Development and verification of whole column imaging detection-capillary isoelectric focusing method for determination of isoelectric point of pertactin / 中国生物制品学杂志

@#Objective To develop and verify a whole column imaging detection-capillary isoelectric focusing(WCID-CIEF)method for the determination of isoelectric point(pI)of pertactin(PRN).Methods The WCID-CIEF method for the determination of PRN antigen was developed by optimizing the parameters such as the focusing time and final concentration of samples in the WCID-CIEF process,and verified for the specificity,accuracy,repeatability,intermediate precision,durability and inter-batch consistency.Results The optimal focusing time of WCID-CIEF for the determination of PRN antigen pI was 1 min at 1 500 V and 3 min at 3 000 V. The optimal final concentration of PRN antigen was 300 μg/mL. The PRN antigen pI was about 6. 035,and the blank matrix showed no interference peak in the position of each peak of antigen. The method had good specificity,accuracy,repeatability,intermediate precision,durability and consistency among batches.Conclusion The developed WCID-CIEF method is suitable for the pI detection and charge heterogeneity analysis of PRN antigen,which can provide basis for the characterization of PRN antigen and reference for the quality control in the process of development and production of related vaccines.

Development and verification of whole-column imaging capillary isoelectric focusing electrophoresis analysis method for charge heterogeneity of human growth hormone Fc fusion protein / 中国生物制品学杂志

@#Objective To develop and verify a whole-column image capillary isoelectric focusing(iCIEF) electrophoresis method to analyze the charge heterogeneity of recombinant human growth hormone Fc fusion protein(Fc-rhGH).Methods The iCIEF analysis method of Fc-rhGH was developed by optimizing the target protein concentration,cosolvent(urea)concentration and focusing time.The target protein was simultaneously analyzed by this method and traditional flat plate isoelectric focusing(IEF) electrophoresis,and the results were compared;The specificity,accuracy,precision,limit of quantitation(LOQ) and durability of the developed method were verified.Results The optimized method was using the mixed solution of 8 mol/L urea,0.35% methyl cellulose(MC),4% amphoteric electrolyte and 0.5% isoelectric point marker as the sample buffer,and the focusing condition was 1 500 V 1 min,3 000 V 5.5 min.IEF was not suitable for analyzing the charge heterogeneity of Fc-rhGH solution.Using the optimized iCIEF for analysis,the target protein was significantly different from the unrelated protein,and the baseline of blank reagent was stable;The recovery rate of accuracy verification was within 90%～110%,and the linear range was 0.25～0.75 mg/mL(50%～150% of the target loading volume);The RSD of each isomer pI in the repeatability verification was less than 0.3%,and the RSD of peak area percentage was less than 5%;The LOQ was 0.04 mg/ml.The sample storage time durability,amphoteric electrolyte pharmalyte 3-10 durability and MC durability of this method were good.Using this method to analyze the charge heterogeneity of Fc-rhGH physicochemical reference substance,eight charge heterogeneities of the reference substance were effectively separated,and the pI ranged from 5.9 to 6.4.Conclusion The developed iCIEF method had good specificity,accuracy,precision and durability,and was more suitable for efficient analysis of charge heterogeneity of Fc-rhGH than traditional flat plate IEF,which was of great significance for the quality control of Fc-rhGH and other Fc fusion proteins.

Development and verification of capillary isoelectric focusing-wholecolumn imaging detection method for isoelectric point analysis of recombinant human brain natriuretic peptide / 中国生物制品学杂志

@#Objective To establish and verify a capillary isoelectric focusing-whole column imaging detection(CIEFWCID) method for the analysis of isoelectric point(pI) of recombinant human brain natriuretic peptide.Methods The ampholyte,space-occupying agent,protein concentration,focusing time were optimized by CIEF-WCID method,and the best condition for the detection of recombinant human brain natriuretic peptide was obtained.The repeatability,precision and durability of the developed method were verified,and three batches of recombinant human brain natriuretic peptide produced continuously were analyzed for pI.Results HR AESlyte 8-10.5 was selected as ampholyte,while 25 mmol/L sodium hydroxide as the space-occupying agent;The final concentration of the sample was 87.5 μg/mL and the focusing time was 8min.The relative standard deviation RSD of pI detection was 0.1% after six consecutive injections of the same sample;The RSD of pI detection of six samples was 0.1%;The pI RSD of the main peak was 0.1% at different final concentrations of the sample,and the pI RSD of the sample was 0.1% at different storage time,while the pI markers could not be changed arbitrarily.The pI was detected in three consecutive batches of recombinant human brain natriuretic peptide samples.Conclusion The developed CIEF-WCID method for pI analysis of recombinant human brain natriuretic peptide had good repeatability and precision and might be used for follow-up quality control of recombinant human brain natriuretic peptide.

