Basal level of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay

A solid-phase enzyme immunoassay for prostaglandin D₂ (PGD₂) was developed in which PGD₂ was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labelled and free PGD₂, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)- propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3 – 100 pg for PGD₂. The IC₅₀ value for PGD₂ in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enyzme immunoassay was applied to the measurement of PGD₂ content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD₂. PGD₂ contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay (r = 0.966) after raising its contents by intravenous administration of PGD₂. The level of PGD₂ in rat brain was extremely low but determined to be 0.11 ± 0.03 ng/g tissue (mean ± S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change.     

Separation of recombinant antibodies from DNA using divalent cations

New downstream methods for the purification of antibodies are required to meet the demands of current and future antibody applications, e.g. for mass production as cancer therapeutic. The standard chromatographic methods suffer from high material costs and mass transfer limitations. In this study, we established and characterized a method for DNA precipitation for antibody purification using divalent cations, particularly CaCl₂, using four different antibodies. By implementing high‐throughput screening using a factorial design plan, we determined that CaCl₂ concentration and PO₄^3? concentration were significant factors, while temperature and pH were not significant. We detected DNA precipitation as well as host‐cell protein (HCP) reduction. Two‐dimensional difference gel electrophoresis (2D‐DIGE) revealed that improved HCP removal does not occur via an unspecific random mechanism such as the enclosure of proteins in the precipitate. CaCl₂ precipitation of DNA and HCP can be combined with nonchromatographic methods such as precipitation and protein A affinity chromatography. This additional purification method not only enhances DNA removal, but also the removal of HCP and antibody multimers, which will reduce immunogenicity and increase homogeneity of the resulting drug.     

Polymerization of lignin fragments contained in a model effluent by polyphenoloxidases and horseradish peroxidase/hydrogen peroxide system*

An effluent containing soluble lignin fragments was treated with potato-polyphenoloxidases (PPO) or horseradish peroxidase/hydrogen peroxide system (HRP/H(2)O(2)). In both cases the reaction was evidenced by the formation of a brown precipitate that was a consequence of the polymerization of lignin fragments. The effect of reaction time, pH, and amount of soluble lignin per unit of enzyme activity on the insolubilization yield was evaluated for PPO-initiated reactions. For HRP-initiated reactions, the amount of H(2)O(2) per unit of enzyme activity was also evaluated. Mathematical models were calculated to predict the insolubilization yield as a function of the significant variables. Based on these models, the insolubilization reaction was optimized and reached maximal values of ca. 50% in both reaction systems. Higher insolubilization yields were not achieved. Chemical characterization of the soluble lignin fragments indicated that the insolubilization yield could not be improved, because the lignin fragments had limited amounts of free phenolic substructures available for the initial steps of the polymerization reaction.     

The elicitor-induced oxidative burst in cultured chickpea cells drives the rapid insolubilization of two cell wall structural proteins

Elicitation of cultured chickpea cells caused rapid insolubilization of two cell wall structural proteins, p190, a putative hydroxyproline-rich glycoprotein and p80, a putative proline-rich protein. This process appeared to result from an H₂O₂-mediated oxidative cross-linking mechanism and was initiated within 5 min and complete within 20 min. Further, elicitation of cells induced a rapid, transient generation of H₂O₂ (oxidative burst), with an onset after 5 min and a maximum H₂O₂-release after 20 min, as measured by a luminol-dependent chemiluminescence assay. Both chemiluminescence and protein insolubilization were suppressed by exogenous application of catalase or diphenylene iodonium, an inhibitor of plasma-membrane NADPH oxidase, respectively. In contrast, exogenous H₂O₂ mimicked the effect of the elicitor, suggesting that the putative oxidative crosslinking of the proteins depends directly on H₂O₂ from the oxidative burst. The peroxidase inhibitor salicylhydroxamic acid blocked both the elicitor- and the exogenous-H₂O₂-stimulated insolubilization, indicating that a peroxidase activity downstream of H₂O₂-supply is required. The protein kinase inhibitor staurosporine blocked the elicitation of the oxidative burst and protein insolubilization. In contrast, the protein phosphatase 2A inhibitor cantharidin accelerated, potentiated and extended the elicited oxidative burst. Cantharidin even stimulated the responses in the absence of the elicitor. The competitive effect of both inhibitors confirms that a coordinated activation of (i) protein kinase(s) and (ii) counteracting protein phosphates(s) is a poised signal transduction step for the induction of an NADPH-oxidase-dependent oxidative burst, which drives the putative peroxidase-catalyzed cross-linking of the cell wall proteins.Abbreviations DPI diphenylene iodonium - Ext-1 extensin-1 - gE1 anti-glycosylated extensin-1 antibodies - HRGP hydroxyp-roline-rich glycoprotein - LDC luminol-dependent chemiluminescence - POD peroxidase - PA polyacrylamide - PRP proline-rich proteins - SHAM salicylhydroxamic acid Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. We thank Dr. C.J. Lamb (Salk Institute, La Jolla, Calif., USA) and Dr. L.A. Staehelin (University of Colorado, Boulder, Colo., USA) for their kind gifts of antibodies.     

