A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin fromClostridium septicum

Clostridium septicumproduces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced byClostridium septicum.This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin ofClostridium septicumand produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot.

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A new, Modern, COST-Saving micro/macro method for the determination of serum fructosamine

Acta Veterinaria Hungarica ◽

10.1556/avet.48.2000.3.5 ◽

2000 ◽

Vol 48 (3) ◽

pp. 285-291 ◽

Cited By ~ 4

Author(s):

Klára Oppel ◽

L. Bárdos ◽

A. Ferencz ◽

Hajnalka Lakner ◽

Judit Simon ◽

...

Keyword(s):

Diabetic Control ◽

Test Tube ◽

Farm Animals ◽

Human Blood Plasma ◽

Plasma Samples ◽

Time Saving ◽

Serum Fructosamine ◽

One Year ◽

Manual Test

Serum/plasma fructosamine (SeFa) concentration is a reliable indicator used in human diabetic control. Tests for monitoring the carbohydrate/energy metabolism of (farm) animals are less commonly performed in veterinary laboratories, since most of the reliable determinations, both automated and manual, are relatively expensive. The aim of this study was to develop a precise, money- (and time-) saving automated micro method for measuring SeFa. ELISA microplates (20 µL samples and 200 µL reagents) and an automatic microplate autoreader were used. The classical nitroblue tetrazolium (NBT) stain reagent solution of Johnson et al. (1982) was modified using a SIGMA reagent to render it stable for up to one year. SeFa concentrations measured by the new method in 30 human blood plasma samples were compared with values obtained by the standard (generally used) LaRoche kit procedure. Fifteen cow, 13 dog and 18 chicken plasma samples were assayed by the new automated ‘micro’ method as well as by the manual test tube ‘macro’ method commonly used earlier. The modified reagent was applied for both methods. The coefficient of correlation (r) between the results obtained by the two methods was consistently between 0.94 and 0.98 (p < 0.001).

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Eco-friendly spectrophotometric methods for assessment of alfuzosin and solifenacin in their new pharmaceutical formulation; Green profile evaluation via eco-scale and GAPI tools

Current Pharmaceutical Analysis ◽

10.2174/1573412916999200730005740 ◽

2020 ◽

Vol 16 ◽

Author(s):

Mamdouh R. Rezk ◽

Mina Wadie ◽

Soheir A. Weshahy ◽

Mahmoud A. Tantawy

Keyword(s):

Minor Component ◽

Time Saving ◽

Prostate Hyperplasia ◽

Spectrophotometric Methods ◽

Ratio Difference ◽

Ich Guidelines ◽

Mean Centering ◽

Benign Prostate ◽

Urological Diseases

Background: Alfuzosin is recently co-formulated with solifenacin for relieving two coincident urological diseases, namely; benign prostate hyperplasia and overactive bladder Objective: Herein, green, simple and rapid spectrophotometric methods were firstly developed for simultaneous determination of the two cited drugs in their co-formulated pharmaceutical capsule Methods: Alfuzosin, which is the major component in the dosage form, was directly assayed at its extended wavelength at 330.0 nm. The challenging spectrum of the minor component, solifenacin, was resolved by five spectrophotometric methods, namely; dual wavelength (DW) at 210.0 & 230.0 nm, first derivative (1D) at 222.0 nm, ratio difference (RD) at 217.0 - 271.0 nm , derivative ratio (1DD) at 223.0 and mean centering of ratio spectra (MC) at 217.0 nm Results: The Proposed methods were successfully validated as per ICH guidelines. Alfuzosin showed linearity over the range of 4.0 - 70.0 μg/mL, while that of solifenacin were 4.0 - 50.0 μg/mL for DW, 2.0 - 70.0 μg/mL for 1D and RD methods, 1.0 - 70.0 μg/mL for 1DD and 4.0 - 70.0 μg/mL for MC method. Statistical comparison with their official ones showed no noticeable differences. The methods showed good applicability for assaying drugs in their newly combination. Besides eco-scale, the greenness profile of the methods was assessed and compared with the reported spectrophotometric one via the newest metric tool; green analytical procedure index (GAPI). Conclusions: The proposed methods are superior in not only being smart, accurate, selective, robust and time-saving, but also in using distilled water as an eco-friendly and cheap solvent

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A Toolbox for the Determination of Nitroaromatic Explosives in Marine Water, Sediment, and Biota Samples on Femtogram Levels by GC-MS/MS

