Macroprolactin in subjects with hyperprolactinaemia: clinical observations and relations between free PRL and PRL complexed with IgG

In some patients with hyperprolactinaemia a large portion of circulating prolactin is bound to authologous gammaglobulin and therefore it is called macroprolactin or Big-Big-Prolactin (BB-PRL). THE AIM: of the study was to select patients with predominance of macroprolactin and to learn more about the natural course of this disorder, in particular about the possible dependence of the presence of clinical features from the amount of circulating "free" PRL level, and also to search whether the quantitative proportions of both forms of PRL are stable or they change parallel to changes of the total serum PRL level. MATERIAL AND METHODS: We identified 58 patients with hyperprolactinaemia, in whom BB-PRL consisted>or=60% of the total PRL concentration. The predominance of macroprolactin was settled using the well accepted method of polyethylene glycol (PEG) precipitation of large m.w. serum proteins, followed by contemporary immunoradiometric measurement of the total and free PRL levels, and calculation of BB-PRL. Repeating such measurements during the long term observation lasting 6-66 months (mean 33 months), which was possible in 18 our patients (13--with idiopathic hyperprolactinaemia and 5--with pituitary adenoma), we could analyze the relations between both forms of PRL during the specific treatment, after it's cessation and, in few cases--during pregnancy. Apart of that, in 18 patients selected from 53 with idiopathic hyperprolactinaemia, we analyzed the shortterm alterations in the ratio between free and complexed PRL during the metoclopramide PRL stimulation test. RESULTS AND CONCLUSIONS: 1. In hyperprolactinaemic patients with predominance of BB-PRL, there was no direct correlation between the presence of clinical features and the concentration of residual "free" PRL. 2. During the long-term observation, in spite of moderate changes in the total PRL concentration induced by the treatment or it's cessation (excluding pregnancy), the ratio of "free" PRL and BB-PRL remained stable. 3. During the short time of metoclopramide stimulation test, there was a marked rise mainly of the total and "free" PRL concentrations, and, in some tested subjects, the predominance of BB-PRL was lost temporally for 1 to 2 hours.     

New Diagnostic Cutoffs for Adrenal Insufficiency After Cosyntropin Stimulation Using Abbott Architect Cortisol Immunoassay

IntroductionThe accurate interpretation of the cosyntropin (adrenocorticotropic hormone [ACTH]) stimulation test requires method- and assay-specific cutoffs of the level of cortisol. Compared with a historical cutoff (18 μg/dL) for polyclonal antibody-based immunoassays, lower thresholds were proposed for the Roche Elecsys II assay, which uses a monoclonal antibody. However, cutoffs for other commonly adopted, monoclonal antibody-based cortisol assays were not yet available. Here, we established the thresholds for the level of cortisol specific to the Abbott Architect immunoassay by comparing the measurements of the level of cortisol using 3 immunoassays.MethodsThe ACTH stimulation test was performed in patients with suspected adrenal insufficiency (n = 50). The serum cortisol level was measured using the Abbott Architect, Roche Elecsys II, and Siemens Centaur assays. The results of the Abbott assay were also compared with those of liquid chromatography-tandem mass spectrometry. The receiver operating characteristic analysis was performed to derive new diagnostic thresholds for the Abbott assay using the polyclonal antibody-based Siemens assay as the reference method.ResultsThe concentrations of cortisol measured using the Abbott assay were similar to those measured using liquid chromatography-tandem mass spectrometry and the Roche Elecsys II assay but significantly lower than those measured using the Siemens assay. The optimized threshold for cortisol using the Abbott assay was 14.6 μg/dL at 60 minutes after stimulation (sensitivity, 92%; specificity, 96%) and 13.2 μg/dL at 30 minutes after stimulation (sensitivity, 100%; specificity, 89%).ConclusionWe recommend a threshold of 14.6 μg/dL for the level of cortisol at 60 minutes after ACTH stimulation for the Abbott assay. In comparison with the historical threshold of 18 μg/dL, the application of the new cutoff may significantly decrease false-positive results due to ACTH stimulation testing. The use of assay-specific cutoffs will be essential for reducing misclassification and overtreatment in patients with suspected adrenal insufficiency.     

