Identification and characterization of DNA aptamers specific to VP2 protein of canine parvovirus.

Canine parvovirus-2 (CPV-2) is ubiquitously distributed in dog population worldwide causing a severe and often fatal gastroenteritis. Owing to its highly contagious nature, rapid detection of CPV is crucial in effective control of the disease. Aptamers have emerged as potential alternative to antibodies as affinity reagents in diagnostic field. Present study was aimed to select and validate ssDNA aptamers specific to CPV. Systematic evolution of ligands through exponential enrichment (SELEX) method was employed for selection of CPV structural protein (VP2) specific DNA aptamers. SELEX was performed using a pool of ssDNA library comprising 30 random nucleotide region. A total of seven rounds of SELEX were performed using VP2 protein as target antigen which yielded ten aptamers (1A-10A) with distinct sequences. Target binding of all ten aptamers was assessed by dot blot and enzyme-linked oligonucleotide assay (ELONA) which revealed that 5A, 6A, 9A, and 10A were superior binders. In silico analysis of the aptamers revealed the existence of binding site on VP2 protein, and binding pattern was similar to in vitro findings. The affinity (KD) of all these four binders against CPV was evaluated by ELONA indicating relatively higher affinity of 6A and 10A than remaining two DNA sequences. Out of which, aptamer 6A displayed cross-reactivity with canine distemper virus and canine corona virus. Hence, aptamer 10A was considered as better binding sequence having high specificity and affinity for CPV. The study confirms the future utility of selected aptamers in development of a reliable diagnostic for rapid detection of CPV. KEY POINTS: • Canine parvovirus-specific ssDNA aptamers were identified with nanomolar affinity (100-150 nM). • Three aptamers displayed negligible cross-reactivity with other related viruses. • Aptamer 10A displayed high binding affinity and specificity to CPV.

Recent advancements in lateral flow immunoassays: A journey for toxin detection in food.

Biotechnology embraces various physical and chemical phenomena toward advancement of health diagnostics. Toward such advancement, detection of toxins plays an important role. Toxins produce severe health impacts on consumption with high mortality associated in acute cases. The most prominent route of infection and intoxication is through food matrices. Therefore, rapid detection of toxins at low concentrations is the need of modern diagnostics. Lateral flow immunoassays are one of the emergent and popularly used rapid detection technology developed for detecting various kinds of analytes. This review thus focuses on recent advancements in lateral flow immunoassays for detecting different toxins in agricultural food. Appropriate emphasis was given on how the labels, recognition elements, or detection strategy has laid an impact on improvement in immunochromatographic assays for toxins. The paper also discusses the gradual change in sensitivities and specificities of assays in accordance with the method of food processing used. The review concludes with the major challenges faced by this technology and provides an outlook and insight of ideas to improve it in the future.

Methylation of the Sox9 and Oct4 promoters and its correlation with gene expression during testicular development in the laboratory mouse.

Sox9 and Oct4 are two important regulatory factors involved in mammalian development. Sox9, a member of the group E Sox transcription factor family, has a crucial role in the development of the genitourinary system, while Oct4, commonly known as octamer binding transcription factor 4, belongs to class V of the transcription family. The expression of these two proteins exhibits a dynamic pattern with regard to their expression sites and levels. The aim of this study was to investigate the role of de novo methylation in the regulation of the tissue- and site-specific expression of these proteins. The dynamics of the de novo methylation of 15 CpGs and six CpGs in Sox9 and Oct4 respectively, was studied with sodium bisulfite genomic DNA sequencing in mouse testis at different developmental stages. Consistent methylation of three CpGs was observed in adult ovary in which the expression of Sox9 was feeble, while the level of methylation in somatic tissue was greater in Oct4 compared to germinal tissue. The promoter-chromatin status of Sox9 was also studied with a chromatin immune-precipitation assay.

Current strategies for early epithelial ovarian cancer detection using miRNA as a potential tool.

