Direct Sandwich Research Articles

A new enzyme-linked immunosorbent assay recognizing both rat and human activated coagulation Factor XII (FXIIa)

We describe the first sandwich enzyme-linked immunosorbent assay (ELISA) capable of recognizing both rat and human activated coagulation Factor XII (FXIIa). Increased plasma concentrations of FXIIa have been associated with adverse outcomes in several cardiovascular disease states. In humans, the FXIIa antigen in plasma is quantified by a direct sandwich ELISA employing an antibody (mAb 2/215) raised against its β-fragment (β-FXIIa), but this assay is unable to detect rat β-FXIIa antigen. Thus, experimental models are at present limited in their capacity to reveal mechanisms by which FXIIa might contribute to cardiovascular pathology. Consistent with overlap between human and rat FXIIa protein epitope sequences, Western blot analysis and ELISA demonstrate that another previously developed antibody against human FXIIa (mAb 201/9) detects β-FXIIa in both human and rat plasma, with no evidence for cross-reactivity with the FXII zymogen or FXIIa complexed with its endogenous inhibitor. The mAb 201/9 based ELISA identified similar elevations in FXIIa in plasma from rats and humans with chronic renal failure. The capacity of this ELISA to identify rat plasma FXIIa has potential application to a wide range of experimental models of human cardiovascular disease.

Atomistic simulations of the solid-liquid transition of 1-ethyl-3-methyl imidazolium bromide ionic liquid

Achieving melting point around room temperature is important for applications of ionic liquids. In this work, molecular dynamics simulations are carried out to investigate the solid-liquid transition of ionic liquid 1-ethyl-3-methyl imidazolium bromide ([emim]Br) by direct heating, hysteresis, void-nucleation, sandwich, and microcanonical ensemble approaches. Variations of the non-bonded energy, density, diffusion coefficient, and translational order parameter of [emim]Br are analyzed as a function of temperature, and a coexisting solid-liquid system is achieved in the microcanonical ensemble method. The melting points obtained from the first three methods are 547 ± 8 K, 429 ± 8 K, and 370 ± 6 K; while for the sandwich method, the melting points are 403 ± 4 K when merging along the x-axis by anisotropic isothermal-isobaric (NPT) ensemble, 393 ± 4 K when along the y-axis by anisotropic NPT ensemble, and 375 ± 4 K when along the y-axis by isotropic NPT ensemble. For microcanonical ensemble method, when the slabs are merging along different directions (x-axis, y-axis, and z-axis), the melting points are 364 ± 3 K, 365 ± 3 K, and 367 ± 3 K, respectively, the melting points we get by different methods are approximately 55.4%, 21.9%, 5.1%, 14.5%, 11.6%, 6.5%, 3.4%, 3.7%, and 4.3% higher than the experimental value of 352 K. The advantages and disadvantages of each method are discussed. The void-nucleation and microcanonical ensemble methods are most favorable for predicting the solid-liquid transition.

Comparison of four distinct detection platforms using multiple ligand binding assay formats

Several detection platforms are available for ligand binding assays (LBA), each claiming superiority in sensitivity and dynamic range. However, little information exists in the literature directly comparing the various LBA platforms for quantitation. We have tested four common platforms to evaluate and compare the interchangeability of detection platforms by comparing sensitivity and dynamic range to a colorimetric LBA. The detection platforms compared are: colorimetric, chemiluminescence, time-resolved fluorescence (TRF) and electrochemiluminescence (ECL). Five different LBA protocols were tested with each of the detection endpoints. The assay protocols include the following ligand binding assay formats: direct binding, sandwich ELISA, competitive and cell based ELISA. We found that no detection platform consistently performed better than all the others and it was not possible to predict which platform would perform best for a given assay protocol. We also found surprising differences in assays (plate coating efficiency, low signal) which add to difficulty in choosing the best platform ad hoc. We propose here that in developing new assay protocols for detection of biotherapeutic agents, multiple detection platforms should be tested in order to forward the best assays possible and for the right reasons.

