Reagents In Conjunction Research Articles

Characterization of the Function of Carbonic Anhydrase 8

Background/Objective: Severe asthma is a complex pulmonary disease characterized by airway inflammation, bronchoconstriction, and acid-base dysregulation. In the Severe Asthma Research Program bronchoscopies, transcriptomics showed CA8 as a gene that is strongly associated with asthma severity. CA8, however, lacks classical CA enzyme function: it does not catalyze hydration and dehydration of CO2. The function of CA8 in the airway epithelium remains unknown. We hypothesize that CA8 serves a protective role in the airway due to its down regulation in patients with severe asthma. We aim to characterize the function of CA8 bystudying its potential as an enzymatic protein. Methods: We used colorimetric assays to detect and quantify nitrogen oxides. We tested for Snitrosothiolsynthase, denitrosylase, nitrate and nitrite synthase, and nitrite reductase activitiesusing the Griess reagent in conjunction with Saville denitrosylation reagents and with reduction using vanadium chloride. Samples were incubated for 60 minutes. We then went on to design ametabolomic experiment in which products will be identified by NMR: for these, we transfected Chinese hamster ovary (CHO) cells using lentivirus containing GFP-labeled CA8 or emptyvector (negative control). Results: CA8 protein does not have these following enzymatic functions: SNO synthase,denitrosylase, nitrite and nitrate synthase, and nitrite reductase. We successfully transfected with GFP-labeled CA8 and are awaiting results of the metabolomic studies. Conclusion and Potential Impact: Isolated CA8 does not appear to have any nitrogen oxideredox activities relevant to asthma. The next steps include confirmatory western and SNOwestern blots to determine protein s-nitrosylation using transfected CHO whole cell lysate. Extracellular medium pH will also be measured. We will then move on to NMR-basedmetabolomics. This will help us better understand the biochemical mechanisms of CA8.Ultimately, this can provide researchers with a novel approach to asthma treatments.

Development of antibody-based assays for high throughput discovery and mechanistic study of antiviral agents against yellow fever virus

Despite the availability of a highly effective yellow fever virus (YFV) vaccine, outbreaks of yellow fever frequently occur in Africa and South America with significant mortality, highlighting the pressing need for antiviral drugs to manage future outbreaks. To support the discovery and development of antiviral drugs against YFV, we characterized a panel of rabbit polyclonal antibodies against the three YFV structural proteins and five non-structural proteins and demonstrated these antibody reagents in conjunction with viral RNA metabolic labeling, double-stranded RNA staining and membrane floatation assays as powerful tools for investigating YFV polyprotein processing, replication complex formation, viral RNA synthesis and high throughput discovery of antiviral drugs. Specifically, the proteolytic processing of the viral polyprotein can be analyzed by Western blot assays. The predominant nuclear localization of NS5 protein as well as the relationship between intracellular viral non-structural protein distribution and foci of YFV RNA replication can be revealed by immunofluorescence staining and membrane flotation assays. Using an antibody against YFV NS4B protein as an example, in-cell western and high-content imaging assays have been developed for high throughput discovery of antiviral agents. A synergistic antiviral effect of an YFV NS4B-targeting antiviral agent BDAA and a NS5 RNA-dependent RNA polymerase inhibitor (Sofosbuvir) was also demonstrated with the high-content imaging assay. Apparently, the antibody-based assays established herein not only facilitate the discovery and development of antiviral agents against YFV, but also provide valuable tools to dissect the molecular mechanism by which the antiviral agents inhibit YFV replication.

Difunctionalization of Cyclopropyl Amines with N-Iodosuccinimide (NIS) or in Situ Formed Cyanogen Iodide (ICN).

We report herein a 1,3-difunctionalization of cyclopropylamines that serve as a π nucleophile in a two-electron (2e) SN2-like ring opening pathway. N-Iodosuccinimide (NIS) or in situ generated cyanogen iodide (ICN) is employed as electrophilic iodinating reagents in conjunction with TMSCN or succinimide to furnish multiple pairs of functional groups disposed in a 1,3-manner. This 2e ring opening manifold overcomes the constraint of our previously reported 1e protocol as demonstrated by successful activation of monocyclic tertiary cyclopropylamines.