Ovarian cancer is one of the most aggressive and significant malignant tumor forms in the female reproductive system. It is the leading cause of death among gynecological cancers owing to its metastasis. Since its preliminary disease symptoms are lacking, it is imperative to develop early diagnostic biomarkers to aid in treatment optimization and personalization. In this vein, microRNAs, which are short sequence non-coding molecules, displayed great potential as highly specific and sensitive biomarker. miRNAs have been extensively advocated and proven to serve an instrumental part in the clinical management of cancer, especially ovarian cancer, by promoting the cancer cell progression, invasion, delayed apoptosis, epithelial-mesenchymal transition, metastasis of cancer cells, chemosensitivity and resistance and disease therapy. Here, we cover our present comprehension of the most up-to-date microRNA-based approaches to detect ovarian cancer, as well as current diagnostic and treatment strategies, the role of microRNAs as oncogenes or tumor suppressor genes, and their significance in ovarian cancer progression, prognosis, and therapy.

Virtual screening of truncated single stranded DNA aptamers for Staphylococcal enterotoxin type A.

Single stranded DNA (ssDNA)/RNA aptamers, are screened through the labor intensive, iterative Systematic Evolution of Ligand by Exponential Enrichment process (SELEX) method. Complete sequence of screened aptamers never interacts with target or participates in final structure. Hence, in silico tools can be used to redesign a short length aptamer from previously reported aptamers which can have high affinity and specificity to the target. This approach is fast, cost effective, and less laborious than in vitro SELEX towards finding an aptamer sequence with better affinity with the target. Here, Staphylococcal enterotoxin type A (SEA) was used as target. A total of nine aptamers reported for different Staphylococcal food poisoning (SFP) enterotoxins were used as a starting pool. The aptamers were variously truncations and thoroughly analyzed through in silico methods. Three truncated aptamers namely AptSEA1.4, AptSEA2.4 and AptSEA8.4 were found to show higher affinity with target SEA. The computational data was also validated with DOT BLOT assay complemented with image analysis. These results also confirmed that the % specific binding and the dissociation constant (Kd) of truncated aptamers AptSEA1.4, AptSEA2.4 and AptSEA8.4 was better than their original counterparts. The truncated aptamers showed great promise to be used as a capture reagent in developing a sensitive assay for detection of SEA.Communicated by Ramaswamy H. Sarma.

Recent Trends in Composite Nanozymes and Their Pro-Oxidative Role in Therapeutics.

Nanozymes are inorganic nanostructures whose enzyme mimic activities are increasingly explored in disease treatment, taking inspiration from natural enzymes. The catalytic ability of nanozymes to generate reactive oxygen species can be used for designing effective antimicrobials and antitumor therapeutics. In this context, composite nanozymes are advantageous, particularly because they integrate the properties of various nanomaterials to offer a single multifunctional platform combining photodynamic therapy (PDT), photothermal therapy (PTT), and chemodynamic therapy (CDT). Hence, recent years have witnessed great progress in engineering composite nanozymes for enhanced pro-oxidative activity that can be utilized in therapeutics. Therefore, the present review traverses over the newer strategies to design composite nanozymes as pro-oxidative therapeutics. It provides recent trends in the use of composite nanozymes as antibacterial, antibiofilm, and antitumor agents. This review also analyzes various challenges yet to be overcome by pro-oxidative composite nanozymes before being used in the field.

Humic acid-mediated reduction in toxicity of Co3O4 NPs towards freshwater and marine microalgae in surfactant mixed medium.

The ever-increasing applications of Co3O4 nanoparticles (NPs) have posed a serious concern about their discharge in the aquatic environment and ecotoxic implications. Being toxic towards aquatic species, the impact of other aquatic components such as dissolved organic matter (DOM), salinity, and surfactants are not studied sufficiently for their effect on the stability and ecotoxicity of Co3O4 NPs. The present study aims at the influence of humic acid (HA) on the toxicity of Co3O4 NPs in freshwater (C. minutissima) and marine (T. suecica) microalgae under surfactants mixed medium. The measure of % reduction in biomass and photosynthetic pigment were used as toxicity endpoints. Among various tested concentrations of HA, 25 mg/L HA was found suitable to minimize the NP's toxicity with or without the presence of surfactants. Co3O4 NPs mediated reduction in biomass of C. minutissima was significantly minimized by the cumulative effect of HA with T80 (51.68 ± 4.55%) followed by CTAB (46.23 ± 5.62%) and SDS (42.60 ± 2.46%). Similarly, HA with T80 (26.93 ± 6.38%) followed by SDS (17.02 ± 6.64%) and CTAB (13.01 ± 3.81%) were found to minimize the growth inhibitory effect of Co3O4 NPs in T. suecica. The estimation of chlorophyll - a content also indicated that microalgae treated with HA could maintain their photosynthetic ability more than control even in the co-presence of surfactants. Also, the reduced toxicity of Co3O4 NPs were attributed to an increase in hydrodynamic sizes of HA-treated Co3O4 NPs in both marine media (f/2) and freshwater media (BG11) due to increased aggregation and faster sedimentation of Co3O4 NPs.