Cook Your Own Ampholytes For Use In Isoelectric Focusing And Chromatofocusing

Isoelectric focusing and chromatofocusing are techniques to separate amphoteric compounds based on their isoelectric points. The commonest used c ompounds to generate a pH gradient in these techniques are ampholytes. The prohibitively expensive cost of carrier ampholytes limits the application of these techniques in less advantaged parts of the world. A simple and inexpensive method is de scribed in this paper to prepare a broad pH range (pH 3 to 11) ampholytes for use in isoelectric focusing or chromatofocusingin any routine laboratory. The method involves crosslinking polyethylene polyamine to create complex cross-linked polyaminesin the first step. Whereas, the second step involves reacting the cross-linked polyamines with an unsaturated carboxylic acid to produce ampholytes.

Transferrin isoelectric focusing for the investigation of congenital disorders of glycosylation: analysis of a ten-year experience in a Brazilian center / Isoeletrofocalização da transferrina para investigação das doenças congênitasda glicosilação: análise de dez anos de experiência de um centro brasileiro

Abstract Objectives: To characterize cases of suspected congenital disorders of glycosylation (CDG) investigated in a laboratory in southern Brazil using the transferrin isoelectric focusing TfIEF test from 2008 to 2017. Method: Observational, cross-sectional, retrospective study. The laboratory records of 1,546 individuals (median age = 36 months, 25-75 IQR = 10-108; males = 810) submitted to the TfIEF test during the period were reviewed. Results: Fifty-one individuals (3%) had an altered TfIEF pattern (5 ± 2.8 cases/year; median age = 24 months, 25-75 IQR = 11-57 months; males = 27, 53%). For 14 of them, data on diagnosis conclusion were available (classic galactosemia = 4; hereditary fructose intolerance = 4; peroxisomal diseases = 2; PMM2-CDG = 2; MPDU1-CDG = 1; SLC35A2-CDG = 1).Comparing the cases with the normal and altered TfIEF patterns, there was a higher prevalence of altered cases in the age group from 11 months to 3 years. There was an increase in the likelihood of change in TfIEF, especially in the presence of inverted nipples or liver disease. Conclusions: The data suggest that the investigation of a case with suspected CDG is a complex problem, being aggravated by the existence of other IEMs (inborn errors of metabolism) associated with altered TfIEF pattern and lack of access to confirmatory tests. The presence of inverted nipples and liver disease, especially in individuals aged 11 months to 3 years, should suggest the need for TfIEF investigation.

Resumo Objetivos: Caracterizar os casos com suspeita de CDG investigados em laboratório do sul do Brasil pelo exame de IEFTF de 2008 a 2017. Metodologia: Estudo observacional, transversal, retrospectivo. Foram revisadas as fichas laboratoriais de 1.546 indivíduos (mediana de idade = 36 meses, IQ 25-75 = 10-108; sexo masculino = 810) que fizeram o exame de IEFTF no período. Resultados: Cinquenta e um indivíduos (3%) apresentaram padrão alterado na IEFTF (5 ± 2,8 casos/ano; mediana de idade = 24 meses, IQ 25-75 = 11-57 meses; sexo masculino = 27, 53%). Para 14 deles, estavam disponíveis dados sobre a conclusão do diagnóstico (galactosemia clássica = 4; intolerância hereditária à frutose = 4; doenças peroxissomais = 2; PMM2-CDG = 2; MPDU1-CDG = 1; SLC35A2-CDG = 1). Comparando os casos com padrão normal e alterado na IEFTF, houve maior prevalência de casos alterados na faixa etária de 11 meses a 3 anos. Verificou-se um aumento na probabilidade de alteração na IEFTF principalmente na presença de mamilos invertidos ou de hepatopatia. Conclusões: Os nossos dados sugerem que a investigação de um caso com suspeita de CDG é complexa, é agravada pela existência de outros EIM associados a padrão alterado na IEFTF e pela falta de acesso a exames confirmatórios. A presença principalmente de mamilos invertidos e de hepatopatia em indivíduos na faixa etária de 11 meses a 3 anos deve sugerir a necessidade de investigação por IEFTF.

