scholarly journals Enzyme Immunoassay for Aflatoxin B1-Lysine Adduct and Its Validation

2001 ◽  
Vol 84 (5) ◽  
pp. 1465-1474 ◽  
Author(s):  
Nayak Sujatha ◽  
Sarilla Suryakala ◽  
Beedu Sashidhar Rao
Keyword(s):  
Enzyme Immunoassay ◽  
Aflatoxin B1 ◽  
Retention Time ◽  
Simple Procedure ◽  
Molar Ratio ◽  
Rat Serum ◽  
In Vitro Synthesis ◽  
Coating Antigen ◽  
Relative Retention

Abstract A simple procedure was developed for in vitro synthesis and characterization of aflatoxin B1-lysine adduct using aflatoxin B1, N-α-acetyl lysine and m-chloroperbenzoic acid (MCPBA). At a molar ratio of 1:16 (aflatoxin B1:N-α-cetyl lysine), the recovery of adduct was 62%. Analysis of the adduct by thinlayer chromatography showed a single spot (Rf= 0). Absorption spectra of the adduct showed 2 peaks at 275 and 335 nm. Liquid chromatographic (LC) analysis of th AFB1-lysine adduct showed a relative retention time of 2.1 min. Using the same epoxidation procedure, BSA-AFB1 adduct and ovalbumin-AFB1 adduct were synthesized for production of antibodies and as coating antigen, respectively. Control rat serum, spiked with AFB1-lysine adduct and subjected to LC analysis showed a retention time of 2.1 min, which is similar to that of AFB1-lysine reference standard, synthesized. Further, enzymatically hydrolyzed, control rat serum spiked with BSA-AFB1 adduct showed 2 peaks with retention times of 2.1 and 2.7 min. Based on the LC analysis, recovery of BSA-AFB1 in terms of AFB1-lysine adducts was 67 ± 5%. The major peak (2.1 min) accounted for 72% of the adduct; the second minor peak (2.7 min) accounted for 28% of the total AFB1-lysine adducts formed. Stability studies on the AFB1-lysine adduct synthesized, indicated that it was stable for 1 month. Antibody capture assay showed an absorbance of 0.9 to 1.0 at a dilution of 1:50 000 when ovalbumin-AFB1 was used as a coating antigen. Indirect competitive ELISA showed 50% displacement (IC50) of the antibodies at a concentration of 13 ng AFB1-lysine, whereas the IC50 for AFB1 was 7 ng. The recovery of AFB1-lysine adduct spiked to control rat serum followed by enzymatic hydrolysis and immunoanalysis (indirect ELISA) was 93 ± 6%. The enzyme immunoassay was validated by a rodent model, in which the animals were exposed to aflatoxin B1 (20 μg AFB1/kg body mass/day). The level of AFB1-lysine adduct in the rat serum was 27.3 ± 4.37 μg/mg albumin.

scholarly journals Synthesis and Characterization of Mercapturic Acid (N-acetyl-L-cysteine)-Aflatoxin B1 Adduct and Its Quantitation in Rat Urine by an Enzyme Immunoassay

2009 ◽  
Vol 92 (2) ◽  
pp. 487-495 ◽  
Author(s):  
Sujatha Nayak ◽  
Penmatsa Tanuja ◽  
Rao Beedu Sashidhar
Keyword(s):  
Enzyme Immunoassay ◽  
Aflatoxin B1 ◽  
Polyclonal Antibodies ◽  
Mercapturic Acid ◽  
Rat Urine ◽  
In Vitro Synthesis ◽  
Layer Chromatography ◽  
Serum Albumen

Abstract A simple and sensitive indirect noncompetitive enzyme immunoassay to quantitate mercapturic acid-aflatoxin B1 (AFB1) adduct in rat urine is reported. A novel procedure was developed for in vitro synthesis of an immunogen, bovine serum albumen-glutathione-aflatoxin B1 (BSA-GSH-AFB1) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Sulphydryl group's analysis confirmed the conjugation of SH groups to AFB1. Thin-layer chromatography and spectral analysis (absorption, fluorescence, and Fourier transform infrared) of the conjugates further confirmed the formation of the adducts. Polyclonal antibodies specific to mercapturic acid-AFB1 adduct were produced against BSA-GSH-AFB1. The assay was found to be linear in the range of 100 pg100 ng of the analyte (y ab x). A 50 displacement of BSA-GSH-AFB1 antibodies was achieved at an inhibitory concentration (IC50)of 11.9 ng GSH-AFB1 (r2 0.98) and 1.22 ng N-acetyl-L-cysteine (NAC)-AFB1 (r2 0.98). Spiking 5 g/mL of reference standard to the control rat urine showed a recovery of 98 2. The immunoassay was validated in a rodent model exposed to a single oral dose of 1 mg/kg body mass of pure AFB1. The excretion of NAC-AFB1 adduct was quantitated at the end of 24 h. The concentration of the NAC-AFB1 adduct excreted in urine as determined by the immunoassay was found to be in the range of 3.225.97 g/mg creatinine. The present method may find wide application as a biochemical tool in molecular epidemiological and intervention studies with respect to human exposure to dietary aflatoxins.