Gold nanotheranostics: future emblem of cancer nanomedicine.

Cancer nanotheranostics aims at providing alternative approaches to traditional cancer diagnostics and therapies. In this context, plasmonic nanostructures especially gold nanostructures are intensely explored due to their tunable shape, size and surface plasmon resonance (SPR), better photothermal therapy (PTT) and photodynamic therapy (PDT) ability, effective contrast enhancing ability in Magnetic Resonance imaging (MRI) and Computed Tomography (CT) scan. Despite rapid breakthroughs in gold nanostructures based theranostics of cancer, the translation of gold nanostructures from bench side to human applications is still questionable. The major obstacles that have been facing by nanotheranostics are specific targeting, poor resolution and photoinstability during PTT etc. In this regard, various encouraging studies have been carried out recently to overcome few of these obstacles. Use of gold nanocomposites also overcomes the limitations of gold nanostructure probes and emerged as good nanotheranostic probe. Hence, the present article discusses the advances in gold nanostructures based cancer theranostics and mainly emphasizes on the importance of gold nanocomposites which have been designed to decipher the past questions and limitations of in vivo gold nanotheranostics.

Exposure-based ecotoxicity assessment of Co3O4 nanoparticles in marine microalgae.

The exposure-effect study was conducted to evaluate the effect of Co3O4 nanoparticles on Tetraselmis suecica. The growth suppressing effect has been observed during the interaction between nanoparticles and microalgae as indicated by 72 h EC50 (effective concentration of a chemical at which 50% of its effect is observed) value (45.13±3.95 mg/L) of Co3O4 nanoparticles for Tetraselmis suecica. Decline in chlorophyll a content also indicated the compromised photosynthetic ability and physiological state of microalgae. Further biochemical investigation such as increase in extracellular LDH (lactate dehydrogenase) level, ROS (reactive oxygen species), and levels of membrane lipid peroxidation in treated samples signifies the compromised cellular health and membrane disintegration caused by nanoparticles. Parallel to this, the cell entrapment, membrane damage, and attachment of nanoparticles on cell surface were also visualized by SEM-EDX (scanning electron microscope-energy dispersive X-ray) microscopy. The overall results of this study clearly indicated that Co3O4 nanoparticles might have toxic effects on growth of marine microalgae and other aquatic life forms as well. Hence, release of Co3O4 nanoparticles in aquatic ecosystem and resulting ecotoxic effect should be broadly addressed.

AuPeroxidase nanozymes: Promises and applications in biosensing.

Nanozymes or enzyme mimetic nanostructures possessing intrinsic catalytic activity circumvent various limitations of natural enzymes and hence have emerged as their promising alternative. Gold nanostructures or their hybrids with other metals are popular nanozymes because of their easy synthesis, facile surface modifications, stability, and possessing excellent peroxidase activity. Since 2004, when peroxidase activity was reported for the first time in gold nanoparticles, numerous researches have been done on assessing their peroxidase activity with changing morphology, and making hybrid with different metals. Past few years have witnessed a boom in the use of peroxidase gold nanozyme in biosensing applications. To follow the pace of gold peroxidase (Auperoxidase) nanozyme based biosensing strategies, it is apt to meticulously review this subject. Therefore, the present review covers research publications since 2013 and intends to understand the peroxidase activity in gold nanozymes with variable physico-chemical factors. It further dwells into the proposed mechanisms for the intrinsic peroxidase activity in gold nanozymes. With this review, we intend to present an eagle eye's view of various ways in which gold nanozymes have been used in detection or sensing of diverse analytes such as clinical biomarkers, environmental or food contaminants, antibiotics etc.

Exposure of synthesized Co3O4 nanoparticles to Chlorella minutissima: An ecotoxic evaluation in freshwater microalgae.

