RESUMEN
Our study examined the relationship between the expression of matrix metalloproteinases (MMP)-1, MMP-2, and MMP-9 proteins and the pathogenesis of osteoarthritis (OA). We employed rigorous inclusion and exclusion criteria in computer-based bibliographic databases to extract published studies relevant to this investigation. The STATA 12.0 software was used for the statistical analyses. A total of 1408 studies were initially searched, and 10 studies with 458 OA patients and 295 healthy controls were included in this meta-analysis. The meta-analysis results suggested that the protein levels of MMP-1, MMP-2, and MMP-9 were higher in patients with OA than those in the control group. A subgroup analysis according to ethnicity showed that the protein levels of MMP-1 and MMP-2 were higher in Asian patients with OA than in controls. Caucasians showed no statistically significant differences in protein expression of MMP-1 and MMP-2 between the OA patient group and the control group. Interestingly, the protein levels of MMP-9 in patients with OA were higher than those in the control group in both Asians and Caucasians. A sample-source analysis suggested that the serum levels of MMP-2 and MMP-9 proteins were higher in patients with OA than in controls, while MMP-1 and MMP-9 protein expressions were higher in the synovial joint fluid of patients with OA than in controls. In conclusion, our meta-analysis results suggested that the increased expression of MMP-1, MMP-2, and MMP-9 proteins might be associated with the pathogenesis of OA.
Asunto(s)
Metaloproteinasa 1 de la Matriz/sangre , Metaloproteinasa 2 de la Matriz/sangre , Metaloproteinasa 9 de la Matriz/sangre , Osteoartritis/sangre , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Osteoartritis/patología , Líquido Sinovial/metabolismoRESUMEN
Transient luminous events in the atmosphere, such as lighting-induced sprites and upwardly discharging blue jets, were discovered recently in the region between thunderclouds and the ionosphere. In the conventional picture, the main components of Earth's global electric circuit include thunderstorms, the conducting ionosphere, the downward fair-weather currents and the conducting Earth. Thunderstorms serve as one of the generators that drive current upward from cloud tops to the ionosphere, where the electric potential is hundreds of kilovolts higher than Earth's surface. It has not been clear, however, whether all the important components of the global circuit have even been identified. Here we report observations of five gigantic jets that establish a direct link between a thundercloud (altitude approximately 16 km) and the ionosphere at 90 km elevation. Extremely-low-frequency radio waves in four events were detected, while no cloud-to-ground lightning was observed to trigger these events. Our result indicates that the extremely-low-frequency waves were generated by negative cloud-to-ionosphere discharges, which would reduce the electrical potential between ionosphere and ground. Therefore, the conventional picture of the global electric circuit needs to be modified to include the contributions of gigantic jets and possibly sprites.
RESUMEN
Seven forms of a therapeutic recombinant antibody that binds to the her2/neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both light chains is responsible for two acidic forms. A low potency form is due to isomerization of heavy chain aspartate 102; the Asp102 succinimide is also present in a basic peak fraction. Forms with both Asn30 deamidation and Asp102 isomerization modifications were isolated. Deamidation of heavy chain Asn55 to isoaspartate was also detected. Isoelectric focusing in a polyacrylamide gel was used to verify the assignments. All modifications were found in complementarity determining regions.
Asunto(s)
Anticuerpos/química , Receptor ErbB-2/inmunología , Secuencia de Aminoácidos , Anticuerpos/inmunología , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Focalización Isoeléctrica , Datos de Secuencia Molecular , Mapeo Peptídico , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Tripsina/químicaRESUMEN
This paper describes the analysis of recombinant human deoxyribonuclease (rhDNAse), an acidic and complex phosphoglycoprotein, by capillary zone electrophoresis (CZE). Separation performance was found to be dramatically improved by the addition of calcium ions to the CZE running buffer, due to the influence of calcium binding on the charge and the electrophoretic behavior of rhDNAse. The pH dependent calcium binding effects on the electrophoretic separation were demonstrated at both acidic and basic pH, resulting in a two-dimensional (pH 4.8 and 8.0) calcium aided analysis that achieved multipeak resolution of the complex, glycosylation based, charge microheterogeneity of rhDNAse. Two-dimensional investigation of neuraminidase- and alkaline phosphatase-digested protein further demonstrated that the acidic pH resolved acidic charge heterogeneity and that the basic pH discriminated neutral heterogeneity. This work demonstrates the resolving power of CZE for the analysis of a complex microheterogeneous glycoprotein, and emphasizes the importance of employing multiple separation conditions in accordance with known structural characteristics of the protein.
