Post-collection, pre-measurement variables affecting VEGF levels in urine biospecimens

Angiogenesis, the development and recruitment of new blood vessels, plays an important role in tumour growth and metastasis. Vascular endothelial growth factor (VEGF) is an important stimulator of angiogenesis. Circulating and urinary VEGF levels have been suggested as clinically useful predictors of tumour behaviour, and investigations into these associations are ongoing. Despite recent interest in measuring VEGF levels in patients, little is known about the factors that influence VEGF levels in biospecimens. To begin to address this question, urine samples were collected from patients with solid tumours undergoing radiotherapy and healthy volunteers. Four factors were examined for their effects on VEGF concentrations as measured by chemiluminescent immunoassay: time from sample collection to freezing, number of specimen freeze-thaw cycles, specimen storage tube type and the inclusion or exclusion of urinary sediment. The results of this study indicate that time to freeze up to 4 hrs, number of freeze-thaw cycles between one and five, and different types of polypropylene tubes did not have statistically significant effects on measured urinary VEGF levels. Urinary sediment had higher VEGF levels than supernatant in five of six samples from healthy patients.It is not clear whether there is an active agent in the sediment causing this increase or if the sediment particles themselves are affecting the accuracy of the assay.Therefore, we recommend centrifuging urine, isolating the supernatant, and freezing the sample in polypropylene microcentrifuge tubes or cryogenic vials within 4 hrs of collection.In addition, we recommend the use of samples within five freeze-thaw cycles.     

A more sensitive and specific radioenzymatic assay for catecholamines

This modification of the catechol-O-methyltransferase (COMT) based radioenzymatic assay for norepinephrine (NE) and epinephrine (E) improves sensitivity, selectivity and eliminates many inhibitors of COMT. Prior to assay, samples are extracted into heptane with diphenylborate, then into dilute acetic acid. This extraction procedure has an efficiency of 78% for NE but less than 2% for S-adenosylmethionine (SAM). The extraction procedure also excludes calcium and other COMT inhibitors present in urine, plasma and every tissue tested. This eliminates the requirement for individual standardization of tissue and urine samples. Sensitivity of the assay for NE and E is 10 and 6 pg/ml respectively in 1 ml of plasma. The intraassay coefficients of variation for NE and E are 4 and 13% and the interassay coefficients of variation for NE and E are 10 and 16% in a human plasma sample containing low catecholamine levels. The assay permits quantitation of plasma E levels that were undetectable in prior assays.     

A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test

Urinary levels of sulfated metabolites of lithocholic acid (LCA) are expected to be a useful index of liver function. Thus, a sensitive, specific, and feasible enzyme-linked immunosorbent assay (ELISA) of these sulfated LCA metabolites (LCA-Suls) should be established. A newly generated monoclonal antibody specific to glycolithocholic acid sulfate (glycine-amidated LCA-Sul (GLCA-Sul)) was immobilized on microtiter plates via a second antibody. A urine specimen and an alkaline phosphatase-labeled antigen were added to the plate, which was then incubated at room temperature for 3h. After this competitive reaction, bound enzyme activity was measured colorimetrically using p-nitrophenyl phosphate as a substrate. The detection limit for GLCA-Sul was 0.4 pg/assay. Nonamidated LCA-Sul and taurine-conjugated LCA-Sul showed 40 and 11% cross-reactivities, respectively, while 3-sulfates of cholic acid (CA; 0.02%), chenodeoxycholic acid (CDCA; 0.63%), and deoxycholic acid (DCA; 2.2%) exhibited very low cross-reactivities. Applicability of the ELISA system to clinical samples was well validated by parallelism, recovery test, and intra/inter-assay variance. Enzymatic deconjugation with bile acids sulfatase resulted in dramatically decreased urinary levels, supporting the specificity of the ELISA toward GLCA-Sul. The mean GLCA-Sul levels in early morning urine from healthy volunteers were 314 ng/mg Ucre (males: n=16) and 507 ng/mg Ucre (females: n=9). Patients with liver diseases, including chronic hepatitis (CH) and liver cirrhosis (LC) exhibited significantly higher values (mean 5222 ng/mg Ucre: n=21). The present 'monoclonal ELISA' is predicted to be useful as a novel noninvasive diagnostic tool for liver function and hepatobiliary diseases.     

Evaluation of enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine in saliva and urine

While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 ± 0.53 pmol/μmol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 ± 0.39 pmol/μmol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.     

Evaluation of enzyme-linked immunosorbent assay and liquid chromatography–tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine in saliva and urine

While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 ± 0.53 pmol/μmol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 ± 0.39 pmol/μmol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.     

