Prospects for chemiluminescent and bioluminescent immunoassay and nucleic acid assays in food testing and the pharmaceutical industry

The sensitivity, speed and convenience of chemiluminescent (CL) and bioluminescent (BL) immunoassays and probe assays have led to a diverse range of applications for these technologies, mainly in the clinical laboratory. These methods are now being explored by the food and pharmaceutical industries. Demanding detection limits and the complexity of sample preparation for food and pharmaceutical analyses present daunting challenges for the analyst. Immunoassay and nucleic acid amplification technologies have been applied to food testing, but these have mostly favoured non-luminescent endpoints. Food assays with CL or BL endpoints are now emerging, e.g., Clostridium botulinum type A detection using a CL immunosorbent assay; Salmonella and Zygosaccharomyces detection using a combination of PCR and CL detection. The analytical challenges posed by the pharmaceutical industry include testing for contaminants in raw materials and drug products, and drug discovery. The sensitivity and rapid signal acquisition characteristics of CL and BL are advantageous for the high throughput, massively parallel testing of micro-sized samples demanded in drug discovery. Current progress and the prospects for CL and BL immunoassay and nucleic acid technologies in this and other pharmaceutical and food applications is reviewed. © 1998 John Wiley & Sons, Ltd.     

BCL3 gene role in facial morphology

BACKGROUND: Cleft lip (CL) with or without palate (CLP) and isolated cleft palate (CP) are etiologically complex diseases with interactions among various environmental and genetic factors. The aim of the current study was to identify association with genetic markers and phenotypic craniofacial data in patients with CL/CLP/CP parents. METHODS: Posteroanterior and lateral digital radiographs of the cranium were obtained from 74 parents of patients with CL/CLP/CP. One hundred seventy‐three patients with CL/CLP/CP and 190 controls were enrolled in the study for the association test. Five genetic markers of the IRF6 gene and 14 markers of the 19q13 locus were genotyped. Linear regression analysis was performed for the relationship of cephalometric measurements with genotype data adjusted for age, gender, and cleft type. Chi‐square and transmission disequilibrium tests were performed to evaluate differences in alleles of the BCL3 gene. Positive findings were replicated in an independent sample (n = 95) of patients with CL/CLP/CP parents. RESULTS: Genetic markers of the BCL3 gene at 19q13, rs7257231, and rs1979377 in the familial association test and rs10401176 in the case‐control association test, were associated with craniofacial phenotype. Carriers of BCL3 allele rs7257231T had longer posterior cranial bases than noncarriers (p_adjusted = 0.0028), and in the familial‐based association test showed the statistically strongest relationship (p_adjusted = 0.05) to phenotype. Relation of rs7257231 to facial formation was confirmed in the replication group (p = 0.0024). CONCLUSIONS: The results indicate that BCL3, which has functions related to cell adhesion and whose downregulation can cause disruption of ectodermal development, is likely to be important in facial formation. Birth Defects Research (Part A), 2012. © 2012 Wiley Periodicals, Inc.     

Multiple cis-regulatory elements regulate distinct and complex patterns of developmental and wound-induced expression of Arabidopsis thaliana 4CL gene family members

Lignin is an important biopolymer that is deposited in secondary cell walls of plant cells (e.g., tracheary elements) and in response to stresses such as wounding. Biosynthesis of lignin monomers occurs via the phenylpropanoid pathway, in which the enzyme 4-coumarate:CoA ligase (4CL) plays a key role by catalyzing the formation of hydroxycinnamoyl-CoA esters, subsequently reduced to the corresponding monolignols (hydroxycinnamoyl alcohols). 4CL is encoded by a family of four genes in Arabidopsis thaliana (At4CL1-At4CL4), which are developmentally regulated and co-expressed with other phenylpropanoid genes. We investigated in detail the wound-induced expression of At4CL1-At4CL4, and found that At4CL1 and At4CL2 mRNA accumulation follows biphasic kinetics over a period of 72 h, while At4CL4 expression is rapidly activated for a period of at least 12 h before declining. In order to localize cis-regulatory elements involved in the developmental and wound-induced regulation of the At4CL gene family members, At4CL promoter-beta-glucuronidase (GUS) reporter gene fusions were constructed and transferred into Arabidopsis plants. Analysis of these plants revealed that the promoter fragments direct discrete and distinct patterns of expression, some of which did not recapitulate expected patterns of wound-induced expression. The locations of regulatory elements associated with the At4CL2 gene were investigated in detail using a series of transgenic Arabidopsis plants containing promoter fragments and parts of the transcribed region of the gene fused to GUS. Positive and negative regulatory elements effective in modulating developmental expression or wound responsiveness of the gene were located both in the promoter and transcribed regions of the At4CL2 gene.An erratum to this article can be found at     

