Thin-layer chromatography combined with surface-enhanced Raman scattering for rapid detection of benzidine and 4-aminobiphenyl in migration from food contact materials based on gold nanoparticle doped metal-organic framework.

In this work, a rapid and sensitive thin-layer chromatography combined with surface-enhanced Raman spectroscopy method was established for rapid detection of benzidine and 4-aminobiphenyl in migration from food contact materials based on Au nanoparticle doped metal-organic framework. Benzidine and 4-aminobiphenyl were firstly separated by thin-layer chromatography to solve the limitation of their overlapping Raman peaks. Then the target molecules were monitored by adding AuNPs/MIL-101(Cr) on the sample spots. Under the optimum conditions, the concentration of benzidine and 4-aminobiphenyl can be quantitatively measured in the range of 2.0-20.0 and1.0-15.0 µg/L, respectively with good linear relationship, and the limits of detection were 0.21 and 0.23 µg/L, respectively. Furthermore, the developed method was applied to analyze benzidine and 4-aminobiphenyl in migration of different food contact materials. The recoveries of benzidine and 4-aminobiphenyl for migration of food contact materials, including paper cups, polypropylene food containers, and polyethylene glycol terephthalate bottles, were 80.6-116.0 and 80.7-118% with relative standard deviations of 1.1-9.1 and 3.1-9.9%, respectively. Surface-enhanced Raman scattering detection was performed conveniently in the on-plate mode without additional elution process. The method shows great potential in rapid monitoring of hazardous substances with overlapping characteristic Raman peaks in food contact materials.

Hypersensitive electrochemical immunoassays based on highly N-doped silicon carbide (SiC) electrode.

Highly N-doped SiC was presented as an optimal electrode for electrochemical immunoassays with a far higher sensitivity than chemiluminescence detection. As the first step, the electrochemical properties of highly N-doped SiC, such as the double-layer capacitance (Cdl), rate constant for electron transfer (kapp) and ideal polarizable potential range (electrochemical window) were analyzed and compared with those of Au, Pt, and graphite electrodes. The highly N-doped SiC electrode was used for the quantification of oxidized 3,3',5,5'-tetramethylbenzidine (TMB) which was widely used as chromogenic substrate for commercialized immunoassay kits. In order to enhance the sensitivity for the quantification of the oxidized TMB the chronoamperometry was applied to avoid the background current of i-V measurement. Finally, the chronoamperometry based on the highly N-doped SiC electrode was applied to commercial immunoassay kits for the medical diagnosis of the human immunodeficiency virus (HIV) and the human hepatitis B surface antigen (hHBsAg). The chronoamperometric measurement based on the highly N-doped SiC electrode was proved to detect at far lower limits in comparison with the conventional optical density measurement as well as the chemiluminescence assay based on luminol as a chemiluminescent probe.

[Studies on an efficient method for determining 3,3'-dimethylbenzidine in the workplace air]. / Badania nad skuteczna metoda oznaczania 3,3'-dimetylobenzydyny w powietrzu na stanowiskach pracy.

BACKGROUND: 3,3'-Dimethylbenzidene (DMB) is a substance classified into the group of carcinogens. The value of maximum admissible concentration for this substance in the workplace air is not specified in Poland. Bearing in mind that DMB is used in domestic companies there is a need to develop a sensitive method for determining 3,3'-dimethylbenzidine in the work environment. MATERIAL AND METHODS: The method consists in passing DMB-containing air through sulfuric acid-treated glass fiber filters, washing out the substance settled on the filter, using water and solution of sodium hydroxide, liquid-liquid extraction with toluene, replacing dissolvent with acetonitrile and analyzing the obtained solution. Studies were performed using high-performance liquid chromatography (HPLC) technique. An Agilent Technologies chromatograph, series 1200, with a diode-array detector (DAD) and a fluorescence detector (FLD) was used in the experiment. In the test, an Ultra C18 column of dimensions: 250×4.6 mm, particle diameter (dp) = 5 µm (Restek) was applied. RESULTS: The method is linear (r = 0.999) within the investigated working range of concentration 1.08-21.6 µg/ml, which is equivalent to air concentrations 2-40 µg/m3 for a 540 l air sample. The limit of detection (LOD) of quantification determination is 5.4 ng/ml and the limit of quantification (LOQ) - 16.19 ng/ml. CONCLUSIONS: The analytical method described in this paper allows for selective determination of 3,3'-dimethylbenzidine in the workplace air in the presence of 1,4-phenylenediamine, benzidine, aniline, 3,3'-dimethoxybenzidine, 2-nitrotoluene, 3,3'-dichlorobenzidine and azobenzene. The method is characterized by good precision and good accuracy, it also meets the criteria for procedures involving the measurement of chemical agents, listed in EN 482:2012.

