A human urinary trypsin inhibitor (UTI) was purified by 80% amonium sulfat precipitation, affnity chromatography using trypsin cellulofine gel. Specificity inhibitory activity of solution after affinity chromatography was increase from 0.8 to 29.1 and its recovery was 20.8%. The reslt indicated that binding of trypsin to formyl- cellulofine and the affinity chromatography with trypsin cellulofine gel was successful. Capalary electrophoresis of urine, solutions before and after affinity chromatography also supported the above result.
Glycoproteins ; Chromatography, Affinity

Intein is a part of polypeptide in the premature protein with the capability of self-splicing, which is widely applied in protein purification, protein conjuction, cyclopeptide preparation, protein labeling and biosensor. In this review, we summarized the development of intein used in protein purification, discussed intein-mediated chromatographic and non-chromatographic purification systems, and summarized the researches in manipulating intein cleavage reaction. This work is to provide clues for improvement of intein-mediated protein purification.
Chromatography, Affinity ; Inteins ; Protein Splicing ; Proteins ; isolation & purification

Brucella ; Brucellosis/diagnosis* ; Chromatography, Affinity ; Gold Colloid ; Humans

The systematic and in-depth study of phosphoproteome rely on highly reproducible and specific phosphopeptide enrichment methods. At present, a variety of enrichment methods have been developed based on different principles, and these methods often display different selectivity and specificity. It is therefore very important to select the most suitable enrichment method according to different research purposes. This review summarized the phosphopeptide enrichment based on affinity chromatography, immunoprecipitation, chemical derivatization, chromatography and other newly developed methods. The advantages and disadvantages of these methods, as well as the related optimization and improvement strategies, were discussed in detail. In addition, we also briefly summarized the progress of the combination of phosphopeptide enrichment and fractionation methods developed in recent years.
Phosphopeptides/metabolism* ; Proteomics/methods* ; Titanium/chemistry* ; Chromatography, Affinity ; Proteome ; Phosphorylation

This study assessed folate intakes, folate concentrations in plasma and erythrocytes, plasma total homocysteine (tHcy) concentration, and urinary excretion of folate metabolites in Korean women with childbearing potential. A total of 23 women voluntarily participated in this study. Precise dietary intakes for 3 consecutive days were determined by weighing all foods consumed and folate intake was calculated using a computer-aided dietary analysis system. Folate concentration of plasma and erythrocytes was determined by a microbiological method. Plasma tHcy concentration was assayed using an HPLC analysis method. Urine excreted over the same period of time was collected and folate catabolites, para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (ApABG), were evaluated using a reverse-phase HPLC method after affinity chromatography. Young women of 20 and under were likely to consume less folate with low energy intake, had lower folate concentration in plasma and erythrocytes, and excreted a lesser amount of ApABG and total folate catabolites than women of 25 years and over. The results of this study confirmed that young Korean women with childbearing potential, especially those under 21 years of age, might be at risk for compromised folate status due to insufficient folate intakes from inadequate energy consumption.
Chromatography, Affinity ; Chromatography, High Pressure Liquid ; Energy Intake ; Erythrocytes ; Female ; Folic Acid* ; Homocysteine ; Humans ; Plasma

OBJECTIVETo establish a method to test citrinin (CIT) in monascus products by immuno-affinity chromatography (IAC)-high performance liquid chromatography (HPLC), and to detect the content of CIT in monascus products in Fujian province.

METHODSIAC-HPLC was applied to detect the CIT content in monascus products. The conditions to use HPLC were as follows: C(18) reversed-phase chromatographic column, 150.0 mm×4.6mm×3 µm; mobile phase: the volume ratio of acetonitrile and 0.1% phosphoric acid solution at 65:35; isocratic elution; column temperature: 28°C; flow velocity: 0.8 ml/min; fluorescence detector, excitation wavelength (λ(ex)) was 331 nm and emission wavelength (λ(em)) was 500 nm. The standard curved was established by the linear regression of peak area (Y) to CIT content (X, ng/ml). The accuracy and precision of the method would then be verified. And 32 kinds of monascus products were determined and their color values were compared by this method.

RESULTThe standard curve established in this study was Y = 4634.8X-136.42, r = 1.000; whose limits of detection was 20 µg/kg and the limits of qualification was 64 µg/kg. In the range between 200 and 800 µg/kg, the standard recovery rate was 98.9% - 110.0% (n = 3), and the relative standard deviation (RSD) was 0.51% - 1.76%. Out of the 32 samples, CIT was detected from 11 samples of monascus rice, 9 samples of monascus powder and 5 samples of monascus pigments, the content was around 0.212 - 14.500 mg/kg. 4 out of 7 functional monascus samples were detected out CIT, whose content at 0.142 - 0.275 mg/kg.

CONCLUSIONThe method to detect CIT in monascus products by IAC-HPLC has been established.

OBJECTIVETo prepare immunoaffinity column of zearalenone.

METHODSThe zearalenone immunoaffinity column (IAC) was prepared by coupling CNBr-activated Sepharose 4 Fast Flow (4FF) with the anti-zearalenone monoclonal antibody which was purified by caprylic acid-ammonium sulfate method. The coupling reaction was identified by UV-absorbance measurements, and the IAC prepared was evaluated by indirect-competition ELISA and HPLC.