Determination of S-phenylmercapturic acid by GC-MS and ELISA: a comparison of the two methods

Abstract

S-phenylmercapturic acid (PMA) is a specific urinary biomarker of benzene at exposure levels lower than 1?ppm. However, measuring PMA in urine is an expensive task by either GC or HPLC due to the necessity of extensive sample pretreatment. In the present study, a commercial chemiluminescence enzyme-linked immunosorbent assay (ELISA) test for PMA and GC-MS were used for screening urine samples of 60 workers employed in petrochemical settings. The ELISA results were evaluated by comparison with the GC-MS. Overall, the ELISA test proved sensitive (limit of detection?=?0.1?µg?l^?1), rapid, robust and reliable, affording results in good agreement with the GC-MS (54% of measurements) and no false-negatives. On the other hand, 46% of the ELISA assays were assigned as false-positives (arbitrarily established when ELISA >5?µg?l^?1, GC-MS <5?µg?l^?1) and a correlation coefficient of 0.687 was calculated between the two methods. It appears that urinary PMA routine biomonitoring on large numbers of samples is carried out in a cost-effective and rapid approach by preliminary screening with the ELISA assay followed by GC-MS confirmation of concentrations exceeding the biological exposure index for PMA.     

Serum and urinary immunoglobulin in the rat model of acute urinary tract infection

Total levels of urine and serum immunoglobulin IgM, IgG and IgA, and the E. coli-specific bacterial immunoglobulin response were determined by enzyme-linked immunosorbent assay (ELISA) in a rat model of acute urinary tract infection. High levels of urinary IgM were detected as early as day 3 post infection and then decreased to statistically insignificant levels. Peak levels of IgG occurred in the serum and urine on day 14. Urine and serum IgA levels remained low throughout the study period. The results demonstrate that in the rat model of acute urinary tract infection, IgM appears first in the urine and serum, and rapidly decreases. IgG then appears in the serum and urine followed by a late E. coli-specific immunoglobulin serum and urine response. Also, a non-specific component of the immunoglobulin response was noted in both the serum and urine. In the rat, IgA appears to play little or no role in the urine or in the serum response to the infection.     

Urinary MicroRNA-10a and MicroRNA-30d Serve as Novel,Sensitive and Specific Biomarkers for Kidney Injury

The steadily increasing incidence of kidney injury is a significant threat to human health. The current tools available for the early detection of kidney injury, however, have limited sensitivity or specificity. Thus, the development of novel biomarkers to detect early kidney injury is of high importance. Employing mouse renal ischemia-reperfusion and streptozotocin (STZ)-induced renal injury as acute and chronic kidney injury model, respectively, we assessed the alteration of microRNA (miRNA) in mouse urine, serum and kidney tissue by TaqMan probe-based qRT-PCR assay. Our results demonstrated that kidney-enriched microRNA-10a (miR-10a) and microRNA-30d (miR-30d) were readily detected in mouse urine and the levels of urinary miR-10a and miR-30d were positively correlated with the degree of kidney injury induced by renal ischemia-reperfusion or STZ diabetes. In contrast, no such alteration of miR-10a and miR-30d levels was observed in mouse serum after kidney injury. Compared with the blood urea nitrogen (BUN) assay, the test for urinary miR-10a and miR-30d levels was more sensitive for the detection of acute kidney injury. Furthermore, the substantial elevation of the urinary miR-10a and miR-30d levels was also observed in focal segmental glomerulosclerosis (FSGS) patients compared to healthy donors. In conclusion, the present study collectively demonstrates that urinary miR-10a and miR-30d represent a novel noninvasive, sensitive, specific and potentially high-throughput method for detecting renal injury.     

Clinical significance of urinary N1,N12-diacetylspermine levels in patients with hepatocellular carcinoma

BACKGROUND/AIM: N1,N12-diacetylspermine (DiAcSpm), a diacetylpolyamine which was recently identified in urine, appeared to be a useful tumor marker for urogenital cancers. Here we examined the clinical significance of urinary DiAcSpm as a tumor marker for hepatocellular carcinoma (HCC). METHODS: Urine samples were collected from patients with HCC and benign liver diseases. Urinary levels of DiAcSpm were measured by ELISA, which was newly developed in order to analyze large numbers of samples. RESULTS: The appropriate threshold value was set at 325 nM/g x creatinine. The sensitivity of the DiAcSpm assay for HCC was 65.5% and the specificity calculated between HCC and liver cirrhosis was 76.0%. The percentage of DiAcSpm-positive HCC patients was similar to that for AFP or PIVKA-II. At more advanced clinical stages, the positive percentage of these three markers increased but the DiAcSpm levels appeared to move independently of AFP and PIVKA-II. In HCC patients, the DiAcSpm levels reflected the progression of disease or the effect of treatment. CONCLUSIONS: DiAcSpm levels were found to reflect the severity, activity or viability of HCC. Urinary DiAcSpm can therefore be considered one of the useful indexes for patients with HCC.     