Immunomagnetic bead-based recovery and real time quantitative PCR (RT iq-PCR) for sensitive quantification of aflatoxin B1

Aflatoxin B1 is an unavoidable natural mycotoxin that enters the food chain by contamination of food grains and feedstuffs, potentially posing carcinogenic risks to animal and human health. Immuno-PCR methods have the potential to address the need of meeting the regulatory limits by detecting trace levels of toxins present in food and animal feeds. This paper describes a real-time immuno-quantitative PCR (RT-iqPCR) assay for quantification of aflatoxin B1 suspended in methanol:water solution that can also serve as an extraction solvent. Immuno-PCR approaches were examined including direct vs. indirect sandwich assays using monoclonal vs. polyclonal antibodies. Our best approach was obtained using monoclonal antibodies to capture aflatoxin in solution prior to immobilizing the Fc portion of the capture antibodies onto to protein G magnetic beads. This was followed by the addition of a polyclonal ‘signal antibody’ tethered with an oligonucleotide template for a subsequent PCR assay. The RT-iqPCR assay described herein leads to the sensitive detection and quantification of aflatoxin B1 from 10ppb down to 0.1ppb with high correlation (r2=0.97) and efficiency (99.5%). The approach also detected the high-dose ‘hook effect’ phenomenon (excess antigen) which was overcome by the use of dilution protocols to eliminate false negatives that may occur at levels above quantification limits of the assay. The RT-iqPCR approach discussed here is presented as a model system that could easily be adapted for aflatoxin detection in a variety of food or animal feed samples using a simple methanol:water solution as an extraction solvent.

Microfluidic Device for Fluorescence Immunoassays by Using Porous Matrix

The present work presents the availability of using porous matrix in microfluidic devices as a solid phase matrix for immunoassays. Porous matrixes on the surface of the microchannels were microfabricated by MEMS technology and electrochemical etching technology, which were coated on the wall of the rectangular microchannel in the microdevices to provide a surface-enlarging matrix. The microfabrication process of porous matrixes was investigated and optimized. Then the surface morphology of the porous matrixes was characterized by SEM. Both direct method and dual-antibody sandwich method were used for fluorescence immunoassays. Using sandwich immunoassay, 6.25μg/mL - 25μg/mL human IgG in real samples have been detected with a correlation coefficient of 0.9773. These porous microdevices have shown some advantages over its large-scale counterparts, including lower sample and reagent consumption, lower cost, less analytical time and so on, which enables detection for clinical testing.

Expression of recombinant Apple chlorotic leaf spot virus coat protein in heterologous system: production and use in immunodiagnosis

Expression condition for maximum recovery of recombinant Apple chlorotic leaf spot virus (ACLSV) coat protein was standardized. The in vitro expressed fusion protein with 6xHis tag (~43 Kd) was purified from inclusion bodies and used as an antigen for raising polyclonal antiserum in rabbit. This antiserum consistently detected ACLSV in pome and stone fruits as well as herbaceous host plants by direct double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and direct tissue blot immunoassay (DTBIA). The conditions for immuno-capture RT-PCR (IC-RT-PCR) were also standardized.

Kinetics of Salmonella typhimurium binding with antibody by immunoassays using a surface plasmon resonance biosensor

This study examined the kinetics of Salmonella typhimurium binding to an antibody using a direct assay and sandwich assay with a surface plasmon resonance (SPR) biosensor. A kinetic model was proposed based on curve-fitting of the experimental SPR sensorgrams using an integral rate equation. The SPR sensitivity in the sandwich assay was significantly higher than that in the direct assay. The association rate constants and affinities of the cell in the sandwich assay were double those in the direct assay. The enhanced binding attraction between the cell and detection antibody in the sandwich assay improved the lower limit of detection.

