Chemiluminescent enzyme immunoassay for measuring leptin

Leptin is one of the representative adipocyte-derived protein hormones. Measuring the serum leptin concentration gives an important index for preventing and treating diabetes mellitus and other diseases. We constructed in this study a chemiluminescent enzyme immunoassay (CLEIA) for measuring leptin by using the anti-leptin polyclonal antibody and alkaline phosphatase (ALP). The method applies the IgG-conjugated ferrite particle to capture leptin in a sample and the ALP-conjugated Fab fragment to detect the captured leptin. We tested Block ace, CE510, and bovine serum albumin (BSA) for their abilities to block non-specific binding of ALP-conjugated anti-leptin Fab to the ferrite particle and found BSA to be the most effective. The measurable range with this ELISA for leptin was 0.1-1.0 pg/mL of leptin and the detection limit (blank+2SD) was 0.1 pg/mL of leptin. These results demonstrate sufficient sensitivity with our system to measure the serum leptin concentration and its clinical usefulness. The results also suggest that a sensitive enzyme immunoassay can be constructed by using only one polyclonal antibody.     

A rapid, simple immunofluorometric assay: development and characterization

A novel two-site immunofluorometric assay, which includes only one incubation step and one separation step, is described. The assay is based on using small-sized beads (0.5 mum diameter) as a soild support and measuring the unbound fraction of labeled antibody in the liquid. The use of a mixture of the solid-phase and labeled antibodies at different concentrations makes it possible to determine antigen concentration over a wide range, without an initial sample dilution. Two assays were developed: one using anti-IgG polyclonal antibodies and the other using antibovine serum albumin monoclonal antibodies. The detection range of the polyclonal antibody assay using 30-minute incubation was 0.2 to 40 mug/mL for human IgG standard. The detection range of the monoclonal antibody assay was 0.2 to 14 mug/mL for bovine serum albumin (BSA) standard with 2 minutes required for the incubation. The interassay variability for the BSA measurement was 1.9% at 4.0 mug/mL of BSA and intra-assay variability was 2.3% at 3.2 mug/mL of BSA. The principles of this assay can be applied in the measurement of any protein in a rapid and reproducible way. (c) 1992 John Wiley & Sons, Inc.     

Chemiluminescence sandwich enzyme immunoassay for determination of human granulocyte colony stimulating factor (G-CSF)

A chemiluminescence sandwich enzyme immunoassay, using a glucose oxidase (GO) label, was developed for detecting attomole amounts of human granulocyte colony stimulating factor (G-CSF). Purified goat F(ab′)₂ immobilized on a bead and purified goat Fab′ labelled with GO were selected in combination with a chemiluminescent detection system comprising luminol and ferricyanide. The detection limits for G-CSF were 4amol/assay (1 pg/mL) in buffer solution and 10 amol/assay (2.5 pg/mL) in human serum. Coefficients of variation within assay and between assay ranged from 5.5% to 7.8% and from 3.4% to 16.0%, respectively. The G-CSF content of serum from normal healthy individuals was measurable using this method. G-CSF in 24 normal human sera showed a mean value of 19.3 pg/mL and ranged from 3.6 to 83.0 pg/mL.     

Anti-leptin receptor antibody mimics the stimulation of lipolysis induced by leptin in isolated mouse fat pads