Asunto(s)
Calcio/química , Desoxirribonucleasas/análisis , Electroforesis Capilar/métodos , Animales , Bovinos , Desoxirribonucleasas/química , Desoxirribonucleasas/genética , Glicoproteínas/análisis , Humanos , Concentración de Iones de Hidrógeno , Metales/química , Modelos Moleculares , Proteínas Recombinantes/análisisRESUMEN
A capillary isoelectric focusing (cIEF) method has been developed for the purpose of determining the identity and charge distribution of mouse/human chimeric antibody to human CD20 antigen (C2B8). The assay was validated in accordance with ICH guidelines in order to demonstrate that it is suitable for its intended purpose and so that it may be performed as a lot release test for bulk and final product. As a result of the validation process the assay was found to be linear over the concentration range of 2-356 micrograms ml-1 with recovery of 125I-labeled C2B8 at the target sample concentration of 125 micrograms ml-1 equal to 99%. The repeatability and intermediate precision relative standard deviations of the four major peaks for migration time, peak area, and peak area percent ranged from 0.9-4.4%. The specificity of the assay was demonstrated by baseline resolution of the C2B8 main peak from product excipients, and other Genentech monoclonal antibodies. The results of this validation demonstrate that the cIEF assay for the determination of identity and charge distribution of C2B8 is accurate, precise, linear, and highly specific. The assay is rapid and suitably rugged.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos CD20/inmunología , Focalización Isoeléctrica/métodos , Proteínas Recombinantes de Fusión/química , Animales , Anticuerpos Monoclonales/análisis , Ritmo Circadiano , Humanos , Radioisótopos de Yodo , Modelos Lineales , Ratones , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Propiedades de Superficie , Temperatura , Factores de TiempoRESUMEN
A simple and non-radioactive complement-dependent cytotoxicity assay was developed to determine the relative potency of an anti-CD20 mAb, IDEC-C2B8. The assay measures the relative number of viable cells based on the uptake and metabolism of the redox dye, Alamar blue. A linear relationship between the relative fluorescence unit generated and the number of viable cells was demonstrated. The assay is simple, has high throughput (performed in 96-well microtiter plates), and shows reproducible dose-response curves in the concentration range of 0.02-3.3 micrograms/ml. With intra-assay variability of 5-12%, interassay variability of 6-10% and spike recoveries of 101-109%, the assay has high precision and accuracy. Specificity was demonstrated by the lack of activity of immunoglobulins that do not bind CD20, or anti-CD20 antibody isotype (gamma 4) which does not bind complement. The assay is able to detect degradative changes in the molecule caused by heat, light and proteolytic treatments, suggesting its use as a stability-indicating method. Finally, the Alamar blue method compared favorably with other more conventional methods used to assess cell viability. The assay has the desired properties for use as a potency assay for quality control testing of anti-CD20 mAb.
Asunto(s)
Anticuerpos Antiidiotipos/análisis , Anticuerpos Monoclonales/inmunología , Antígenos CD20/inmunología , Pruebas Inmunológicas de Citotoxicidad , Oxazinas , Xantenos , Linfocitos B/inmunología , Colorantes , Proteínas del Sistema Complemento/inmunología , Humanos , Proteínas Recombinantes de FusiónRESUMEN
The microscale techniques of CZE, cIEF and SDS capillary electrophoresis have been evaluated for the analysis of a complex glycoprotein, recombinant tissue plasminogen activator (rtPA). A series of omega-amino acid buffers (pH approximately 5) was found suitable for the CZE separation of rtPA on coated capillaries. rtPA could be resolved into a series of major and minor peaks in an epsilon-aminocaproic acid buffer containing 0.01% (v/v) Tween 80. For cIEF, a two step method with pressure mobilization was utilized. Using a commercial instrument, either a polymer solution with a 50 microns I.D. capillary or narrow bore capillaries without a polymer solution (25 microns I.D.) were employed. rtPA was resolved into at least eight species within a pI range of 6.4-9.2 using Ampholine 3.5-10. Migration time precision for the major peaks ranged from 0.2% for CZE to < or = 2-3% R.S.D. for cIEF. Total recovery of rtPA from the capillary was also demonstrated for both methods. Analysis of rtPA, rtPA Type I, rtPA Type II and the desialylated forms resulted in the expected elution profiles. Finally, the potential of SDS capillary electrophoresis using a coated capillary for an rtPA Type I/Type II purity assay was shown.