Development of a High-specificity Enzyme-linked Immunosorbent Assay (ELISA) System for the Quantification and Validation of Intact Rat Osteocalcin

Osteocalcin (OC) exhibits hard tissue-specific expression and binding activity to hydroxyapatite. Therefore, measurement of secreted OC is a very useful index for evaluating osteoblastic differentiation in regenerative bone. In the present study, we established a high-specificity sandwich enzyme-linked immunosorbent assay (ELISA) system for the quantification of intact rat OC, which could be useful for validating tissue-engineered bone samples nondestructively and continuously. The range of detection with the sandwich ELISA system was 0.1–100 ng OC/mL of cell culture media or rat sera. No cross-reactivities were detected with OCs from other species, including human, bovine and mouse OCs, and other mammalian sera, which would contain the corresponding endogenous OCs. The intra- and inter-assay coefficients of variation were ≤4.9% and ≤5.9%, respectively. Recovery tests only showed variation between 89.4% and 103.7%. Using the newly developed direct sandwich ELISA system, we found that the secreted OC levels from rat bone marrow-derived mesenchymal stem cells during osteogenic differentiation with dexamethasone were significantly higher than those from cells undergoing non-osteogenic or adipogenic differentiation. It was established that this ELISA system would be suitable for quantitative assessment of bone formation by cultured cells with or without scaffolds in rat experimental models.

Zoonotic risk of hepatitis E virus (HEV): A study of HEV infection in animals and humans in suburbs of Beijing

To investigate hepatitis E virus (HEV) infection among different animals and workers in pig farms and slaughterhouses, and analyze the genotype of HEV isolated in this study. Serum samples were collected from adult swine, cows, sheep, younger swine (< 3 months), and workers in pig farms and slaughterhouses (professional group). Fecal samples were collected from younger swine in the south suburbs of Beijing. Anti-HEV antibody was evaluated by direct sandwich enzyme immunoassay. HEV RNA was extracted from fecal samples and amplified by nested reverse transcription polymerase chain reaction (RT-nPCR). The PCR products were sequenced, and the sequence homology and phylogenetics of the HEV strains isolated from swine were analyzed. The anti-HEV positivity rates in adult swine, cows, sheep, younger swine, professional group and general population were 98.23% (222/226), 29.35% (54/184), 9.80% (20/207), 60.73% (99/164), 42.51% (105/247) and 20.29% (522/2572), respectively. The HEV RNA positivity rate of fecal samples was 22.89% (19/83) and 16/19 samples were positive for HEV RNA amplified with both primers, HEV open reading frame (ORF)1 and HEV ORF2. Sequence analysis of these 16 samples showed that there were two groups, designated BJ-1 and BJ-2. The nucleotide homology of BJ-1 and BJ-2 was 99%. Phylogenetic analysis indicated that both of these groups belonged to genotype 4d. Workers in pig farms and slaughterhouses were more likely to contract HEV infection than the general population because of close contact with swine with a high prevalence of anti-HEV.

Production of monoclonal antibodies for sandwich immunoassay detection of Pacific ciguatoxins

Ciguatoxins are the major causative toxins of ciguatera seafood poisoning. Limited availability of ciguatoxins has hampered the development of a reliable and specific immunoassay for detecting these toxins in contaminated fish. Monoclonal antibodies (mAbs) specific against both ends of Pacific ciguatoxins CTX3C and 51-hydroxyCTX3C were prepared by immunization of mice with the protein conjugates of rationally designed synthetic haptens in place of the natural toxin. Haptenic groups that possess a surface area larger than 400 Å 2 were required to produce mAbs that can bind strongly to CTX3C or 51-hydroxyCTX3C. A direct sandwich enzyme-linked immunosorbent assay (ELISA) using these mAbs was established to detect CTX3C and 51-hydroxyCTX3C at the ppb level with no cross-reactivity against the other marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, or maitotoxin.