An anti-leptin receptor polyclonal antibody (receptor antibody), as well as leptin, stimulated the release of free fatty acids from isolated mouse fat pads in a time-dependent manner. Following a 90-min incubation, maximal lipolysis was observed at 6 microg/ml receptor antibody and 0.1 nM leptin. The receptor antibody did not show any additive effect to the stimulation of lipolysis induced by leptin, suggesting that they exert their actions through a similar mechanism involving the leptin receptor. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), quin 2-AM, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and neomycin sulfate (neomycin) all potently inhibited the stimulation of lipolysis by the receptor antibody and leptin. Short-term incubation of the fat pads with the receptor antibody or leptin showed a transient increase in the cellular content of cAMP and myo-inositol 1,4,5-trisphosphate (IP3) in similar concentrations to the free fatty acid release. Quin 2-AM and W-7 also inhibited the increase in cAMP content, suggesting that a Ca(2)+/calmodulin-dependent process may be involved in a part of the mechanism in which the receptor antibody and leptin exert their effects. The increase in cellular IP3 content via phosphoinositide-specific phospholipase C (PLC) sensitive to neomycin appears to be a primary step to initiate intracellular events. Both the receptor antibody and leptin may stimulate the lipolysis through mechanisms involving a transient increase in the cellular IP3 content followed by cAMP production, which leads to the activation of cAMP-dependent protein kinase.     

Human leptin induces angiogenesis in vivo

Leptin is an adipocyte-produced peptide, which plays a crucial role in the regulation of body weight. There is also evidence that leptin stimulates endothelial cell proliferation and the formation of capillary-like tubes in vitro. The disc angiogenesis system was used to measure the angiogenic effect of leptin in vivo. Discs containing 25, 50, 100 and 250 ng/ml of leptin were implanted subcutaneously in Wistar rats, removed after a growth period of 7 and 14 days, and compared with spontaneous growth controls and with positive controls containing equivalent doses of vascular endothelial growth factor (VEGF). Discs were examined morphologically for stroma and vessel development and by immunohistochemistry for quantitative evaluation of angiogenesis. The specificity of the angiogenic effect of leptin was tested by blocking leptin with a polyclonal anti-leptin antibody. Leptin induced a significant level of angiogenesis in a dose-dependent manner both at 7 and 14 days, with a peak at the dose of 100 ng/ml. The angiogenic activity of leptin was completely abolished by the anti-leptin neutralizing antibody. VEGF also induced significant dose-dependent angiogenesis at the same time points with a peak observed at a concentration of 100 ng/ml. The angiogenic response to leptin was significantly higher at 7 days compared with VEGF but not at the 14-day time point. In conclusion, leptin has a specific angiogenic effect in vivo, which is dose- and time-dependent in a concentration range of 25–250 ng/ml. This effect is equivalent to the angiogenic effect of VEGF but is evident earlier compared with VEGF.     

Homogeneous electrogenerated chemiluminescence immunoassay for the determination of digoxin employing Ru(bpy)(2)(dcbpy)NHS and carrier protein.

A highly sensitive homogeneous electrogenerated chemiluminescence (ECL) immunoassay for the determination of anti-digoxin antibody and digoxin hapten was developed employing Ru(bpy)(2)(dcbpy)NHS (bpy = 2,2'-bipyridyl; dcbpy = 2,2'-bipyridine-4,4'-dicarboxylic acid; NHS = N-hydroxysuccinimide ester) as an electrochemiluminescent label and bovine serum albumin (BSA) as a carrier protein. A digoxin hapten was indirectly heavily labelled with Ru(bpy)(2)(dcbpy)NHS through BSA to form Ru(bpy)(2)(dcbpy)NHS-BSA-digoxin conjugate. The ECL intensity of the immunocomplex of the conjugate with anti-digoxin antibody markedly decreased when the immunoreaction between Ru(bpy)(2)(dcbpy)NHS-BSA-digoxin conjugate and anti-digoxin antibody took place. Two formats, direct homogeneous immunoassay for anti-digoxin antibody and competitive immunoassay for digoxin, were developed to determine anti-digoxin antibody and digoxin, respectively. The anti-digoxin antibody concentration in the range 7.6 x 10(-8)-7.6 x 10(-6) g/mL was determined by direct homogeneous format. Digoxin hapten was determined throughout the range 4.0 x 10(-10)-1.0 x 10(-7) g/mL with a detection limit of 1.0 x 10(-10) g/mL by competitive format. The relative standard derivation for 6.0 x 10(-9) g/mL was 4.3%. The method has been applied to assaying digoxin in control human serum.     

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.     