Asunto(s)
Electroforesis/métodos , Activador de Tejido Plasminógeno/análisis , Activador de Tejido Plasminógeno/química , Acetatos/química , Acrilamidas/química , Adsorción , Secuencia de Aminoácidos , Mezclas Anfólitas/química , Animales , Electroforesis Capilar/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Ensayo de Inmunoadsorción Enzimática , Cabras/inmunología , Sueros Inmunes/inmunología , Focalización Isoeléctrica/métodos , Punto Isoeléctrico , Datos de Secuencia Molecular , Neuraminidasa/metabolismo , Plasminógeno/metabolismo , Alcohol Polivinílico/química , Estructura Secundaria de Proteína , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Dodecil Sulfato de Sodio/química , Tensoactivos/química , Activador de Tejido Plasminógeno/inmunología , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Four commercial ampholytes: Ampholine and Pharmalyte (Pharmacia Biotech), Bio-Lyte (Bio-Rad) and Servalyt (Serva) were evaluated for their ability to resolve recombinant tissue-type plasminogen activator (rt-PA) glycoforms by isoelectric focusing (IEF) and capillary IEF (cIEF). Each brand of ampholytes focused rt-PA into 3-4 major and 5-6 minor bands on slab gel electrophoresis. Visually, focused bands stained with Coomassie Blue appeared to be similarly resolved by all the ampholytes except for Ampholines, where the bands were closely grouped and more intensely stained. When cIEF was performed, Pharmalytes and Ampholines resolved rt-PA glycoforms consistent with the slab gels. No discernible peaks were detected during cIEF of rt-PA using Servalyts or Bio-Lytes. UV spectrophotometric scans of the components used for cIEF showed that Servalyts absorbed intensely over a range which overlapped the detector bandpass. Bio-Lytes showed absorption over a narrower UV range but still overlapped the detector bandpass, thus preventing the discernment of protein peaks. For this cIEF system the best ampholytes were Ampholines and Pharmalytes.
Asunto(s)
Mezclas Anfólitas/química , Activador de Tejido Plasminógeno/análisis , Aminoácidos/química , Tampones (Química) , Electrólitos/química , Electroforesis en Gel de Poliacrilamida/métodos , Concentración de Iones de Hidrógeno , Indicadores y Reactivos/química , Focalización Isoeléctrica/métodos , Poliaminas , Polímeros/química , Proteínas Recombinantes/análisis , Espectrofotometría UltravioletaRESUMEN
Capillary isoelectric focusing (cIEF) and IEF of recombinant humanized monoclonal antibody HER2 (rhuMAbHER2) show five charged isoforms with estimated pI values ranging from 8.6-9.1. The cIEF assay demonstrated good precision with relative standard deviations (R.S.D.) 0.7-3.7% and 0.4-4.2% for intra and interassay analysis, respectively. The method was linear for the area of the main peak over the concentration range 2-250 micrograms/ml with a Pearson correlation coefficient > 0.99. The limit of detection for the main peak was determined to be 2 ppm. With both sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) and SDS-polyacrylamide gel electrophoresis, the nonreduced rhuMAbHER2 migrated as a single major peak with minor peaks in the aggregate and clip regions. After reduction, the electropherogram and the slab gel showed the expected heavy chain and light chain fragments with minor peaks in the aggregate and clip regions. The SDS-CGE assay showed good precision with R.S.D. values of 0.1-7.8% and 0.1-8.1% for intra and interassay analysis, respectively. The Pearson correlation coefficient for the area of the main peak was > 0.99 demonstrating linearity for the concentration range 0.5-500 micrograms/ml. The limit of detection for intact rhuMAbHER2 was determined to be 0.5 ppm. The data presented demonstrates the feasibility of replacing the slab gel techniques with capillary electrophoresis in a quality control environment.