Enzyme-immunoassay of thromboxane B2 at the picogram level

A highly sensitive and reproducible enzyme-immunoassay for the measurement of thromboxane B₂ was developed. Thromboxane B₂ (T×B₂) was coupled with β-D- galactosidase by mixed anhydride reaction. Thromboxane B₂-antiserum was generated in rabbits and used at a final dilution of 1:480,000. The separation of immuno- complex from the free form of TxB₂ was accomplished by the double antibody method. The second antibody was sheep anti rabbit IgG. The precipitated enzyme activity was measured fluorometrically with 4-methyl-umbelliferyl-gb-D-galactoside as substrate.This method allowed to measure TxB₂ in the range of 0.002 - 5 picomole per tube. The cross-reactivity of the anti-thromboxane B₂-antiserum with 2,3-dinor thromboxane B₂ was about 20%, but it was less than 0.2% for the other prostanoids tested.TxB₂ extracted from human urine was measured by enzyme-immunoassay (y) and radioimmunoassay (x) which has been found closely correlated to values obtained by gas chromatography-mass spectrometry. Regression analysis of the data comparing enzyme-immunoassay and radioimmunoassay gave the equation y = 0.996 x + 0.470, correlation coefficient r = 0.9947. Inter-assay coefficient of variation was 3.1%.The assay was further simplified by coating the second antibody on glass beads. The regression equation between this solid-phase enzyme immunoassay (y) and radioimmunoassay ( (x) was y = 0.9860 × 1.927, r = 0.9895, and enzyme immunoassay (y) was y = 0.9749 × −0.94808, r = 0.9887. Thus, the enzyme-immunoassay shows specificity and sensitivity comparable to radioimmunoassay making use of radioactive tracer unnecessary.     

Ultrastructural localization of acetylcholinesterase activity by means of the electron dense precipitate derived from Koelle's cuprous thiocholine iodide by treatment with phosphomolybdic acid and osmium tetroxide

Summary An osmium resistant, thermostable and electron dense precipitate was obtained from cuprous thiocholine iodide (Koelle's precipitate) by a joint interaction with phosphomolybdic acid and OsO₄. No diffusion artifact due to the conversion of the primary precipitate to the secondary precipitate was observed, contrary to that seen after (NH₄)₂S or K₃Fe⁺⁺⁺ (CN)₆ posttreatment of the cuprous thiocholine iodide. In addition, phosphomolybdic acid and OsO₄ provided a counterstain effect on the ultrastructural background. By the present modification, Koelle's histochemical method becomes a useful cytochemical method for ultrastructural localization of acetylcholinesterase activity.     

Highly sensitive solid-phase enzyme immunoassay for terminal deoxynucleotidyl transferase

A highly sensitive and specific solid-phase enzyme immunoassay system for terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) has been developed by the use of monospecific antibody against calf thymus TdT and β-d-galactosidase from Escherichia coli as label. The immunoassay system was composed of solid phase (polystyrene beads) with immobilized F(ab′)₂ antibody fragments and the antibody Fab′ fragments labeled with β-d-galactosidase. The minimum detectable concentration of calf TdT was 0.1 ng/ml (0.01 ng/assay), making it more sensitive than the radioimmunoassay or enzyme immunoassay methods that use alkaline phosphatase as label, as reported previously. The assay system cross-reacted with human TdT, and TdT in neoplastic cells or sera from leukemic patients was successfully detected by the present immunoassay method.     