Detection of Salmonella typhimurium using an electrochemical immunosensor

An electrochemical immunosensor based on screen-printed gold working electrode with onboard carbon counter and silver–silver chloride pseudo-reference electrode for Salmonella typhimurium detection is described in this paper. Monoclonal anti-S. typhimurium antibody was immobilized using physical and covalent immobilization via amine coupling of carboxymethyldextran on the surface of the gold working electrode. A direct sandwich enzyme-linked immunosorbent assays (ELISA) format was then developed and optimized using a polyclonal anti-Salmonella antibodies conjugated to horseradish peroxidase (HRP) as the enzyme label. 3,3′,5,5′-Tetramethylbenzidine dihydrochloride (TMB)/H2O2 was used as the enzyme mediator/substrate system. Electrochemical detection was conducted using chronoamperometry at −200mV vs. onboard screen-printed Ag–AgCl pseudo-reference electrode. The applied potential was selected through the study of the electrochemical behaviour of bare gold electrode with TMB–H2O2–IgG–HRP system. S. typhimurium detection of 5×103cellsml−1 and ∼20cellsml−1 was achieved respectively for physical and covalent antibody immobilization. The developed sensor was then compared to a commercial ELISA kit and a chromogenic agar plating method for meat samples analysis. The sensor format shows a promising technology for simple and sensitive detection system for Salmonella contamination. Rapid detection of Salmonella is a key to the prevention and identification of problems related to health and safety.

Detection of Citrus tristeza virus (CTV) from Satsuma Owari mandarins (Citris unshiu) by direct tissue blot immunoassay (DTBIA), DAS‐ELISA, and biological indexing

Three different citrus‐producing regions of Turkey—Edremit Gulf, Coastal Aegean, and eastern Black Sea—were surveyed in 2005 and 2006. A total of 119 samples were collected from Satsuma Owari mandarin (Citrus unshiu) trees grafted on Citrus tristeza virus (CTV)‐resistant Poncirus trifoliata rootstocks in commercial groves and home gardens. All samples were tested for the presence of CTV by direct tissue blot immunoassay (DTBIA) and double antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) using young shoots. The samples that tested positive were indexed for biological properties. The results obtained from DTBIA tests showed that 20 of 119 (16.8%) tested trees were infected with CTV and 99 trees were virus free. All samples that tested positive in DTBIA were also positive in DAS‐ELISA. After biological indexing, none of these 20 isolates showed any symptoms on sour orange, grapefruit, and sweet orange plants, but all isolates induced vein‐clearing symptoms on Mexican lime (Citrus aurantifoli) in 9 months. Some isolates also caused leaf‐cupping and chlorosis in Mexican lime. However, 15 months after initial grafting, all isolates induced mild to moderate stem‐pitting on Mexican lime. In addition, young tissues, petioles, and leaf samples were collected periodically at monthly intervals for 1 year (2006) from a 25‐year‐old Satsuma Owari mandarin grafted on P. trifoliata in the Edremit Gulf. CTV was readily detected in tissue blots of young shoots and petioles from CTV‐infected plants during the autumn, winter, and spring seasons. Similarly, the highest ELISA values were obtained in April; the lowest values were noted in September. This study showed that DTBIA is a rapid, sensitive, and reliable procedure for detecting CTV under field conditions.

Naturally Acquired IgM Antibody Response to the C-Terminal Region of the Merozoite Surface Protein 1 of Plasmodium vivax in Korea: Use for Serodiagnosis of Vivax Malaria

The antibody levels against the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1c) were measured in 276 patients with P. vivax malaria (patient group), 320 malaria-naïve healthy individuals (control group 1), and 70 malaria-naïve individuals with various disorders (control group 2) using the immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the direct sandwich ELISA. To evaluate the antibody response during relapse, 5 relapsed patients were tested using the IgM capture ELISA. The IgM antibodies were negative in 99.7% of control group 1 and in 100% of control group 2; they were positive in 90.6% of the patient group. The total antibody levels were positive in 88.4% of the patient group with the direct sandwich ELISA. The sera from the second malaria episode, i.e., relapsed patients, were 100% positive on the IgM capture ELISA. The results of this study suggest that the IgM capture ELISA may be a useful diagnostic method for P. vivax malaria for both primary infection and relapse.