scholarly journals Some mass-spectral and n.m.r. analytical studies of a glutathione conjugate of aflatoxin B1

1983 ◽  
Vol 210 (1) ◽  
pp. 227-233 ◽  
Author(s):  
E J Moss ◽  
D J Judah ◽  
M Przybylski ◽  
G E Neal
Keyword(s):  
Aflatoxin B1 ◽  
Reduced Glutathione ◽  
Spectroscopic Studies ◽  
Single Step ◽  
Molar Ratio ◽  
Reverse Phase ◽  
Glutathione Conjugate ◽  
Mass Spectral ◽  
Liquid Chromatography System

A system for the formation of an aflatoxin B1-reduced glutathione conjugate in vitro was developed, capable of yielding 80% conversion of aflatoxin B1 into the conjugate. A reverse-phase high-pressure-liquid-chromatography system was also devised that not only facilitates improved resolution of the compound but that, by manipulation of the pH, is also capable of an extensive purification of the compound from other aflatoxin B1 metabolites in a single step. Material produced by these techniques, after further purification, has been used in 1H-n.m.r. and mass-spectroscopic studies. Results were obtained that support the proposed linkage of the aflatoxin B1 to reduced glutathione in a 1:1 molar ratio via a thioether linkage. Amino acid analyses were also consistent with this structure. The absence of a Schiff-base linkage of aflatoxin B1 8,9-dihydrodiol to glutamate was further demonstrated by the presence of a gamma-glutamyltransferase-catalysed-transferable glutamate moiety. These data are consistent with the structure 8,9-dihydro-8-(S-glutathionyl)-9-hydroxy-aflatoxin B1.

A Monoclonal Antibody-Based Enzyme Immunoassay for Fibrin Degradation Products in Plasma

1988 ◽  
Vol 59 (02) ◽  
pp. 310-315 ◽  
Author(s):  
P W Koppert ◽  
E Hoegee-de Nobel ◽  
W Nieuwenhuizen
Keyword(s):  
Enzyme Immunoassay ◽  
Degradation Products ◽  
Blood Clot ◽  
Tissue Type ◽  
Fibrin Degradation Products ◽  
Type Plasminogen Activator ◽  
Strong Positive Reaction ◽  
Sandwich Type ◽  
Capture Antibody

SummaryWe have developed a sandwich-type enzyme immunoassay (EIA) for the quantitation of fibrin degradation products (FbDP) in plasma with a time-to-result of only 45 minutes.* The assay is based on the combination of the specificities of two monoclonal antibodies (FDP-14 and DD-13), developed in our institute. FDP-14, the capture antibody, binds both fibrinogen degradation products (FbgDP) and FbDP, but does not react with the parent fibrin(ogen) molecules. It has its epitope in the E-domain of the fibrinogen molecule on the Bβ-chain between amino acids 54-118. Antibody DD-13 was raised using D-dimer as antigen and is used as a tagging antibody, conjugated with horse-radish peroxidase. A strong positive reaction is obtained with a whole blood clot lysate (lysis induced by tissue-type plasminogen activator) which is used as a standard. The EIA does virtually not detect FbgDP i. e. purified fragments X, Y, or FbgDP generated in vitro in plasma by streptokinase treatment. This indicates that the assay is specific for fibrin degradation products.We have successfully applied this assay to the plasma of patients with a variety of diseased states. In combination with the assay previously developed by us for FbgDP and for the total amount of FbgDP + FbDP (TDP) in plasma, we are now able to study the composition of TDP in patients plasma in terms of FbgDP and FbDP.

Influence of β-Cyclodextrin and Hydroxypropyl-β-Cyclodextrin on Enhancement of Solubility and Dissolution of Isradipine

2017 ◽  
Vol 10 (3) ◽  
pp. 3752-3757
Author(s):  
Narendar D ◽  
Ettireddy S
Keyword(s):  
Inclusion Complex ◽  
Dissolution Rate ◽  
Solid Dispersions ◽  
Physical Stability ◽  
Molecular Complexes ◽  
In Vitro Dissolution ◽  
Molar Ratio ◽  
Phase Solubility ◽  
Cyclodextrin Complexation