Analysis of WCID-cIEF Spectrogram of Recombinant Interleukin-15 Fusion Protein / 中国药学杂志

OBJECTIVE: To analyze the glycosylated chains of recombinant interleukin-15 fusion protein using capillary isoelectric focusing-whole column imaging detection (WCID-cIEF) spectrograms. METHODS: Using established corresponding mathematical models and the least square method, the WCID-cIEF spectrograms of whole protein, de-salicylic-acid protein and de-N-glycosylation-chain protein were analyzed. Among the mathematical models, the interval-1-peak model was selected. And according to the model, the relationship between isoform peak-areas and isoelectric points was listed. RESULTS: The rationality of the interval-1-peak model was confirmed and a series of basic data was obtained according to the model as follows:the apparent m value of the protein was 25.53 reference(R), the apparent n value of the protein was 28.83R, the apparent m value of sialic acid was 0.86 (0.855) R, the apparent n value was 0.12 (0.119) R, the apparent n value of N-acetylglucosamine (undifferentiated from N-acetylgalactosamine) was 0.06(0.061) R, and the apparent m value of formed carboxyl after N-chain removal was 0.19 (0.186) R. Some information of protein sugar composition was also obtained: the sialylation degree was about 1.83 mol•mol-1, the percentage of prototype protein was about 8.3%, the percentage of single N-glycosated modification protein was about 19.8%, the percentage of double N-glycosated modification protein was about 28.4%, the percentage of triple N-glycosated modification protein was about 23.7%, and the percentage of O-glycosated modification (with sialic acid) protein was about 19.8%. The main sugar types should be G0 (F), G1 (F), G2 (F), G1A1 (F), and G2A1 (F). CONCLUSION: The structure of sugar chain is complex, but it also has some repeatability and regularity. We hope that through this study, the glycoprotein sugar chain can be quickly outlined, the understanding of glycoprotein and the study of protein interaction can be improved.

Protein biomarkers of external root resorption: A new protein extraction protocol. Are we going in the right direction?

OBJECTIVE: The aim of this study is to determine a protocol of gingival crevicular fluid protein extraction used for the first dimension of 2-DE gels. It also aims at conducting a review on the current candidates for protein markers of this pathology, all of which may be used to prevent the disease. METHODS: Gingival crevicular fluid was collected from two groups of 60 patients each, with and without external root resorption. Samples were extracted by means of various methods of protein extraction. SDS-PAGE gels were used to assess the quality of the method which was subsequently tested during isoelectric focusing of 2-DE gels taken from samples of patients with and without the disease. RESULTS: Milli-Q ultrapure ice cold water, without precipitation for gingival crevicular fluid protein extraction, proved the method with greatest sharpness to detect protein bands. Additionally, it allowed two-dimensional electrophoresis to be performed. CONCLUSION: The new protein extraction protocol does not interfere in isoeletric focusing of 2-DE gels. Furthermore, it provides the greatest sharpness in detecting protein bands of SDS-PAGE gels. This will allow mapping and searching of new external root resorption markers, particularly due to the difficulty in carrying out molecular tests with the current candidates for protein markers. .

OBJETIVO: o objetivo desse trabalho foi determinar o protocolo de extração proteica do fluido crevicular gengival, que pudesse ser utilizado para a realização da primeira dimensão dos géis 2-DE, bem como fazer uma revisão dos atuais candidatos a marcadores proteicos dessa patologia que podem ser utilizados na prevenção dessa doença. MÉTODOS: foi coletado o fluido crevicular gengival de dois grupos de 60 pacientes, com e sem a reabsorção radicular externa. As amostras foram extraídas por diversos métodos de extração proteica e utilizados géis SDS-PAGE para aferir a qualidade do método, que posteriormente foi testado durante a realização da focalização isoelétrica dos géis 2-DE, de amostras de pacientes com e sem a patologia. RESULTADOS: a utilização de água Milli-Q gelada ultrapura, sem nenhuma precipitação para a extração proteica do fluido crevicular gengival, foi o método com maior nitidez das bandas proteicas, além de permitir a realização da eletroforese bidimensional. CONCLUSÕES: o novo protocolo de extração proteica não interfere na focalização durante a realização dos géis 2-DE, além de maior nitidez na resolução das bandas proteicas dos géis SDS-PAGE. Isso permitirá o mapeamento e busca de novos marcadores da reabsorção radicular externa, tendo em vista a dificuldade de realização de testes moleculares com os atuais candidatos a marcadores proteicos. .

