Relevance of tumor markers for prognosis and predicting therapy response in non-small cell lung cancer patients: A CEPAC-TDM biomarker substudy.

BACKGROUND: Protein tumor markers are released in high amounts into the blood in advanced non-small cell lung cancer (NSCLC). OBJECTIVE: To investigate the relevance of serum tumor markers (STM) for prognosis, prediction and monitoring of therapy response in NSCLC patients receiving chemotherapy. METHODS: In a biomarker substudy of a prospective, multicentric clinical trial (CEPAC-TDM) on 261 advanced NSCLC patients, CYFRA 21-1, CEA, SCC, NSE, ProGRP, CA125, CA15-3 and HE4 were assessed in serial serum samples and correlated with radiological response after two cycles of chemotherapy and overall (OS) and progression-free survival (PFS). RESULTS: While pretherapeutic STM levels at staging did not discriminate between progressive and non-progressive patients, CYFRA 21-1, CA125, NSE and SCC at time of staging did, and yielded AUCs of 0.75, 0.70, 0.69 and 0.67 in ROC curves, respectively. High pretherapeutic CA15-3 and CA125 as well as high CYFRA 21-1, SCC, CA125 and CA15-3 levels at staging were prognostic for shorter PFS and OS -also when clinical variables were added to the models. CONCLUSIONS: STM at the time of first radiological staging and pretherapeutic CA15-3, CA125 are predictive for first-line treatment response and highly prognostic in patients with advanced NSCLC.

Missing prognostic value of soluble PD-1, PD-L1 and PD-L2 in lung cancer patients undergoing chemotherapy - A CEPAC-TDM biomarker substudy.

BACKGROUND: Programmed cell death receptors and ligands in cancer tissue samples are established companion diagnostics for immune checkpoint inhibitor (ICI) therapies. OBJECTIVE: To investigate the relevance of soluble PD-1, PD-L1 and PD-L2 for estimating therapy response and prognosis in non-small cell lung cancer patients (NSCLC) undergoing platin-based combination chemotherapies. METHODS: In a biomarker substudy of a prospective, multicentric clinical trial (CEPAC-TDM) on advanced NSCLC patients, soluble PD-1, PD-L1 and PD-L2 were assessed in serial serum samples by highly sensitive enzyme-linked immunosorbent assays and correlated with radiological response after two cycles of chemotherapy and with overall survival (OS). RESULTS: Among 243 NSCLC patients, 185 achieved response (partial remission and stable disease) and 58 non-response (progression). The distribution of PD-1, PD-L1 and PD-L2 at baseline (C1), prior to staging (C3) and the relative changes (C3/C1) greatly overlapped between the patient groups with response and non-response, thus hindering the discrimination between the two groups. None of the PD markers had prognostic value regarding OS. CONCLUSIONS: Neither soluble PD-1, PD-L1 nor PD-L2 did provide clinical utility for predicting response to chemotherapy and prognosis. Studies on the relevance of PD markers in ICI therapies are warranted.

Diagnostic Potential of Exosomal microRNAs in Colorectal Cancer.

Background: Despite the significance of colonoscopy for early diagnosis of colorectal adenocarcinoma (CRC), population-wide screening remains challenging, mainly because of low acceptance rates. Herein, exosomal (exo-miR) and free circulating microRNA (c-miR) may be used as liquid biopsies in CRC to identify individuals at risk. Direct comparison of both compartments has shown inconclusive results, which is why we directly compared a panel of 10 microRNAs in this entity. Methods: Exo-miR and c-miR levels were measured using real-time quantitative PCR after isolation from serum specimens in a cohort of 69 patients. Furthermore, results were compared to established tumor markers CEA and CA 19-9. Results: Direct comparison of exo- and c-miR biopsy results showed significantly higher microRNA levels in the exosomal compartment (p < 0.001). Exo-Let7, exo-miR-16 and exo-miR-23 significantly differed between CRC and healthy controls (all p < 0.05), while no c-miR showed this potential. Sensitivity and specificity can be further enhanced using combinations of multiple exosomal miRNAs. Conclusions: Exosomal microRNA should be considered as a promising biomarker in CRC for future studies. Nonetheless, results may show interference with common comorbidities, which must be taken into account in future studies.

Development and In-House Validation of an LC-MS and LC-MS/MS Assay for the Determination of Food Fraud for Different Fish Species.