The current study focuses on the ecotoxicity of cobalt oxide nanoparticles (Co3O4 NPs) in the aquatic environment towards freshwater microalgae, Chlorella minutissima. The interaction of Co3O4 NPs with microalgae shows the growth suppressing effect. The 72 h EC 50 (effective concentration of a chemical having 50% of its impact) values of Co3O4 NPs for C. minutissima was 38.16 ± 1.99 mg/L. The decline in chlorophyll a content and increase in reactive oxygen species (ROS) also indicated the compromised physiological state of microalgae. An increased LDH (lactate dehydrogenase) level in treated samples suggests membrane disintegration by Co3O4 NPs. Light microscopy, scanning electron microscopy (SEM) and Energy Dispersive X-Ray-Scanning electron microscopy (EDX-SEM) further confirm cell entrapment and deposition of Co3O4 NPs on the cell surface. Cellular internalization of NPs, as shown by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES), also contributes towards the toxicity of NPs. The findings suggest the role of extracellular as well as intracellular nanoparticles (NPs) in exerting a toxic effect on the C. minutissima.

Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential.

The in-vitro diagnostic industry is always striving to explore specific and high-affinity recognition entities, sensitive probes, and newer technology or platforms to develop disease detection methods with lower production and time costs and with minimum interference or variability. Aptamers suffice as a reliable recognition element by addressing the issues mentioned earlier. Hence, this work focuses on screening high-affinity ssDNA ligands to capture an exemplary biomarker CA125 using membrane-SELEX technology coupled with aptainformatics (translational bioinformatics using aptamers). The ssDNA ligands or aptamers have been screened and characterized extensively through an array of assays to ensure a valuable diagnostic potential with KD (dissociation constant) of 166 nM. The robustness of the selected aptamer ligand 2.26 and its complex with target CA125 is investigated in the presence of serum and extreme salt concentrations. Its diagnostic potential is convincingly demonstrated by running a competitive nucleic acid lateral flow assay at various sample concentrations. The ssDNA ligand reported in this manuscript holds immense potential in the detection and specific targeting of CA125 biomarker.

Aptamer-gold nanozyme based competitive lateral flow assay for rapid detection of CA125 in human serum.

For several decades, point-of-care technology (POCT) has proven its potential regarding swift and cost-efficient detection of analytes. Lateral flow assay is a highly popular POC technology that needs improvisation to increase its sensitivity, cost effectiveness and quantification so that it becomes more user friendly and affordable technology. In this context, the present study has investigated the use of aptamers and nanozymes together for the first time in developing an Aptamer-nanozyme lateral flow assay (ALFA). The present study uses a specific aptamer for CA125 as capture reagent and peroxidase mimetic gold nanoparticles as label for detection of CA125 in human serum through developed competitive ALFA. The assay was specific and has a limit of detection of 3.71 U/mL. The ALFA test was in house validated for its precision, recovery and showed a significant correlation with established CA125 chemiluminiscent ELISA with P-value<0.0001. In summary, this assay quantitatively detects an analyte by using an aptamer and peroxidase mimetic gold nanoparticles that ensures circumventing the use of antibodies and incorporating enzyme mimetic activity in assay systems.

Development of ELISA for measurement of progesterone employing 17-alpha-OH-P-HRP as enzyme label.

The present study was aimed to develop a highly specific and sensitive Enzyme Linked Immunosorbent Assay (ELISA) to measure progesterone in human serum using a heterologous combination of immunogen and enzyme conjugate. The antiserum was raised against Progesterone-3-O-carboxymethyloxime bovine serum albumin (P-3-O-CMO-BSA) in New Zealand white rabbits. The enzyme conjugate was prepared by labeling 17-alpha-hydroxy-progesterone-3-O-carboxymethyloxime (17-alpha-OH-P-3-O-CMO) with Horseradish Peroxidase (HRP) to form 17-alpha-OH-P-3-CMO-HRP. A Checkerboard assay was performed to determine the working dilutions of antiserum and enzyme conjugate. Dose-response studies were carried out by incubating 100microL enzyme conjugate along with 50microL of standards in the primary antibody coated wells for 1 hour. The bound enzyme activity was measured colorimetrically using tetramethyl benzidine/hydrogen peroxide (TMB/H(2)O(2)) as substrate. The enzyme substrate reaction was terminated with 100microL of 0.5 M H(2)SO(4) after 20 min and the intensity of the color was measured using Tecan ELISA reader at 450 nm. The assay was validated in terms of sensitivity, specificity, precision and recovery. The lowest detection limit of the assay was 0.2 ng/mL. Cross-reaction with analogous steroids pregnenolone and 17-alpha-OH-P were found to be 6.8 and 6.1%, respectively. For other analogous steroids, it was less than 0.1%. The intra- and inter-assay coefficient of variation ranges from 4.52-7.39% and 4.65-9.55%, respectively. The developed ELISA correlated well with established RIA, with a correlation coefficient of 0.91 (n = 40).