De novo alpha 2 hemoglobin gene (HBA2) mutation in a child with hemoglobin M Iwate and symptomatic methemoglobinemia since birth

Cyanosis in an apparently healthy newborn baby may be caused by hemoglobin variants associated with the formation of methemoglobin, collectively known as M hemoglobins. They should not be confused with genetic alterations in methemoglobin reductase enzyme systems of red cells since treatment and prognosis are completely different. A newborn male child was noted to be significantly cyanotic at birth and is the basis for this report. Hemoglobin isoelectric focusing, acid and alkaline gel electrophoresis, and HBA/HBB gene sequencing were performed for the child, both parents and a sister. The newborn child was treated with methylene blue in an intensive care unit fearing that he had a defective reductase system and exposure to oxidant drugs or toxins. Newborn hemoglobin screening with high performance liquid chromatography was abnormal on the 10th and 45th days but no conclusive diagnosis was reached. Cyanosis persisted up to four years of age with no other symptoms. Hemoglobin M Iwate [alpha2 87(F8) His>Tyr, HBA2:c.262C>T] was detected. It was not present in the child's presumed mother, father, sister, and brother. The analysis of 15 short tandem repeats in the trio demonstrated a de novo mutation occurrence (p-value < 1 × 10 -8). The family was reassured that no further action was necessary and genetic counseling was provided. Methemoglobins should be considered for differential diagnosis of cyanosis in newborns even if no familial cases are detected. Except for cosmetic consequences, the clinical course of patients with hemoglobin M Iwate is unremarkable.

Análisis de hemoglobinopatías en regiones afrocolombianas usando muestras de sangre seca de cordón umbilical / Analysis of hemoglobinopathies in afro-colombian regions using dried blood samples of umbilical cord

Introducción: las hemoglobinopatías constituyen el grupo de trastornos genéticos más frecuentes a nivel mundial, con mayor prevalencia en población negra. En Colombia hay regiones de alta densidad poblacional de afrodescendientes, lo cual plantea la necesidad de establecer estrategias para la identificación temprana de los portadores y enfermos con propósitos de prevención. Objetivo: realizar la identificación temprana de hemoglobinopatías en muestras de sangre seca de cordón umbilical. Material y métodos: para las muestras de sangre seca se utilizaron tarjetas de tamizaje neonatal rutinario de hipotiroidismo congénito. La sangre es eluida por medio de soluciones buffer acuosas y luego se aplican para separación por electroforesis en geles de isoelectroenfoque y en columna de electroforesis capilar. Resultados: de 5141 muestras recibidas de sangre seca de cordón umbilical, se descartaron 1294 por problemas de calidad, se procesaron 3847 (75%) y de éstas 3244 (84%) fueron útiles para el estudio. Se encontraron 288 (7%) con alguna variante de hemoglobina, FC (1,88%), FS(4,65%), SC(0.06%), SS(0,12%), Bart (0,18%), FC/otro (0,12%). Conclusiones: las muestras de sangre seca de cordón umbilical permiten realizar el estudio por electroforesis capilar y por isoelectroenfoque de las diferentes variantes de hemoglobinas anormales, que puede ser aplicable al diagnóstico temprano, siempre que se cumplan requisitos de calidad, en la aplicación de la muestra y en el tiempo de entrega para análisis. (Acta Med Colomb 2012: 37: 118-124).