Background: Fish and fish products are one of the most important food sources of high commercial interest. The global food trade and the associated risks are constantly presenting new challenges to consumer protection and public authorities, which, among other things, demand state-of-the-art analytical methods to ensure food authenticity. Objective: The establishment of MS-based strategies plays a decisive role alongside the (further) development of ELISA- or DNA-oriented methods. Methods: In the present work, therefore, the development and in-house validation of an LC-MS and LC-MS/MS-based assay for authenticity testing of certain fish species is described. Results: Based on the execution of a validated bottom-up LC-electrospray-MS and MS/MS assay and multivariate analysis, the commercially available species Lutjanus malabaricus (red snapper) and Sebastes spp. (redfish) are distinguished from each other, whereas an additional 68 samples [nine additional marine species such as pangasius (Pangasianodon hypophthalmus), salmon (Salmo salar), turbot (Scophthalmus maximus), plaice (Pleuronectes platessa), sole (Solea solea), lemon sole (Glyptocephalus cynoglossus), halibut (Reinhardtius hypoglossoides), red salmon (Oncorhynchus nerka), and great scallop (Pecten jacobaeus)] served as blinded negative controls to ensure the specificity of the assay. Conclusions and Highlights: A promising LC-MS and LC-MSMS based assay has been developed that could enable the detection of fish fraud at the protein level in the future.

Improved sensitivity for detection of breast cancer by combination of miR-34a and tumor markers CA 15-3 or CEA.

BACKGROUND: MicroRNAs biomarkers have shown value for diagnosis and prognosis of various cancers. Combination with established tumor markers has rarely been done. RESULTS: Breast cancer patients had significantly higher serum RNA loads (AUC 0.665), lower miR-34a (AUC 0.772), higher CEA and CA 15-3 levels (AUCs 0.717 and 0.721) than healthy controls. miR-34a correlated with tumor stage and hormone receptor status. There was no significant difference between groups for all other miRNAs. Combination of miR-34a with CEA or CA 15-3 led to improved AUCs of 0.844 and 0.800, respectively. Sensitivity of miR-34a and CA 15-3 reached 56.1% at 95% specificity. When compared with benign breast diseases, combination of miR-34a (AUC 0.719) and CEA (0.623) or CA 15-3 (0.619) resulted in improved performances (0.794 and 0.741). Sensitivity of miR-34a and CA 15-3 reached 53.7% at 95% specificity. CONCLUSION: While miR-34a provides valuable information for diagnosis and staging, combination with tumor markers CA15-3 or CEA improves the sensitivity for breast cancer detection. PATIENTS AND METHODS: The diagnostic relevance of the miR-21, miR-34a, miR-92a, miR-155, miR-222 and miR-let-7c was tested in sera of 103 individuals (55 breast cancer, 20 benign breast diseases, 28 healthy controls). MiRNAs were detected by quantitative rt-PCR after extraction and reverse transcription. Cel-miR-39 and miR-16 were used for normalization. Established tumor markers CEA, CA 15-3, CA 19-9 and CA 125 were measured by automatized immunoassays. Diagnostic performance was tested by areas under the curve (AUC) of receiver operating characteristic (ROC) curves and sensitivities at 90% and 95% specificity.

Validation of Arxula Yeast Estrogen Screen assay for detection of estrogenic activity in water samples: Results of an international interlaboratory study.

Endocrine-active substances can adversely impact the aquatic ecosystems. A special emphasis is laid, among others, on the effects of estrogens and estrogen mimicking compounds. Effect-based screening methods like in vitro bioassays are suitable tools to detect and quantify endocrine activities of known and unknown mixtures. This study describes the validation of the Arxula-Yeast Estrogen Screen (A-YES®) assay, an effect-based method for the detection of the estrogenic potential of water and waste water. This reporter gene assay, provided in ready to use format, is based on the activation of the human estrogen receptor alpha. The user-friendly A-YES® enables inexperienced operators to rapidly become competent with the assay. Fourteen laboratories from four countries with different training levels analyzed 17ß-estradiol equivalent concentrations (EEQ) in spiked and unspiked waste water effluent and surface water samples, in waste water influent and spiked salt water samples and in a mixture of three bisphenols. The limit of detection (LOD) for untreated samples was 1.8ng/L 17ß-estradiol (E2). Relative repeatability and reproducibility standard deviation for samples with EEQ above the LOD (mean EEQ values between 6.3 and 20.4ng/L) ranged from 7.5 to 21.4% and 16.6 to 28.0%, respectively. Precision results are comparable to other frequently used analytical methods for estrogens. The A-YES® has been demonstrated to be an accurate, precise and robust bioassay. The results have been included in the ISO draft standard. The assay was shown to be applicable for testing of typical waste water influent, effluent and saline water. Other studies have shown that the assay can be used with enriched samples, which lower the LOD to the pg/L range. The validation of the A-YES® and the development of a corresponding international standard constitute a step further towards harmonized and reliable bioassays for the effect-based analysis of estrogens and estrogen-like compounds in water samples.

