in patients with positive patch test reactions to gentamicin. PATIENTS AND METHODS: About 7814 patients were patch tested with a baseline patch test series and 620 of them were further tested with gentamicin. The clinical histories, concurrent contact sensitivities, and sources of sensitization are analysed among these patients. RESULTS: Positive patch test reactions to gentamicin were seen in 29/620 patients, most of whom (18/29) also reacted to neomycin and to kanamycin (7/29). Mean age of the gentamicin-positive patients was 62 years, but three young operating room nurses with hand dermatitis had a history of gentamicin exposure from bone cement. Among the 11/29 neomycin-negative patients, a history of exposure to different aminoglycosides was apparent, and one patient had a history of systemic netilmicin-medication-associated exanthema. CONCLUSIONS: Positive patch test reactions to gentamicin reflect sensitization to different aminoglycosides for which gentamicin seems to represent a sensitive indicator. Gentamicin sensitization may result from occupational exposure to gentamicin containing bone cements or from systemic medication with aminoglycosides.

The deposition of C5b-9 complexes and its precursors on E. coli J5 during complement activation enhances uptake and toxicities of gentamicin.

The complement system provides the host with protection against pathogenic agents and in some cases can result in damage to host tissue. However, the exact mechanism of how complement kills gram-negative bacteria in lysozyme-neutralized and or lysozyme-depleted serum is still under active investigation. In previous studies, it has been demonstrated that inner membrane damage by the membrane attack complex contributes to depolarization and the subsequent collapse of the membrane potential. In these studies we have shown that the membrane attack complex and its precursors provide additional protective effect by the enhanced uptake of antibiotics in the death of E. coli J5. Specifically, the deposition of C5b fragments from C6 neutralized Pooled Normal Human Serum (PNHS) and C5b6 complexes from C7 neutralized PNHS on E. coli J5 contribute to antibiotic uptake and killing. Since C5b and C5b6 do not form pores, we suggest that disturbances and or cracks in the outer membrane by the deposited complexes accelerates uptake of the antibiotics and enhanced killing of E. coli J5 employed in these studies.

[Immunogenic properties of the colloidal gold]. / Immunogennye svoistva kolloidnogo zolota.

We studied the capacity of colloidal gold for enhancing specific and nonspecific immune response in laboratory animals (rabbits, rats, and mice) immunized with antigens of various nature. The antibody titers obtained with colloidal gold as a carrier were higher as compared to the standard immunization techniques (free antigen or Freund's adjuvant). Application of colloidal gold increased nonspecific immune responses as well: lysozyme concentration in the blood, activity of the complement system proteins, as well as phagocytic and bactericidal activities. The obtained antibodies were tested by immunodot assay using gold markers. Immunization of the animals with colloidal gold conjugates with haptens as well as complete antigens was shown to induce formation of highly active antibodies without using other antigens such as complete Freund's adjuvant. In addition, antigen quantities for animal immunization with colloidal gold was by one order of magnitude lower as compared to the complete Freund's adjuvant immunization. This fact can point to direct adjuvant activity of colloidal gold.

[Hentaxan as an immunoregulator in purulent wounds in parturient women]. / Hentaksan iak imunorehuliator pry hniinykh ranakh u porodil'.

Hentaxan, a new silicon sorbent, is a complex drug preparation containing hentamycin sulfate and zinc-tryptophan, endowed with antioxidant and immunomodulating activities. We used it for treating suppurating wounds in those women in labour. As many as 65 parturient women were examined. The conclusion drawn from the obtained results is that the immunomodulating potential of hentaxan is not very high, for which reason we recommend that hentaxan be combined with laferon which effects the T-link of immunity. The proposed method is at present under study.

Automated homogeneous immunoassay for gentamicin on the dimension clinical chemistry system.