Cardiac troponin I is a sensitive, specific biomarker of cardiac injury in laboratory animals

This study directly demonstrates that cardiac troponin I (cTnI) is a sensitive, specific, and persistent biomarker in laboratory animals. Histopathological and pathophysiological cardiac changes in dogs, rats and mice correlated with increased serum cTnI with various cardiac inotropic agents, and cardiotoxic drugs and with cardiac arrhythmias, tachycardia, cardiac effusion with dyspnoea, and ageing. A comparison of six immunoassays for cTnI and cardiac troponin T (cTnT) to detect and monitor cardiac injury in a rodent model indicated that enzyme-linked immunosorbent (Life Diagnostics Inc and TriChem Resources Inc, West Chester, Philadelphia, USA) and Immulite (Diagnostic Products Corporation, Llanberis, UK) assays had low sensitivity and less than 1% of the dynamic range of Centaur (Bayer Healthcare Diagnostics, Newbury, UK) cTnI and Elecsys (Roche Diagnostics, Basel, Switzerland) and M8 (Bioveris Europe, Whitney, UK) cTnT assays. In dogs, however, the Immulite assay was effective and correlated with the Centaur. Serum concentrations were highly correlated but 10-fold lower for cTnT compared with cTnI with cardiac injury. Centaur assay also detected cTnI in myocardium from marmosets, swine, cattle, and guinea pigs, indicating it to be candidate cardiac biomarker for these species as well. Purified rat cTnI was 50% more reactive than purified human cTnI in the Centaur assay. In the rat, an age- and gender-dependent variation in serum cTnI was found. Male rats aged six and eight months had a 10-fold greater serum cTnI than age-matched females and three-month-old rats. These increases correlated with minimal histopathological change. Isoproterenol-induced serum cTnI increased up to 760-fold the minimal detectable concentration of 0.07 microg/L, within 4-6 h and decreased with a half-life of 6 h, with an expected return to baseline of 60 h. Severity of histopathological change correlated with serum cTnI during the ongoing injury.     

Clinical Consequences of a Change in Anti-Thyroglobulin Antibody Assays During the Follow-up of Patients with Differentiated Thyroid Cancer

ObjectiveThyroglobulin (Tg) quantitation by immunometric assays is compromised by anti-thyroglobulin antibodies (TgAbs), potentially resulting in falsely low Tg concentrations. TgAb screening is recommended when measuring Tg, but current TgAb immunoassays do not detect all possible TgAbs in circulation. We assessed the impact of a change in TgAb assay on apparent disease status of patients with differentiated thyroid carcinoma (DTC).MethodsPatients seen at the Mayo Clinic, Rochester, Minnesota, for follow-up of DTC, who had been tested using 2 different TgAb assays (Beckman Access and Roche Elecsys) were identified. Electronic medical records were reviewed to evaluate any impact the change in TgAb assay had on clinical disease status assessment and follow-up.ResultsA total of 1,457 patients were tested using both assays. A change in TgAb status was found in 124 (8.5%) patients; a total of 117 patients who were TgAbnegative on the Beckman assay became TgAb-positive with the Roche assay. Additional testing was performed in 5 of these patients. Seven patients previously considered TgAb-positive were now TgAb-negative. In all 7 cases, physicians documented that they considered these patients now to be truly TgAb-negative and free of disease.ConclusionDiscrepancies in TgAb status are seen when using different TgAb assays. Relying on Tg and TgAb measurements to determine disease status may lead to underestimation of residual cancer. A multimodal (clinical, biochemical, and radiologic) approach to follow up on patients with DTC should be continued, pending the development of Tg quantitation methods that are highly sensitive and not affected by TgAb interference. (Endocr Pract. 2014;20:1032-1036)     

American Association of Clinical Endocrinologists,American College of Endocrinology Disease State Clinical Review: Clinical Relevance of Macroprolactin in the Absence or Presence of True Hyperprolactinemia

Objective: To review the current literature regarding the prevalence of macroprolactin (macroPRL) in hyperprolactinemic patients and determine recommendations for testing.Methods: An electronic United States National Library of Medicine PubMed search was conducted for search term “macroprolactin.” Only English-language articles were considered.Results: MacroPRL is an under-recognized cause of elevated prolactin (PRL) and is present in approximately 4% to 40% of hyperprolactinemic patients depending on the referral population. Clinical findings which could be due to hyperprolactinemia are the impetus for testing for PRL. Because of this there is significant overlap in the clinical presentation of patients with true hyperprolactinemia and those with macroPRL, differentiation cannot always be made on the basis of symptoms. A lack of recognition of the presence of macroPRL can lead to unnecessary laboratory investigations, imaging, and pharmacologic or surgical treatment.Conclusion: Until there is a commercially available PRL assay that is not subject to interference by macroPRL, clinicians should consider the possibility of macroPRL, especially if the clinical presentation, imaging findings, and/or response to therapy reveal inconsistenciesAbbreviations:DA = dopamine agonistGFC = gel filtration chromatographyIgG = immunoglobulin GmacroPRL = macroprolactinMMP3 = matrix metalloproteinase-3NS = nonsignificantPEG = polyethylene glycolPRL = prolactinRA = rheumatoid arthritisSLE = systemic lupus erythematosus     