Improving Performance and Stability of Planar Perovskite Solar Cells through Grain Boundary Passivation with Block Copolymers

Organic–inorganic metal halide perovskite solar cells (PSCs) exhibit excellent photovoltaic performance but have the drawbacks of instabilities against moisture and heat due to the inherent hydroscopic nature and volatility of their organic components. Herein, it is reported that using the block copolymer F127 as the passivation reagent in conjunction with the solvent annealing process can efficiently improve the performance and stability of corresponding organic–inorganic PSCs. It is anticipated that the hydrophilic poly(ethylene oxide) tails of F127 polymers connect with contiguous perovskite crystals and passivate defects at perovskite grain boundaries, whereas the dangling hydrophobic poly(phenyl oxide) centers suppress perovskite decomposition caused by moisture and heat. After the optimization of the F127 additive, planar PSCs with champion power conversion efficiencies of 21.01% and 18.71% are achieved on rigid and flexible substrates, respectively. The F127 passivation strategy provides an effective approach for fabricating high‐efficiency and stable PSCs.

COD removal from biologically stabilized landfill leachate using Advanced Oxidation Processes (AOPs)

The removal of soluble organic matter from mature landfill leachate by means of biological, chemical, and combined biological-chemical methods was investigated in this study. Aerobic sequencing batch reactor (SBR) with activated sludge was used as the biological treatment to remove ammonium. The chemical treatments included ozone (O3), ozone + hydrogen peroxide (O3 + H2O2), Fenton’s reagents (H2O2 + Fe2+), and ozone + Fenton’s reagents (O3 + H2O2 + Fe2+). Results indicated that the SBR achieved approximately 25% Chemical Oxygen Demand (COD) removal with an initial COD of 1276 mg/L. Ozonation of raw leachate achieved 16% COD removal after 240 min of treatment while O3 + H2O2 achieved 33% COD removal of raw leachate with 900 mg/L H2O2 in 120 min. Moreover, Fenton’s process also achieved 33% COD removal from raw leachate. Results suggest that the biological treatment followed by chemical treatments had the most efficient COD removal. After biological stabilization, ozonation removed 52% of the COD within 240 min. The Fenton’s process achieved 67% COD removal using equal doses of H2O2 and Fe2+. However, Fenton’s reagents in conjunction with O3 removed 69% and 72% COD in 120 min and 240 min, respectively. The dosage test also revealed that the 2:1 ratio of H2O2 and Fe2+ had the best COD removal. Therefore, these results indicate that the combination of biological and post-chemical treatment methods effectively remove organic pollutants from mature landfill leachate.

Ir(III)/MPAA-Catalyzed Mild and Selective C-H Amidation of N-Sulfonyl Ketimines: Access To Benzosultam-Fused Quinazolines/Quinazolinones.

Ir(III)-catalyzed unprecedented mild C-H amidation for weakly coordinating cyclic N-sulfonyl ketimines, accelerated by a mono protected l-amino acid, has been developed. The method uses 1,4,2-dioxazol-5-ones as the robust amidating reagent in conjunction with a catalytic amount of silver triflate. It is highly selective and does not require a stoichiometric amount of oxidants or additives. A series of mechanistic experiments was performed to gain some insights into the reaction mechanism. The strategy provides easy access to novel benzosultam-quinazoline and benzosultam-quinazolinone hybrid scaffolds endowed with pharmaceutically relevant features.

Qualitative and Quantitative Proteome Analysis of Oral Fluids in Health and Periodontal Disease by Mass Spectrometry.

The significance of protein identification and characterization by classical protein chemistry approaches is clearly highlighted by our detailed understanding of the biological systems assembled over a time period of almost a century. The advent of state-of-the-art mass spectrometry (MS) with sensitivity, speed, and global protein analysis capacity without individual protein purification has transformed the classical protein chemistry with premise to accelerate discovery. These combined with the ability of the oral fluids such as whole saliva (WS) and gingival crevicular fluid (GCF) to reflect both systemic and locally derived proteins have generated significant interest to characterize these fluids more extensively by MS technology. This chapter deals with the experimental details of preanalytical steps using multidimensional protein separation combined with MS analysis of WS and GCF to achieve detailed protein composition at qualitative and quantitative levels. These approaches are interfaced with gold standard "stable-isotope" labeling technologies for large-scale quantitative MS analysis which is a prerequisite to determine accurate alterations in protein levels as a function of disease progression. The latter incorporates two stable-isotope chemistries one specific for cysteine containing proteins and the other universal amine-specific reagent in conjunction with oral fluids in health and periodontal disease to perform quantitative MS analysis. In addition, specific preanalytical steps demanded by the oral fluids such as GCF and WS for sample preparations to overcome limitations and uncertainties are elaborated for reliable large-scale quantitative MS analysis.