Separation and identification of hormone-active compounds using a combination of chromatographic separation and yeast-based reporter assay.

Arxula adeninivorans-based yeast cell assays for the detection of steroid hormones demonstrated their efficiency for the determination of total hormone activity in a variety of samples using a microtiter plate format. In this study, a preliminary chromatographic separation using thin-layer chromatography plates is introduced in order to allow a rapid identification of the compounds responsible for this hormonal activity. The yeast whole cell assay can then be performed on the plate, producing a detectable signal where a steroid hormone is present. Simultaneous detection of estrogens, progestogens and androgens on the same plate in the picogram range was achieved, while keeping the assay as simple and affordable as possible. The assay requires a single incubation of the thin-layer chromatography plate and the detection of reporter protein production can be performed by fluorescence scanning of the plate at different wavelengths. The chromatographic separation allows the separation of several estrogens, androgens and progestogens, thus making its application for 'real world' samples very useful. In this work, different water-based samples from environmental origins were used to demonstrate the capacity of this new bioassay. Trials showed that most samples, with the exception of complex samples such as wastewater influent, can be assayed.

Diagnostic relevance of a novel multiplex immunoassay panel in breast cancer.

Multiple factors contribute to the development and progression of breast cancer. Markers of tumor growth and invasion, cell death, immune activation, and angiogenesis can be assessed in parallel by a novel multiplex immunoassay panel. The diagnostic performance of a multiplex cancer biomarker magnetic bead panel comprising 24 tumor associated parameters was evaluated in sera of 154 women including 77 patients with breast cancer, 10 with precancerous lesions, 31 with benign breast diseases, and 36 healthy controls. Marker levels were log-transformed for variance stabilization. Significance testing was done using t-test or Wilcoxon rank-sum test with correction of p values for multiple testing. Furthermore, receiver operating characteristic analyses were performed. Serum levels of several biomarkers were significantly (p ≤ 0.001) higher in cancer patients than in healthy controls, particularly alpha-fetoprotein, cancer antigen 15-3, cancer antigen 19-9, migration inhibitory factor, carcinoembryonic antigen, cancer antigen 125, hepatocyte growth factor, soluble Fas, tumor necrosis factor-α, stem cell factor, and osteopontin. As most markers were also elevated in benign breast diseases, only cancer antigen 15-3 showed significant differences to cancer patients (p ≤ 0.001). The resulting areas under the curve in receiver operating characteristic curves for discrimination between benign and malignant breast diseases achieved 0.71 with a sensitivity of 33.8% at 95% specificity. Multiplexing enables parallel analysis of different biomarker classes for cancer detection. Established cancer antigen 15-3 proved to be most relevant for differential diagnosis.

Diagnostic Performance of a Novel Multiplex Immunoassay in Colorectal Cancer.

BACKGROUND/AIM: We evaluated the diagnostic performance of a newly-launched magnetic bead-based multiplex immunoassay panel including cancer, apoptotic, immunological and angiogenesis biomarkers for differential diagnosis of colorectal cancer (CRC). PATIENTS AND METHODS: Serum samples of 106 individuals comprising of 35 patients with CRC (23 colon cancer, 12 rectal cancer), 20 with respective benign colorectal diseases and 51 healthy controls were analyzed by the Milliplex™ MAP Human Circulating Cancer Biomarker Panel 1 run on the Bio-Plex™ 200 System. RESULTS: IL-8, CEA, HGF, TNFα, CYFRA 21-1, OPN, TGFα, CA 19-9, CA 125, AFP and sFas showed significantly higher levels in cancer samples compared to healthy controls. It is noteworthy that comparing CRC and benign colorectal disease samples, many immunological and cell death markers were elevated as well. Exclusively, six markers were distinguished significantly between both groups: CEA showed the best performance in differential diagnosis reaching an AUC of 0.859 in ROC curve followed by CA 19-9, CYFRA 21-1, IL-8, CA 125 and OPN reaching AUCs between 0.696 and 0.744. Correlation with tumor stage was found for CEA, sFas and CYFRA 21-1. Finally marker scores were assembled showing that a combination of CEA and CA 19-9 had a higher AUC (0.893) compared to the biomarkers alone. CONCLUSION: Differential diagnosis of CRC can be improved by new biomarker classes and their combination assessed by novel multiplex immunoassay.

