Surface Of Microplate Wells Research Articles

A whole area scanning-enabled direct-counting strategy for studying blocking efficiency in mitigating protein-solid surface binding.

The study of protein-solid surface binding as well as blocking efficiency of blocking agents plays an important role in the development of high-performance immunoassays. Although conventional colorimetric based assays are widely employed to monitor protein non-specific binding on the surface of microplate wells and evaluate the performance of blocking agents, there is still a great need to develop new methods to achieve the same goal from a new perspective. In this study, an innovative whole area scanning (WAS)-enabled direct-counting strategy was developed and validated through studying the blocking efficiency of different blocking agents on the non-specific binding of streptavidin-alkaline phosphatase conjugate (Strep-ALP, a model protein) to the surface of 96-well microplates. After non-specific binding of Strep-ALP in wells with or without blocking agents' treatment and loading of ELF™ 97 phosphate (ELFP), ALP in Strep-ALP conjugates converts ELFP to water-insoluble ELF™ 97 alcohol (ELFA), which precipitates locally, self-assembles into large needle structures, and glows green fluorescence upon excitation. After quenching the reaction, WAS of the whole wells allows us to directly count the number of individual fluorescent precipitates, which can be used to calculate and compare the blocking efficiency of three commonly used blocking agents (BSA, casein, and dry milk) based on mitigating the non-specific binding of Strep-ALP. WAS-enabled counting of individual needle-type precipitates opens a new avenue to investigate protein-solid surface binding as well as the efficiency of blocking agents with high sensitivity.

Influence of nicotine on the adherence of Candida albicans ATCC 14053 and Candida parapsilosis ATCC 22019 and expression of selected binding-related genes

ABSTRACTSmoking is considered one of the main factors for development of dental plaque. Yeasts have been described to contribute to the plaque community. The aim of this study was to investigate the influence of nicotine on the growth of planktonic cells and biofilm, cell aggregation, surface hydrophobicity, cell adherence and binding-related genes expressed by Candida albicans and Candida parapsilosis in vitro. The relative number of viable fungal cells was determined based on viable plate counts. The biofilm growth was quantified using the crystal violet assay. Reverse transcription-polymerase chain reaction was used to evaluate the regulation of selected genes associated with adherence. The results indicated that nicotine enhanced the growth of both planktonic and biofilm oral fungal cells. Cell surface hydrophobicity and the expression of Hyphal Wall Protein 1 (HWP1) and agglutinin-like sequences 3 (ALS3) of C. albicans and C. parapsilosis were found to increase relative to the nicotine concentrations. The results suggest that nicotine can enhance the growth of C. albicans and C. parapsilosis in vitro and influence their adherence to the surface of microplate wells that mimic as the tooth surface.

Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

A Sensitive and Specific IgM-ELISA for the Serological Diagnosis of Human Leptospirosis Using a rLipL32/1-LipL21-OmpL1/2 Fusion Protein

Objective To construct a lipL32/1-lipL21-OmpL1/2 fusion gene and its prokaryotic expression system, and to establish an enzyme-linked immunosorbent assay (ELISA) using the rLipL32/1-LipL21-OmpL1/2 fusion antigen of Leptospira interrogans for sensitive and specific detection of IgM in the serum of patients with leptospirosis. Methods lipL32/1-lipL21-OmpL1/2 fusion genes were constructed using a primer-linking PCR. The target recombinant protein antigens, rLipL32/1, rLipL21, rOmpL1/2 and rLipL32/1-LipL21-OmpL1/2, were expressed and the purified antigens were then immobilized to the surface of microplate wells for ELISA-based detection of IgM in the sera of leptospirosis patients. Results Of 493 acute leptospirosis patients, 95.7% and 97.8% were positive by rLipL32/1-LipL21- OmpL1/2-IgM-ELISA using different serum dilutions, which was higher than the rLipL32/1-IgM-ELISA (93.1% and 90.3%), rLipL21-IgM-ELISA (90.3% and 87.0%), and rOmpL1-IgM-ELISA (85.6% and 81.1%) ( P<0.01). All IgM-ELISAs tested negative against 56 non-leptospirosis patients with typhoid fever, hemorrhagic fever or dengue fever. Conclusion Trigeminal fusion antigen increases ELISA sensitivity and the rLipL32/1-LipL21-OmpL1/2- IgM-ELISA is a sensitive and specific serological diagnostic method for clinical leptospirosis.

