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

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.

Chromatography, Affinity ; methods ; Chromatography, High Pressure Liquid ; methods ; Citrinin ; analysis ; Drug Contamination ; Monascus

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 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.

Antibodies, Monoclonal ; isolation & purification ; Chromatography, Affinity ; methods ; Chromatography, High Pressure Liquid ; Zearalenone ; antagonists & inhibitors ; immunology

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*

As a novel bioaffinity chromatography technique, cell membrane chromatography (CMC) was first established by Professor He in 1996, with which combined high performance liquid chromatography, cytobiology, and receptor pharmacology. The cell membrane stationary phase (CMSP) consists of porous silica coated with active cell membranes. By immersing silica into a suspension of cell membranes, the whole surface of silica was covered by the cell membranes. In CMC, the interaction of drugs or compounds with the immobilized cell membrane or its receptors is investigated using liquid chromatography. In general, with the aim to provide scientific foundation for further development and application, this paper mainly focuses on the characteristics of the cell membrane stationary phase (CMSP), the CMC analytical system, and its applications in traditional Chinese medicines (TCMs) about CMC. With the development of CMC, the breakthrough progress of it in studying active components of TCMs field is expectant.
Animals ; Cell Membrane ; chemistry ; Chromatography, Affinity ; methods ; Drugs, Chinese Herbal ; analysis ; Humans ; Medicine, Chinese Traditional ; methods

OBJECTIVETo identify specific serum glycoprotein profiles that correspond to the carcinogenic process of primary liver cancer (PLC) by analyzing a population with high-incidence of PLC using lectin affinity microarray.

METHODSSerum samples were collected from individuals classified as high risk for PLC (including patients with liver cirrhosis and hepatitis B) and development of PLC was recorded. Healthy individuals served as normal controls. The serum samples were subjected to glycoprotein profling by using lectin microarrays and the results were confirmed by lectin blot. Between-group differences were statistically analyzed.

RESULTSPLC carcinogenesis was found to be correlated with enhanced affinity for AAL, ACL, ConA, LCA, MPL, NML, PHA-E, PHA-L, PSA, RCA-I, STL, VAL,WGA, and SNA (P less than 0.05). These data implied that changes in specific glycan structures, such as aFuc, GlcNAc, GalNAc, mannose, bisecting GlcNAc and terminal beta1-4 Gal, may be involved in PLC carcinogenesis . The PLC group showed significantly different results for all detected lectins, except SNA (P less than 0.05). However, among the PLC group, the SNA affinity was not significantly different for the hepatitis B group (P =0.443, P more than 0.05).

CONCLUSIONGlycans may be associated with the carcinogenic process of PLC and may be developed as diagnostic and prognostic biomarkers of PLC in the future.

Carcinogenesis ; Chromatography, Affinity ; Cohort Studies ; Glycoproteins ; blood ; Humans ; Lectins ; blood ; Liver Neoplasms ; blood ; pathology