Asunto(s)
Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Receptor ErbB-2/inmunología , Electroforesis Capilar , Electroforesis en Gel de Poliacrilamida , Humanos , Focalización Isoeléctrica , Punto Isoeléctrico , Mercaptoetanol/química , Proteínas Recombinantes/análisis , Proteínas Recombinantes/inmunología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Dodecil Sulfato de Sodio/química , TemperaturaRESUMEN
Two methods, High-Performance Receptor Binding Chromatography (HPRBC) and Cell Proliferation (CP), have been developed as alternatives to the classical hypophysectomized rat weight gain bioassay for the determination of potency for recombinant human growth hormone (rhGH). In the HPRBC assay, rhGH is combined with an excess of the soluble extracellular domain of the recombinant human growth hormone receptor (referred to as 'receptor' in the discussion of the HPRBC assay). Nondenaturing size-exclusion chromatography is used to analyzed the resulting complex, which forms in a 2:1 receptor to rhGH ratio. The 2:1 complex is assayed at a concentration near the Kd (approximately 0.4 nM), providing high specificity for rhGH and detection of rhGH variants with reduced activity. In the CP assay, a mouse myeloid leukaemia cell line (FDC-P1) transfected with the full-length receptor is exposed to varying levels of rhGH for 16-20 h. The incorporation of 3H-thymidine into DNA is used as an index of cell proliferation. The results show that the HPRBC assay provides significantly improved precision with a relative standard deviation (RSD) of < or = 5% vs. an RSD of 23% for the rat bioassay. The CP assay has RSDs of 4-16%. Analysis of rhGH variants and mutants shows that the potencies measured by both the HPRBC and CP assays are in general agreement with the rat weight gain bioassay. Both of the HPRBC and CP assays are sufficiently rugged for operating in a Good Manufacturing Practices (GMP) routine batch release testing environment. In vitro alternatives such as the HPRBC and CP assays build a foundation for replacing the hypophysectomized rat weight gain bioassay by correlating receptor dimerization, binding specificity and signal transduction with the biological activity of rhGH.
Asunto(s)
Hormona del Crecimiento/metabolismo , Receptores de Somatotropina/metabolismo , Aumento de Peso , Secuencia de Aminoácidos , Animales , Bioensayo , Bovinos , Femenino , Hormona del Crecimiento/química , Hormona del Crecimiento/normas , Humanos , Hidrólisis , Hipofisectomía , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/normas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Attempts were made to validate a capillary isoelectric focusing (cIEF) method for a recombinant glycoprotein as an alternative technique to slab gel isoelectric focusing methods routinely used to monitor such charge heterogeneity. The cIEF method principally separates the charged glycoforms of recombinant tissue-type plasminogen activator (rt-PA) on the basis of their sialic acid content. Nine to ten distinct peaks were consistently resolved, with the profile dependent on the class of ampholyte used. The pI of rt-PA measured with synthetic pI standards was in the range pH 6.5-7.5 with the migration of the standards affected by the presence of the protein. The method showed an acceptable recovery of > 100% and had good sensitivity where 25 ng of protein could be resolved into constituent peaks. Recovery of both major peaks and total protein measured by peak areas was linear over a wide range from 50-1000 micrograms/mL. A detailed study showed that when a capillary had been used for some time, capillary age affected peak migration times and, to a lesser extent, resolution. Peak migration times were stable over a temperature range of 15-30 degrees C, and decreased predictably with increasing voltages (400-600 V/cm) and decreasing N,N,N',N'-tetramethylethylene diamine (TEMED) concentrations (0.4-1.5% v/v). Overall the data indicated that this methodology has the potential to be used in the commercial release of protein pharmaceuticals if variability resulting from capillary age and lot were resolved. Even in its present format the method equals the performance of slab gel IEF whilst offering significant improvements in ease of operation and in time and reagent use.