Copper-catalyzed asymmetric addition of olefin-derived nucleophiles to ketones.

Enantioenriched alcohols found in an array of bioactive natural products and pharmaceutical agents are often synthesized by asymmetric nucleophilic addition to carbonyls. However, this approach generally shows limited functional-group compatibility, requiring the use of preformed organometallic reagents in conjunction with a stoichiometric or substoichiometric amount of chiral controller to deliver optically active alcohols. Herein we report a copper-catalyzed strategy for the stereoselective nucleophilic addition of propargylic and other alkyl groups to ketones, using easily accessible (poly)unsaturated hydrocarbons as latent carbanion equivalents. Our method features the catalytic generation of highly enantioenriched organocopper intermediates and their subsequent diastereoselective addition to ketones, allowing for the effective construction of highly substituted stereochemical dyads with excellent stereocontrol. Moreover, this process is general, scalable, and occurs at ambient temperature.

Reversed-phase ion-pair ultra-high-performance-liquid chromatography–mass spectrometry for fingerprinting low-molecular-weight heparins

Heparin is a complex mixture of sulfated linear carbohydrate polymers. It is widely used as an antithrombotic drug, though it has been shown to have a myriad of additional biological activities. Heparin is often partially depolymerized in order to decrease the average molecular weight, as it has been shown that low molecular weight heparins (LMWH) possess more desirable pharmacokinetic and pharmacodynamic properties than unfractionated heparin (UFH). Due to the prevalence of LMWHs in the market and the emerging availability of generic LMWH products, it is important that analytical methods be developed to ensure the drug quality. This work explores the use of tributylamine (TrBA), dibutylamine (DBA), and pentylamine (PTA) as ion-pairing reagents in conjunction with acetonitrile and methanol modified mobile phases for reversed-phase ion-pairing ultraperformance liquid chromatography coupled to mass spectrometry (RPIP-UPLC–MS) for fingerprint analysis of LMWH preparations. RPIP-UPLC–MS fingerprints are presented and compared for tinzaparinand enoxaparin.

Significance of chemical conditioning to improve dewaterability of biosludge generated from tanneries

To improve the dewaterability, biosludge generated from activated sludge process and membrane bioreactor operated for treatment of tannery wastewater was conditioned with Fenton’s reagent alone and Fenton’s reagent in conjunction with polyelectrolyte, respectively. Studies were carried out at an optimum pH of 3.0 with a fixed Fe2+ concentration of 3,000 mg/L. Hydrogen peroxide and polyelectrolyte dosages were varied for the sludge generated from membrane bioreactor and activated sludge process. Optimum dosages were ascertained for hydrogen peroxide and polyelectrolyte. Hydrogen peroxide dosage of 900 and 750 mg/L were observed as optimum for membrane bioreactor sludge and activated process sludge with respect to volume of settled sludge. Polyelectrolyte dosage of 7.5 mg/L for membrane bioreactor sludge and 5 mg/L for activated process sludge in conjunction with Fenton’s reagent was found to be the optimum with respect to volume of settled sludge. For the optimum dosage of Fenton’s reagent in conjunction with polyelectrolyte, results were compared with respect to capillary suction time and release of bound water.

Nanotextured Material for Applications in CSF Sample Screening and Characterization

ABSTRACTNeurodegenerative disease is primarily characterized by protein misfolding and the resultant protein aggregation. Presence of soluble oligomeric aggregates of proteins including various Aβ and α-syn aggregate species can be correlated to the onset and progression of many neurodegenerative diseases. The ability to detect protein misfolding requires the design of a diagnostics assay the will enable molecular level probing. The use of nanoporous ceramic templates enables size based immobilization of the target proteins and by leveraging the principle of “macromolecular crowding” protein association can be mapped with a high degree of resolution. By tailoring the surface functionalization within nanoporous ceramic templates, macromolecular immobilization can be selectively controlled, which in turn significantly enhances the perturbation to the electrical double layer/. The changes to the electrical double layer are measured with a high degree of sensitivity through impedance spectroscopy.Pre symptomatic diagnosis and distinction between Alzheimer’s and Parkinson’s diseases can be achieved by the specific detection and quantification of levels of each of these different toxic protein species in cerebrospinal fluid (CSF). Detection using highly selective morphology specific reagents in conjunction with the ultrasensitive nanoporous electronic biosensor showed the presence of different protein morphologies in human CSF samples. Detection is primarily achieved by identifying the specific association of the protein with its receptor using electrochemical impedance spectroscopy. Furthermore, we show that these morphology specific reagents can readily classify between post-mortem CSF samples from AD, PD and cognitively normal sources. These studies suggest that detection of specific oligomeric aggregate species holds great promise as sensitive biomarkers for neurodegenerative disease.