A new radioimmunoassay for human chorionic gonadotropin using monoclonal antibody

A new radioimmunoassay (RIA) for human Chorionic Gonadotropin (hCG) was developed using murine monoclonal antibody to the beta-subunit of hCG (beta-hCG). The IgG fraction of the monoclonal antibody which did not react with 125I-beta-hCG was purified from hybridoma ascites, and covalently coupled to Sepharose 4B. This solid-phase antibody was incubated with standard hCG or serum sampled for 48 hours. The reaction medium was then removed by centrifugation and 125I-beta-hCG and anti-beta-hCG rabbit polyclonal antibody were added to the precipitate. The alcohol precipitation method was used for separating "bound" and "free" forms in the second reaction. The sensitivity for hCG in this assay system was 0.5 mIU/ml serum and the cross-reactivity with human Luteinizing Hormone (hLH) was 0.4%. This assay system was shown to be clinically applicable. Serial serum samples from two patients with trophoblastic disease were assayed and minute amounts of hCG, which could not be determined by conventional assay methods, could be assayed by this new RIA.     

“Cleavable” hapten-biotin conjugates: Preparation and use for the generation of anti-steroid single-domain antibody fragments

Antibody engineering technology has the potential to provide artificial antibodies with higher performance than conventional antibodies. Filamentous phage particles are often used to express a vast diversity of mutated antibody fragments from which clones displaying improved fragments can be isolated. We recently showed that hapten-biotin conjugates, combined via a linker involving a reductively cleavable disulfide bond, are useful for isolating phage clones displaying high-affinity anti-hapten antibody fragments. Here we prepare cleavable hapten-biotin conjugates and use them to isolate anti-hapten antibody fragments with relatively low affinities. Three diagnostically important steroids (estradiol-17β [E₂], cortisol, and 17α-hydroxyprogesterone) were each coupled with a biotin derivative containing a disulfide bond. These conjugates could be bound simultaneously by their relevant anti-steroid antibody and NeutrAvidin, and their linkers were easily cleaved by dithiothreitol (DTT) treatment. The E₂-biotin conjugate was used to generate anti-E₂ single-domain antibody fragments (sdAbs). Random point mutations were introduced by error-prone PCR into the gene fragment encoding the V_H domain of a mouse anti-E₂ antibody, and these products were expressed as phagemid particles that were reacted with the E₂-biotin conjugates that had already been immobilized on a solid-phase via NeutrAvidin. Thorough washing off of nonspecific phages and subsequent DTT treatment provided a phagemid clone that displayed a mutated sdAb with improved binding properties.     

Fasciola hepatica: some properties of a precipitate which forms when metacercariae are cultured in immune rat serum.

When Fasciola hepatica metacercariae are cultured in immune rat serum a precipitate forms on their teguments which is a complex of parasite metabolic antigen and rat immunoglobulin, possibly IgG. Continuous secretion of the antigen by the parasite and its precipitation with antibody leads to the eventual appearance of precipitate in the culture medium. Ouchterlony double diffusion tests indicate that only one antigen/antibody interaction appears to be involved in precipitate formation. When rats were vaccinated with precipitate emulsified with Freund's complete adjuvant then given an oral challenge with metacercariae, there was a reduction of as much as 50% in their worm burdens compared with controls. This evidence suggests that the precipitate contains a functional antigen of the parasite. The rat immunoglobulin component is therefore presumed to be protective antibody.     