甘草提取物通过调控miR-212-5p/BCL2L2 基因抑制甲状腺肿瘤细胞的增殖、迁移和侵袭

目的探讨甘草提取物GL-1对甲状腺肿瘤细胞增殖、迁移和侵袭的影响及其分子机制。方法以10、20、30 μg/mL GL-1处理甲状腺肿瘤细胞CAL-62,或在CAL-62细胞中转染miR-212-5p mimics、anti-miR-212-5p、si-BCL2L2、pcDNA-BCL2L2。其中转染pcDNA-BCL2L2细胞并以30 μg/mL GL-1处理。噻唑蓝比色法 (MTT)检测CAL-62细胞增殖,Transwell小室法检测CAL-62细胞迁移和侵袭,实时定量PCR (qPCR)检测CAL-62细胞中miR-212-5p表达,Western blot检测相关蛋白Bcl-2样蛋白2 (BCL2L2)、细胞周期蛋白D1 (Cyclin D1)和基质金属蛋白酶-2 (MMP-2)表达。生物学信息预测miR-212-5p的下游靶基因,双荧光素酶基因报告实验进一步验证。数据采用单因素方差分析、Tukey’s事后检验和t检验。结果与对照组相比,10、20、30 μg/mL浓度GL-1降低CAL-62细胞24、48、72 h的细胞活性 (P < 0.05),并呈剂量、时间依赖性。与对照组相比,10、20、30 μg/mL浓度GL-1干预后,CAL-62细胞侵袭数[(143.56±14.22)个、(100.32±10.23)个、(68.23±6.49)个比(189.65±15.23)个]、迁移数[(198.56±14.35)个、(141.35±12.58)个、(89.56±8.95)个比 (295.36±17.56)个]和BCL2L2蛋白表达量 (0.76±0.08、0.51±0.06、0.24±0.02比1.00±0.12)均降低 (P 均< 0.05),而miR-212-5p水平 (1.61±0.11、1.99±0.13、2.28±0.15比1.00±0.07)升高(P < 0.05),并呈剂量依赖性。过表达miR-212-5p和沉默BCL2L2表达在24、48、72 h时CAL-62细胞活性、细胞迁移数、侵袭数和Cyclin D1、MMP-2蛋白表达量降低 (P < 0.05)。生物学信息预测和双荧光素酶基因报告实验证实BCL2L2是miR-212-5p的靶基因。过表达miR-212-5p抑制BCL2L2蛋白水平,沉默miR-212-5p促进BCL2L2蛋白表达 (P < 0.05)。过表达BCL2L2可逆转GL-1对CAL-62细胞增殖、迁移、侵袭及Cyclin D1、MMP-2蛋白表达的抑制作用。结论 GL-1通过miR-212-5p/BCL2L2抑制甲状腺肿瘤细胞的增殖、迁移和侵袭。     

Nonsyndromic cleft lip with or without cleft palate: evidence of linkage to BCL3 in 17 multigenerational families.

Nonsyndromic cleft lip with or without cleft palate (CL/P) is a common craniofacial developmental defect. Recent segregation analyses have suggested that major genes play a role in the etiology of CL/P. Linkage to 22 candidate genes was tested in 11 multigenerational families with CL/P, and 21 of these candidates were excluded. APOC2, 19q13.1, which is linked to the proto-oncogene BCL3, gave suggestive evidence for linkage to CL/P. The study was expanded to include a total of 39 multigenerational CL/P families. Linkage was tested in all families, using an anonymous marker, D19S178, and intragenic markers in BCL3 and APOC2. Linkage was tested under two models, autosomal dominant with reduced penetrance and affecteds only. Homogeneity testing on the two-point data gave evidence of heterogeneity at APOC2 under the affecteds-only model. Both models showed evidence of heterogeneity, with 43% of families linked at zero recombination to BCL3 when marker data from BCL3 and APOC2 were included. A maximum multipoint LOD score of 7.00 at BCL3 was found among the 17 families that had posterior probabilities > = 50% in favor of linkage. The transmission disequilibrium test provided additional evidence for linkage with the 3 allele of BCL3 more often transmitted to affected children. These results suggest that BCL3, or a nearby gene, plays a role in the etiology of CL/P in some families.     