CSF levels of oligomeric alpha-synuclein and beta-amyloid as biomarkers for neurodegenerative disease

Protein misfolding and aggregation is a critically important feature in many devastating neurodegenerative diseases, therefore characterization of the CSF concentration profiles of selected key forms and morphologies of proteins involved in these diseases, including β-amyloid (Aβ) and α-synuclein (a-syn), can be an effective diagnostic assay for these diseases. CSF levels of tau and Aβ have been shown to have great promise as biomarkers for Alzheimer's disease. However since the onset and progression of many neurodegenerative diseases have been strongly correlated with the presence of soluble oligomeric aggregates of proteins including various Aβ and a-syn aggregate species, specific detection and quantification of levels of each of these different toxic protein species in CSF may provide a simple and accurate means to presymptomatically diagnose and distinguish between these diseases. Here we show that the presence of different protein morphologies in human CSF samples can be readily detected using highly selective morphology specific reagents in conjunction with a sensitive electronic biosensor. We further show that these morphology specific reagents can readily distinguish between post-mortem CSF samples from AD, PD and cognitively normal sources. These studies suggest that detection of specific oligomeric aggregate species holds great promise as sensitive biomarkers for neurodegenerative disease.

ChemInform Abstract: The Case for the Use of Unrefined Natural Reagents in Analytical Chemistry: A Green Chemical Perspective

AbstractReview: 86 refs.

Nuclear-delimited Angiotensin Receptor-mediated Signaling Regulates Cardiomyocyte Gene Expression

Angiotensin-II (Ang-II) from extracardiac sources and intracardiac synthesis regulates cardiac homeostasis, with mitogenic and growth-promoting effects largely due to altered gene expression. Here, we assessed the possibility that angiotensin-1 (AT1R) or angiotensin-2 (AT2R) receptors on the nuclear envelope mediate effects on cardiomyocyte gene expression. Immunoblots of nucleus-enriched fractions from isolated cardiomyocytes indicated the presence of AT1R and AT2R proteins that copurified with the nuclear membrane marker nucleoporin-62 and histone-3, but not markers of plasma (calpactin-I), Golgi (GRP-78), or endoplasmic reticulum (GM130) membranes. Confocal microscopy revealed AT1R and AT2R proteins on nuclear membranes. Microinjected Ang-II preferentially bound to nuclear sites of isolated cardiomyocytes. AT1R and AT2R ligands enhanced de novo RNA synthesis in isolated cardiomyocyte nuclei incubated with [alpha-(32)P]UTP (e.g. 36.0 +/- 6.0 cpm/ng of DNA control versus 246.4 +/- 15.4 cpm/ng of DNA Ang-II, 390.1 +/- 15.5 cpm/ng of DNA L-162313 (AT1), 180.9 +/- 7.2 cpm/ng of DNA CGP42112A (AT2), p < 0.001). Ang-II application to cardiomyocyte nuclei enhanced NFkappaB mRNA expression, a response that was suppressed by co-administration of AT1R (valsartan) and/or AT2R (PD123177) blockers. Dose-response experiments with Ang-II applied to purified cardiomyocyte nuclei versus intact cardiomyocytes showed greater increases in NFkappaB mRNA levels at saturating concentrations with approximately 2-fold greater affinity upon nuclear application, suggesting preferential nuclear signaling. AT1R, but not AT2R, stimulation increased [Ca(2+)] in isolated cardiomyocyte nuclei. Inositol 1,4,5-trisphosphate receptor blockade by 2-aminoethoxydiphenyl borate prevented AT1R-mediated Ca(2+) release and attenuated AT1R-mediated transcription initiation responses. We conclude that cardiomyocyte nuclear membranes possess angiotensin receptors that couple to nuclear signaling pathways and regulate transcription. Signaling within the nuclear envelope (e.g. from intracellularly synthesized Ang-II) may play a role in Ang-II-mediated changes in cardiac gene expression, with potentially important mechanistic and therapeutic implications.