Comparison of four different assays for determination of serum S-100B

BACKGROUND: S-100B determination has been shown to be clinically useful in the management of melanoma patients. After the development of a test for determination of the isoforms S100-A1B and S100-BB in serum (S-100B), several sensitive assays for the detection of serum S-100B have become available. We compared four S-100B assays, two automated (LIAISON Sangtec 100 and Elecsys S100) and two manual ones (Sangtec 100 ELISA and CanAg S100 EIA), with respect to clinical data, reference values and correlation. METHODS: In a total of 280 samples from 155 melanoma patients and 98 healthy individuals S-100B values were measured simultaneously with the different assays. RESULTS: The inter and intra coefficients of variation were best for the automated assays. The functional sensitivity of both manual assays was 0.15 microg/L. Method comparison revealed satisfactory correlation coefficients of 0.9 or higher, but the slopes ranged from 0.29 to 3.36. Except for the Sangtec 100 ELISA, the linearity between the assays was acceptable. The overall sensitivity for melanoma ranged from 37% (Elecsys S100) to 47% (LIAISON Sangtec 100) and the sensitivity increased with stage. ROC curves showed the best accuracy for the LIAISON Sangtec 100 assay. CONCLUSIONS: All assays gave satisfactory results, but it is advisable to improve the performance of the manual assays for better sensitivity. Agreement about an international reference standard is needed.     

Serum prolactin and growth hormone determined by radioimmunoassay and a two-site immunoradiometric assay: comparison with the Nb2 cell bioassay

Serum levels of the two lactogenic hormones prolactin (PRL) and growth hormone (GH) were compared when determined by radioimmunoassay (RIA) and two-site immunoradiometric (IRMA) assays in 83 normal premenopausal women. The mean values for the PRL and GH results determined by RIA were higher than those obtained by IRMA, despite strong correlations between the two (PRL, r = 0.92; GH, r = 0.79). The lactogenic hormones were also determined together by the Nb2 cell bioassay (BA) in 38 of these same women, and the results compared with the sum of the PRL and GH immunoassays. There was a strong correlation between the BA and RIA (r = 0.75), and the BA/PRL+GH RIA ratio averaged 1.6 +/- 0.5. Corresponding values for IRMA were r = 0.66, and BA/PRL + GH IRMA 3.3 +/- 1.1. Thus, the polyclonal RIA antisera appeared to recognize bioactive hormone components not determined by the double monoclonal antibody IRMA. Another 23 women at risk for familial breast cancer, and 14 cystic breast disease patients were also studied. High BA, but normal RIA results, giving mean ratios of 2.4 +/- 1.1 and 3.6 +/- 3.0 respectively, suggest the presence of a further variant with high bioactivity not detected by RIA in these two clinical situations.     

Hyperprolactinaemia--pitfalls in PRL assessment

Prolactin (PRL) is one of the most commonly assessed hormones, and hyperprolactinaemia seems to be often endocrine disorder. Hyperprolactinaemia is not a disease, but only a symptom indicating relevant medical conditions to be diagnosed and properly treated. Laboratory findings should be always cautiously interpreted with reference to clinical context. Possible problems could be evoked by errors during sampling and assessment itself. While interpreting laboratory results, one have to pay attention on pulsate secretion profile of PRL (within hours), and shows marked diurnal cycle (with maximum during sleep period). PRL level depends also on emotional status (stress amplifies PRL secretion), and also on dietary habits and stimulants. Lastly, a growing body of evidence proven that in some cases elevated PRL level could be caused by presence of polymeric form of PRL--so called "macroprolactin". This form has diminished receptor-binding specificity and weak, if any, biological effect while immunoreactivity is preserved. In clinical practice, in cases of macroprolactinaemia high level of circulating hormone does not correlate with slight, if even, clinical symptoms. To avoid errors in prolactin assessment blood should be drawn fasting, preferentially in series or during dynamic test after dopaminergic blockade with metoclopramide. Interpretation must parallel include clinical data. It is essential that PRL level is proportional to pituitary lactotroph tumor size. Extremely high PRL concentration could exceed technical capability of laboratory equipment and remain underestimated, or even undiagnosed. Beneath presented algorithm could be useful in planning diagnostic and therapeutic procedures.     