Identification and functional analysis of the Pm4CL1 gene in transgenic tobacco plant as the basis for regulating lignin biosynthesis in forest trees

Reducing the lignin content of trees could provide both economic and environmental benefits. To this end, the coumarate:coenzyme A ligase 1 gene (4CL1) was isolated from Pinus massoniana Lamb (Pm4CL1). The sequence of the full-length Pm4CL1 cDNA (accession no. FJ810495) contained an entire open reading frame (ORF) of 1,614 bp, which encoded a polypeptide of 537 amino acid residues. Tobacco (Nicotiana tabacum L.) as a model plant was used for functional characterization of the Pm4CL1 gene in transgenic plants. Results revealed that 4CL1 enzyme activity and lignin content in most antisense Pm4CL1 transgenic tobacco lines were decreased as compared to wild-type; the average 4CL1 enzyme activity was decreased by 48.75% and lignin content was decreased by 24.5%. In contrast, in the sense Pm4CL1 transgenic tobacco lines, average 4CL1 enzyme activity was increased by 72.3% and lignin content was increased by 27.6%. These results suggest that the Pm4CL1 gene from P. massoniana could be applied to regulate lignin biosynthesis in transgenic trees.     

Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells

The firefly enzyme luciferase catalyzes the luminescent reaction of luciferin with ATP and oxygen. The luciferase gene has recently been cloned and proposed as a reporter gene in procaryotic and eucaryotic cells. We present here a luciferase activity assay which relies on luminescence detection using a standard scintillation counter. This technique is simple, fast, inexpensive, and still very sensitive: as little as 0.02 pg (250,000 molecules) of enzyme is readily detected. The technique is optimized for the luciferase assay in mammalian cell lysates. Thus, the luciferase gene may become a very useful tool for gene regulation studies.     

Current trends in mapping human genes

The human is estimated to have at least 50,000 expressed genes (gene loci). Some information is available concerning about 5000 of these gene loci and about 1900 have been mapped, i.e., assigned to specific chromosomes (and in most instances particular chromosome regions). Progress has been achieved by a combination of physical mapping (e.g., study of somatic cell hybrids and chromosomal in situ hybridization) and genetic mapping (e.g., genetic linkage studies). New methods for both physical and genetic mapping are expanding the armamentarium. The usefulness of the mapping information is already evident; the spin-off from the Human Genome Project (HGP) begins immediately. The complete nucleotide sequence is the ultimate map of the human genome. Sequencing, although already under way for limited segments of the genome, will await further progress in gene mapping, and in particular creation of contig maps for each chromosome. Meanwhile the technology of sequencing and sequence information handling will be developed. It is argued that the HGP is a new form of coordinated, interdisciplinary science; that its primary objective must be seen as the creation of a tool for biomedical research--a source book that will be the basis of study of variation and function for a long time; that the impact on scientist training will be salutary by relieving graduate students of useless drudgery and by training scientists competent in both molecular genetics and computational science; and that the funding of the HGP will have an insignificant negative effect on science funding generally, and indeed may have a beneficial effect through economy of scale and a focusing of attention on the excitement of biology and medical science.     

Identifying a Cinnamoyl Coenzyme A Reductase (CCR) Activity with 4-Coumaric Acid: Coenzyme A Ligase (4CL) Reaction Products in <Emphasis Type="BoldItalic">Populus tomentosa</Emphasis>