Asunto(s)
Electroforesis Capilar/métodos , Focalización Isoeléctrica/métodos , Activadores Plasminogénicos/análisis , Glicosilación , Modelos Lineales , Proteínas Recombinantes/análisis , Estándares de Referencia , Sensibilidad y EspecificidadRESUMEN
A rapid (< 10 min) one-step capillary isoelectric focusing (cIEF) method was developed to monitor charged glycoforms of recombinant human tissue-type plasminogen activator (rt-PA). Focusing takes place between the detector and the anode and the electro-osmotic flow (EOF) sweeps the separated glycoforms past the detector, towards the cathode. The separation uses a neutral coated capillary and hydroxypropylmethylcellulose (HPMC) to reduce the EOF to a constant and reproducible value. The method uses an ampholyte mix with a 50:50 ratio of pH 5-8 and pH 3-10 ampholytes in 4 M urea and 0.1% HPMC to produce maximal resolution whilst maintaining protein solubility during focusing. The electropherograms were compared to isoelectric focusing (IEF) slab gels of samples of intact rt-PA. In both cases approximately ten charged species could be detected. Data analysis indicated that the intra-assay precision was < 5% for peak migration times and < 10% for normalized peak areas. The number of charged species detected by each of the two methods was consistent for samples of intact rt-PA, rt-PA types I and II and for neuraminidase-digested rt-PA. Overall the data indicate that the automated cIEF method can be an adjunct to slab-gel IEF in the characterization and routine analysis of recombinant glycoproteins.
Asunto(s)
Focalización Isoeléctrica/métodos , Activador de Tejido Plasminógeno/química , Animales , Células CHO , Cricetinae , Glicosilación , Humanos , Proteínas Recombinantes/químicaRESUMEN
Maintenance of CD4+ T helper lymphocyte counts has been used as a surrogate marker of efficacy for drugs in the treatment of AIDS. In a multicenter clinical trial, subtle improvement of CD4+ T cell counts may be masked and misinterpreted if care is not paid to likely sources that can contribute to the variability of measurement of CD4+ T lymphocytes. This review addresses major areas that can contribute to the variability of measurement of CD4+ T lymphocytes, with emphasis on applications to multicenter clinical trials, and proposes areas of improvement that may not be well recognized by the medical community. Whereas there are excellent guidelines for immunophenotyping, equal attention is needed in hematologic enumeration of WBC and absolute lymphocytes. In particular, allowing the margin of error acceptable to blood cell standards for HIV-infected specimens is unsatisfactory. Special attention should also be given to the stability of lymphocytes in the anticoagulant during storage, the lysing method, the quality assurance programs as well as intrasubject fluctuations which may be derived from exercise, medications and diurnal variations. Awareness of these contributing factors by physicians and technical analysts will expedite the discovery of potential therapy in the treatment of AIDS. For a multicenter clinical trial, it is advisable to select a centralized laboratory adopting a uniform protocol with regard to sample preparation and handling, using more stringent quality controls for hematologic analysers, calibration of instruments and immunophenotyping. Pending a true reference standard that can monitor the variation of the entire analytical procedure, we anticipate that future interlaboratory quality assurance programs will include absolute T lymphocyte count, an important parameter for assessing the accuracy and consistency of CD4+ T helper cell counts generated from a laboratory.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Recuento de Leucocitos , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida/patología , Humanos , Estudios Multicéntricos como Asunto/normas , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
Cyclic RGD-containing peptides caused a dose-dependent inhibition of binding of human embryonic kidney cells transfected with recombinant GpIIb/IIIa (r293 clone B) to human fibrinogen coated on to non-tissue culture plates. The inhibitory activity, IC50, of a panel of seventeen RGD-containing peptides ranged from 0.12 to 89.2 microM. These IC50 values correlated with those determined by the inhibition of platelet aggregation (r = 0.99). Even though there was a correlation, there were differences between the platelet aggregation and the bioadhesion assay. The binding of r293 clone B to fibrinogen was not increased by ADP suggesting that GpIIb/IIIa expressed on the surface of r293 clone B cells may be in the 'activated' form. Moreover, preincubation of r293 clone B cells with a monoclonal antibody (mAb) specific for GpIIIa (4B12) resulted in a dose-dependent decrease of binding to fibrinogen while a mAb specific for GPIIb (2D2) had no effect. Neither of these mAbs inhibited platelet aggregation. The binding of r293 clone B cells to fibrinogen required Ca2+ or Mg2+. This cell-based bioadhesion method can provide a tool for screening potential GpIIb/IIIa antagonists and investigating the interaction of GpIIb/IIIa and fibrinogen not possible with platelet aggregation.