[Method for determining o-tolidine in workplace air by gas chromatography].

OBJECTIVE: To establish a method for determining o-tolidine in workplace air by gas chromatography. METHODS: o-tolidine in workplace air was collected with a glass fiber filter, desorbed with methanol, and determined by gas chromatography-flame ionization detector. RESULTS: The concentration of o-tolidine showed a linear relationship with peak area within 0.04∼9.00 µg/ml; the detection limit was 0.04 µg/ml; the minimum detectable concentration was 0.0002 mg/m(3) (calculated by 375 L air sample); the sampling efficiency was 93%∼100%; the elution efficiency was 94%∼96%; the relative standard deviation was 0.8-2.5%. Sample could be stored at 4 °C for at least 8 days and at room temperature for as long as 6 days. CONCLUSION: This determination method meets the requirements of Guide for establishing occupational heath standards-Part 4 Determination methods of air chemicals in workplace (GBZ/T 210.4-2008) and is suitable for determination of o-tolidine in workplace air.

Biological activity resulting from exposure to aquatic environmental genotoxic pollutants in northern Egypt.

We estimated pollution in Lake Edku and the Mediterranean Sea, El-Maadiya Region, with 3 aromatic amines (1-naphthylamine, 2-naphthylamine and benzidine) in the muscle tissue of fish. There were marked seasonal variations in the aromatic amine levels. We also determined oxidative stress (blood glutathione, and catalase activity) and genotoxic effects (chromosomal aberrations and urinary metabolites) in fishermen from each area. The fishermen suffered from oxidative stress and had high levels of the urinary metabolite sulfanilamide [mean (microg/mg creatinine): Lake Edku 20.7, Mediterranean 14.5, controls 5.3]. Frequencies for total chromosomal aberrations were significantly raised in the peripheral blood lymphocytes of fishermen in both areas [frequency (per 100 metaphases): Mediterranean 67, Lake Edku 45, controls 14].

Rapid and sensitive method for simultaneous determination of six carcinogenic aromatic amines in mainstream cigarette smoke by liquid chromatography/electrospray ionization tandem mass spectrometry.

Aromatic amines are one of the sources of carcinogenicity in cigarette and tobacco smoke. Accurate quantification of these chemicals is needed to assess public health risk. A new validated rapid, sensitive and analyte specific liquid chromatography/electrospray ionization tandem mass spectrometric (LC/MS/MS) method has been developed for the simultaneous determination of six aromatic amines in mainstream cigarette smoke using research reference cigarette 2R4F. Three popular Indian brand cigarettes were also analyzed using the same procedure. The limit of detection of this method ranged from 0.04 to 0.59 ng/cig using an injection volume of 7 microl. The identification of each amine was established by chromatographic retention times, analyte specific fragmentation pattern and relative peak area ratios of two product/precursor ion pairs. The method showed excellent reproducibility and was also rapid, selective and robust for aromatic amine determination from cigarette smoke.

Detoxification of benzidine-based azo dye by E. gallinarum: time-course study.

Direct black 38 (DB38) dye is a well-established toxic and carcinogenic compound. Present investigation reports isolation of an Enterococcus gallinarum strain capable of decolorizing and degrading it. Changes in toxicity and mutagenicity of DB38 and its metabolites were also determined using a battery of carefully selected tests (cytotoxicity, respiration inhibition test and Ames test). Toxicity assays were carried out on E. gallinarum itself as this also gave information about suitability of this strain for the dye decolorization operation. The strain was found to reduce both toxicity and mutagenicity of DB38 metabolites. Benzidine and 4-aminobiphenyl (4-ABP) were identified as the DB38 metabolites, responsible for its toxic and mutagenic properties, by HPLC-MS analysis. Further degradation of benzidine and 4-ABP was found to result in the decrease in toxicity and mutagenicity.

Comparison of gene expression profiles in erythroid-like cells derived from mouse embryonic stem cells differentiated in simple and co-culture systems.