Self-binding antibodies (autobodies) form specific complexes in solution

In this report we have shown that members of the murine self-binding antibody family, S107, form soluble complexes and precipitate under conditions in which non-self-binding antibodies remain in solution. Two approaches were used to demonstrate the self-association of autobodies: size-exclusion column chromatography and polyethylene glycol (PEG)-mediated precipitation assay. The anti-phosphorylcholine antibody T15 and two somatic variants, U4, which binds DNA, and U10, which has no identified specificity, produced larger precipitates in 10% PEG than other non-self-binding antibodies. The selectivity of PEG-mediated precipitation of self-binding antibodies is demonstrated by reduction of precipitation with specific haptens known to inhibit self-binding in solid-phase assays. Phosphorylcholine and nucleotides reduced precipitation of T15 and U4, respectively, but not U10. To rule out Fc-Fc mediated self-association in solution, we have also demonstrated self-complexing of F(ab')2 fragments of T15 using PEG. The self-binding locus was further dissected using peptides derived from V regions. A 24-residue peptide derived from the second hypervariable region of the VH of S107 specifically enhanced precipitation of T15, U4, and U10, but not other antibodies. These results provide evidence of a dormant potential of self-binding antibodies to precipitate under conditions that reduce the solubility of proteins. The implication of this potential is discussed with respect to pathological complex formation.     

The HDL proteome in acute coronary syndromes shifts to an inflammatory profile

Inflammation is a major factor underlying acute coronary syndromes (ACS). HDL particles may be remodeled, becoming functionally defective, under the inflammatory conditions seen in ACS. Shotgun proteomics was used to monitor changes in the HDL proteome between male age-matched control, stable CAD, and ACS subjects (n = 10/group). HDL was isolated by ultracentrifugation and separated by 1D-gel followed by LC-MS/MS. We identified 67 HDL-associated proteins, 20 of which validated recently identified proteins including vitronectin and complement C4B, and 5 of which were novel. Using gene ontology analysis, we found that the HDL-proteome consisted of proteins involved in cholesterol homeostasis (~ 50%), with significant contributions by proteins involved in lipid binding, antioxidant, acute-phase response, immune response, and endopeptidase/protease inhibition. Importantly, levels of apoA-IV were significantly reduced in ACS patients, whereas levels of serum amyloid A (SAA) and complement C3 (C3) were significantly increased (spectral counting; t-test p ≤ 0.05), as confirmed by immunoblot or ELISA. Despite differences in protein composition, ABCA1, ABCG1, and SR-BI mediated cholesterol efflux assays did not indicate that HDL from ACS patients is functionally deficient as compared to controls, when corrected for apoA-I mass. Our results support that the HDL proteome differs between control, CAD and ACS patients. Increased abundance of SAA, C3, and other inflammatory proteins in HDL from ACS patients suggests that HDL reflects a shift to an inflammatory profile which, in turn, might alter the protective effects of HDL on the atherosclerotic plaque. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

冠脉PCI治疗后血浆脂蛋白相关磷脂酶A2与基质金属蛋白酶-9水平的变化及临床意义

目的:探讨冠脉经皮冠状动脉介入(PCI)治疗后血浆脂蛋白相关磷脂酶A2(LP-PLA2)与基质金属蛋白酶-9(MMP-9)水平的变化及其临床意义。方法:选择150例急性冠脉综合征(ACS)患者、128例稳定型心绞痛(SAP)患者及100例健康者分为作为ACS组、SAP组及对照组。比较三组入院时血浆MMP-9、LP-PLA2水平及ACS组经PCI治疗前后血浆MMP-9、LP-PLA2水平的变化。结果:与对照组比较,SAP组与ACS组的血浆MMP-9、LP-PLA2水平均明显增高(P0.05);与SAP组比较,ACS组明显增高(P0.05)。与术前比较,ACS组术后血浆MMP-9、LP-PLA2水平明显降低(P0.05)。MMP-9与LP-PLA2在ACS组血浆中呈显著正相关(r=0.617,P0.05),在SAP组与对照组中无相关性(P0.05)。结论:冠脉病变程度越严重,血浆MMP-9和LP-PLA2水平更高;PCI治疗后冠脉斑块趋于稳定,血浆MMP-9和LP-PLA2水平降低,且二者有相关性,提示MMP-9和LP-PLA2参与冠状动脉粥样硬化的发病与发展,且对预测ACS高危人群及评价疗效有一定的临床价值。     