Micro-quantitative determination of ciprostene in plasma by gas chromatography—mass spectrometry coupled with an antibody extraction

A simple and highly sensitive method has been developed for the determination in plasma of ciprostene, 9β-methyl-6α-carbaprostaglandin I₂, using gas chromatography—mass spectrometry following solid-phase extraction on an immobilized antibody column. The anti-ciprostene antibody obtained from rabbit serum was coupled to an agarose support matrix, and the immobilized antibody thus prepared was used as an extraction phase for sample clean-up. The extracted drug was treated with pentafluorobenzyl bromide followed by bis(trimethylsilyl)trifluoroacetamide. The derivative was quantitatively analysed by negative-ion chemical ionization gas chromatography—mass spectrometry. The lower limit of quantitation was 50 pg/ml when 1 ml of human plasma was used. The plasma concentration of ciprostene in a dog treated with ciprostene at 2.5 μg/kg was determined successfully by this method.     

Study of circulating immune complexes in three hematologic diseases: Acute myeloid leukemia,acute lymphatic leukemia,and hematosarcoma

Summary Circulating immune complexes (CIC) were detected by means of the polyethylen glycol (PEG) precipitation method, in the serum of untreated patients with hematologic diseases including acute myeloid leukemia (AML), acute lymphatic leukemia (ALL) and hematosarcoma (HS). Immunoglobulins (Ig) and the C ₄ fraction of complement were quantitated in the precipitate dissociated by potassium cyanate (KCNO) and in the serum. When compared to control sera, the results showed a simultaneous increase of both precipitate and Ig component. The proportion of each Ig in the precipitate was stable for the controls but variable for the patients. On the whole precipitated proteins, 30% were systematically quantitated in patients and controls. In the remaining portion were noticed Clq and C ₃ fractions of complement as well as haptoglobin and albumin. The nature of the antigen was discussed.     

Coagulation factor V exists uncomplexed in bovine plasma

Bovine coagulation factor V has been examined immunochemically to ascertain whether the coagulant polypeptide (h) with M_r = 290 000–330 000 is complexed in plasma with a second immunochemically distinct polypeptide (I₂) of M_r = 400 000. Antiserum containing antibodies to h and l₂ detects the l₂ polypeptide eluting earlier than the h chain on gel filtration of plasma with either added calcium or EDTA, consistent with the behavior of a higher molecular weight noninteracting species. An immobilized monospecific antibody to l₂ removes only the l₂ polypeptide from a purified factor V preparation containing both h and l₂. Moreover, while a monospecific antibody to the h chain was able to precipitate purified radioactively labelled h chain alone or mixed with plasma, the l₂ antibody was unable to precipitate radioactively labelled h chain even after attempted recombination of the h chain with l₂ present in plasma. These studies indicate that the l₂ polypeptide is not complexed to the h chain in a purified system or in plasma and reinforce the conclusion that factor V is a single polypeptide chain uncomplexed in plasma.     

Leishmania donovani: physicochemical, immunological, and biological characterization of excreted factor from promastigotes.

Leishmanial excreted factor (EF) from promastigote cultures was enriched from the crude product by differential precipitation with ammonium sulfate and perchloric acid, followed by column chromatography; and by boiling EF-antibody complex. Boiling destroyed the antibody, releasing the EF, which retained its ability to precipitate antibody. Enriched EF from Leishmania donovani promastigotes was found to be a highly negatively charged, carbohydrate-like material with a molecular weight approximating to 33,000, when monitored against a series of protein markers by gel filtration. Its ability to precipitate with antibody was unimpaired by boiling, lyophilization, pH changes from 1 to 11, treatment with high concentrations of NaCl, 10% phosphotungstic acid in 10% HCl, 0.6 M perchloric acid, 5% H₂SO₄, acetone, or dioxan. It did not absorb at wavelengths between 220 and 750 nm. Treatment with trypsin, Pronase, neuraminidase, and hyaluronidase did not affect its activity. Biochemical analysis showed that enriched EF contains carbohydrates but, at our level of detection, no protein, lipid, triglycerides, fatty acids, DNA, RNA, pentoses, amino sugars, sialic or uronic acid. Precipitation of EF by antibody was studied and the optimal molecular proportions for complete precipitation determined. EF-antibody complex, prepared at optimal proportions, and EF complexed with methylated bovine serum albumin, like EF alone, did not elicit antibody production in rabbits. EF in 0.5% phenol-saline elicited a delayed skin response of induration and erythema in guinea pigs cured of L. enriettii. Elevated temperature increased the release of EF from promastigotes, while the presence of trypsin acting at 37 C seemed to inhibit this effect slightly. Fractionation of mechanically broken promastigotes, by differential centrifugation and stepwise sucrose gradients, revealed a factor that precipitated rabbit antibody against whole promastigotes. This factor was associated with the soluble, organelle-free fraction and resembled EF when monitored by gel diffusion. This factor did not migrate when the complete extract from the broken promastigotes was run in immunoelectrophoresis. Boiling the extract for 5 min released a factor, which migrated to the anode. This factor appeared to be associated with another component in the promastigote, from which it dissociated on boiling. Boiling hamster tissues infected with leishmanial amastigotes, i.e., spleens containing L. donovani and epididymides containing L. tropica, also released factors similar to EF. These precipitated antibody in the same way, producing precipitation arcs that were continuous with those formed by EF from the homologous promastigotes. EF acted as a conditioner for culture promastigotes. Conditioned cultures showed maximal growth before similar, unconditioned cultures. However, both types of culture produced equal numbers of promastigotes per unit volume by the end of exponential growth.     