A Multi-Analyte Assay for the Non-Invasive Detection of Bladder Cancer

Accurate urinary assays for bladder cancer (BCa) detection would benefit both patients and healthcare systems. Through genomic and proteomic profiling of urine components, we have previously identified a panel of biomarkers that can outperform current urine-based biomarkers for the non-invasive detection of BCa. Herein, we report the diagnostic utility of various multivariate combinations of these biomarkers. We performed a case-controlled validation study in which voided urines from 127 patients (64 tumor bearing subjects) were analyzed. The urinary concentrations of 14 biomarkers (IL-8, MMP-9, MMP-10, SDC1, CCL18, PAI-1, CD44, VEGF, ANG, CA9, A1AT, OPN, PTX3, and APOE) were assessed by enzyme-linked immunosorbent assay (ELISA). Diagnostic performance of each biomarker and multivariate models were compared using receiver operating characteristic curves and the chi-square test. An 8-biomarker model achieved the most accurate BCa diagnosis (sensitivity 92%, specificity 97%), but a combination of 3 of the 8 biomarkers (IL-8, VEGF, and APOE) was also highly accurate (sensitivity 90%, specificity 97%). For comparison, the commercial BTA-Trak ELISA test achieved a sensitivity of 79% and a specificity of 83%, and voided urine cytology detected only 33% of BCa cases in the same cohort. These datashow that a multivariate urine-based assay can markedly improve the accuracy of non-invasive BCa detection. Further validation studies are under way to investigate the clinical utility of this panel of biomarkers for BCa diagnosis and disease monitoring.     

Systematic evaluation of sample preparation methods for gel-based human urinary proteomics: quantity, quality, and variability

We performed systematic evaluation of 38 protocols to concentrate normal human urinary proteins prior to 2D-PAGE analysis. Recovery yield and pattern of resolved protein spots were compared among different methods and intra-/inter-individual variabilities were examined. Precipitation with 90% ethanol provided the greatest protein recovery yield (92.99%), whereas precipitation with 10% acetic acid had the least protein recovery (1.91%). In most of precipitation protocols, the higher percentage of applied organic compounds provided the greater recovery yield. With a fixed concentration at 75%, the urine precipitated with acetonitrile had the greatest number of protein spots visualized in 2D gel, whereas the acetic-precipitated sample had the smallest number of spots. For the intra-individual variability, the first morning urine had the greatest amount of total protein but provided the smallest number of protein spots visualized. Excessive water drinking, not caffeine ingestion, caused alterations in the urinary proteome profile with newly presenting spots and also proteins with decreased excretion levels. As expected, there was a considerable degree of inter-individual variability. Coefficients of variation for albumin and transferrin expression were greatest by inter-individual variables. Male urine had greater amount of total protein but provided smaller number of protein spots compared to female urine. These data offer a wealth of useful information for designing a high-quality, large-scale human urine proteome project.     

Analysis of urinary 8-oxo-7,8-dihydro-purine-2’-deoxyribonucleosides by LC-MS/MS and improved ELISA

Non-invasive monitoring of oxidative stress is highly desirable. Urinary 7,8-8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) is a biologically relevant and convenient analytical target. However, immunoassays can over-estimate levels of urinary 8-oxodG. Measurement of more than one DNA oxidation product in urine would be advantageous in terms of mechanistic information. Urines samples were analysed for 8-oxodG by solid-phase extraction/LC-MS/MS and ELISA. The solid-phase extraction/LC-MS/MS assay was also applied to the analysis of urinary 7,8-dihydro-8-oxo-2’-deoxyadenosine (8-oxodA). Concurring with previous reports, urinary 8-oxodG measured by ELISA was significantly higher than levels measured by LC-MS/MS. However, apparent improvement in the specificity of the commercially available Japanese Institute for the Control of Ageing (JaICA) ELISA brought mean LC-MS/MS and ELISA measurements of urinary 8-oxodG into agreement. Urinary 8-oxodA was undetectable in all urines, despite efficient recovery by solid phase extraction. Exploitation of the advantages of ELISA may be enhanced by a simple modification to the assay procedure, although chromatographic techniques still remain the ‘gold standard’ techniques for analysis of urinary 8-oxodG. Urinary 8-oxodA is either not present or below the limit of detection of the instrumentation.     