The feeder layer and the presence of specific growth factors are thought to induce the differentiation of embryonic stem cells (ESCs) in culture. The aim of this study was to evaluate the effect of erythropoietin (EPO) on the differentiation of ESCs into erythroid colonies in simple and co-culture systems. Embryoid bodies were dissociated and replated in semisolid medium in simple culture or in a co-culture system with bone-marrow stromal cells (BMSCs), both in the presence or absence of EPO. Colony assays, benzidine staining, and ultrastructural studies were carried out until day 10 of culture. Expression of the epsilon globin, betaH1 globin, runt-related transcription factor 1 (RUNX1), betamajor globin, and erythropoietin receptor (EPOR) genes was evaluated using semi-quantitative RT-PCR. A comparison with the corresponding controls showed that colony size increased in both systems (P

Short-term interactions of aniline and benzidine with three soils in both natural and artificial matrices.

The fate of aromatic amines in natural systems is important to understand due to the persistence and toxicity of these chemicals. Laboratory experiments were performed to elucidate aniline and benzidine behavior in silty-clay, sandy loam, and sandy soils, and six background matrices (rainwater, 12.5 mM CaCl(2), 25 mM CaCl(2), and each passed through soil columns). The goals of this study were to test the validity of using CaCl(2) as a laboratory simulation for rainwater and to observe how short-term sorption (24 h) of aniline and benzidine changed when these solutions were passed through soil columns. Results indicated that neither CaCl(2) solution exactly predicted the sorption of these chemicals in corresponding rainwater solutions, likely due to varying soil properties that influenced the sorption mechanisms. Statistical analyses revealed that the passage of rainwater or CaCl(2) solutions through soil columns did not significantly affect the sorption of aniline or benzidine. Cation exchange and solubility plots were created to identify the sorption mechanisms taking place in the short-term batch experiments. These plots indicated that cation exchange played a role in the sorption of both aniline and benzidine under all conditions, while solubility plots showed higher correlations for benzidine, a consequence of its lower aqueous solubility.

Sorption of benzidine and 3,3'-dichlorobenzidine to lake sediments. 1. Conceptualization and development of a multiparameter model.

Aromatic amines, such as benzidine and 3,3'-dichlorobenzidine (DCB), are part of the dyes and pigments manufacturing process. The prolonged use of these carcinogenic chemicals in the past generation has introduced a significant amount of contamination to the environment. Their persistency in several mediums has sparked a number of studies in an attempt to develop predictive tools of their fate and transport in the environment. In this study, benzidine and DCB batch isotherms were developed and evaluated. The sediment samples were variable in composition, ranging from sandy to silty-clay sediment samples. The batch isotherms were then analyzed using high-performance liquid chromatography. Subsequently, a multiparameter model (MPM) that accounted for partitioning, covalent bonding, and cation exchange was developed and tested in an effort to understand the various mechanisms. Results proved the proposed model to be effective in predicting sorption of aromatic amines to lake sediments. The findings suggest that the MPM can provide a better understanding of the sorption process of aromatic amines than more conventional models.

Benzidine photodegradation: a mass spectrometry and UV spectroscopy combined study.

The benzidine photodegradation process was studied using UV/Vis spectroscopy and electrospray ionization mass spectrometry (ESI-MS) combined with collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS). Mass spectrometry was used to characterize benzidine and to identify and characterize possible degradation products and intermediates. The MS data showed that benzidine is quite persistent in aqueous medium. Moreover, the MS analysis enabled us to propose the following three degradation products/intermediates: 4'-nitro-4-biphenylamine, tetrahydroxybiphenyl and 4,4'-dinitrobiphenyl. For the benzidine molecular ion and protonated molecule and for the protonated molecules of the degradation products/intermediates detected, fragmentation patterns are proposed based on CID and MS/MS data. For the photodegradation process different catalysts were used, namely the commercial TiO2 Degussa P25, and the laboratory-prepared ZnO, TiO2 anatase and a titanium-zinc oxide with a perovskite type structure. Comparison of the different catalysts showed that degradation was favoured with the commercial TiO2. Nevertheless, the other catalysts appear to be promising and economic alternatives for potential future remediation studies.

Concentrations and distribution of 3,3'-dichlorobenzidine and its congeners in environmental samples from Lake Macatawa.