Theranostic Nanostructures for Ovarian Cancer.

Ovarian cancer (OC) has emerged as one of the leading causes of death in women due to the lack of early-stage diagnosis resulting in impairment and delay in treatment of malignancy, which raises the morality rate. Existing diagnostic (pelvic examination, CA125, and enzyme-linked immunosorbent assay) or therapeutic modalities (radiotherapy, abdominal pelvic radiation therapy, and chemotherapy) are insufficient to decrease the 5-year survival rate. Nanoparticles (NPs) have been extensively explored as probes for imaging or therapy of cancer. As an extension of this, probes have been designed to possess both imaging and therapeutic modality in a single molecule and this has emerged as the science of nanotheranostics. This review presents the existing diagnostic and therapeutic strategies in use for OC and discusses their loopholes that limit the prognosis of OC. The review presents a general description of important properties of nanostructures and the type of nanostructures that have been used as imaging/therapeutic probe in cancer. The state-of-the-art nanotheranostics probe for targeting OC is presented. Systematic and complete studies that can correlate the findings of researchers from different global areas are lacking. The current status of nanostructures in various phases of clinical trials and those approved by U.S. Food and Drug Administration (FDA) has been presented. No specific targeted theranostic probe for OC has yet been approved by the FDA. Here, the underlying reasons and the challenges faced for nanotheranostics of OC are discussed, along with its future prospects.

Gold nanostructures as cancer theranostic probe: promises and hurdles.

Gold nanostructures (GNSts) have emerged as substitute for conventional contrast agents in imaging techniques and therapeutic probes due to their tunable surface plasmon resonance and optical properties in near-infrared region. Thus GNSts provide platform for the amalgamation of diagnosis and treatment (theranostics) into a single molecule for a more precise treatment. Hence, the article talks about the application of GNSts in imaging techniques and provide a holistic view on differently shaped GNSts in cancer theranostics. However, with promises GNSts also face various hurdles for their use as theranostic probe which are primarily associated with toxicity. Finally, the article attempts to discuss the challenges faced by GNSts and the way ahead that need to be traversed to place them in nanomedicine.

Development of rapid and sensitive one-step direct enzyme linked immunosorbent assay for 17-alpha-OH-progesterone in serum.

Using a homologous combination of immunogen and enzyme conjugate, a highly specific and sensitive Enzyme Linked Immunosorbent Assay (ELISA) was developed to measure 17-alpha-hydroxy-progesterone (17-alpha-OH-P) in human serum. The antiserum was raised against 17-alpha-hydroxy-progesterone-3-O-carboxymethyloxime bovine serum albumin (17-alpha-OH-P-3-O-CMO-BSA) in New Zealand white rabbits. The enzyme conjugate was prepared by labeling 17-alpha-hydroxy-progesterone-3-O-carboxymethyloxime with horseradish peroxidase (HRP). Checkerboard assay was performed to determine the working dilutions of antiserum and enzyme conjugate. Dose-response studies were carried out by incubating 25 microL enzyme conjugate along with 50 microL of standards on the primary antibody coated wells for 1 hour. The bound enzyme activity was measured colorimetrically using Tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. The enzyme substrate reaction was terminated with 100 microL of 0.5 M H2SO4 after 20 min and the intensity of the color was measured using Tecan ELISA reader at 450 nm. The assay was validated in terms of sensitivity, specificity, precision and recovery. The detection limit of the assay was 180 pg/mL. The assay was more specific as compared to most other reported immunoassays for 17-alpha-OH-P. Cross reaction with analogous C18, C19, and C21 steroids was less than 0.1% except for progesterone which showed 2.1% cross reaction. The intra- and inter-assay coefficients of variation ranges from 3.7-7.5% and 6.9-11.7%, respectively. The developed ELISA correlated well with established RIA, with a correlation coefficient of 0.9 (n=30).