Evaluation of an on-line solid-phase extraction method for determination of almokalant, an antiarrhythmic drug, by liquid chromatography

A fully automated liquid chromatographic method based on a Prospekt solid-phase extraction unit is described for determination of the antiarrhythmic drug almokalant in plasma. The assay comprises solid-phase extraction on a C₂ phase and separation on a C₁₈ column with fluorometric detection. In the original procedure 40 samples a day could be run unattended but by modifying the sequence in the solid-phase extraction process it was possible to increase this number to 70. The method gives an absolute recovery of 92% and a repeatability (C.V.) of 2.9% at 75 nmol/1 of plasma. The limit of quantitation is 2 nmol/1 of plasma (C.V. < 20%). As regards accuracy and precision the performance of the method is as good as the manual method based on liquid-liquid extraction. The Prospekt method is, above all, faster and requires far less manual effort.     

Investigation of the mechanisms affecting Cu and Fe bioavailability from legumes

Chemical composition and content in polyphenols, phytic acid, and dietary fiber of whole cooked common bean (Phaseolus vulgaris L.) and faba bean (Vicia faba L.) and of soluble and insoluble fractions separated from them were determined. Simultaneous determination of Cu, Fe, and protein bioavailability in the small intestine of rat was carried out in single-dose, short-term (1 h) experiments. After cooking, about 80% of seed components (on a weight basis) of either legume was recovered in the precipitate (insoluble fraction) after extraction with water. Protein, lipid, starch, dietary fiber, and polyphenols underwent the most severe insolubilization, together with more than 70% of total Cu and Fe. Cu, Fe, and protein bioavailability showed a similar trend (i.e., the lower the protein, the lower the Cu and Fe availability). Availability of proteins, Cu, and Fe in the insoluble fractions were the lowest, but Cu bioavailability was higher than that of Fe in all fractions. The results provide evidence that the heat-induced insolubilization process adversely affects not only protein but also Cu and Fe bioavailability from legumes and that polyphenols are likely to be a major inhibitor on absorption.     

An Improved Procedure of Specific Polysome Precipitation with Antibody

The specific immunoprecipitation of polysomes prepared from a mouse myeloma, 31C, synthesizing an IgG₁ immunoglobulin has been investigated. A reported method in which polysomes were coprecipitated by sequential addition of antibody to 31C myeloma protein, antigen (i.e., the 31C protein) and again the antibody, was used. Salt concentration greatly affected the immunoprecipitation of polysomes. In the presence of 100 mm KCl or NaCl, 10–20% of myeloma polysomes and only 1% of mouse liver polysomes were precipitated with the antibody to myeloma protein. On the other hand, 90% of the both polysomes were precipitated by the same antibody at a salt concentration of 10 mm. Triton X-100 and sucrose had little effect on preventing nonspecific binding of immunoglobulin to ribosomes. Experiments were carried out to obtain an optimal ratio of the amount of polysome to that of antibody and antigen to be added for the coprecipitation of polysomes. To date we have tried 25 μg of the first antibody, 14 μg of antigen added second to the polysomes and 38 μg of the antibody added finally and these were found to precipitate most efficiently one A₂₆₀ unit of 31C polysomes.     