Release of 3,3'-dichlorobenzidine (DCB), an intermediate in dye manufacturing processes, is of environmental concern due to its carcinogenic nature. An 11-year field study has been conducted to elucidate the fate and behavior of DCB and its congeners in the Lake Macatawa (Holland, MI, USA) sediment-water system. The sediments were variable in composition, ranging from sandy sediments with 1-8.7% total organic carbon (OC) to silty-clay sediments with 7.5-20.6% total OC. The pH ranged from 6.3 to 7.4. The findings from this field study confirmed that DCB and its congener, benzidine, are transported over long distances. The concentration of DCB in the water phase was found to be from non-detectable to approximately 1300 times greater than the water quality criteria established for DCB (0.021 microg/l). Its congener, benzidine, which is substantially more toxic for humans than its parent compound has been found up to approximately 12300 times higher levels than the EPA guidelines suggest (0.000086 microg/l). Both DCB and benzidine were found in the sediment phase. DCB was observed at almost 70 mg/kg in a sample collected in 1993. The concentrations in both phases have declined in recent years. To assess the threat of the released pollutants, it is necessary to understand how pollutants tend to behave in various media. Therefore, it is crucial to identify and quantify all sources of the chemical and its congeners in order to meaningfully predict the fate (and transport) of a hydrophobic organic compound (HOC) in the environment.

Trace-level determination of benzidine and 3,3'-dichlorobenzidine in aqueous environmental samples by online solid-phase extraction and liquid chromatography with electrochemical detection.

A simple, rapid, and reliable online methodology for the determination of benzidine and 3,3'-dichlorobenzidine (3,3'-DCB) in natural waters is proposed. The analytes are extracted and preconcentrated from aqueous samples in a small stainless steel precolumn packed with a polymeric PLRP-S phase. The precolumn is further online-analyzed by reversed-phase gradient-elution chromatography with a highly sensitive and selective coulometric detection at E = 700 mV. Recoveries greater than 90% and a relative standard deviation of approximately 5% are achieved with samples spiked at low micrograms-per-liter concentration levels. The detection limits of the method in fortified reagent water samples are 100 ng/L for benzidine and 50 ng/L for 3,3'-DCB.

N-Acetylbenzidine-DNA adduct formation by phorbol 12-myristate-stimulated human polymorphonuclear neutrophils.

N'-(3'-Monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is the major adduct in exfoliated urothelial cells and in peripheral white blood cells of workers exposed to benzidine. This study was designed to assess the metabolic pathways leading to dGp-ABZ formation in human peripheral white blood cells. [(3)H]-N-Acetylbenzidine (ABZ) transformation was assessed using myeloperoxidase (MPO), hypochlorous acid (HOCl), and human peripheral white blood cells in the absence and presence of DNA or dGp. MPO metabolism required H(2)O(2), but not NaCl. While transformation by HOCl was completely inhibited by 10 mM taurine, the level of metabolism of ABZ by MPO was only reduced 56%. Transformation by either MPO or HOCl was inhibited by 100 mM DMPO, 1 mM glutathione, and 1 mM ascorbic acid. Glutathione formed a new product with MPO, but not with HOCl. Previously identified oxidation products of ABZ, N'-hydroxy-N-acetylbenzidine or 4'-nitro-4-acetylaminobiphenyl, were not detected. With DNA or dGp present, a new product was observed that corresponded to synthetic dGp-ABZ in its HPLC elution profile, in nuclease P(1) hydrolysis to dG-ABZ, and in (32)P-postlabeling analysis. The HOCl-derived adduct was identified by electrospray ionization mass spectrometry, with collision-activated dissociation, as dGp-ABZ. Metabolism of [(3)H]ABZ by peripheral blood cells was stimulated about 3-fold with 30 ng/mL beta-phorbol 12-myristate 13-acetate (PMA). Using (32)P-postlabeling, dGp-ABZ was detected only in the presence of PMA and its level was increased more than 300-fold if either 0.7 mg/mL DNA or dGp was present. Indomethacin (0.1 mM) did not alter adduct formation. With dGp, dGp-ABZ formation could be detected with as little as 0.12 x 10(6) neutrophils. Using specific chromatographic and enzymatic techniques, neutrophil-derived dGp-ABZ was identical to the synthetic standard. Thus, these results are consistent with human polymorphonuclear neutrophils forming dGp-ABZ by a peroxidatic mechanism involving MPO.

Sulfinamide formation following peroxidatic metabolism of N-acetylbenzidine.