Analysis of urinary 8-oxo-7,8-dihydro-purine-2'-deoxyribonucleosides by LC-MS/MS and improved ELISA

Non-invasive monitoring of oxidative stress is highly desirable. Urinary 7,8-8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a biologically relevant and convenient analytical target. However, immunoassays can over-estimate levels of urinary 8-oxodG. Measurement of more than one DNA oxidation product in urine would be advantageous in terms of mechanistic information. Urines samples were analysed for 8-oxodG by solid-phase extraction/LC-MS/MS and ELISA. The solid-phase extraction/LC-MS/MS assay was also applied to the analysis of urinary 7,8-dihydro-8-oxo-2'-deoxyadenosine (8-oxodA). Concurring with previous reports, urinary 8-oxodG measured by ELISA was significantly higher than levels measured by LC-MS/MS. However, apparent improvement in the specificity of the commercially available Japanese Institute for the Control of Ageing (JaICA) ELISA brought mean LC-MS/MS and ELISA measurements of urinary 8-oxodG into agreement. Urinary 8-oxodA was undetectable in all urines, despite efficient recovery by solid phase extraction. Exploitation of the advantages of ELISA may be enhanced by a simple modification to the assay procedure, although chromatographic techniques still remain the 'gold standard' techniques for analysis of urinary 8-oxodG. Urinary 8-oxodA is either not present or below the limit of detection of the instrumentation.     

慢性肾小球肾炎患者治疗前后血清CRP和VEGF浓度变化及临床意义

目的：研究慢性肾小球肾炎（CNG）中血清C反应蛋白（CRP）和血管内皮生长因子（VEGF）的浓度变化及其临床意义。方法：采用ELISA法测定35例正常对照组与41例慢性肾小球肾炎患者治疗前后血清IL-6和VEGF的浓度,同时放射免疫分析法测定血清TNF-α浓度,免疫比浊法测定血清CRP与尿Alb浓度。结果：①治疗前后CNG患者血清中IL-6、TNF-α和CRP较正常对照组均显著升高（P〈0.05或P〈0.01）,但治疗后IL-6、TNF-α和CRP水平显著低于治疗前（P〈0.01）,且血清CRP与IL-6和TNF-α呈正相关（P〈0.01）。②治疗后,CNG患者血清VEGF水平与尿Alb含量较治疗前明显降低（P〈0.05或P〈0.01）,仍显著高于正常对照组（P〈0.05或P〈0.01）,且血清VEGF与尿Alb水平呈正相关（P〈0.01）。结论：CRP、IL-6和TNF-α参与了CNG患者慢性炎症反应,VEGF则与蛋白尿的产生密切相关,治疗前后血清CRP和VEGF检测对于慢性肾小球肾炎的病情了解及临床疗效评估均具有重要的临床价值。     

Validation of an ELISA for urinary dopamine: applications in monitoring treatment of dopamine‐related disorders

Dopamine is a catecholamine that serves as a neurotransmitter in the central and peripheral nervous system. Non‐invasive, reliable, and high‐throughput techniques for its quantification are needed to assess dysfunctions of the dopaminergic system and monitor therapies. We developed and validated a competitive ELISA for direct determination of dopamine in urine samples. The method provides high specificity, good accuracy, and precision (average inter‐assay variation < 12%). The analysis is not affected by general urinary components and structurally related drugs and metabolites. The correlation between ELISA and LC‐MS/MS analyses was very good (r = 0.986, n = 28). The reference range was 64–261 μg/g Cr (n = 64). Week‐to‐week biological variations of second morning urinary dopamine under free‐living conditions were 23.9% for within‐ and 35.5% for between‐subject variation (n = 10). The assay is applied in monitoring Parkinson's disease patients under different treatments. Urinary dopamine levels significantly increase in a dose‐dependent manner for Parkinson's disease patients under l ‐DOPA treatment. The present ELISA provides a cost‐effective alternative to chromatographic methods to monitor patients receiving dopamine restoring treatment to ensure appropriate dosing and clinical efficacy. The method can be used in pathological research for the assessment of possible peripheral biological markers for disorders related to the dopaminergic system.     