BACKGROUND: Monitoring of the concentration of gentamicin in serum and plasma during therapy is widely recommended and practiced in hospitals. Our aim was to develop a homogeneous immunoassay based on particle-enhanced turbidimetric inhibition immunoassay technology to quantify gentamicin on the Dimension clinical chemistry system. METHODS: Assay performance was assessed on each of the Dimension models in a 15-instrument interlaboratory comparison study. A split-sample comparison (n = 1171) was also performed between the gentamicin methods on the Dimension system and the Abbott TDx analyzer, using multiple reagent and calibrator lots on multiple instruments. RESULTS: The Dimension method was linear to 25.1 micromol/L (12.0 microg/mL) with a detection limit of 0.63 micromol/L (0.3 microg/mL). Calibration was stable for 30 days. The within-run imprecision (CV) was <1.3%, and total imprecision ranged from 1.8% to 3.2% between 4.2 micromol/L (2.0 microg/mL) and 16.7 micromol/L (8.0 microg/mL) gentamicin. Linear regression analysis of the results on the Dimension method (DM) vs the Abbott TDx yielded the following equation: DM = 0.98TDx - 0.42; r = 0.987. Minimal interference was observed from structurally related compounds such as sagamicin, netilmicin, and sisomicin. CONCLUSION: The monoclonal antibody used in this method has similar reactivities toward the individual gentamicin subspecies C1, C1a, and C2, thus providing analytical recovery not significantly dependent on relative subspecies concentrations.

Effects of Ginkgo biloba extract on the cochlear damage induced by local gentamicin installation in guinea pigs.

Investigations evaluating the protective effect of Ginkgo biloba extract (EGb) on gentamicin (GM) ototoxicity were undertaken. Guinea pigs treated with 5 mg/kg gentamicin sulfate on the round window niche (RWN) showed acute changes on electrocochleogram and hair cell or microvilli damage on scanning electron microscopy (SEM). There was accumulation of GM in the whole cochlea, especially in the organ of Corti, stria vascularis, and type III fibrocyte on immunohistochemical study. However, the guinea pigs pretreated with local or systemic EGb revealed no significant changes by local GM installation. From these results, we concluded that EGb has a protective effect on the development of GM ototoxicity in the cochlea.

Aminoglycoside detection using a universal ELISA binding procedure onto polystyrene microtiter plates in comparison with HPLC analysis and microbiological agar-diffusion assay.

The use of enzyme-linked immunosorbent assay for the detection of aminoglycosides has been hindered due to low molecular weight compound adsorption to solid phases. Here, we describe an enzyme-linked immunosorbent assay based on the treatment of polystyrene microtiter plates with Alcian blue prepared in acetic acid prior to coating with the antibiotic. Whereas no detection of tobramycin was possible on commercially treated or untreated enzyme-linked immunosorbent assay plates, the Alcian blue treatment permitted detection of 0.025 and 0.05 microg ml(-1) of tobramycin respectively using 0.05 and 0.1% of Alcian blue with a coefficient of variation of 1.85 and 7.69%, respectively. Comparative studies of five tobramycin samples of unknown quantity using enzyme-linked immunosorbent assay and high-performance liquid chromatography gave equivalent results while those done via microbiological agar-diffusion assay were an overestimation of the actual quantity. The use of the Alcian blue pretreatment enzyme-linked immunosorbent assay procedure has permitted, in previous studies, the measure of antibodies against synthetic peptides and phospholipids. Subsequently, our demonstration of the sensitivity and reliability of this method in the quantification of tobramycin strongly suggests that the use of Alcian blue pretreatment in enzyme-linked immunosorbent assay can be applied universally to avert molecule immobilization problems on solid phases.

Crystallization of intact monoclonal antibodies.

Attempts were made to crystallize four monoclonal antibodies, one IgG2a kappa and three IgG1 kappa. Using a PEG 3350 screen combined with detergents, and developed from our experiments with an IgG2a kappa antibody specific for canine lymphoma cells, crystals have now been obtained of two of these four immunoglobulins, an antiphenytoin and an antiphenobarbital antibody. A complex between the antiphenobarbital antibody and its drug antigen crystallized as well. The antibody for phenytoin has, to this point, produced only clustered microcrystals, marginally suitable for X-ray analysis. Single crystals of the IgG1 kappa antibody against phenobarbital, however, were characterized by X-ray diffraction to be primitive monoclinic, with unit cell dimensions a = 67 A, b = 193 A, c = 74 A, and beta = 110 degrees. These crystals have an entire IgG1 kappa molecule as the asymmetric unit and they diffract to at least 3.2 A resolution.

Dot-ELISA for the rapid detection of gentamicin in milk.