Carcinoembryonic antigen and cytokeratin-19 fragments for assessment of therapy response in non-small cell lung cancer: a systematic review and meta-analysis.

BACKGROUND: This meta-analysis evaluated whether pretherapy serum levels of carcinoembryonic antigen (CEA) and cytokeratin-19 fragments (CYFRA 21-1) are predictive of response to therapy in non-small cell lung cancer (NSCLC) and whether changes in these markers during vs pretherapy are indicative of response. METHODS: Original peer-reviewed studies enrolling adults with untreated advanced NSCLC were identified using PubMed. Two reviewers independently extracted data from eligible studies and assessed study heterogeneity and the risk of study bias. RESULTS: Fourteen studies were eligible; 11 had objective response as an end point and three evaluated clinical benefit (i.e., response and stable disease). Study bias was relatively low. Both markers showed comparable modest predictive value across studies, with baseline CYFRA 21-1 numerically better in predicting treatment benefit. A good performance in identifying objective response during treatment was seen (AUC 0.724 (95% CI 0.667-0.785) for CYFRA 21-1 and 0.728 (95% CI, 0.599-0.871) for CEA). A decline in CYFRA 21-1 levels during treatment was highly indicative for objective response (sensitivity 79.1% (95% CI 71.5-85.1)). CONCLUSIONS: Comprehensive analysis of study heterogeneity and bias provides a high level of evidence for the clinical utility of CEA and CYFRA 21-1 for the prediction and monitoring of response in NSCLC.

Simultaneous detection of three sex steroid hormone classes using a novel yeast-based biosensor.

A biosensor detecting estrogens, progestogens, and androgens in complex samples and in a single step is described. Three Arxula adeninivorans yeast strains were created, each strain producing a different recombinant human hormone receptor and a different fluorescent reporter protein. These strains were then mixed to create G1212/YRC102-hHR-fluo, the biological component of the biosensor. During incubation with G1212/YRC102-hHR-fluo, hormones present in a sample bind to their target receptor, which leads to the production of a specific fluorescent protein. Three fluorescence scans of the yeast suspension determine which fluorescence protein has been produced, thus revealing which hormone receptor (estrogen, progesterone, and androgen) has been activated by the hormones or hormone mimics present in the sample. The biosensor has similar sensitivities to the existing A. adeninivorans cell-based assays. The detection of the three hormone classes in one single experiment reduces the labor and time required to assay for the three hormone classes. The biosensor was also trialed with animal serum samples for the detection of progestogens, androgens, and estrogens and gave results that correlated well with ELISA analysis in case of progestogens. These results highlight the potential usefulness of the biosensor for comprehensive determination of hormone status in samples from veterinary origin. Biotechnol. Bioeng. 2017;114: 1539-1549. © 2017 Wiley Periodicals, Inc.

Diagnostic Performance of Risk of Ovarian Malignancy Algorithm Against CA125 and HE4 in Connection With Ovarian Cancer: A Meta-analysis.