A novel enzyme-linked immunosorbent assay for cortisol using a long-chain biotinylated cortisol-3-CMO derivative.

An enzyme-linked immunosorbent assay (ELISA) using streptavidin-biotin system as a bridge between antibodies bound antigen and reporter molecule (horseradish peroxidase enzyme) has been described. The cortisol antiserum was generated against cortisol-3-O-carboxylmethyl oxime-bovine serum albumin (F-3-CMO-BSA). We have prepared biotin-labelled cortisol as a primary probe and utilized streptavidin-labelled horseradish peroxidase (SA-HRP) as secondary probe to monitor the antigen-antibody interaction. To the cortisol antibody coated micro wells, 25 microL of standard or samples, along with 100 microL of biotinylated cortisol, were kept for 1 h at room temperature. Thereafter, wells were washed and 100 microL of SA-HRP was added to all wells and kept again for 20 min at room temperature. Bound enzyme activity was measured using tetramethyl benzidine/hydrogen peroxidase (TMB/H2O2) as substrate. The incorporation of streptavidin-biotin system as a bridge between antibody bound antigen and reporter molecule (horseradish peroxidase enzyme) increased sensitivity and specificity of the cortisol assay. The use of low molecular weight primary label (F-3-CMO-biotin) might have facilitated the easy and selective access of the analyte present in serum to compete with the antigen-binding pocket of antibody, thereby detecting as low as 3.42 ng/mL of analyte specifically.

Colorimetric sensing of malathion using palladium-gold bimetallic nanozyme.

In this work, a simple, sensitive and selective label free colorimetric assay using palladium-gold nanorod as nanozyme is reported for malathion detection. Study investigates the peroxidase potential of the nanozyme on colorimetric substrates and explores the effect of selected organophosphates on their enzyme mimetic activity. Palladium-gold nanozyme shows excellent peroxidase mimetic activity with O-phenylenediamine in the presence of hydrogen peroxide. Its Kinetic parameters Km and kcat are better than horseradish peroxidase which makes it a superior enzyme. Nanozyme is stable over a broad temperature range (4-70°C) and shows high peroxidase activity from 2 to 6pH. The peroxidase activity of nanozyme is selectively quenched with increasing concentration of malathion and is the principle of developed assay. Assay has a lowest detection limit of 60ng/ml and shows no cross-reaction with other analogous organophosphates or metal salts. Validation on tap water samples spiked with different concentrations of malathion shows good recovery in the range of 80-106%. Assay also displays good intra and inter-assay precision which lie in the range of 2.7-6.1% and 3.2-5.9% respectively. This study demonstrated the catalytic potential of palladium-gold nanorods, which can be employed as nanozyme for developing highly sensitive detection methods.

The influence of spacer-containing enzyme conjugate on the sensitivity and specificity of enzyme immunoassays for hapten.

BACKGROUND: In hapten enzyme immunoassays (EIA), there is an increase or decrease of labeled hapten recognition by antibody that affects sensitivity of the assay. We incorporated a spacer between a hapten derivative and enzyme to test its influence on the sensitivity and specificity of enzyme immunoassays. METHOD: Antibodies were generated against cortisol-3-O-carboxymethyl-oxime-bovine serum albumin (cortisol-3-O-CMO-BSA) and cortisol-21-hemisuccinate-bovine serum albumin (cortisol-21-HS-BSA) as an immunogen. Four cortisol horseradish peroxidase (HRP) enzyme conjugates were prepared using 2 cortisol derivatives (cortisol-3-O-CMO and cortisol-21-HS) with and without adipic acid dihydrazide (ADH) as a spacer. Eight combinations of homologous and heterologous assays were evaluated. RESULT: The incorporation of ADH spacer in cortisol-enzyme conjugate improved the sensitivity in heterologous (bridge and site plus bridge) EIA systems. In heterologous assays (site plus bridge), the presence of spacer in enzyme conjugate reduced the cross-reactivity with cross-reacting steroids. CONCLUSION: Spacer in the enzyme conjugate for hapten ELISA can improve the sensitivity of heterologous assay of hapten-like steroids. It may also reduce the cross-reactivity for some assays.