Introduction: the hemoglobinopathies represent the most common group of genetic disorders worldwide, with higher prevalence in black population. In Colombia there are regions with high density population of African descent, which raises the need to develop strategies for early identification of carriers and patients with preventive purposes. Objective: realize early identification of hemoglobinopathies in dried blood samples from the umbilical cord. Material and methods: for the dried blood samples we used routine neonatal screening cards for congenital hypothyroidism. The blood is eluted by means of aqueous buffer solutions and then applied for separation by electrophoresis in isoelectric focusing gels and in capillary electrophoresis column. Results: of the 5141cord dried blood samples received, 1294 were discarded due to quality problems; 3847 (75%) were processed and of these, 3244 (84%) were appropriate for the study. We found 288 (7%) with some hemoglobin variant, FC (1.88%), FS (4.65%), SC (0.06%), SS (0.12%), Bart (0.18%) , FC / other (0.12%). Conclusions: dried blood samples of umbilical cord allow the study of the different variants of abnormal hemoglobins by capillary electrophoresis and isoelectric focusing, which may be applicable to an early diagnosis in the neonatal screening, provided that quality requirements in the sample application and in the delivery time for analysis are accomplished. (Acta Med Colomb 2012: 37: 118-124).

Pilot study of hemoglobinopathies in newborns of the Rafael Calvo maternity clinic of Cartagena, Colombia / Estudio piloto de hemoglobinopatías en recién nacidos de la Clínica Maternidad Rafael Calvo de Cartagena, Colombia

Introduction: The hemoglobinopathies are a heterogeneous group of congenital anemias from Africa, Asia and the Mediterranean. Due to the migration of this population have spread worldwide, especially in Latin America and the Caribbean region, which Cartagena de Indias is included, with a large proportion of people of African descent. The lack of routine programs that include an appropriate methodology for precise identification of those affected and carriers, impossible to know the real behavior of this disease in our country and an early and appropriate to the patients before the disease manifests itself and produce its serious consequences. Objective: To estimate the incidence and describe the epidemiological profile of hemoglobinopathies in newborns Rafael Calvo Maternity Clinic of Cartagena, in the period from January to June 2010. Methods: Prospective descriptive study of a population of 1729 newborns. Samples were collected cord blood on filter paper. Isoelectric focusing electrophoresis (IEF )was used to separate the haemoglobins. Results: 94.4% (1633samples) were normal (hemoglobinFA), 4.5% (78samples) were heterozygous for haemoglobin S (HbFAS), 1% (17samples) were heterozygous for haemoglobin C (hemoglobinFAC) and 0.1% (1 sample) was double heterozygous SC (hemoglobinFSC). Conclusion: Due to the high incidence of hemoglobinopathies found in this pilot study highlights the importance and necessity of establishing an obligatory neonatal screening in the city of Cartagena, in order to make a timely diagnosis and monitoring of affected and carrier.

Introducción:Las hemoglobinopatías comprenden un grupo heterogéneo de anemias congénitas originarias de África, Asia y la cuenca mediterránea. Debido a las migraciones de este grupo poblacional se han esparcido en todo el mundo, especialmente en América Latina y la región Caribe, en la cual está incluida Cartagena de Indias, con una gran proporción de población afrodescendiente.La inexistencia de programas rutinarios para la identificación precisa de los afectados y portadores, imposibilita conocer el comportamiento real de esta enfermedad en nuestro medio, el manejo temprano y adecuado a los pacientes, antes que la enfermedad se manifieste y produzca sus graves secuelas. Objetivo: Estimar la incidencia y describir el perfil epidemiológico de las hemoglobinopatías, en recién nacidos de la Clínica Maternidad Rafael Calvo de Cartagena, en el período de enero a junio del año 2010. Método: Estudio descriptivo prospectivo. Se tomó muestra de cordón umbilical recolectada en papel de filtro, a una población de 1729 recién nacidos, empleando la técnica de electroforesis de punto isoeléctrico. Resultados: El 94.4 % (1633 muestras) fueron normales (hemoglobina FA), el 4.5% (78 muestras) fueron heterocigotos para hemoglobina S (hemoglobina FAS), el 1% (17 muestras) fueron heterocigotos para hemoglobina C (hemoglobina FAC) y el 0.1% (1 muestra) fue doble heterocigoto SC (hemoglobina FSC). Conclusión: debido a la alta incidencia de hemoglobinopatías encontrada en este estudio piloto, se resalta la importancia de establecer un programa de tamizaje neonatal en la población de Cartagena, a fin de realizar un oportuno diagnóstico y seguimiento de los afectados y portadores.