A study of the ionic liquid mediated microwave heating for the synthesis of new thermally stable and optically active aromatic polyamides under green procedure

Ionic liquids (ILs) of the 1,3-dialkylimidazolium-type have great potential for the innovative application of microwaves in synthesis and separation. The use of ILs as reagents in conjunction with microwave heating makes it possible to prepare new organsoluble optically active aromatic polyamides (PAs) by a direct polycondensation reaction of our synthesized dicarboxylic acid: (2S)-4-[(4-methyl-2-phthalimidylpentanoylamino)benzoylamino]- isophthalic acid (9), with several aromatic diamines. The polymerization reactions provided chiral PAs in high yield and inherent viscosities in the range of 0.43–0.85 dLg−1. The chemical structures of some of these samples as representatives were characterized by 1H NMR and elemental analysis. All polymers were characterized by FTIR and specific rotation tools. Their thermal properties were determined by differential scanning calorimetry and thermogravimetric analysis. The efficiency of microwave irradiation conjugated with ILs was compared with that of the polycondensation of this monomer in ILs using conventional heating. The results showed that much shorter reaction times, higher yields and inherent viscosities were obtained under microwave assisted conditions. Furthermore, the use of ILs as a media and catalyst for the above polymerization reactions will eliminate the need of volatile and toxic solvents and reagents as condensing agents and provide a environmentally benign process.

The case for the use of unrefined natural reagents in analytical chemistry—A green chemical perspective

An important part of the Green Chemistry philosophy is the need to develop and adopt green analytical techniques and procedures. These include the reduction of reagent and solvent usage, the minimization of solid, liquid and gaseous materials produced by analytical processes, the replacement of reagents and solvents of high occupational or environmental toxicity with much more innocuous materials, and the reduction of energy use in analytical processes. One aspect that has received little attention in this context is the use of unrefined natural reagents derived from plant and animal tissues or microbial cells. Crude plant extracts may contain chemical compounds that enable their use as indicators in acid–base or redox titrations, or as chromogenic or fluorogenic agents. Enzymes extracted from plants may be used directly in soluble form, or incorporated in biosensors or solid phase reactors, for analytical measurements, or as a means of removing interferences or performing speciation studies. The use of natural reagents in conjunction with a flow injection system can confer a number of advantages. The enhanced kinetic control that flow analysis offers may assist in avoiding undesirable side reactions that would otherwise occur using unpurified reagents. The lifetime of natural reagents may be prolonged when used in a flow analysis system because their exposure to light or air can be controlled. Any changes in response that do occur may be readily corrected by regular standard checking and recalibration. This article reviews the use of natural reagents with an emphasis on flow-based analytical systems, and makes the case for further research in this latent area of green analytical chemistry.

Analysis of Major Protein−Protein and Protein−Metal Complexes of Erythrocytes Directly from Cell Lysate Utilizing Capillary Electrophoresis Mass Spectrometry

The separation and detection of the major protein-protein and protein-metal complexes of erythrocytes directly from cell lysate under native conditions has been accomplished for the first time using capillary electrophoresis electrospray ionization-mass spectrometry (CE/ESI-MS). All three major protein-protein and protein-metal complexes in human red blood cells (RBCs) with a concentration dynamic range of approximately 3 orders of magnitude were successfully detected. Intact complexes detected in lysed RBCs included carbonic anhydrase II (CAII-Zn at approximately 0.8 amol/cell) complexed with its zinc cofactor, carbonic anhydrase I (CAI-Zn at approximately 7 amol/cell) complexed with its zinc cofactor, and hemoglobin A (Hb-tetramer at approximately 450 amol/cell)a tetramer formed by two alpha-beta-subunits and four heme groups. The average molecular weights measured for these complexes were consistent with their theoretical values within 0.01% mass accuracy. The use of Polybrene as a self-coating reagent in conjunction with ammonium acetate at pH approximately 7.4, narrow capillary for high separation efficiency, and forward polarity CE to avoid acid production at the tip of the capillary were overriding experimental factors for successful analysis of protein complexes. Diluting the lysed blood sample in ammonium acetate for a minimum of 6 h before injecting the sample into the CE was essential for obtaining the mass accuracy consistent with their theoretical average molecular weights. At physiological pH, the mass spectrum of the electrophoretic peak of Hb-tetramer included a small amount of the monomers and Hb-dimer. The migration time and peak profile of these species were almost identical to that of the tetramer, indicating that they are formed from decomposition of the Hb-tetramer during the ESI process. A separate electrophoretic peak for the Hb-dimer was only detected when the pH of the BGE was lowered from 7.4 to approximately 6.6. Running CE in forward polarity mode was essential for detection of the intact Hb-tetramer as well as CAI-Zn and CAII-Zn complexes. Under forward polarity mode, CE outlet/ESI shared electrode acts as the cathode of the CE circuit and the anode (positive voltage for positive ions) of the ESI circuit, thereby maintaining approximately neutral pH at the CE outlet/ESI electrode. In addition, under forward polarity mode, CAII-Zn and CAI-Zn migrated ahead of Hb-tetramer, avoiding being masked by 562x and 64x, respectively, molar excess of Hb-tetramer.