Fluorescent microplate-based analysis of protein-DNA interactions I: immobilized protein

A simple protein-DNA interaction analysis has been developed using a high-affinity/high-specificity zinc finger protein. In essence, purified protein samples are immobilized directly onto the surface of microplate wells, and fluorescently labeled DNA is added in solution. After incubation and washing, bound DNA is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.2 nM DNA. Since the detection of bound DNA is noninvasive and the protein-DNA interaction is not disrupted during detection, iterative readings may be taken from the same well, after successive alterations in interaction conditions, if required. In this respect, the assay may therefore be considered real time and permits appropriate interaction conditions to be determined quantitatively. The assay format is ideally suited to investigate the interactions of purified unlabeled DNA binding proteins in a high-throughput format.

Simultaneous Fluorescent Detection of Two mRNA Targets by High Performance Signal Amplification

Development of drug candidates, which modulate cytokine responses or metabolic pathways by targeting gene expression, requires analytical systems that measure specific messenger RNAs rapidly and accurately and that can be adapted to high throughput screening operations. Chromagen’s High Performance Signal Amplification (HPSA), system, a DNA probe hybridization method, has been enhanced to measure two different mRNA targets simultaneously in the same micro-plate well. Multi-target testing using HPSA takes advantage of a family of low molecular weight fluorescent dyes with large quantum yields, which exhibit distinct excitation and emission wavelengths. A two-target model system using cultured human THP-1 cells examined IL-1b mRNA induction in response to bacterial lipopolysaccharide (LPS) exposure and also monitored intrinsic b-actin mRNA as a housekeeping gene transcript. An oligonucleotide DNA probe complementary to IL-1b RNA was labeled with a reporter ligand while a b-actin DNA probe was linked directly to an enzyme. Sample mRNA was captured onto the surface of micro-plate wells and hybridized to both DNA probes. After removing nonhybridized probe, a second enzyme conjugate that specifically recognizes the IL-1b DNA probe reporter ligand was allowed to bind. The two enzyme systems were distinguished by using substrates labeled with different fluorescent tags (one for IL-1b and the other for b-actin). Resolution of the two fluorescent products was carried out with Chromagen’s high-sensitivity, photoncounting fluorometer. This system was capable of detecting synthetic RNA targets for IL-1b and b-actin in the attomole range. Authentic b-actin mRNA was measured in 50,000 uninduced cells and this signal could be specifically competed away by addition of excess unlabeled b-actin probe during hybridization. A time course of IL-1b mRNA induction in THP-1 cells by LPS revealed that peak induction occurred after 2-3 hours. The bactin mRNA level showed an initial decrease, but remained relatively constant throughout the remaining time points. Results obtained with the dual detection format paralleled those generated when each target was measured separately in its single-target gene expression assay. Chromagen’s HPSA two-target system not only augments screening information, but also saves testing time and conserves reagents. The potential of this system is being explored with different gene targets of therapeutic value and other housekeeping genes. This presentation was given at the 1999 International Symposium for Laboratory Automation and Robotics held in Boston, MA, October 17-20, 1999. The full manuscript is available on CD-Rom and can be acquired by contacting Christine O’Neil, 508-497-2224; email islar@islar.com or access the ISLAR website at www.islar.com.

Simultaneous Fluorescent Detection of Two mRNA Targets by High Performance Signal Amplification

Development of drug candidates, which modulate cytokine responses or metabolic pathways by targeting gene expression, requires analytical systems that measure specific messenger RNAs rapidly and accurately and that can be adapted to high throughput screening operations. Chromagen's High Performance Signal Amplification (HPSA), system, a DNA probe hybridization method, has been enhanced to measure two different mRNA targets simultaneously in the same micro-plate well. Multi-target testing using HPSA takes advantage of a family of low molecular weight fluorescent dyes with large quantum yields, which exhibit distinct excitation and emission wavelengths. A two-target model system using cultured human THP-1 cells examined IL-1β mRNA induction in response to bacterial lipopolysaccharide (LPS) exposure and also monitored intrinsic β-actin mRNA as a housekeeping gene transcript. An oligonucleotide DNA probe complementary to IL-1β RNA was labeled with a reporter ligand while a β-actin DNA probe was linked directly to an enzyme. Sample mRNA was captured onto the surface of micro-plate wells and hybridized to both DNA probes. After removing non-hybridized probe, a second enzyme conjugate that specifically recognizes the IL-1β DNA probe reporter ligand was allowed to bind. The two enzyme systems were distinguished by using substrates labeled with different fluorescent tags (one for IL-1β and the other for β-actin). Resolution of the two fluorescent products was carried out with Chromagen's high-sensitivity, photon-counting fluorometer. This system was capable of detecting synthetic RNA targets for IL-1β and β-actin in the attomole range. Authentic β-actin mRNA was measured in 50,000 uninduced cells and this signal could be specifically competed away by addition of excess unlabeled β-actin probe during hybridization. A time course of IL-1β mRNA induction in THP-1 cells by LPS revealed that peak induction occurred after 2–3 hours. The β-actin mRNA level showed an initial decrease, but remained relatively constant throughout the remaining time points. Results obtained with the dual detection format paralleled those generated when each target was measured separately in its single-target gene expression assay. Chromagen's HPSA two-target system not only augments screening information, but also saves testing time and conserves reagents. The potential of this system is being explored with different gene targets of therapeutic value and other housekeeping genes.