不同剂量阿司匹林干预对非ST 段抬高性急性冠脉综合征患者机体炎症 水平及血管内皮功能的影响

目的:探讨不同剂量阿司匹林对接受保守治疗的非ST段抬高性ACS患者机体炎症水平及血管内皮功能的影响,为ACS患者阿司匹林的应用提供更充分的临床依据。方法:200例患者以不稳定型心绞痛和非ST段抬高性心肌梗死分层后随机分2组,阿司匹林高剂量治疗组,阿司匹林低剂量治疗组。阿司匹林高剂量组患者在入院给予300mg阿司匹林负荷后继以150mg/d治疗。阿司匹林低剂量组患者入院给予150mg负荷后继以75mg/d维持,两组患者其余基础的治疗一致。患者在入院时及干预1周后,抽取静脉血检测患者血炎症因子hs-CRP、IL-6、TNF-α及反应血管内皮功能的NO、ET-1的变化。结果:①经治疗后,2组患者的炎症因子水平均显著下降,P<0.05或P<0.01;同时发现高剂量阿司匹林具有更显著的抗炎及改善血管内皮功能的作用,两组间指标比较,差异显著,P<0.05。②在因出现不良反应而终止试验的人数方面,两组间无显著性差异,P>0.05。结论:高剂量阿司匹林具有更显著拮抗机体炎症及改善血管内皮功能的作用,值得在ACS患者的治疗中进行推广应用。     

Interchangeability and diagnostic accuracy of two assays for total and free prostate-specific antigen: two not always related items

The variation between different PSA assays seems to influence the interpretation of individual PSA values and the clinical decisions about prostate cancer. One reason for this variability could be the different reactivity of antibodies for the various molecular forms of serum PSA; as a result, samples containing the same amount of tPSA but different proportions of fPSA can produce very different values. In this study, serum samples were collected prospectively from 152 consecutive patients referred to 2 institutions (Regional Hospital, Venice, 90 subjects; San Bortolo Hospital, Vicenza, 62 subjects) for PSA elevation and/or symptoms. Serum samples were assessed according to the manufacturers' instructions on the following 2 analyzers: the Immulite 2000 assay (Diagnostic Products Corporation, Los Angeles, USA), which measures tPSA and fPSA, and the ADVIA Centaur (Bayer Diagnostics, Tarrytown, USA), which assays tPSA and cPSA. cPSA values were transformed into fPSA by the equation fPSA=tPSA-cPSA. When taking Immulite tPSA and f/tPSA values as 100%, ADVIA Centaur values were 92.6% and 122%, respectively, which means that 20% of patients would be classified differently according to the traditional biopsy cutoff. In conclusion, there are considerable differences between the 2 methods, which could affect clinical decisions.     

Mass spectrometry-based immunoassays for the next phase of clinical applications

Recent applications of affinity mass spectrometry into clinical laboratories brought a renewed interest in immunoaffinity mass spectrometry as a more specific affinity method capable of selectively targeting and studying protein biomarkers. In mass spectrometry-based immunoassays, proteins are affinity retrieved from biological samples via surface-immobilized antibodies, and are then detected via mass spectrometric analysis. The assays benefit from dual specificity, which is brought about by the affinity of the antibody and the protein mass readout. The mass spectrometry aspect of the assays enables single-step detection of protein isoforms and their individual quantification. This review offers a comprehensive review of mass spectrometry-based immunoassays, from historical perspectives in the development of the immunoaffinity mass spectrometry, to current applications of the assays in clinical and population proteomic endeavors. Described in more detail are two types of mass spectrometry-based immunoassays, one of which incorporates surface plasmon resonance detection for protein quantification. All mass spectrometry-based immunoassays offer high-throughput targeted protein investigation, with clear implications in clinical research, encompassing biomarker discovery and validation, and in diagnostic settings as the next-generation immunoassays.     