Toxics ◽

10.3390/toxics9030060 ◽

2021 ◽

Vol 9 (3) ◽

pp. 60

Author(s):

Tobias Hartwig Bünning ◽

Jennifer Susanne Strehse ◽

Ann Christin Hollmann ◽

Tom Bötticher ◽

Edmund Maser

Keyword(s):

Sample Preparation ◽

Freeze Drying ◽

Solid Phase ◽

Phase Extraction ◽

Direct Extraction ◽

Time Saving ◽

Marine Samples ◽

Volume Method ◽

Biota Samples

To determine the amount of the explosives 1,3-dinitrobenzene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene, and its metabolites in marine samples, a toolbox of methods was developed to enhance sample preparation and analysis of various types of marine samples, such as water, sediment, and different kinds of biota. To achieve this, established methods were adapted, improved, and combined. As a result, if explosive concentrations in sediment or mussel samples are greater than 10 ng per g, direct extraction allows for time-saving sample preparation; if concentrations are below 10 ng per g, techniques such as freeze-drying, ultrasonic, and solid-phase extraction can help to detect even picogram amounts. Two different GC-MS/MS methods were developed to enable the detection of these explosives in femtogram per microliter. With a splitless injector, limits of detection (LODs) between 77 and 333 fg/µL could be achieved in only 6.25 min. With the 5 µL programmable temperature vaporization—large volume method (PTV-LVI), LODs between 8 and 47 fg/µL could be achieved in less than 7 min. The detection limits achieved by these methods are among the lowest published to date. Their reliability has been tested and confirmed by measuring large and diverse sample sets.

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Determination of Flavonoids Compounds of Three Species and Different Harvesting Periods in Crataegi folium Based on LC-MS/MS

Molecules ◽

10.3390/molecules26061602 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1602

Author(s):

Ya-Ping Guo ◽

Hong Yang ◽

Ya-Li Wang ◽

Xiao-Xiang Chen ◽

Ke Zhang ◽

...

Keyword(s):

Quantitative Analysis ◽

Qualitative Analysis ◽

Quantitative Method ◽

Total Content ◽

Qualitative And Quantitative Analysis ◽

Pharmacological Activities ◽

Time Saving ◽

Qualitative And Quantitative ◽

First Time

Crataegi folium have been used as medicinal and food materials worldwide due to its pharmacological activities. Although the leaves of Crataegus songorica (CS), Crataegus altaica (CA) and Crataegus kansuensis (CK) have rich resources in Xinjiang, China, they can not provide insights into edible and medicinal aspects. Few reports are available on the qualitative and quantitative analysis of flavonoids compounds of their leaves. Therefore, it is necessary to develop efficient methods to determine qualitative and quantitative flavonoids compounds in leaves of CS, CA and CK. In the study, 28 unique compounds were identified in CS versus CK by qualitative analysis. The validated quantitative method was employed to determine the content of eight flavonoids of the leaves of CS, CA and CK within 6 min. The total content of eight flavonoids was 7.8–15.1 mg/g, 0.1–9.1 mg/g and 4.8–10.7 mg/g in the leaves of CS, CA and CK respectively. Besides, the best harvesting periods of the three species were from 17th to 26th September for CS, from 30th September to 15th October for CA and CK. The validated and time-saving method was successfully implemented for the analysis of the content of eight flavonoids compounds in CS, CA and CK for the first time.

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Determination of platelet antigens and glycoproteins in the human fetus

Blood ◽

10.1182/blood.v68.2.488.488 ◽

1986 ◽

Vol 68 (2) ◽

pp. 488-492 ◽

Cited By ~ 100

Author(s):