The content of this investigation was to study the influence of β-cyclodextrin and hydroxy propyl-β-cyclodextrin complexation on enhancement of solubility and dissolution rate of isradipine. Based on preliminary phase solubility studies, solid complexes prepared by freeze drying method in 1:1 molar ratio were selected and characterized by DSC for confirmation of complex formation. Prepared solid dispersions were evaluated for drug content, solubility and in vitro dissolution. The physical stability of optimized formulation was studied at refrigerated and room temperature for 2 months. Solid state characterization of optimized complex performed by DSC and XRD studies.  Dissolution rate of isradipine was increased compared with pure drug and more with HP-β-CD inclusion complex than β-CD. DSC and XRD analyzes that drug was in amorphous form, when the drug was incorporated as isradipine β-CD and HP-β-CD inclusion complex. Stability studies resulted in low or no variations in the percentage of complexation efficiency suggesting good stability of molecular complexes. The results conclusively demonstrated that the enhancement of solubility and dissolution rate of isradipine by drug-cyclodextrin complexation was achieved.   

Complexation with Hydroxypropyl-gama-Cyclodextrin of Birch Tree Extract Physico-chemical Characterisation of their Binary Products

2008 ◽  
Vol 59 (6) ◽  
Author(s):  
Codruta Soica ◽  
Cristina A. Dehelean ◽  
Valentin Ordodi ◽  
Diana Antal ◽  
Vicentiu Vlaia
Keyword(s):  
Inclusion Complexes ◽  
Water Solubility ◽  
In Vitro Dissolution ◽  
Molar Ratio ◽  
Bark Extract ◽  
Birch Bark ◽  
Preparation Methods ◽  
Birch Tree ◽  
Physico Chemical

Birch bark contains important pentacyclic triterpens that determine an anticancer, anti-inflammatory and antiviral activity. The compounds can be extracted by simple procedures with organic solvents. The major problem of this type of triterpens is their low water solubility which can be increased by physical procedures like cyclodextrin complexation. The aim of present study was to analyse the products between birch bark extract and hydroxypropyl-g -cyclodextrin. Hydroxypropyl-g -cyclodextrin (HPGCD) was used as a host to improve its solubility in water, via inclusion complex formation. In order to obtain the inclusion complexes, 1:2 molar ratio and two preparation methods (physical mixing, kneading) were used. The inclusion complexes were analyzed by in vitro dissolution tests, thermal analysis and X-ray diffraction.

Apoprotein C effect on triglyceride transport in tandem-perfused rat hind end and liver

1984 ◽  
Vol 247 (3) ◽  
pp. G305-G310
Author(s):  
W. J. Kortz ◽  
J. R. Nashold ◽  
M. R. Greenfield ◽  
H. Hilderman ◽  
S. H. Quarfordt
Keyword(s):  
Fatty Acid ◽  
Free Fatty Acid ◽  
Fatty Acyl ◽  
Liver Fat ◽  
Molar Ratio ◽  
Perfusion System ◽  
In Vitro Perfusion ◽  
Arterial Inflow ◽  
High Concentrations

The metabolism of double-labeled triglyceride in a synthetic emulsion was defined in an in vitro perfusion system of rat hind end and liver described previously [Am. J. Physiol. 245 (Gastrointest. Liver Physiol. 8): G106-G112, 1983]. The metabolism of [3H]glycerol-[14C]triolein was defined in the absence of added apoproteins and with additions of human CII and both CII and CIII. Without apoprotein, a pronounced lipolysis of the triglyceride was recognized by high concentrations of radiolabeled glycerol and free fatty acid in the perfusate. The removal of an aliquot of hind-end venous effluent 5 min after adding the labeled triglyceride emulsion to the arterial inflow demonstrated a brisk lipolysis of the substrate when incubated outside the perfusion system. The addition of CII protein to the emulsion before its introduction into the tandem system eliminated perfusate lipolysis, both within the perfusion system and in incubations of aliquots withdrawn from the system. Intravascular lipolysis was not seen with triglyceride emulsions containing both CII and CIH or when an aliquot of hind-end venous effluent was incubated with triglycerides that had not been exposed to the perfusion system. The intravascular lipolysis observed for the [14C]triglyceride added to the tandem system without apoproteins was associated with relatively greater recoveries of 14C-fatty acyl in liver, fat, and muscle and relatively greater recoveries of 14CO2 than when CII alone or both CII and CIII were added with the triglyceride. The addition of CIII to CII in a 1:1 molar ratio increased the recovery of 14C-fatty acyl in muscle and the recovery as 14CO2.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro synthesis of enzymatically active HIV-1 protease for rapid phenotypic resistance profiling

2005 ◽  
Vol 32 (4) ◽  
pp. 294-299 ◽  
Author(s):  
Dieter Hoffmann ◽  
Bernd Buchberger ◽  
Cordula Nemetz
Keyword(s):  
In Vitro Synthesis ◽  
Phenotypic Resistance ◽  
Hiv 1
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