Soybean allergen detection methods – A comparison study

Soybean containing products are widely consumed, thus reliable methods for detection of soy in foods are needed in order to make appropriate risk assessment studies to adequately protect soy allergic patients. Six methods were compared using eight food products with a declared content of soy: a direct sandwich ELISA based on polyclonal rabbit antibody (ab) to raw soy flakes, a commercial and an in-house competitive ELISA both based on ab to denatured, 'renatured' soy protein, an enzyme-allergosorbent test (EAST) inhibition based on two sera from soy allergic patients, histamine release (HR) using basophils passively sensitized with patient serum and a PCR method detecting soy DNA. Eight food products were selected as model foods to test the performance of the methods. There was an overall good agreement between the methods in terms of ranks of soy content but not the quantity. The sandwich ELISA aimed at native soy proteins had the lowest detection limit of 0.05 ppm, but only identified soy in 5/8 products, and generally in lower amounts compared to other methods. The competitive ELISA had a higher detection limit of 21 ppm, but seemed more successful in detecting processed soy. Only HR, EAST inhibition and PCR detected soy in all eight products. In spite of a general good correlation in terms of ranks of soy content, more than a single method may be necessary to confirm the presence of soy in foods.

Soluble lectin-like oxidized low density lipoprotein receptor-1 in type 2 diabetes mellitus

The lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) can be proteolytically cleaved and released as soluble forms (sLOX-1). We have determined serums LOX-1 in type 2 diabetes and evaluated the effect of glucose and advanced glycation end products (AGEs) on sLOX-1 in vitro and in vivo. Endothelial cells were incubated with glucose or AGEs, and sLOX-1 in cell medium was measured. Serum sLOX-1 was measured in 219 diabetic patients and 187 controls by ELISA. The effect of lowering glucose and AGEs on sLOX-1 was determined in 38 poorly controlled diabetic patients after improvement in glycemic control. Incubation of endothelial cells with AGE-BSA led to a dose-dependent increase in sLOX-1, whereas the effect of glucose on sLOX-1 was less marked. Serum sLOX-1 was 9% higher in diabetic patients compared with controls (P<0.01). In the poorly controlled patients, serum sLOX-1 decreased by 12.5% after improvement in glycemic control (P<0.05). The magnitude of reduction in sLOX-1 correlated with the improvement in hemoglobin A1c and AGEs but not with the reduction in oxidized LDL. sLOX-1 level is increased in type 2 diabetes. Both glucose and AGEs are important determinants of LOX-1 expression, and lowering glucose and AGEs leads to a reduction in sLOX-1.

Cardiometabolic risk factors assessed by a finger stick dried blood spot method.

Cardiovascular and metabolic risk factors are gaining attention as potential indicators for intervention in the prevention and treatment of cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM). Blood spot technology offers an efficient, convenient method to measure these risk factors in an at-risk population. Simple and convenient methods to assess cardiometabolic risk factors will allow clinicians to formulate treatment strategies for effective prevention and management of these conditions. Insulin and high sensitivity C-reactive protein (hs-CRP) were measured in dried blood spot and corresponding serum samples using conventional commercial serum kits based on a direct sandwich ELISA technique. The triglyceride assay involved enzymatic hydrolysis of triglycerides by lipase to glycerol and free fatty acids. The glycerol produced was then measured by coupled enzyme reactions. Blood spot assays were modified from previously published methods to give improved accuracy and turnaround time. Blood spot levels correlated well with serum levels of hs-CRP (r = 0.99), triglycerides (fasting, r = 0.95; nonfasting, r = 0.94), and insulin (fasting, r = 0.93; nonfasting, r = 0.97). Blood spot testing for insulin, hs-CRP, and triglycerides may be helpful in the cardiometabolic screening or monitoring of patients at risk of developing CVD, T2DM, or metabolic syndrome.

A high sensitivity assay for the inflammatory marker C-Reactive protein employing acoustic biosensing.