Hyperoxia increases leptin production: a mechanism mediated through endogenous elevation of corticosterone

Leptin, a cytokine involved in the regulation of food intake, has been reported to be decreased in lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis and increased in critically ill patients with sepsis. We investigated the role of leptin during hyperoxia in mice, which results in alveolar edema, severe weight loss, and death within 3-4 days. In oxygen-breathing mice, serum leptin was increased six- to sevenfold and its mRNA was upregulated in white adipose tissue. Leptin elevation could not be attributed to changes in circulating tumor necrosis factor-alpha but was completely dependent on endogenous corticosterone elevation because adrenalectomized mice did not exhibit any increase in leptin levels. Using leptin-deficient mice and wild-type mice treated with anti-leptin antibody, we demonstrate that weight loss was leptin independent. Lung damage was moderately attenuated in leptin-deficient mice but was not modified by anti-leptin antibody or leptin administration, suggesting that leptin does not play an essential role in the direct and short-term effects of oxygen-induced injury.     

Functional properties of proteins immobilized on albumin microspheres

Bovine serum albumin (BSA) microspheres with an average diameter of 12.5 micron were prepared by crosslinking of BSA molecules with glutaraldehyde in the presence of polymethylmethacrylate dissolved in chloroform-toluene. Trypsin and anti-human IgG antibody were immobilized onto their surfaces by the glutaraldehyde-activation method. The catalytic activity and storage stability of the immobilized trypsin were satisfactorily high. The enzyme immunoassay (EIA) method using BSA-microspheres as a solid phase has a high sensitivity (the minimum concentration of detectable antigen in the sample: 0.2 ng/ml) and a wide concentration range (final concentration 0.027-3000 ng/ml) for the detection of human IgG.     

Adaptation of fluorescence polarization immunoassay to the assay of macromolecules

This paper describes an original methodology for determining macromolecular antigen levels by polarization of fluorescence. it involves the use of fluorescent derivatives of Fab fragments of a monoclonal antibody (Mr 50,000), whose fluorescence polarization rises significantly when it combines with a macromolecular antigen. An experimental system (Fab anti-aldosterone and aldosterone--bovine serum albumin (BSA)) is studied to test this methodology, which was then used to develop an immunoassay for human immunoglobulin M (IgM), using anti-mu chain Fabs. In the two assays, the binding stoichiometry of Fab/antigen was 10/1 and 8/1 for aldosterone--BSA and IgM, respectively. The lower limit of detection of the IgM assay was 0.8 microgram/ml and thus it was applicable to clinical detection of IgM concentrations.     

Leptin-leptin receptor are involved in angiogenesis in human hepatocellular carcinoma

Recent evidence in vitro and in vivo indicates that leptin, an adipose tissue-secreted hormone which is involved in the regulation of satiety, metabolic rate and thermogenesis, is implicated in angiogenesis. However, the role of leptin-mediated angiogenesis in hepatic carcinogenesis has not yet been completely elucidated. In this study, we have correlated microvascular density and leptin/leptin receptor (Ob-R) expression in endothelial and tumor cells with the histopathological type in human hepatocellular carcinoma (HCC). For this purpose, specimens of 40 primary HCC were submitted to immunohistochemical investigation using anti-CD31, anti-leptin and anti-Ob-R antibodies. Poorly-differentiated HCC had a higher degree of vascularization than other stages and leptin/Ob-R expression in both tumor and endothelial cells increased in parallel with the grade of malignancy and was highly correlated with the degree of angiogenesis. In the chick embryo chorioallantoic membrane in vivo assay, HCC biopsy specimens induced a strong angiogenic response, which was counteracted by an anti-leptin antibody. Taken together, these findings indicate that leptin/Ob-R correlate with angiogenesis and tumor progression in patients with HCC and that an anti-leptin antibody exerts an angiostatic activity in HCC.     