Asunto(s)
Bioensayo , Adhesión Celular/efectos de los fármacos , Oligopéptidos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/fisiología , Adenosina Difosfato/farmacología , Anticuerpos Monoclonales/farmacología , Calcio , Células Clonales , Ácido Edético/farmacología , Ácido Egtácico/farmacología , Fibrinógeno , Humanos , Magnesio , Modelos Biológicos , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Glicoproteínas de Membrana Plaquetaria/genética , Proteínas Recombinantes/efectos de los fármacos , TransfecciónRESUMEN
Although prorelaxin has a similar structure as proinsulin, the posttranslational processing of prorelaxin seems to be quite different from that of proinsulin. There are no pairs of basic residues flanking the relaxin moiety in most prorelaxins studied so far. Instead, the prorelaxins of many species contains a tetrabasic sequence (Arg-Lys-Lys-Arg) between the connecting peptide and the A-chain. This is the recognition sequence of furin. In order to study this possible processing by furin, we express the recombinant porcine prorelaxin in Chinese hamster ovary cells. The expected 19 kDa recombinant porcine prorelaxin was found to be constitutively secreted into the medium at a level of approximately 250 ng/ml. No conversion of the 19 kDa prorelaxin into the 6 kDa relaxin was observed. Unlike most prohormones which are biologically inactive, the recombinant prorelaxin was found to be biologically active in an in vitro bioassay.
Asunto(s)
Precursores de Proteínas/biosíntesis , Relaxina/biosíntesis , Transfección , Secuencia de Aminoácidos , Animales , Bioensayo , Células CHO , Cricetinae , Endometrio/efectos de los fármacos , Endometrio/fisiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Vectores Genéticos , Humanos , Técnicas In Vitro , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Precursores de Proteínas/genética , Precursores de Proteínas/farmacología , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Relaxina/genética , Relaxina/farmacología , Mapeo Restrictivo , PorcinosRESUMEN
A novel assay for antibody captured bioactivity (ACB) has been developed to quantitate deoxyribonuclease I (DNase) in human serum samples. The procedure is simple, sensitive, reproducible and has a high throughput. Serum samples are diluted a minimum of 1/4 and assayed in 96-well microtiter plates coated with polyclonal antibodies specific to DNase. The serum is removed from the wells, the plates are washed and the antibody bound DNase is incubated at 37 degrees C with a DNA-methyl green substrate. The assay is sensitive to 0.8 ng/ml with a range to 10 +/- 2 ng/ml, depending upon the time of incubation (48 +/- 2 h). The recovery of rhDNase spiked into human serum samples averaged 84.4% +/- 6.7% in sera diluted 1/4 and 97.8% +/- 7.2% at a 1/8 serum dilution. Intra-assay precision ranged from 3.0 to 7.5% coefficient of variation (% CV) and interassay precision ranged from 5.0 to 10.2% CV for spiked serum controls. Endogenous DNase concentrations in 27 normal human sera were found to range from < 2.0 to 11.4 ng/ml. Endogenous DNase-like activity was found in Cynomolgus and Rhesus monkey sera; this activity diluted linearly and did not interfere with accurate quantitation of added rh DNase. No endogenous DNase-like activity could be detected in ten Sprague-Dawley rat sera. Bovine pancreatic DNase was found to have only very low cross-reactivity in this assay system. The ACB assay format can potentially be applied to the quantitation of other enzymes in serum and other biological samples.