Y Gruel ◽

B Boizard ◽

F Daffos ◽

F Forestier ◽

J Caen ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Human Fetus ◽

Antenatal Diagnosis ◽

Polyclonal Antibodies ◽

Umbilical Vein ◽

Membrane Surface ◽

Platelet Suspension ◽

Direct Puncture ◽

Normal Human

Abstract The autosomal recessive transmission of Glanzmann's thrombasthenia (GT) and Bernard-Soulier syndrome (BSS), together with requests of families who already had children with these diseases, prompted us to investigate the feasibility of their antenatal diagnosis. The preliminary step leading to the early detection of GT or BSS was to characterize, in the normal human fetus, the platelet antigens and glycoproteins (GPs) and to define their normal amounts on the membrane surface. Blood samples from 32 fetuses between 18 to 26 weeks of gestation were collected by direct puncture of the umbilical vein using an ultrasound-guided needle. Polyclonal antibodies from human origin directed against PLA1, Leka antigens, and the GPIIb IIIa complex (IgGL), or murine monoclonal antibodies specific for GPIb (AN51, 6D1), GPIIIa (AP-3), or GPIIb IIIa (AP-2) were studied using platelet suspension immunofluorescence tests. The binding of each antibody was quantified using a cytofluorograph (Ortho 50H). PLA1 and Leka antigens were expressed in normal amounts on fetal platelets as early as 16 weeks of intrauterine life. The GPIIb IIIa complex quantified by polyclonal or monoclonal antibodies was in the same range in fetuses (IgGL = 427 +/- 23 AUF, AP-2 = 459.5 +/- 8.5; AP-3 = 536 +/- 14) and in adults (IgGL = 420 +/- 30; AP-2 = 498 +/- 11; AP-3 = 515 +/- 13). The platelet binding of antibodies that recognized GPIb was higher in fetuses (AN51 = 491.5 +/- 14; 6D1 = 479 +/- 15) than in adults (AN51 = 426.5 +/- 9; 6D1 = 449 +/- 8.7). These results suggest that immunological techniques can be applied as early as 18 weeks of gestation for the antenatal diagnosis of GT and BSS.

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Activation and mechanism of Clostridium septicum alpha toxin

Molecular Microbiology ◽

10.1111/j.1365-2958.1993.tb00934.x ◽

1993 ◽

Vol 10 (3) ◽

pp. 627-634 ◽

Cited By ~ 58

Author(s):

J. Ballard ◽

Y. Sokolov ◽

W.-L. Yuan ◽

B. L. Kagan ◽

R. K. Tweten

Keyword(s):

Alpha Toxin ◽

Clostridium Septicum

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Determination of Ochratoxin A in Feed by Immunoaffinity Cleanup and Liquid Chromatography

Journal of AOAC International ◽

10.5740/jaoacint.11-334 ◽

2013 ◽

Vol 96 (3) ◽

pp. 599-602 ◽

Cited By ~ 2

Author(s):

Ping Ding ◽

Ziyou Mi ◽

Yali Hou ◽

Yigang He ◽

Jianhua Xie

Keyword(s):

Liquid Chromatography ◽

Detection Limit ◽

Ochratoxin A ◽

Cyanogen Bromide ◽

Polyclonal Antibodies ◽

Immunoaffinity Column ◽

Low Levels ◽

Sepharose 4B ◽

Feed Samples

Abstract A method using LC was developed for determination of ochratoxin A (OTA) in feeds. The extracted samples were cleaned up by an immunoaffinity column prepared by covalently coupling polyclonal antibodies against OTA to cyanogen bromide-activated Sepharose 4B. The eluates were determined by LC with fluorescence detection. Recoveries of OTA from fortified samples of 1–10 μg/kg levels ranged from 84.3 to 90.0%, with CVs of 3.3–7.8%. The detection limit was 0.045 μg/kg based on an S/N of 3:1. A total of 65 feed samples were screened for OTA with the proposed method. The results showed that only nine samples were contaminated with OTAs at low levels. The presented method was successfully applied to quantify OTAs in real feed samples.

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RADIOIMMUNOASSAY FOR 2-METHOXYOESTRONE IN HUMAN PLASMA

Acta Endocrinologica ◽

10.1530/acta.0.0910158 ◽

1979 ◽

Vol 91 (1) ◽

pp. 158-166 ◽

Cited By ~ 7

Author(s):

Günter Emons ◽

Peter Ball ◽

Gertrud v. Postel ◽

Rudolf Knuppen

Keyword(s):

Human Plasma ◽

Plasma Concentrations ◽

Cross Reactivity ◽

Elderly Men ◽

Specific Antibodies ◽

Assay Procedure ◽

Menopausal Women ◽

Bovine Serum Albumin Conjugate ◽

Post Menopausal

ABSTRACT A bovine serum albumin conjugate of 2-methoxyoestrone was used for the preparation of highly specific antibodies in rabbits. Cross-reactivity for catecholoestrogens and monophenolic steroids was below 0.3 %. Only 2-methoxyoestradiol cross-reacted with 44 %. An assay procedure for the determination of unconjugated and conjugated 2-methoxyoestrone in human plasma is described. The following mean plasma concentrations (pg/ml) were found (unconjugated/conjugated): children 61/1130, young men 74/1320, elderly men 109/1260, cycling women 131/1040, post-menopausal women 102/1420, and pregnant women 3980/5850.

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