Nanoscale fabrication of protein A on self-assembled monolayer and its application to surface plasmon resonance immunosensor

The fabrication of protein A film on self-assembled monolayer was done for the construction of immunosensor using surface plasmon resonance (SPR) measurement. The layer of heterobifunctional linker, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) was self-assembled on the gold (Au) surface. Due to the succinimidyl functional group in SPDP to be reacted with amine (NH₂) group of protein A, the covalent immobilization of protein A was subsequently induced toward Au surface. The characteristics of film formation were investigated using SPR with respect to the various concentrations of SPDP and protein A. The optimal concentration for the film formation was found to be 0.1 mg/mL of SPDP and 0.1 mg/mL of protein A, respectively. The surface topography of protein A layer using atomic force microscopy showed that the heteromolecular layer was formed successfully. The antibody, anti-bovine serum albumin (BSA), was immobilized onto protein A layer, and the fabricated antibody layer was applied for the detection of BSA. The extent of BSA–antibody binding was measured using SPR and its lower detection limit of BSA was 100 pM.     

Proinflammatory cytokines and leptin are increased in serum of prepubertal obese children

It has not yet been shown in prepubertal children how cytokines, leptin, and body mass, as well as parameters of obesity are interrelated. The aim of this study was to explore the relation between circulating levels of some cytokines with leptin and body mass index. A case control study was carried out in obese children of both sexes. An obese group was carried out with 63 school prepubertal children and a control group comprised the same number of nonobese children paired by age and by sex. Mean serum leptin concentration was significantly higher in the obese children at 19.9 +/- 7.4 ng/mL, than the control group (7.9 +/- 5.1 ng/mL). Serum IL-1beta, IL-6, and TNF-alpha levels were also significantly higher in the obese group than controls (33.0 +/- 8.9, 45.2 +/- 11.8, and 9.2 +/- 2.3 pg/mL, versus 3.6 +/- 1.0, 13.1 +/- 3.9, and 3.9 +/- 1.0 pg/mL, resp). In controversy, serum IL-2 level was diminished in the obese group as 0.4 +/- 0.1 versus 0.9 +/- 0.1 U/L. Obesity may be a low-grade systemic inflammatory disease. Obese prepubertal children have elevated serum levels of IL-1beta , IL-6, and TNF-alpha which are known as markers of inflammation.     

Movement of proteins across the digestive system of the tobacco budworm, Heliothis virescens

Bovine serum albumin (BSA) and anti‐BSA polyclonal antibody were used as model polypeptides to examine the movement of foreign proteins across the insect digestive system and their accumulation in hemolymph of fourth stadium tobacco budworms, Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae). Hydrateable meal pads were developed in these studies as a method for easily introducing compounds into the insect digestive system. When insects were allowed to feed continuously on hydrated meal pads containing 0.8 mg of anti‐BSA per gram diet, the level of antibody found in hemolymph was 2.4 ± 0.1 and 3.4 ± 0.1 µg ml^?1 (average  1 SEM) after 8 and 16 h, respectively, as determined by enzyme‐linked immunosorbant assay (ELISA). Continuous feeding on hydrated meal pads containing the same concentration of BSA produced hemolymph concentrations of 1.5 ± 0.1 and 1.6 ± 0.1 µg ml^?1 hemolymph at 8 and 16 h, respectively. Western blot analyses demonstrated that BSA and anti‐BSA both retained their primary and multimeric structure and that anti‐BSA maintained its antigenic activity in the meal pads and after movement from meal pads into the hemolymph. When 1 µg of anti‐BSA or BSA was injected into the hemocoel of fourth instars, the concentrations decreased with time and 120 min after injection were 20% and 0.6% of the original concentration, respectively. When added at the same concentration to plasma in vitro, the decrease was 81.5% and 57.5%, respectively, at 2 h. The accumulation of native anti‐BSA and BSA protein in insect hemolymph is the result of their rate of movement across the gut and their rate of turnover in hemolymph. Movement of anti‐BSA and BSA across the digestive system was also noted in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), Acheta domesticus (L.) (Orthoptera: Gryllidae), and Gromphadorhina portentosa (Schaum) (Blattaria: Blattellidae). Anti‐BSA and BSA were not detected in the hemolymph of Manduca sexta (L.) (Lepidoptera: Sphingidae) after feeding.     