Bandas oligoclonales en líquido cefalorraquídeo de pacientes con esclerosis múltiple del Hospital Militar Central, Bogotá DC / Oligoclonal bands in cerebrospinal ñuid of patients with multiple sclerosis at the Hospital Militar Central- Bogotá DC

INTRODUCCIÓN: la esclerosis múltiple es la enfermedad neurológica más común y discapacitante en adultos jóvenes. Hasta el momento no existe ninguna prueba certera para su diagnóstico. El laboratorio clínico ha tomado gran importancia al incluir la valoración de la producción intratecal de anticuerpos en el proceso diagnóstico de esta patología. OBJETIVO: describir el comportamiento de las bandas oligoclonales del líquido cefaloraquídeo en un grupo de pacientes. MATERIALES Y METODOS: se realizó un estudio observacional descriptivo tipo serie de casos para describir el comportamiento del resultado de las bandas oligoclonales (BO) en el líquido cefalorraquídeo, en pacientes con esclerosis múltiple (EM) del Hospital Militar Central (HMC) de Bogotá que estaban en tratamiento. Se calcularon las frecuencias absolutas y medidas de tendencia central de las variables estudiadas. RESULTADOS: entre la población de usuarios del Hospital Militar Central hubo 26 pacientes con esclerosis multiple, con edad media de 43 años (DS = 8,54). El 75,1 % (19 pacientes) mujeres y el 26.9% (7 pacientes) hombres. El 50 % (13 pacientes) presentaron bandas oligoclonales positivas.. CONCLUSIÓN: al utilizar la electroforesis de proteínas de alta resolución como técnica para la determinación de las bandas oligoclonales en el líquido cefalorraquídeo se obtuvieron resultados similares a los reportados en la literatura. Sin embargo, se deben hacer todos los esfuerzos para introducir la detección de bandas oligoclonales por la técnica recomendada internacionalmente: el isoelectroenfoque comparando simultáneamente el suero y el líquido cefalorraquídeo, con lo cual se puede obtener una gran sensibilidad; allí radica su importancia y utilidad como valor predictivo negativo.

INTRODUCTION: multiple sclerosis is the most common disabling neurological disease in youth adults. So far there is no accurate test for diagnosis. The laboratory tests have become very important because include assessment of intrathecal antibody production in the diagnosis of this disease. OBJETIVE: describe the behavior of cerebrospinal fluid oligoclonal bands in a group of patients MATERIAL AND METHODS: a descriptive case series, with the population of MS patients currently receiving specific treatment in the Hospital Militar Central in Bogota. The absolute frequencies and measures of central tendency of the variables studied were calculated. RESULTS: among the population described there are 26 patients with MS, with a mean age of 43 years old (SD +/- 8.542). 75.1% (19) were females and 26.9% (7) male. 50% (13) of patients had positive oligoclonal bands.. CONCLUSION: with a standardized technique for determining oligoclonal bands in cerebrospinal fluid, as the high-resolution agarose gel electrophoresis the authors obtained similar results as reported in the international literature. It should make all efforts to introduce the detection of BO with the internationally recommended technique named "isoelectric focusing", who simultaneously compared serum and cerebrospinal fluid, that can get a great sensitivity and a high negative predictive value.

Neonatal Screening for Hemoglobinopathies.

A pilot study was undertaken to develop a feasible neonatal screening strategy for hemoglobinopathies. Isoelectric focusing using dried blood spots samples as a primary screening technique was standardized for the first time in India. The screened positives were confirmed by high performance liquid chromatography followed by parental screening, confirmation, and education.

Diagnostic value of oligoclonal band detected by isoelectric focusing with immunoblotting in inflammatory demyelinating diseases in nervous system / 中华神经科杂志