OBJECTIVES: The aim of this study was to determine whether the Risk of Ovarian Malignancy Algorithm (ROMA) is more accurate than the human epididymis 4 (HE4) or carbohydrate antigen 125 (CA125) biomarkers with respect to the differential diagnosis of women with a pelvic mass. The secondary objective is to assess the performance of ROMA in early-stage ovarian cancer (OC) and late-stage OC, as well as premenopausal and postmenopausal patient populations. METHODS/MATERIALS: The PubMed and Google Scholar databases were searched for relevant clinical studies. Eligibility criteria included comparison of ROMA with both HE4 and CA125 levels in OC (unspecified, epithelial, and borderline ovarian tumors), use of only validated ROMA assays, presentation of area under the curve and sensitivity/specificity data, and results from early-stage OC, late-stage OC and premenopausal and postmenopausal women. Area under the curve (AUC), sensitivity/specificity, and the diagnostic odds ratio (DOR) results were summarized. RESULTS: Five studies were selected comprising 1975 patients (premenopausal, n = 1033; postmenopausal, n = 925; benign, n = 1387; early stage, n = 192; and late stage, n = 313). On the basis of the AUC (95% confidence interval) data for all patients, ROMA (0.921 [0.855-0.960]) had a numerically greater diagnostic performance than CA125 (0.883 [0.771-0.950]) and HE4 (0.899 [0.835-0.943]). This was also observed in each of the subgroup populations, in particular, the postmenopausal patients and patients with early OC. The sensitivity and specificity (95% confidence interval) results showed ROMA (sensitivity, 0.873 [0.752-0.940]; specificity, 0.855 [0.719-0.932]) to be numerically superior to CA125 (sensitivity, 0.796 [0.663-0.885]; specificity, 0.825 [0.662-0.919]) and HE4 (sensitivity, 0.817 [0.683-0.902]; specificity, 0.851 [0.716-0.928]) in all patients and for the early- and late-stage OC subgroups. Finally, the ROMA log DOR results were better than HE4 and CA125 log DOR results especially for the early-stage patient group. CONCLUSIONS: The results presented support the use of ROMA to improve clinical decision making, most notably in patients with early OC.

Development of a recombinant Arxula adeninivorans cell bioassay for the detection of molecules with progesterone activity in wastewater.

This study describes the development of a bioassay to detect the presence of progesterone and progesterone-like molecules in wastewater samples. The basis of the bioassay is the integration of the human progesterone receptor gene into the yeast Arxula adeninivorans for the constitutive synthesis of the receptor. After incubation, binding of the analyte to the receptor induces the production of a reporter protein. Two reporter proteins were compared for detection parameters such as half-maximal activity (EC50), limit of detection (LoD) and limit of quantification (LoQ). When the extracellular phytase K was used, an EC50 value of 155 ng L(-1) and a LoD of 27 ng L(-1) progesterone were obtained after 4 h incubation, while use of the fluorescent dsRED as the reporter protein, resulted in an EC50 of 320 ng L(-1) and a LoD of 65 ng L(-1) after 20 h incubation. Use of phytase K as the reporter protein offers decreased incubation time and increased sensitivity; however the dsRED reporter system is less labor-intensive. Additionally, the affinity of known agonists and antagonists of the human progesterone receptor was determined. The utility of this bioassay was confirmed by measuring total progesterone equivalent concentration of samples from a wastewater treatment plant. The A. adeninivorans-based transactivation assay was able to measure concentrations of about 311 ng L(-1) in the influent stream but could not detect progesterone activity in effluent. One key feature of the assay is the robustness of A. adeninivorans, which allows sample measurement without any sample preparation.

Purification and immunodetection of the complete recombinant HER-2[neu] receptor produced in yeast.

For the first time, the full length recombinant HER-2[neu] receptor has been produced in a yeast (Arxula adeninivorans). It is one of the most studied membrane receptors in oncology and is involved in aggressive tumor formation. A yeast integration rDNA cassette containing the human gene coding for the HER-2[neu] protein was constructed and a screening procedure was performed to select the most productive transformant. Different detergents were tested for efficient solubilization of the membrane bound protein, with CHAPS giving the best results. To increase the yield of the recombinant protein from HER-2[neu] producing A. adeninivorans, optimal culture parameters were established for cultivation in bioreactor. The recombinant protein was subsequently assayed using ELISA and SPR immunoassays systems with antibodies raised against two different epitopes of the human receptor. In both cases, elution fractions containing the recombinant HER-2[neu] receptor successfully reacted with the immunoassays with limits of quantification below 100ngml(-1). These results demonstrate that the full length recombinant HER-2[neu] reported here has the potential to be a new standard for the detection of HER-2 type cancer.

Novel serum nucleosomics biomarkers for the detection of colorectal cancer.