Measurement of glial fibrillary acidic protein by a two-site immunoradiometric assay

The two-site immunoradiometric assay (two-site IRMA) for the brain-specific glial fibrillary acidic protein (GFA protein) is carried out by reaction of the GFA protein solution with a solid-phase anti(GFA) followed by a second reaction in which the insoluble product is incubated with purified, radioactive anti-(GFA). Unreacted labeled antibodies remain in solution and are washed away. As the amount of GFA increases, the radioactivity in the solid-phase increases. The most significant assay variables include (a) stability and reactivity of the solid-phase antibody, (b) washing the solid-phase, (c) nonspecific interference by serum proteins, and (d) a paradoxical fall in tube radioactivity which occurs at high dose (the “high-dose hook effect”). The assay becomes more sensitive and precise and the serum effect is minimized when the solid-phase antibody is separated from the matrix by an immunoglobulin “spacer-arm”. For a triplicate determination, the minimal detectable dose averaged $\text{73}\text{pg}\text{200 μ}\text{l}$ incubation. The assay precision enables a 500-fold assay range. GFA activity found in aged crude tissue or tissue-culture extracts, CSF, and used tissueculture media, often did not appear to be immunologically identical to the purified standard GFA protein. This may be explained by the known tendency of GFA protein to aggregate. The assay does not cross-react significantly with other common CNS proteins. Assay of various rat tissues confirms the localization of GFA protein only to the CNS.     

Association between anti-nucleophosmin and anti-cardiolipin antibodies in (NZW × BXSB)F1 mice and human systemic lupus erythematosus

We showed previously that nucleophosmin (NPM), a nucleolar phosphoprotein, is recognized by sera from (NZW × BXSB)F₁ (WB) mice, a model of systemic lupus erythematosus (SLE) and anti-phospholipid syndrome. In the present study we analysed the prevalence and kinetics of anti-NPM autoantibodies in WB mice by a solid-phase ELISA with recombinant human (rh) NPM as the antigen and showed that most male WB mouse sera had anti-NPM antibodies that were responsible for their indirect immunofluorescence staining pattern on Hep-2 cells. Anti-NPM antibodies were significantly associated with anti-cardiolipin (aCL) antibodies. This antibody profile mirrored that observed in certain human SLE sera because anti-NPM antibodies were detected in 28% of the sera from patients with SLE and were similarly associated with aCL antibodies. The demonstration that rhNPM bound to cardiolipin (CL) in vitro and increased the CL-binding activity of a WB-derived aCL monoclonal antibody indicates that NPM can interact with CL to form SLE-related immunogenic particles that might be responsible for the concomitant production of anti-NPM and aCL antibodies.     

A peptide-based fluorescent ratiometric sensor for quantitative detection of proteins

A ratiometric fluorescent sensor was obtained by solid-phase synthesis of a peptide singly labeled at its N-terminus with a 3-hydroxychromone (3HC) derivative, an environmentally sensitive fluorophore with a two-band emission. The construct contains the binding site recognized by an antibody fragment, scFv1F4_Q34S, with nanomolar (nM) affinity. The dye only marginally affected the kinetic and equilibrium binding parameters of the scFv-peptide interaction, as measured by surface plasmon resonance. On interaction with the antibody fragment, the sensor showed up to 47% change in the ratio of its two emission bands, indicating an enhanced screening of the 3HC fluorophore from bulk water. Competition with two unlabeled peptides of different lengths led to a dynamic displacement of the construct governed by the relative binding constants. Calibration showed that the response is proportional to the ratio of scFv1F4_Q34S to labeled peptide. The detection limit of scFv1F4_Q34S was 15 nM. In a more complex medium (100 μg/ml bovine serum albumin), the scFv could be detected in the 50- to 100-nM range. This work demonstrates that, with the perspective of further improvements of the dye spectroscopic properties, fluorescent ratiometric sensing based on small synthetic peptides represents a promising tool for quantitative target detection.