Elevated levels of plasma VEGF in patients with dengue hemorrhagic fever

Vascular endothelial growth factor (VEGF) can be produced by monocytes and endothelial cells. It plays important role in angiogenesis and vascular permeability. The phenomenon of extensive plasma leakage into various serous cavities of the body is a cardinal symptom of dengue hemorrhagic fever (DHF). This study was performed to investigate the role of VEGF in patients with DHF. Plasma samples collected from the 53 dengue fever (DF) patients (including 14 patients with DHF), and 5 additional subjects with non-dengue febrile illness as controls were tested for VEGF levels using commercial enzyme-linked immunosorbent assay (ELISA) kits. The results showed that median plasma levels of VEGF in the patients with DHF (54.6 pg ml(-1)) were significantly higher than those of DF (14.6 pg ml(-1)) and control group (27.1 pg ml(-1)) (P<0.05). In addition, VEGF levels in DF patients were not significantly different from those of control patients with non-dengue febrile illness (P=0.17). Multiple regression analysis was used to analyze the clinical variables independently associated with VEGF levels. The data showed that D-dimer levels were significantly associated with VEGF levels. In this study, plasma VEGF levels in patients with DHF were significantly higher than values from DF patients. The association between increased plasma VEGF levels and increased plasma D-dimer levels in the patients with dengue illness suggests that activation of the fibrinolytic system may play a role in VEGF production in the patients with DF.     

Quantitative determination of urinary 8-hydroxy-2'-deoxyguanosine level in healthy Japanese volunteers

8-hydroxy-2'-deoxyguanosine (8-OHdG), as a measure of oxidative stress, was measured in healthy Japanese volunteers using an ELISA (New 8-OHdG Check, JICA). Analysis of daytime spot urine of 83 healthy male subjects and smoking habit, exercise and age revealed significant correlation only between the urinary level of 8-OHdG and age. As the inter-individual variation of 8-OHdG of the daytime spot urine was relatively high, we next determined inter-and intra-individual variation of 5 healthy volunteers. The levels of 8-OHdG/creatinine in morning spot urine significantly correlated with 8-OHdG levels in 24-h pool urine. Thus, a morning spot urine sample can be used for the measurement of 8-OHdG instead of inconvenient 24-h sampling.     

Influence of jasmonic acid as potential activator of induced resistance against Karnal bunt in developing spikes of wheat

Induction of defense response against Karnal bunt (KB) by suppressing the pathogenesis was observed upon exogenous application of jasmonic acid (JA) as evident from decrease in the coefficient of infection and overall response value in both susceptible and resistant varieties of wheat. The ultra-structural changes during disease progression showed the signs of programmed cell death (PCD). However, JA strengthened the defense barrier by enhancing the lignifications of cell walls as observed in spikes of both varieties by histochemical analysis. Compared to the plants inoculated with pathogen alone, plants of resistant line (RJP) first treated with JA followed by inoculation with pathogen showed more lignifications and extracellular deposition of other metabolites on cells, which is supposed to prevent mycelial invasions. Contrary to this, susceptible (SJP) lines also showed lignifications but the invasion was more compared to resistant line. Induction of protease activity was higher in resistant variety than its corresponding susceptible variety. The protease activity induced during the colonization of the pathogen and its proliferation inside the host system gets inhibited by JA treatment as demonstrated by the quantitative and in-gel protease assay. The results indicate the role of JA signalling in inhibiting the proteases due to expression of certain protease inhibitor genes. SDS-PAGE analysis shows differential gene expression through induction and/or suppression of different proteins in wheat spikes of resistant and susceptible varieties under the influence of JA. Thus, exogenously applied JA provides the conditioning effect prior to the challenge of infection and induces defense against KB probably by maintaining a critical balance between proteases and protease inhibitors and/or coordinating induction of different families of new proteins.     

An immunoenzymatic solid-phase assay for quantitative determination of HIV-1 protease activity

A novel immunoenzymatic procedure for the quantitative determination of HIV protease activity is provided. An N-terminal biotinylated peptide (DU1) that comprises an HIV-1 protease (HIV-PR) cleavage sequence was bound to streptavidin-coated microtiter plates. The bound peptide can be quantified by an immunoenzymatic procedure (enzyme-linked immunosorbent assay, ELISA) that includes a monoclonal antibody (Mab 332) against the peptide (DU1) C-terminal. The incubation of the bound peptide with HIV-PR in solution resulted in a signal decrement, as the peptide was hydrolyzed and the released C-terminal segment washed away. An equation that relates the amount of added enzyme to the kinetics of the reaction was written in order to describe this heterogeneous enzyme-quasi-saturable system. This equation allows quantitative determination of protease activity, a feature widely underrated in previous similar assays. The assay also allows evaluation of the inhibitory activity of HIV-PR inhibitors. Due to the intrinsic advantages of the ELISA format, this method could be used in high-throughput screening of HIV protease inhibitors. The assay can be extended to other proteolytic enzymes.