RESULTSThe column capacity was determined to be 0.40 microg when using 0.5 ml of CNBr activated Sepharose 4FF and 350 microg of purified anti-zearalenone monoclonal antibody. The mean true recoveries were in the range 76.33% - 90.10% and RSD was 6.68% - 10.93% at levels of 60 microg/kg - 300 microg/kg. 30 samples of wheat and maize were detected by the anti-ZEN IAC produced by the laboratory, 17 samples were observed to be contaminated in a comparable range from 31.33 microg/kg - 377.84 microg/kg. Detection limit based on a signal-to-noise ratio 3:1 was 10. 00 microg/kg for ZEN in wheat and maize.

CONCLUSIONIAC, a simple separating method which is used in ZEN extraction from cereals, is able to purify and condense ZEN in one step. The cost of detection can be lowered down because the IAC developed is hopefully to substitute the imported IAC.

The contamination of aflatoxin B_1,B_2,G_1,G_2,M_1 and M_2 in Eupolyphaga Steleophaga was determined by immunoaffinity column clean-up and HPLC-FLD with post-column photochemical derivatization. Chromatographic separations were carried out using a Cloversil C_(18) column( 4. 6 mm×250 mm,5 μm) that were eluted in isocratic with methanol-acetonitrile-water( 20 ∶ 20 ∶ 60) as the mobile phase. The excitation wavelength and the emission wavelength of fluorescence detector were maintained at 360 nm and 450 nm,respectively. The flow rate was 0. 8 m L·min~(-1),and the column temperature was 30 ℃ ． The sample was prepared using the immunoaffinity column,then the recovery was measured with 75. 47%-101. 8% with RSD values lower than 6. 7%. A total of 20 batches of Eupolyphaga Steleophaga samples were assayed. According to the Chinese Pharmacopoeia( 2015 edition,part 1),the aflatoxin B_1 limit should be less than 5 μg·kg~(-1),and the sum of aflatoxins( AFB_1,AFB2,AFG_1,AFG_2) should be less than 10 μg·kg-1. Therefore,the positive rate of the 20 samples was 50. 0%,and 7 batches of samples exceeded the standard,and the over-standard rate was as high as 70. 0%. Among them,aflatoxins B_1,B_2,G_1,G_2,M_1,and M_2 were detected in three batches( SD-1,AH-1,AH-3),and aflatoxins B_1,B2,G1,G2,and M1 were detected in one batch( AH-7). The results showed that the newly developed method in this work is suitable for the simultaneous determination of six aflatoxins in Eupolyphaga Steleophaga,and also suggested that it should be of high values to take the contamination with aflatoxins into concerns.
Aflatoxins/analysis* ; Animals ; Chromatography, Affinity ; Chromatography, High Pressure Liquid ; Cockroaches/chemistry*

"It is not clear yet how the laminin is made in cytoplasm and is secreted by cells, though the significance of the interaction between laminin and cells during the development, cell differentiation, tumor growth, and metastasis is well known. Furthermore, it has been reported that aberrant expression. and secretion of laminin in malignant tumor cells, In this study I found a major cytoplasmic laminin binding protein (LBP), which was revealed as the heat shock cognate protein 70 (hsc70), in human colon carcinoma cells by cell surface labeling with '""I, laminin and gelatin affinity chromatography, peptide microsequencing, and immunoblot experiments. This suggests another function of hsc70 as the cytoplasmic LBP, which may play an important role in biosynthesis, assembly, and secretion of laminin in tumor cells."
Carrier Proteins* ; Cell Differentiation ; Chromatography, Affinity ; Colon* ; Cytoplasm* ; Gelatin ; Hot Temperature* ; Humans* ; Laminin* ; Neoplasm Metastasis ; Shock*

This study was undertaken to purify cystic fluid (CF) antigen of Taenia solium metacestodes by affinity chromatography using specific monoclonal antibody (McAb) and to characterize the antigenicity of the purified antigen. The hybridoma cell lines, prepared by fusion between mouse plasmacytoma and spleen cells from BALB/c mice immunized with CF, secrected antibodies reacting to various helminthic antigens. Majority of cell lines reacted to CF only but some also reacted to parenchymal antigen of T. solium metacestodes, adult T. saginata, sparganum, hydatid cystic fluid, Paragonimus westermani and Clonorchis sinensis, either in combination with CF, other antigens or independently. Cloned cells derived from monoclonal lines also produced antibodies reacting either to CF only or to other helminthes in combination or independently. These results indicated that CF of T. solium metacestodes contained proteins which possessed antigenic determinants not only specific to CF but also cross reactive with the afore-mentioned helminthes. CF of T. solium metacestodes was purified by affinity chromatography using the McAb which reacted to CF and parenchymal antigens. The affinity-purified antigen(A-Ag) and unbound pool(U-Ag) were separated. A-Ag showed 2 protein bands by disc-PAGE whereas CF exhibited 6 bands and U-Ag consisted of all bands CF had. The diagnostic significance of A-Ag was evaluated by ELISA in human neurocysticercosis and other helminthic and neurologic diseases. By A-Ag, the levels of the specific IgG antibody, as shown by absorbance in sera and CSF, were lower than those of CF and U-Ag. Accordingly, the sensitiviy was about 70 % of CF and U-Ag. However, the nonspecific positive reactions to CF and U-Ag, observed in sparganosis, T. saginata infection and paragonimiasis did not occur when A-Ag was used. These results indicated that the affinity-purified A-Ag had the higher specificity but the lower sensitivity as a diagnostic antigen in cysticercosis, probably because it only detected a single or limited numbers of monospecific antibodies among the diverse polyclonal antibodies produced in the patients with neurocysticercosis.
parasitology-helminth-cestoda ; Taenia solium ; cysticercus ; antigen ; affinity chromatography ; monoclonal antibody