Conjugate addition of organocopper reagents in dichloromethane to α,β-unsaturated esters

Organocopper reagents in conjunction with Lewis acid activators provide greater stability than traditional cuprate reagents while maintaining the reactivity needed for conjugate addition reactions in dichloromethane. Whereas cuprates engage in cross-coupling pathways, organocopper nucleophiles are more selective for conjugate addition. The utility of organocopper reagents in dichloromethane for the conjugate addition to α,β-unsaturated esters is expanded upon herein.

On-line derivatization gas chromatography with furan chemical ionization tandem mass spectrometry for screening of amphetamines in urine

A simple alternative method with minimal sample pretreatment is investigated for screening of amphetamines in small volume (using only 20 μL) of urine sample. The method is sensitive and selective. The method uses gas chromatography (GC) direct sample introduction (DSI) for on-line derivatization (acylation) of amphetamines to improve sensitivity. Furan as chemical ionization (CI) reagent in conjunction with tandem mass spectrometry (MS/MS) is used to improve selectivity. Low background with sharp protonated molecular ion peaks of analytes is the evidence of improvement in sensitivity and selectivity. Blank urine samples spiked with known amounts of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyethylamphetamine is analyzed. Selected ion monitoring of the characteristic product ions ( m/ z 119 + 136 + 150 + 163) using furan CI-MS/MS in positive ion mode is used for quantification. Limits of detection (LOD) between 0.4 and 1.0 ng mL −1 and limits of quantitation (LOQ) between 1.0 and 2.0 ng mL −1 are established. Linear response over the range of 1–1000 ng mL −1 ( r 2 > 0.997) is observed for all analytes, except for methamphetamine (2.0–1000 ng mL −1). Good accuracy between 86 and 113% and precision ranging from 4 to 18% is obtained. The method is also tested on real samples of urine from suspected drug abusers. This method could be used for screening and determination of amphetamines in urine samples, however needs additional work for full validation.

Low cost CD4 enumeration using generic monoclonal antibody reagents and a two‐color user‐defined MultiSET™ protocol

The standard three-tube, three-color flow cytometric method utilizing the TriTEST reagents in conjunction with the MultiSET software commonly used in most laboratories in Thailand for CD4 enumeration is expensive and thus unavailable to most HIV-infected patients. A more affordable method, i.e., the PanLeucogating protocol using only two monoclonal antibody reagents, has been described but requires the use of the CellQUEST software that does not have automatic gating and reporting facilities. We describe a simple protocol that utilizes a two-color user-defined protocol with the automated MultiSET software for the acquisition, analysis, and reporting of CD4 results. A two-color user-defined protocol was set up following instructions in the Becton Dickinson Biosciences MultiSET manual, adhering strictly to the information regarding the Gate and Attractor Hierarchy for analyzing various reagent combinations. This simple two-color user-defined MultiSET software was evaluated using generic monoclonal reagents in comparison with the standard TriTEST/MultiSET protocol. The two-color user-defined MultiSET software is easy to use. It requires only modification of the original MultiSET program and the results obtained are comparable with those derived from the standard TriTEST/MultiSET protocol. The use of this easy and reliable two-color user-defined MultiSET protocol represents an affordable alternative to CD4 testing in resource-poor settings.