A cinnamoyl coenzyme A reductase (CCR, EC 1.2.1.44), one of the key enzyme involved in lignin biosynthesis, was cloned from Populus tomentosa (Chinese white poplar). At the same time, a 4CL1 gene was cloned from P. tomentosa, too. The two genes were subcloned in pQE31 vector and expressed in Escherichia coli M15. Both of them were purified by Ni-NTA. Purified CCR protein was digested by trypsin and analyzed by HPLC-MS; the peptide segments had 27% similarity with the sequence of the CCR protein. 4CL was thought to be a neighbor enzyme of CCR in lignin biosynthesis. In this paper, a 4CL1 from P. tomentosa was cloned, and its enzyme reaction products were extracted for the substrates of CCR. Three 4CL1 enzyme reaction products were monitored by HPLC-MS and then the CCR enzyme reaction was detected by GC-MS. In the CCR reaction, the three corresponding aldehyde (p-coumaraldehyde, caffealdehyde, and coniferaldehyde) were detected and identified by Frontier3 software. The results showed that the CCR that we cloned from P. tomentosa had affinities with 4CL1 enzyme reaction products and a ptCCR that was cloned from aspen (Li et al., Plant Cell Physiol 46(7):1073–1082, 2005) only had affinity with feruloyl-CoA. The different results maybe depend on the different study method. The method of exacting 4CL enzyme products as the substrates of CCR in the paper was reliable and can be used in lignin biosynthesis network to detect the enzymes in the neighborhood that depended on the polarity of the substrates and products. This CCR gene had eight homology sequence CCR gene when a BLAST was conducted in Populus trichocarpa genome database. The CCR homology genes in Populus suggested that some CCRs may take part in the lignin biosynthesis, too. The gene family would be the hot spot in the future study.     

Enzymes as versatile labels and signal amplifiers for monitoring immunochemical reactions

Summary Enzymes have proven to be sensitive and versatile labels for immunochemical assays. The sensitivity of an enzyme label stems from its extraordinary catalytic power which in turn provides a great amplification of signals. Its versatility, however, stems from the fact that enzyme activity can be modulated by a number of substances.Enzyme labeled immunoassays can be divided into two categories: (a) heterogeneous and (b) homogeneous (non-separation). In the heterogeneous systems, the quantitation of the antibody bound and unbound fractions requires a physical separation of these two fractions, whereas the homogeneous or non-separation systems do not require such a separation. In the homogeneous systems, the unbound and antibody bound fractions can be distinguished functionally.A total of 11 unique principles used in the development of enzyme labeled immunoassays are described. The advantages, disadvantages and limitations of them are considered, as well as the future paths for research and developments.     

A lab‐on‐a‐chip device for analysis of amlodipine in biological fluids using peroxyoxalate chemiluminescence system

A highly sensitive, rapid and economical method for the determination of amlodipine (AM) in biological fluids was developed using a peroxyoxalate chemiluminescence (CL) system in a lab‐on‐a‐chip device. Peroxyoxalate‐CL is an indirect type of CL that allows the detection of native fluorophores or compounds derivatized with fluorescent labels. Here, fluorescamine was reacted with AM, and the derivatization product was used in a bis‐(2,4,6‐trichlorophenyl)oxalate‐CL system. Fluorescamine reacts selectively with aliphatic primary amine at neutral or basic pH. As most of the calcium channel blocker and many cardiovascular drugs do not contain primary amine, the developed method is highly selective. The parameters that influenced the CL signal intensity were studied carefully. These included the chip geometry, pH, concentration of reagents used and flow rates. Moreover, we confirmed our previous observation about the effects of imidazole, which is commonly used in the bis‐(2,4,6‐trichlorophenyl)oxalate‐CL system as a catalyst, and found that the signal was significantly improved when imidazole was absent. Under optimized conditions, a calibration curve was obtained with a linear range (10–100 µg/L). The limit of detection was 3 µg/L, while the limit of quantification was 10 µg/L. Finally the method was applied for the determination of AM in biological fluids successfully. Copyright © 2014 John Wiley & Sons, Ltd.     

Regulation of Arabidopsis thaliana 4-coumarate:coenzyme-A ligase-1 expression by artificial zinc finger chimeras