A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor

Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than ¹²⁵I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.     

Method for printing functional protein microarrays

Piezoelectric dispensing of proteins from borosilicate glass capillaries is a popular method of protein biochip fabrication that offers the advantages of sample recovery and noncontact with the printing substrate. However, little regard has been given to the quantitative aspects of dispensing minute volumes (1 nL or less) at the low protein concentrations (20 micrograms/mL or less) typically used in microprinting. Specifically, loss of protein sample due to nonspecific adsorption to the glass surface of the dispensing capillaries can limit the amount of protein delivered to the substrate. We demonstrate the benefits of a low ionic strength buffer containing the carrier protein BSA that effectively minimizes the ionic strength-dependent phenomenon of nonspecific protein adsorption to borosilicate glass. Over the concentration range of 20-2.5 micrograms/mL, the dispensing of a reference IgG in 10 mM PBS including 0.1% BSA resulted in the deposition of 3.6- to 44-fold more IgG compared to the deposition of IgG in standard 150 mM PBS in the absence of BSA. Furthermore, when the IgG was dispensed with carrier protein, the resulting spots exhibited a more uniform morphology. In a direct immunoassay for cholera toxin, capture antibody spots dispensed in 10 mM PBS containing 0.1% BSA produced fluorescent signals that were 2.8- to 4.3-fold more intense than antibody spots that were dispensed in 150 mM PBS without BSA. Interestingly, no differences were observed in the specific activities of the capture antibodies as a result of printing in the different buffers. The implications of these results on the future development of protein biochips are discussed.     

Sensitive Chemiluminescence Enzyme Immunoassay for Vascular Endothelial Growth Factor/Vascular Permeability Factor in Human Serum

A sandwich chemiluminescence enzyme immunoassay for measuring the level of VEGF /VPF in serum was constructed. The detectability of the assay is very low (1.0pg/ml) and the measurable range of the assay was very wide (1–1000 pg/ml). The assay showed that the average level of VEGF /VPF in human sera from healthy blood donors was approximately 19 pg/ml.     

Neutralizing chimeric mouse-human antibodies against Burkholderia pseudomallei protease: expression, purification and characterization

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2 % glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.     

A highly sensitive enzyme immunoassay (EIA) system for mouse epidermal growth factor (mEGF)

A highly sensitive two-site enzyme immunoassay system for mouse epidermal growth factor (mEGF) was developed, based on the sandwiching of an antigen between anti-mouse EGF IgG antibody-coated on a polystyrene bead and anti-mouse EGF Fab' antibody-linked peroxidase (horseradish peroxidase, EC. 1.11.1.7). The procedure is simple and rapid compared to a bioassay. Also, the Fab' antibody-peroxidase complex is more stable than the 125I-labeled antibody. Purified mEGF is detectable at a concentration as low as 3 pg/ml. The detection range was 0.3 to 680 pg/sample with 0.1 ml samples. Levels of immunoreactive mEGF in extracts from adult male mice well agreed with those determined by a radioimmunoassay and a radioreceptor assay. The submaxillary gland contained an extremely high concentration of EGF, while other tissues had low levels of EGF.     

Production and analytical properties of antibodies with high specificity to zearalenone

The time course of production, specificity, and analytical potential of anti-zearalenone polyclonal rabbit antibody synthesized by formaldehyde condensation and conjugated to bovine serum albumin were investigated. The relative cross-reactivities with natural analogues were: zearalenone, 100%; alpha-zearalenol, 0.15%; and beta-zearalenol, < 0.02%. With synthetic analogues: zearalanone, 31.7% and alpha-zearalanol, 0.12%. An enzyme immunoassay for zearalenone with a sensitivity of 0.1 ng/ml was developed on the basis of these antibodies and solid-phase conjugates homologous to the immunogen in the method of synthesis.