Objective To explore the diagnostic value of oligoclonal band (OB) detected by isoelectric focusing (IEF) with immunoblotting in inflammatory demyelinating diseases (IDD) in nervous system.Methods Serum and cerebrospinal fluid (CSF) OB was detected by IEF with immunoblotting in 112 patients with IDD ( multiple sclerosis ( MS):n = 48;neuromyelitis optica ( NMO):n = 21:acute disseminated encephalomyelitis ( ADEM):n = 4;secondary IDD from systemic autoimmune diseases:n = 19;peripheral nervous system IDD:n =20) and 24 patients with non-inflammatory neurological disease (NIND).Results CSF-restricted OB was detected in 91.7% (44/48) of MS patients,23.8% (5/21) of NMO patients(x2nmO vs MS= 32.679),1/4 of ADEM patients (Fisher' s excact test),15.8% (3/19) of secondary IDD patients (x2secondary IDD vs MS = 37.425 ),0 of peripheral nervous system IDD patients (x2peripheral nervous system IDD vs MS =37.425) and 0 of NIND patients (x2NIND vs MS =37.425).MS patients had significantly higher percentage of patients with CSF-restricted OB ( all P ＜0.01),compared with NIND and other IDD patients.The sensitivity and specificity of OB detected by IEF with immunoblotting for MS were 91.7% and 89.8%,which were higher than that of OB detected by other methods.Identical serum and CSF OB ( mirror pattern ) was detected in 2 of 4 ADEM patients and 1 of 48 MS patients.Conclusion IEF with immunoblotting to detect OB is a reliable method of diagnosis for MS.

Comparison of isoelectric focusing and polymerase chain reaction for the detection of β-lactamases.

Extended spectrum β-lactamases (ESBLs) have been observed in virtually all the species of family Enterobacteriaceae. Threat posed by antibiotic resistance because of ESBLs is more serious as a number of technical problems are associated with the detection of these enzymes. Although a number of detection methods have been designed for ESBLs, every method has its own benefits and shortcomings as well. In earlier days, isoelectric focusing (IEF) was used as the gold standard for ESBL detection. This study was undertaken to compare IEF with polymerase chain reaction, a method which has been extensively used for ESBL detection these days.

Cloning and prokaryotic expression of LEN-5 β-lactamase gene / 中国人兽共患病学报

In order to express the gene of LEN-5 β-lactamase from a Klebsiella pneumoniae strain,plasmids in the strain were extracted and an 879bp product of LEN-5 gene was obtained with PCR.After being digested with Nde I and Xho I,LEN-5 gene was cloned into pET-26b (+) vector.Then it was confirmed by digestion and DNA sequencing in recombinant plasmid before transformed into E.coli BL21 (DE3).After inducing by IPTG,LEN-5 β-lactamase was expressed.Protein extraction was processed by ultrasonic and protein activity was detected by nitrocefin.The isoelectric focusing electrophoresis showed a pI of 7.6.These results indicated that the LEN-5 gene has been cloned and expressed in prokaryote cell successfully.

Karyological and electrophoretic differences between Pomacea flagellata and P. patula catemacensis: Caenogastropoda: Ampullariidae

The widespread Mexican apple snail Pomacea flagellata (Say 1827) and the strictly endemic "tegogolo" P. patula catemacensis (Baker 1922) (restricted to Lake Catemaco), are the only known American Ampullariidae that have haploid complements n=13. Pomacea patula catemacensis has suffered a critical reduction in abundance due to immoderate fishing for human consumption. Chromosome slides were obtained from colchicine-injected Pomacea snails collected from nine locations along the coastal zone of the Gulf of Mexico, including Lake Catemaco, for use in principal component analysis (PCA). Total proteins in foot homogenates were analyzed through isoelectric focusing (IEF) and native-PAGE electrophoresis on polyacrylamide gels. The chromosome number 2n=26 was confirmed for snails from all locations, with a uniform 9 m + 4 sm formula. However, P. patula catemacensis showed significantly larger chromosomes (absolute size) than any population of P. flagellata. Pomacea patula catemacensis also differed from all populations of P. flagellata in a PCA with standardized data, i.e., independently of the absolute size difference between species. Proteins with an acid isoelectric point were dominant in the foot of both species. The electrophoresis analysis showed that P. flagellata has 17 protein bands, with an upper bound at IEF=7.6, while P. patula catemacensis has only 15 bands, with an upper bound at IEF=7 and a more evenly spaced band pattern. Molecular weights ranged from 40 to approximately 130 kDa in both species. Proteins with high values (>94 kDa) were the most abundant. Pomacea patula catemacensis showed a band of 93 kDa, which was absent from all specimens of P. flagellata. Samples of P. flagellata did not cluster according to any geographical pattern in the statistical analyses, nor did they show any taxonomically useful differences in their electrophoretic patterns that merit sub-specific discrimination.