Arylamine-hemoglobin conjugates identified as sulfinamides are considered dosimeters for the bioavailability of metabolically formed N-oxidation products. This report considers peroxidation as an alternative pathway for aromatic amine metabolism and examines horseradish peroxidase metabolism of N-acetylbenzidine (ABZ) in the presence of glutathione. When 0.06 mM [(3)H]ABZ was incubated with 1 mM glutathione, a decrease in the total extent of metabolism was observed along with detection of a new metabolite (ABZ-SG), representing 12% of the total radioactivity. Optimum ABZ-SG formation occurred at 0.3 mM glutathione with higher concentrations (10 mM) being inhibitory. In the absence of glutathione, a molar ratio of H(2)O(2) to ABZ of 1:1 resulted in complete metabolism of ABZ. This ratio increased to >2:1 in the presence of 0.3 mM glutathione. N-Oxidation products of ABZ metabolism, such as N'-hydroxy-N-acetylbenzidine, were not detected using a variety of incubation conditions. ABZ-SG was sensitive to gamma-glutamyltranspeptidase, and completely hydrolyzed by 0.1 N HC1 or 0.1 N NaOH in 10 min at room temperature. ABZ-SG was identified by mass spectrometry and NMR to be N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide. ABZ-SG formation, but not total ABZ metabolism, was prevented by 0.3 mM NaN(3), 50 mM DMPO, 1.0 mM thiourea, and 1.0 mM histidine. Cyanide (50 mM) and ascorbic acid (0.1 mM) completely inhibited ABZ metabolism. The lack of effect of 50 mM mannitol and 2 microgram of superoxide dismutase suggests that neither hydroxyl radical nor superoxide is involved in the reaction. Studies also indicated that molecular oxygen is not a source of the sulfinamide oxygen. Formation of an ABZ sulfinamide conjugate with hemoglobin was demonstrated. The proposed mechanism for sulfinamide formation, involving two consecutive one-electron oxidations with subsequent rearrangement to a sulfur-stabilized nitrenium ion, suggests that oxygen may be derived from water. The results demonstrate that while arylamine-hemoglobin conjugates serve as useful biomarkers of exposure, their mechanism of formation may be complex, perhaps involving peroxidation as in the case of N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide.

Selective on-line immunoextraction coupled to liquid chromatography for the trace determination of benzidine, congeners and related azo dyes in surface water and industrial effluents.

A new extraction immunosorbent involving antigen-antibody interactions was coupled on-line to liquid chromatography for the selective extraction in aqueous samples of benzidine and congeners, widely used as intermediate compounds in the manufacturing of dyes and pigments. Due to the cross-reactivity of the antibodies for analytes with chemical structures closely related to that of the analyte used for immunization, the immunoextraction sorbent was shown to be able to extract aminoazobenzene and related azo dyes with good recoveries. The on-line coupling was optimized for the trace determination of benzidine, dichlorobenzidine, aminoazobenzene and some azo dyes with detection limits in the range 0.1 to 1 microgram/l. The high selectivity of the immunoextraction was shown by comparing the analysis of an industrial textile effluent obtained using precolumns packed either with a non-selective polymeric sorbent or with the anti-benzidine immunosorbent. In such complex samples, extraction and clean-up are achieved in the same step.

Determination of benzidine in the food colours tartrazine and sunset yellow FCF, by reduction and derivatization followed by high-performance liquid chromatography.

Free and bound benzidine, a non-sulphonated aromatic amine (NSAA), were determined in the food colours tartrazine and sunset yellow FCF. Bound amines were released by reducing with sodium dithionite, then total NSAAs were extracted into chloroform, transferred to aqueous acid solution and diazotized with sodium nitrite before coupling with 2-naphthol-3,6-disulphonic acid, disodium salt (R-salt). Coloured benzidine and aniline derivatives (BZDRS and ANRS) were analysed using reversed-phase ion pair high-performance liquid chromatography (HPLC) and an absorbance detector set at 548 nm. Levels of total benzidine were similar to those reported in studies conducted in the USA, and ranged from < 5 to 270 ng/g. Total aniline was also determined (0.2-188 micrograms/g). Recoveries of benzidine with this method were found to be lower than expected (average ca 46%), but were reproducible. Detection limits were 15-20 ng BZDRS/g (3-4 ng benzidine/g). Mass spectrometry (LC-MS) was evaluated for identifying and determining purity of the standards, but difficulties were encountered when the methodology was applied to commercial samples.