C-Reactive Protein (CRP) is an acute phase reactant routinely used as a biomarker to assess either infection or inflammatory processes such as autoimmune diseases. CRP also has demonstrated utility as a predictive marker of future risk of cardiovascular disease. A new method of immunoassay for the detection of C-Reactive Protein has been developed using Resonant Acoustic Profiling™ (RAP™) with comparable sensitivity to a high sensitivity CRP ELISA (hsCRP) but with considerable time efficiency (12 minutes turnaround time to result). In one method, standard solutions of CRP (0 to 231 ng/mL) or diluted spiked horse serum sample are injected through two sensor channels of a RAP™ biosensor. One contains a surface with sheep antibody to CRP, the other a control surface containing purified Sheep IgG. At the end of a 5-minute injection the initial rate of change in resonant frequency was proportional to CRP concentration. The initial rates of a second sandwich step of anti-CRP binding were also proportional to the sample CRP concentration and provided a more sensitive method for quantification of CRP. The lower limit of detection for the direct assay and the homogenous sandwich assay were both 20 ng/mL whereas for the direct sandwich assay the lower limit was 3 ng/mL. In a step towards a rapid clinical assay, diluted horse blood spiked with human CRP was passed over one sensor channel whilst a reference standard solution at the borderline cardiovascular risk level was passed over the other. A semi-quantities ratio was thus obtained indicative of sample CRP status. Overall, the present study revealed that CRP concentrations in serum that might be expected in both normal and pathological conditions can be detected in a time-efficient, label-free immunoassay with RAP™ detection technology with determined CRP concentrations in close agreement with those determined using a commercially available high sensitivity ELISA.

Brain Natriuretic Peptide (BNP) Level as Marker of Cardiac Function in Imatinib - Treated Chronic Myeloid Leukemia Patients: No Evidence of Cardiotoxicity of Imatinib Therapy.

In the last year, the issue of cardiotoxicity of imatinib mesylate (IM) was on focus. Emerging data seem to deny an increased risk of cardiac events in patients treated with IM, which is the frontline therapy in chronic myeloid leukemia (CML). B-type natriuretic peptide (BNP) is released by the heart in response to myocardial tension and is considered an accurate test for the diagnosis of heart failure. The measurement of BNP in the serum is a rapid and easy tool for evaluation of ventricular function, also in asymptomatic patients. We have measured BNP level in 50 consecutive patients (33 males and 17 females) with IM treated chronic phase CML. Patients were enrolled, after informed consent was obtained, as they presented for a follow-up visit at our Institution. BNP was measured using a direct chemiluminescent sandwich immunoassay: the analytical range extends from 0 to 5000 pg/ml, with a sensitivity <2 pg/ml. Normal range is as follows: <100 pg/dl. Median age was 59 (range: 23–82). Median duration of IM therapy was 38.5 months (range: 1–81). IM mean daily dose at time of sample collection was 404 mg (SD ±121). Thirty three patients (66%) were receiving 400 mg/day, 13 (26%) a lower daily dose (200 mg in 1 case, 300 mg in 12) and 5 patients (10%) had higher IM doses (600 mg in 2, 800 mg in 3). The mean level of BNP in the whole population was 22.0 pg/ml (SD ±26.4); only two patients had values >100 pg/ml. There was a linear correlation between age and BNP levels (t–value=3.850, p=0.0003). Nine out of 24 (37%) patients aged ≥60 had BNP >22 pg/ml, compared to only 1/25 (4%) in the cohort <60 years old (χ2=19.7, p<0.0001). BNP level was not affected by IM daily dose (<400 mg = 34.1±28.6, 400 mg = 18.5±26.2, >400 = 13.5±9.3) or by therapy duration (<36 months = 25.2±30.4, ≥36 months = 19.4±22.6). Considering cardiovascular risk factors, 18 patients (36%) had hypertension, while diabetes and hyperlipemia were present in 2 and 4 cases, respectively. Patients with hypertension had higher levels of BNP (34.9±35.1 vs 14.8±16.5, p=0.03), despite an equivalent IM dose (378±81 vs 419±138 mg) and treatment duration (39.5 vs 30.5 months). No patient experienced major cardiac adverse event during IM therapy. An echocardiogram was performed in the two patients with higher BNP values: both of them were older than 60 and suffered from hypertension, but echocardiogram revealed a normal left ventricular ejective function (LVEF ≥65%). To further assess IM impact on BNP levels, five consecutive patients, who were diagnosed with CML during study period, were tested for BNP before start of IM therapy and after 1, 2 and three months of therapy. No significant modification in BNP level was observed during treatment with IM. In conclusion, Imatinib therapy does not cause an increase in BNP levels. This gives an indirect confirm to the cardiac safety profile of the drug, as indicated also by the lack of major cardiac toxicities in our patients. BNP levels were affected by hypertension and by advanced age, but the latter could be a bias due to a higher incidence of hypertension in the elderly cohort.