A dipstick dot-ELISA for the detection of gentamicin in milk of dairy cattle is reported for the first time. The test is based on a sandwich ELISA using high affinity monoclonal antibodies to gentamicin. Antibodies were adsorbed to nitrocellulose filters, blocked, dried, and stored for several weeks before use. The dipstick ELISA detected gentamicin at a concentration of 0.1 microgram/ml and produced strongly positive results at 0.2 microgram-0.3 microgram/ml. This ELISA is highly specific and no false positives were detected when tested against various aminoglycoside analogs including streptomycin, kanamycin, bekanamycin, amikacin, neomycin, and tobramycin. Further, the elimination in cow milk of gentamicin residues following intramammary administration of the drug was studied in two dairy cattle using dot-ELISA. Milk gentamicin levels were detected at post injection hours up to 120 hr in each of the two dairy cattle. It therefore, appears that gentamicin residues can still be detected in milk after 5 days using dot-ELISA. Based on the simplicity of performance and the economical nature of the test system, dipstick is recommended as a suitable method for wide scale use in field studies and diagnostic laboratories.

[Gentamycin resistant Enterobacteriaceae at a neonatal care unit]. / Gentamycinresistente enterobakterier på en neonatalafdeling.

A case of septicaemia in a seven-day-old infant with a gentamicin-resistant strain of Enterobacter cloacae prompted an epidemiological survey in a neonatal unit. Another 18 patients harboured gentamicin-resistant Enterobacteriaceae without symptoms. Control of the outbreak was achieved by cohort nursing, strict hygiene and reduction of aminoglycoside consumption by 50%. Efficiency of the measures was monitored by weekly faecal samples from all patients. All strains produced the aminoglycoside modifying enzyme AAC(3)II. The resistance was plasmid-mediated.

Serovars and multiple drug resistant Salmonella sp. isolated from children in Rio de Janeiro-Brazil

Foram caracterizadas sorologicamente e testadas para a susceptibilidade e antimicrobianos, 116 amostras de Salmonella isoladas de coproculturas e hemoculturas efetuadas de crianças durante o período de maio de 1987-junho 1992 no Instituto Fernando Figueira, Brasil. Detectou-se seis sorogrupos (04; 07; 08; 09; 03; 10 e 035) representados por 18 serovares, sendo a S. typhimurium (62,93 por cento) a mais frequente, seguida da S. agona (7,75 por cento). Os antimicrobianos utilizados foram a ampicilina (Ap), cefalotina (Cf), cefoxitina (Cx), ceftriaxona (Cro), pefloxacin (Pf), gentamicina (Ge), amicacina (Am), sulfametoxazoletrimetropim (SxT), cloranfenicol (CL) e tetraciclina (Te). 94.52 por cento das cepas de S. typhimurium representaram determinantes de resistência. Foram encontrados 29 padröes de resistência diferentes, sendo o mais frequente para S. typhimurium o Ap, Cf, Ge, Am, Sxt, Cl, Te, Pf (40,47 por cento) e para os outros serovares o Te (20.83 por cento). A determinaçäo da concentraçäo mínima inibitória realizada para cinco dos antimicrobianos citados evidenciou altos níveis de resistência para Ap e Cl, contrapondo-se a níveis mais baixos obtidos para Ge, Sxt e Cro

[Immunochemical determination of gentamicin in serum. II. Preparation of polyclonal gentamicin antibodies]. / Imunochemické stanovenie gentamicínu v sére. II. Príprava polyklónových protilátok proti gentamicínu.

The author describes the preparation of polyclonal rabbit antibodies against gentamicin. As immunogens gentamicin conjugates with bovine serum albumin were used, or else with thyroglobulin and haemocyanin. The immunization pattern involved combined administration of immunogens by the intravenous route, or by the intramuscular or intradermal route, using liposomes and complete Freund adjuvant. The highest antibody titres against gentamicin found by the ELISA method were obtained when procedures were used where the carrier was serum albumin and thyroglobulin and as adjuvant complete Freund adjuvant was used.

[Immunocorrecting properties of antibiotics in secondary immunodeficiency]. / Immunokorrigiruiushchie svoistva antibiotikov v usloviiakh vtorichnogo immunodefitsita.

The study investigated immunocorrecting properties of penicillin G, streptomycin, gentamycin in cyclophosphamide--induced immunodeficiency in mice. It was determined, that antibiotics in sub-bactericidal doses possess pronounced immunocorrecting properties. This effect was observed in both humoral and cell-mediated immune response.