The use of artificial zinc finger chimeras to manipulate the expression of a gene of interest is a promising approach because zinc finger proteins can be engineered to bind any given DNA sequence in the genome. We have previously shown that a zinc finger chimera with a VP16 activation domain can activate a reporter gene in transgenic Arabidopsis thaliana (Sánchez, J.P., Ullman, C., Moore, M., Choo, Y. and Chua, N.H. (2002) Regulation of gene expression in Arabidopsis thaliana by artificial zinc finger chimeras. Plant Cell Physiol . 43 , 1465–1472). Here, we report the use of artificial zinc finger chimeras to specifically regulate the 4-coumarate:coenzyme-A ligase-1 ( At4CL1 ) gene in A. thaliana . At4CL1 is a key enzyme in lignin biosynthesis and the down-regulation of At4CL1 can lead to a decrease in lignin content, which has a significant commercial value for the paper industry. To this end, we designed zinc finger chimeras containing either an activation or a repression domain, which bind specifically to the At4CL1 promoter region. Transgenic lines expressing a zinc finger chimera with the VP16 activation domain showed an increase in At4CL1 expression and enzyme activity. In contrast, transgenic lines expressing a chimera with the KOX (KRAB) repression domain displayed repression of At4CL1 expression and enzyme activity. The activation of At4CL1 expression produced an increase in lignin content, and transgenic plant stems showed ectopic lignin distribution. Repression of the At4CL1 gene resulted in reduced lignin content, and lignin distribution in transgenic stems was severely diminished. Our results confirm and extend previous studies of gene regulation using various artificial zinc finger chimeras in animal and plant systems, and show that this system can be used to up- and down-regulate the expression of an endogenous plant gene such as At4CL1.     

Secondary metabolism in Fusarium fujikuroi: strategies to unravel the function of biosynthetic pathways

The fungus Fusarium fujikuroi causes bakanae disease of rice due to its ability to produce the plant hormones, the gibberellins. The fungus is also known for producing harmful mycotoxins (e.g., fusaric acid and fusarins) and pigments (e.g., bikaverin and fusarubins). However, for a long time, most of these well-known products could not be linked to biosynthetic gene clusters. Recent genome sequencing has revealed altogether 47 putative gene clusters. Most of them were orphan clusters for which the encoded natural product(s) were unknown. In this review, we describe the current status of our research on identification and functional characterizations of novel secondary metabolite gene clusters. We present several examples where linking known metabolites to the respective biosynthetic genes has been achieved and describe recent strategies and methods to access new natural products, e.g., by genetic manipulation of pathway-specific or global transcritption factors. In addition, we demonstrate that deletion and over-expression of histone-modifying genes is a powerful tool to activate silent gene clusters and to discover their products.

     

Water‐miscible ionic liquids as novel effectors for the firefly luciferase reaction

Ionic liquids (IL) are used as a new class of solvents for various reactions. Especially using IL in biocatalysis in an aqueous milieu has attracted considerable attention because enzymes show remarkable differences in their catalytic features in IL‐containing reaction media. Firefly luciferase is widely used in many analytical techniques, because light production of firefly luciferase is one of the most sensitive analytical measures in the ultrasensitive detection of adenosine‐5′‐triphosphate, e.g. for measuring microbial contamination and monitoring gene expression, as well as for monitoring tumor growth and metastasis in whole animals. Firefly luciferase is an unstable enzyme and its inactivation can lead to low sensitivity in the above‐mentioned assays. The present study addresses the comparative influence of six different water‐immiscible IL, the 3‐methylimidazolium derivatives [BMIM]Cl, [HMIM]Cl, [BMIM]Br, [EMIM]Br, [HMIM]Br, and [BMIM]BF₄, on the kinetic properties, structural stability, and function of firefly luciferase from Photinus pyralis using circular dichroism, fluorescence spectroscopy, and a bioluminescence assay. The incubation of luciferase with various IL showed that, with the exception of [BMIM]BF₄, the activity and stability of luciferase was considerably increased in the presence of IL, compared to luciferase in aqueous medium. Moreover, K_m for the substrate adenosine‐5′‐triphosphate in the presence of IL (except for [BMIM]BF₄) decreased while K_m for luciferin remained constant.     

Impact of infections and normal flora in nonhuman primates on drug development

Preclinical safety studies that are required for the marketing approval of a pharmaceutical include single and repeat dose studies in rodent and nonrodent species. The use of nonhuman primates (NHPs), primarily macaques, as the nonrodent species has increased in recent years, in part due to the increase in development of biopharmaceuticals and immunomodulatory agents. Depending on the source of the macaques, they may vary in genetic background, normal flora, and/or the incidence of preexisting pathogens and inflammatory conditions. As the use of alternative sources of macaques rises to meet the increased demand for these animals in biomedical research, the toxicologic pathologist should be well versed in NHP pathology to adequately assess potential drug-related effects in the context of these variations. Such knowledge is particularly important in studies involving immunomodulatory drugs as the toxicologic pathologist should anticipate which type(s) of infections are most likely to arise depending on which arm of the immune system is modulated. The purpose of this review is to discuss the immunosuppressive (e.g., simian type D retrovirus, simian immunodeficiency virus) and opportunistic viruses (e.g., cytomegalovirus, adenovirus, simian virus 40, rhesus rhadinovirus, and lymphocryptovirus), primary and opportunistic bacteria (e.g., Campylobacter spp., Shigella flexneri, Yersinia enterocolitica, Moraxella catarrhalis, Mycobacterium avium complex, enteropathogenic Escherichia coli), and parasites (e.g., Plasmodium spp., Schistosoma spp., Strongyloides fulleborni) that have had the most profound impact on the interpretation of drug safety studies and/or that may reemerge as alternative sources of NHPs are used for drug safety studies.     