Surveillance of bacterial resistance and study on ?-lactamases in cefoxitin-resistant Klebsiella pneumoniae isolates / 中国医师进修杂志

Objective To investigate the resistance and ?-lactamase of cefoxitin-resistant Klebsiella pneumoniae. Methods The minimal inhibitory concentrations were determined by standard agar dilution.Isoelectric focusing was used to measure the PI(s) of an isolate,s ?-lactamase,AmpC and ESBLs activity was confirmed by a three-dimensional extract method. Results The resistant rates of 40 strains were as follows: imipenem and meropenem 0.0%,cefepime 20.6%,cefotaxime(22.5%),ceftazidime 60.0%.The most isolates were demonstrated two or more ?-lactamase bands by IEF.Of 39 strains tested,ESBLs was detected in 31(70.5%) strains and AmpC-type?-lactamase in 16(41.0%) strains by three dimensional extract test. Conclusions These cefoxitin-resistant Klebsiella pneumoniae produced two or more ?-lactamases.It is imperative for clinical microbiology laboratories to detect and research ?-lactamases,especially AmpC enzyme.

Study on the Identification and Potency Determination of L-asparaginases from 2 Kinds of Strain / 中国药房

OBJECTIVE: To discuss the identification method of L-asparaginases prepared from E.coliASI.357 and Erwinia carotovora and the optimal enzymatic reaction conditions of potency determination.METHODS: HPLC method and isoelectric focusing electrophoresis were applied for the identification of L-asparaginase from 2 kinds of strain.The effects of category of buffer solution and pH value on enzymatic reaction of potency determination of L-asparaginase were investigated.RESULTS: HPLC chromatogram of L-asparaginases from E.coli ASI.357 was different from that from Erwinia carotovora.The retention time of the peaks were 11.0 min and 11.8 min.The isoelectric point (PI) of L-asparaginase produced from E.coliASI.357 was within 4.65～5.1 and that produced from Erwinia carotovora was within 7.1～8.20.The optimal enzymatic reaction conditions of potency determination of L-asparaginase produced from E.coliASI.35 were Tris-HCl (pH=9.0) as buffer and that produced from Erwinia carotovora was 0.2 mol?L-1 phosphate (pH=8.0) as buffer.CONCLUSION: The isoelectric point (PI) of L-asparaginases produced from 2 kinds of strain is different from each other as well as their optimal enzymatic reaction conditions of potency determination.The L-asparaginases from 2 kinds of strains should be controlled as 2 different categories.

Whole salivary protein compositions in the patients with periodontitis or head and neck cancer / 实用口腔医学杂志

7;there were 32 b ands in tumor group. ③SDS PAGE showed that the number of protein bands with relative molecular mass of 77 000,50 000-52 000,38 000-30 000 increased in the tumour group. Conclusion: In the saliva of periodontitis indivauals there are more basic proteins,the relative molecular mass of the prot ein in the saliva of patients with tumor is different from that of health contro ls.

Molecular epidemiologic study of extended-spectrum beta-lactamase in escherichia coli and Klebsiella pnenmoniae / 中华检验医学杂志

Objective To investigate the prevalence of the genotypes of extended spectrum ? lactamses (ESBLs) in klebsiella pneumoniae and eschrichia coli, which were isolated in Hangzhou City from 2000 to 2001. Methods Total of 198 isolates of E. coil and K. pneumoniae were isolated from five hospitals in Hangzhou. ESBLs-producing isolates were detected by inhibitor-potentiated disc diffusion test, Agar dilution method was used to determine the minimum inhibitory concentrations (MIC) of cefotaxime and ceftazidime.ESBLs genes were analysed by isoelectric focusing (IEF) assay and PCR initially,genotypes were determined by sequencing. Results 51 isolates (25.76%) from 198 E. coil and K. pneumoniae strains were obtained. In ESBLs producers, the ESBLs types of SHV, TEM, CTX-M were 17.65%,84.31% and 80.39% respectively. genotypes were: SHV 11,12;TEM 1;CTX M 3and CTX M 3 like, CTX M 13,14. respectively. Conclusions CTX M type were the major genotypes of ESBLs in klebsiella pneumoniae and eschrichia coli isolated in Hangzhou city.