BACKGROUND: To improve prognosis of patients with colorectal cancer, powerful blood-based biomarkers enabling for early detection are needed. As genome-wide DNA hypomethylation is associated with carcinogenesis, and cell-free DNA, thought to be of tumor origin, is found in the circulation of patients with cancer, we investigated the relevance of 5-methylcytosine-modified DNA present in cell-free circulating nucleosomes as a serum biomarker using a convenient enzyme-linked immunosorbent assay (ELISA) technique. MATERIALS AND METHODS: Serum samples from 90 individuals [24 with colorectal cancer (CRC), 10 with benign colorectal diseases (BCD) and 56 healthy controls (HC)] were tested for the differential diagnostic performance of a novel ELISA for nucleosome-bound methylated DNA. Methodical features, including intra- and interassay imprecision, were tested using serum pools. To minimize interassay variability, values were transformed to adjusted optical densities and robust statistics were applied for clinical evaluation. Findings were later re-evaluated on a set of 113 patients (49 CRC, 26 BCD and 38 HC). RESULTS: Intra- and interassay reproducibility were 3.4% and 15.3%, respectively. Levels of circulating methylated DNA were significantly decreased in CRC and BCD when compared to HC (p<0.05), although there was no difference between BCD and CRC. For discrimination of CRC from HC, the area under the curve in receiver operating characteristic curve was 0.78 and sensitivities were 33% at 95% specificity and 75% at 70% specificity, respectively. The findings were generally confirmed when validated in the second set of patients. CONCLUSION: Reduced methylation of DNA on circulating nucleosomes detected by ELISA can potentially serve as a diagnostic tool in patients with CRC.

Evaluation and validation of a novel Arxula adeninivorans estrogen screen (nAES) assay and its application in analysis of wastewater, seawater, brackish water and urine.

A novel Arxula adeninivorans yeast estrogen screen (nAES) assay has been developed for detection of estrogenic activity in various liquid samples such as wastewater, seawater, brackish water and swine urine. Two bio-components were engineered to co-express the human estrogen receptor α (hERα) and an inducible reporter gene; either the non-conventional phytase gene (phyK, derived from Klebsiella sp. ASR1) or the non-conventional tannase gene (ATAN1, derived from Arxula). Both reporters were put under the control of an Arxula derived glucoamylase (GAA) promoter, which was modified by the insertion of two estrogen-responsive elements (EREs). The Arxula transformation/expression platform Xplor® 2, which lacks resistance markers and E. coli elements, was used to select stable mitotic transformants. They were then analyzed for robustness and suitability as the bio-component for the nAES assay. Two types of the nAES assay based on the reporter proteins phytase and tannase (nAES-P, nAES-T) were used in this work. The nAES-P type is more suitable for the analysis of seawater, brackish water and urine whereas the nAES-T type exhibited higher robustness to NaCl. Both assay types have similar characteristics for the determination of estrogen in sewage and urine samples e.g. 6-25 h assay period with detection and determination limits and EC(50) values for 17ß-estradiol of 2.8 ng L(-1), 5.9 ng L(-1), 33.2 ng L(-1) (nAES-P) and 3.1 ng L(-1), 6.7 ng L(-1) and 39.4 ng L(-1) (nAES-T). Substrate specificity and analytical measurement range (AMR) for both assay types are also similar. These characteristics show that the nAES assay based on non-conventional salt tolerant yeast is applicable for a high throughput estrogen analysis in the environmental and regulatory control sectors.

A novel estrogen sensor based on recombinant Arxula adeninivorans cells.

A novel yeast cell-based assay was developed for the detection of estrogenic activity in wastewater. Recombinant Arxula adeninivorans strains were engineered to co-express the human estrogen receptor alpha (hERalpha) and a Klebsiella-derived phytase (phyK) reporter gene under the control of an A. adeninivorans-derived glucoamylase (GAA) promoter which had been modified by the insertion of estrogen-responsive elements (EREs). In the presence of estrogenic compounds, hERalpha dimerizes and binds to the estrogen. Reporter gene expression is induced by subsequent binding of the hERalpha-dimer/estrogen complex to estrogen responsive elements (ERE) in the promoter. The insertion of different numbers of EREs in three alternative promoter positions and its effect on reporter gene expression were assessed. In one of the constructs, a detection limit of 5 ng l(-1) and a determination limit of 10 ng l(-1) for 17beta-estradiol-like activity was achieved. The photometric assay used enabled estrogen determination in sewage samples within 30 h.