Gonadotropin and steroid regulation of matrix metalloproteinases and their endogenous tissue inhibitors in the developed corpus luteum of the rhesus monkey during the menstrual cycle

The factors regulating the dynamic expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the primate corpus luteum (CL) during the menstrual cycle are unknown. We hypothesized that LH or progesterone (P) regulate interstitial-collagenase (MMP-1), the gelatinases (MMP-2 and -9), TIMP-1, and TIMP-2 in the CL. Hormone ablation/replacement was performed in rhesus monkeys on Days 9-11 of the luteal phase in five treatment groups (n = 4/group): control (no treatment), antide (GnRH antagonist), antide + LH; antide + LH + trilostane (TRL; 3beta-hydroxysteroid dehydrogenase inhibitor), and antide + LH + TRL + R5020 (nonmetabolizable progestin). On Day 12, the CL was removed and the RNA and protein isolated for real-time polymerase chain reaction and immunoassays, respectively. The MMP-1 mRNA increased 20-fold with antide, whereas LH replacement maintained MMP-1 mRNA at control levels. Likewise, TRL increased MMP-1 mRNA 54-fold, and R5020 prevented this effect. Immunodetectable MMP-1 protein also increased with antide or TRL; these increases were abated with LH or R5020. Gelatinase mRNA and/or protein levels increased with antide (e.g., 3-fold, MMP-2 mRNA), and LH replacement reduced protein levels (e.g., 11-fold, MMP-2). The TRL increased MMP-9, but not MMP-2, expression; however, R5020 replacement had no effect on mRNA or protein levels. The LH treatment increased TIMP-1 and -2 mRNA and TIMP-1 protein expression compared to controls and antide groups, whereas R5020 enhanced only immunodetectable TIMP-1. These data strongly suggest that LH suppresses MMP-1 in the primate CL via P and that it also suppresses gelatinases, either at the mRNA (MMP-2) or protein (MMP-2 and -9) levels, perhaps in part via steroids, including P. In contrast, LH promotes TIMP expression, perhaps via steroids, including P.     

Immunoenzyme demonstration of viruses and virus-specific antibodies by using conjugates based on gene engineering-produced beta-lactamase

Enzyme immunoassays for the detection of viral antigens and virus-specific antibodies in biological samples have been described. Molecular complexes of antibodies and beta-lactamase (penicillinase) have been used as anti-specific conjugates. To synthesize the conjugate, the enzyme obtained with the aid of genetic engineering has been used. Enzyme immunoassays have been tested for the indication of the influenza virus and virus-induced specific antibodies. Enzyme immunoassays were shown to possess certain advantages (e.g., the use of simple and nontoxic substrate) along with the sensitivity identical to that of other methods, employing peroxidase-based conjugates.     

Bioluminescent enzyme immunoassay for estriol. Use of reversibly inactivated bacterial luciferase as label

A bioluminescent enzyme immunoassay using estriol labeled with reversibly inactivated bacterial luciferase is described. An estriol derivative bearing an alkylthiolsulfonate is linked to the cysteinyl thiols of luciferase by formation of mixed disulfide linkages; thus, luciferase becomes inactive. After immunoassay, the inactive luciferase of the label bound to the immunoprecipitate is reactivated by incubation with dithiothreitol and the luciferase activity then is quantitated by a 20-s reaction performed with an automated luminometer (LKB 1251). Under the defined conditions, the labels are stable for at least 14 days as tested at 4 degrees C. A standard curve with a wide linear range from 50 to 6000 pg is demonstrated. This unique technology discussed here, therefore, offers exciting possibilities as a sensitive and rapid enzyme immunoassay for estriol.