Asunto(s)
Desoxirribonucleasas/sangre , Animales , Especificidad de Anticuerpos , Bioensayo , Humanos , Técnicas Inmunológicas , Macaca fascicularis , Macaca mulatta , Proteínas Recombinantes/inmunologíaRESUMEN
A multiple antigen ELISA for E. coli proteins (ECPs) that may be present in purified recombinant human interferon-gamma (rIFN-gamma) was developed. SDS-PAGE and Western blotting analyses showed that the assay antibodies reacted with a wide spectrum of ECPs in the standard and with ECPs in a production run. In spike recovery studies, rIFN-gamma at concentrations of 0.05 mg/mL and higher augmented the immunoreactivity of the ECPs in the standard curve (1.3-40.0 ng ECPs/mL) by approx 50%. To determine ECP content in purified rIFN-gamma, 0.2 mg/mL of rIFN-gamma was added to the standard curve diluent to compensate for enhanced immunoreactivity. The assay was precise (interassay precision of ECP controls < or = 4.1 %CV) and accurate with recoveries of 111-115% of expected for ECPs (15-40 ng/mL) spiked into purified rIFN-gamma (1 mg/mL). Linearity of dilution for ECPs spiked into rIFN-gamma was obtained (r = 0.999). Moreover, linearity of dilution was obtained for ECPs in "in-process" samples, demonstrating the required condition of antibody excess for this type of multiple antigen ELISA. ECPs were not detectable in several purified lots of rIFN-gamma. Therefore, these lots contained < 1.3 ppm ECPs.
Asunto(s)
Proteínas Bacterianas/análisis , Contaminación de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/química , Interferón gamma/aislamiento & purificación , Anticuerpos Monoclonales , Western Blotting , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Humanos , Interferón gamma/química , Proteínas RecombinantesRESUMEN
In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin.
Asunto(s)
Escherichia coli/genética , Precursores de Proteínas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Relaxina/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Western Blotting , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Clonación Molecular/métodos , AMP Cíclico/metabolismo , Endometrio/efectos de los fármacos , Endometrio/metabolismo , Endopeptidasas/metabolismo , Femenino , Humanos , Datos de Secuencia Molecular , Plásmidos , Precursores de Proteínas/metabolismo , Precursores de Proteínas/farmacología , Proteínas Recombinantes/farmacología , Relaxina/metabolismo , Relaxina/farmacología , Mapeo Restrictivo , PorcinosRESUMEN
Chicken antibodies were used to develop an ELISA for the quantitation of parts-per-million levels of protein A in the purification of immunoglobulins or immunoglobulin-like molecules. Quantitation of protein A in the presence of excess human or murine immunoglobulins in this assay was compared with that obtained in ELISAs developed with rabbit antibodies specific either to protein A or to other molecules. Experiments demonstrate that protein A is bound to the immunoglobulins being purified and that this binding affects subsequent recognition by the antibodies used for the assay. Because of these effects and because fragments of protein A might not be detected in assays which rely on Fc binding of protein A, chicken antibodies that bind protein A specifically are an advantage for the quantitation of this protein by ELISA. In addition, comparison of the effect of different types of immunoglobulins on the protein A standard curve suggests that alternatives to including the immunoglobulin under purification with the standards can be utilized.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulinas/aislamiento & purificación , Proteína Estafilocócica A/análisis , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Contaminación de Medicamentos , Ensayo de Inmunoadsorción Enzimática/normas , Ensayo de Inmunoadsorción Enzimática/estadística & datos numéricos , Humanos , Inmunoglobulina G/aislamiento & purificación , Estándares de Referencia , Sensibilidad y Especificidad , Proteína Estafilocócica A/inmunologíaRESUMEN
Sera from over 1,600 patients who received recombinant human tissue plasminogen activator (rt-PA) during clinical trials were assessed for the presence of antibodies to this therapeutic agent. The rt-PA was administered by a variety of dosage regimens for several different indications. Two different forms of rt-PA were used; one was predominantly two chain form, and the other was a predominantly one chain form. A sensitive radioimmunoprecipitation assay was used to measure antibodies to rt-PA in patients' serum before and after treatment. Of 932 patients tested with this assay, 929 were negative for antibodies to t-PA. Three patients developed low titers after treatment. Additional serum samples were obtained from these three patients within 2 years after rt-PA therapy and were negative for antibodies to t-PA. With the limited number of positive samples, no correlation could be found with dose or type of rt-PA, dosing regimen or clinical diagnosis. The virtual absence of antibody formation was confirmed in an additional 754 patients using a novel competitive two-site ELISA. It can be concluded that a single infusion of rt-PA was virtually unassociated with antibody formation, suggesting that repeat treatments could be given when necessary without the risk of immunologic complications as are seen with streptokinase or its derivatives.