Mass spectrometry-based immunoassays for the next phase of clinical applications

Recent applications of affinity mass spectrometry into clinical laboratories brought a renewed interest in immunoaffinity mass spectrometry as a more specific affinity method capable of selectively targeting and studying protein biomarkers. In mass spectrometry-based immunoassays, proteins are affinity retrieved from biological samples via surface-immobilized antibodies, and are then detected via mass spectrometric analysis. The assays benefit from dual specificity, which is brought about by the affinity of the antibody and the protein mass readout. The mass spectrometry aspect of the assays enables single-step detection of protein isoforms and their individual quantification. This review offers a comprehensive review of mass spectrometry-based immunoassays, from historical perspectives in the development of the immunoaffinity mass spectrometry, to current applications of the assays in clinical and population proteomic endeavors. Described in more detail are two types of mass spectrometry-based immunoassays, one of which incorporates surface plasmon resonance detection for protein quantification. All mass spectrometry-based immunoassays offer high-throughput targeted protein investigation, with clear implications in clinical research, encompassing biomarker discovery and validation, and in diagnostic settings as the next-generation immunoassays.     

Development of pure prolactin receptor antagonists

Prolactin (PRL) promotes tumor growth in various experimental models and leads to prostate hyperplasia and mammary neoplasia in PRL transgenic mice. Increasing experimental evidence argues for the involvement of autocrine PRL in this process. PRL receptor antagonists have been developed to counteract these undesired proliferative actions of PRL. However, all forms of PRL receptor antagonists obtained to date exhibit partial agonism, preventing their therapeutic use as full antagonists. In the present study, we describe the development of new human PRL antagonists devoid of agonistic properties and therefore able to act as pure antagonists. This was demonstrated using several in vitro bioassays, including highly sensitive assays able to detect extremely low levels of receptor activation. These new compounds also act as pure antagonists in vivo, as assessed by analyzing their ability to competitively inhibit PRL-triggered signaling cascades in various target tissues (liver, mammary gland, and prostate). Finally, by using transgenic mice expressing PRL specifically in the prostate, which exhibit constitutively activated signaling cascades paralleling hyperplasia, we show that these new PRL analogs are able to completely revert PRL-activated events. These second generation human PRL antagonists are good candidates to be used as inhibitors of growth-promoting actions of PRL.     

A chemical method to isolate hypothalamic nonapeptides by coupling cyst(e)in with bimane

Vasopressin, oxytocin as well as other active nonapeptides (vasotocin, etc) are difficult to isolate from tissues. Traditionally they were identified using cumbersome biological assays or immunoassays, commercially unavailable, and with some cross reactivity. Based on the fact that all these peptides have two Cysteins in their molecules we developed a simple, sensitive and specific method to detect them by HPLC after pre-column fluorescent derivatization with monobromobimane (mBBr). The peptides were separated on a Vydac C18 column after reduction with Tris (2-carboxyethyl) phosphine (TCEP) and derivatization with mBBr for 5 minutes in dark. Using this method we were able to detect specific peaks for arginine-, lysine-vasopressin, and vasotocin at levels as low as 10 pmol. The method can be used to detect other active peptides with cyst(e)ins in their molecule, as well.     

Rapid detection and identification of biological and chemical agents by immunoassay, gene probe assay and enzyme inhibition using a silicon-based biosensor

A rapid biosensor assay procedure that utilizes biotin streptavidin mediated filtration capture onto nitrocellulose membrane, in conjunction with a silicon-based light-addressable potentiometric sensor (LAPS) was developed for detection and identification of biological and chemical threat agents. Sandwich immunoassays, nucleic acid hybridization assays and enzyme inhibition assays are described. For immunoassays, the lower limits of detection (LOD) per 100-microl sample were approximately 5 pg/ml for protein (Staphylococcal enterotoxin B), 2 ng/ml for virus (Newcastle disease virus), and 20 ng/ml for vegetative bacteria (Brucella melitensis). In a dual gene probe assay format, the LOD was 0.30 fmol (1.8 x 10(8) copies per 60-microl) of single stranded target DNA. Enzyme inhibition assays on the LAPS using acetylcholinesterase were able to detect soman and sarin in aqueous samples at 2 and 8 pg (100 and 600 pM), respectively. The assays were easy to perform and required a total time equal to the reaction period plus about 15 min for filtering, washing and sensing. The assay format is suitable for detection of a wide range of infectious and toxic substances. New assays can be developed and optimized readily, often within 1 or 2 days.