A Unique and Sensitive ELISA Technique for Typing ABH Antigens in Bloodstains Using UEA-I Lectin—The Removal of Detergent with a Sephadex G-25 Mini-Column Improves Sensitivity

Abstract A unique sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of ABH antigens in bloodstains has been developed. Human anti-A and -B antisera and Ulex europaeus anti-H lectin were coated on the inner surface of microplate wells. The sample antigens from bloodstains, solubilized with n-octyl-β-D-glucopyranoside which was then removed by passing through a Sephadex G-25 (G-25) mini-column, were placed in the wells. After washing the wells repeatedly, peroxidase-conjugated Ulex europaeus lectin I was added and incubated. Antigen activities were determined by the development of colors using o-phenylenediamine/H2O2. This technique permitted clear detection of all ABH antigens corresponding to the antisera and lectin with high sensitivities. The A and B antigens were solubilized as aggregates with H antigen from the erythrocyte membrane. Excess detergent remaining in the sample reduced the sensitivity and accuracy of this ELISA, probably due to the removal of antibody from the wells by the effect of the surfactant. The treatment of solubilized antigens with G-25, an indispensable step, eliminated the adverse effect of the detergent on the ELISA. The ELISA method reported here was proved to be easy, economical and sensitive, and this technique should be useful in the forensic practice.

A novel assay for typing Rh antigens in blood-stains using a lectin specific to the bisecting [formula omitted] side chain of glycoprotein

A unique sandwich enzyme-linked immunosorbent assay for the determination of Rh antigens in blood stains has been developed using Rh antisera and phaseolus vulgaris E4/peroxidase conjugate (PHAE4/PO). The appropriate antiserum for detecting Rh C, c, D, E or e was coated on the inner surface of microplate wells, and the sample antigens from blood stains, solubilized with n- octyl-β- D-glucopyranoside , were then placed in the wells. After washing the wells repeatedly, PHAE4/PO was added. Bound PHAE4/PO was detected by the development of colors using o-phenylenediamine/H 2O 2. All Rh antigens corresponding to the antisera were clearly detected using this technique. The detection limit expressed by sample dilution was more than 2 × 10 5 times (volume/dried blood weight) for the various antigens from the fresh 5 × 5 mm 2 blood stain. Even when the blood stain samples were left beside a sunny window at room temperature for 2 months, Rh antigens were still detected. When the ABH, MN, P 1, Kidd, Duffy and Lewis blood grouping systems were tested with similar ELISA procedures PHAE4 did not recognize any antigen. Since PHAE4 specifically recognizes and combines with the bisecting N- acetyl- D-glucosamine side chain, it was concluded that the glycoprotein was a component of all Rh antigens immune complexes.

The quantitative estimation of antibody to Pasteurella haemolytica in sheep sera using a micro-enzyme-linked immunosorbent assay (ELISA)

The standardization of a micro-method of enzyme-linked immunosorbent assay (ELISA) coupled to a flow system of reading is described for the quantitation of antibodies to Pasteurella haemolytica in sheep. This indirect ELISA is simple to perform, highly sensitive and possesses a degree of reproducibility superior to more commonly used tests. A capsular extract of P. haemolytica biotype A serotype 1 was adsorbed to the surface of microplate wells and levels of specific immunoglobulin G (IgG) in test sera were assayed using a pig anti-sheep IgG serum conjugated with alkaline phosphatase. After reaction of the enzyme, substrate colour intensities in individual wells were measured using a “flow-through” system resulting in accurate, objective assessments.