Bioorganic Studies Utilizing Rationally Designed Synthetic Molecules: Absolute Configuration of Ciguatoxin and Development of Immunoassay Systems

Abstract Ciguatoxins are the major causative toxins in ciguatera seafood poisoning. The limited availability of ciguatoxins has precluded structural elucidation and development of a reliable and specific immunoassay for detecting the toxins in contaminated fish. To seek a solution for the longstanding problem of ciguatera, we addressed the synthetic challenge by utilizing rationally designed model compounds of ciguatoxins. The C2 configuration and entire absolute configuration of ciguatoxin were successfully elucidated with minutest amounts of natural toxins, using an approach which combined partial structure synthesis, microscale chemical transformations, and the CD exciton chirality method. On the basis of the absolute configuration, the partial structures of ciguatoxins were designed and synthesized as haptens for the preparation of anti-ciguatoxin antibodies. Monoclonal antibodies (mAbs) against both ends of ciguatoxin CTX3C were prepared by immunization of mice with protein conjugates of synthetic haptens of the ABCDE-ring and the IJKLM-ring, in place of the natural toxin. Haptenic groups with surface areas larger than 400 Å2 were required to produce mAbs, which could bind strongly to CTX3C itself. A direct sandwich enzyme-linked immunosorbent assay (ELISA) using these mAbs was shown to detect CTX3C at the ppb level with no cross-reactivity against other related marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, or maitotoxin. In order to make the sandwich immunoassay protocol a general method for detecting other ciguatoxins congeners, the preparation of mAbs for the left-end of ciguatoxin was investigated. Expeditious synthesis of the left end of ciguatoxin with installation of the 3-butene-1,2-diol side-chain of the A-ring as well as surface plasmon resonance (SPR) analysis of the antibody–hapten interactions are also described.

A New 4.0‐Generation Dendrimer Phosphorescence Labeling Reagent and Its Application to Determination of Trace Alkaline Phosphatase by Affinity Adsorption Solid Substrate‐room Temperature Phosphorimetry

AbstractA Triton X‐100‐4.0G‐D (4.0G‐D refers to a 4.0‐generation dendrimer) was brought forward as a new phosphorescence labeling reagent. Two types of specific affinity adsorption (AA) reactions (direct method and sandwich method) were carried out between the labeling product of Triton X‐100‐4.0G‐D‐Wheat germ agglutinin (WGA) and alkaline phosphatase (ALP), the product of AA reaction preserved the good characteristics of room temperature phosphorescence (RTP) of 4.0G‐D and ΔIP of the product was proportional to the content of ALP. According to the fact stated above, a new method for the determination of trace ALP by affinity adsorption solid substrate‐room temperature phosphorimetry (AA‐SS‐RTP) was established on the basis of WGA labeled with the Triton X‐100‐4.0G‐D. The detection limits were 0.20 ag·spot−1 (corresponding concentration: 5.0×10−16 g·mL−1, namely 5.0×10−18 mol·L−1) for a direct method and 0.14 ag·spot−1 (corresponding concentration: 3.5×10−16 g·mL−1, namely 3.5×10−18 mol·L−1) for a sandwich method, respectively. For their high sensitivity, good repeatability and high accuracy, the direct method and sandwich method have been successfully applied to determine the content of ALP in human serum, and the results were coincided with the clinical detection results of the enzyme‐linked immunosorbent assay method by the Zhangzhou Hospital of Traditional Chinese Medicine. Meanwhile, the mechanism for the determination of trace ALP by AA‐SS‐RTP was discussed.