Biological effects of a relatively low concentration of 1-beta-D-arabinofuranosylcytosine in K562 cells: alterations of the cell cycle, erythroid-differentiation, and apoptosis.

Therapeutic strategies for leukemia are directed to induction of differentiation and apoptosis as well as growth inhibition. One of the key antileukemic agents, 1-beta-D-arabinofuranosylcytosine (ara C), is clinically applied according to these therapeutic aims. However, the molecular effects of 0.1 microg/ml of ara C, a concentration that corresponds to the serum level in leukemic patients on a conventional dose of ara C, have not been well disclosed. Here, we addressed these issues using K562 cells which derived from a blastic crisis of chronic myeloid leukemia. DNA synthesis of treated cells was suppressed from 1-6 h. But, it recovered at 12 h and no further inhibition was observed. The number of cells was not decreased but DNA fragmentation was observed at 72 h. The number of erythroid-differentiated cells also increased to 30% at 72 h. Along with treatment, no marked alteration of mRNAs for cell cycle-regulating genes was found and the retinoblastoma gene product remained hyperphosphorylated throughout treatment. The expression of mRNAs for apoptosis-regulating genes also remained unchanged, except for slight down-regulation of Bax. c-myc protein was not found later than 48 h, and Max mRNA was downregulated. c-jun was immediately induced, followed by the fluctuated expression level along with treatment. These findings suggest that the 0.1 microg/ml ara C changed the proliferation, differentiation and death of K562 cells in a biphasic manner. In the early phase, DNA synthesis was inhibited without altering the expression of cell cycle regulating-genes. In the latter phase, cell death and erythroid- differentiation occurred in accordance with the down-regulation of c-myc.

Implantation of collagen IV/poly(2-hydroxyethyl methacrylate) hydrogels containing Schwann cells into the lesioned rat optic tract.

Poly (2-hydroxyethylmethacrylate) (PolyHEMA) hydrogels, when combined with extracellular matrix molecules and infiltrated with cultured Schwann cells, have the capability to induce CNS axonal regrowth after injury. We have further investigated these PolyHEMA hydrogels and their potential to bridge CNS injury sites. Collagen IV-impregnated hydrogels containing Schwann cells were implanted into the lesioned optic tract in 14 rats. On examination 2-4 months later, there was good adherence between the implants and CNS tissue, and large numbers of viable Schwann cells (S100+, GFAP+, Laminin+, and LNGFR+) were seen within the hydrogel matrices. Immunohistochemical analysis showed that the collagen IV-impregnated PolyHEMA hydrogels preferentially supported the transplanted Schwann cells and not host glial cells such as astrocytes (GFAP+) or oligodendroglia (CAII+). Macrophages (ED1+) were also seen within the sponge structure. Eighty-three percent of the implanted hydrogels contained RT97+ axons within their trabecular networks. Regrowing axons were associated with the transplanted Schwann cells and not with the small number of infiltrating astrocytes. RT97+ axons were traced up to 510 microm from the nearest host neuropil. These axons were sometimes myelinated by the transplanted Schwann cells and expressed the peripheral myelin marker Po+. WGA/HRP-labeled retinal axons were seen within transplanted hydrogel sponges, with 40% of the cases growing for distances up to 350-450 microm within the polymer network. The data indicate that impregnating PolyHEMA sponges with collagen IV can modify the host glial reaction and support the survival of transplanted Schwann cells. This study thus provides new information on how biomaterials could be used to modify and bridge CNS injury sites.

p24 antigen capture assay for quantification of human immunodeficiency virus using readily available inexpensive reagents.

Antigen capture enzyme-linked immunosorbent assay (ELISA) for quantitation of the p24 gag protein of human immunodeficiency virus type-1 (HIV-1) is currently the most common method used to demonstrate virus replication both in vivo and in vitro. The present paper describes an ELISA employing readily available inexpensive reagents and gives detailed suggestions for optimizing the variables for specific purposes. The assay is as sensitive as commercial kits (25 pg/ml) and has a linear dose response over a wide range of p24 concentrations (25-1000 pg/ml). For these reasons, as well as its low cost, this assay has proven useful in a variety of research applications. This assay also has been found to be effective in detecting the gag protein of human immunodeficiency virus type-2 and simian immunodeficiency virus.