Proteins In Serum Samples Research Articles

Characterization and application of monoclonal antibodies against N protein of SARS-coronavirus

Severe acute respiratory syndrome-coronavirus (SARS-CoV) causes an infectious disease through respiratory route. Diagnosing the disease effectively and accurately at early stage is essential for preventing the disease transmission and performing antiviral treatment. In this study, we raised monoclonal antibodies (mAbs) against the nucleocapsid (N) protein of SARS-CoV and mapped epitopes by using different truncated N protein fragments. The mapping of those epitopes was valuable for constructing pair-Abs used in serological diagnosis. The results showed that all of the six raised mAbs were divided into two groups recognizing the region of amino acids 249–317 (A group) or 317–395 (B group). This region spanning amino acids 249–395 contains predominant B cell epitopes located at the C-terminus of N protein. One pair-Abs, consisting of N protein-specific rabbit polyclonal antibody and SARS-CoV N protein-specific mAb, was selected to construct a sandwich ELISA-kit. The kit was able to specifically detect SARS-CoV N proteins in serum samples.

Prevalence of parvovirus B19-specific antibodies and of viral DNA in patients with endogenous uveitis

Parvovirus B19 causes erythema infectiosum (fifth disease) in children. The virus has been associated with several autoimmune diseases and has been found in synovium or serum from patients with long-standing arthritis or other rheumatic diseases. We investigated serum and aqueous fluid samples of patients with endogenous uveitis. Sera of 70 consecutive uveitis patients were investigated for IgG against structural proteins VP1 and VP2 and non-structural protein NS1 by western blot technique. Viral DNA in serum was assessed by quantitative PCR. Simultaneous DNA analysis was also performed on aqueous fluid samples from 11 uveitis patients and 10 patients without uveitis during routine cataract extraction. In 15 (21.4%; healthy population: 16 of 10,000) of the 70 serum samples (mean age 42.69 years, range 9-82 years; 48 women, 22 men), viral DNA was detected. IgG directed against structural protein VP1 or VP2 was found in 60 patients (85.7%; healthy population: 80%) and IgG against non-structural protein NS1 in 33 patients (47.1%; healthy population: 22%). In none of the patients was IgM antibody as a serological marker for acute parvovirus infection found. Patients with posterior uveitis (n=11) had detectable serum DNA in 54.5% (p=0.021). This group displayed the highest rate of deterioration of visual acuity (>2 Snellen lines), at 72.7% (all patients: 58.6%; p=0.219). In 2 of the 11 aqueous fluid samples, viral DNA was detected. Antibodies against parvovirus B19 were found in both samples, whereas viral DNA was absent in the serum. In the control group of 10 healthy cataract patients, 3 had detectable DNA in the aqueous humor; 1 of these 3 also had detectable levels of viral DNA in serum. Prevalence of IgG antibodies against NS1 protein in serum samples and DNA in serum is increased in uveitis patients compared with the normal population, suggesting a persistent infection. The presence of DNA in aqueous humor samples shows, for the first time, persistence of parvovirus B19 in the eye in uveitis and also in healthy patients. The role of parvovirus DNA in the aqueous fluid and of IgG antibodies against NS1 in the pathogenesis of uveitis cannot be defined at present.

Neurotrophin Overexpression in Lower Airways of Infants with Respiratory Syncytial Virus Infection

Nerve growth factor and its receptors are upregulated in the lungs of weanling rats with lower respiratory tract infection caused by the respiratory syncytial virus (RSV), and this is a major mechanism of the inflammatory response against the virus. However, no information is available in humans. We sought to determine whether the expression of neurotrophic factors and receptors is increased in human infants infected with RSV. We used a highly sensitive immunoassay to measure the concentrations of nerve growth factor and brain-derived neurotrophic factor proteins in serum samples and in the supernatants and cell fractions of nonbronchoscopic bronchoalveolar lavage fluid. We also used immunofluorescence to detect expression in airway cells of the tyrosine kinase receptor TrkA, which binds nerve growth factor with high affinity. Samples were obtained at 24 and 48 hours postintubation from 31 mechanically ventilated infants: 15 patients with RSV infection, 5 patients with respiratory infection negative for RSV, and 11 postsurgical patients without respiratory infection. Data show significantly higher concentrations of both neurotrophins in the cell fractions of bronchoalveolar lavage samples obtained from infants with RSV infection compared with control infants, whereas no significant difference was found in supernatants or serum samples. We also detected tyrosine kinase receptor immunoreactivity only in cells from airways infected with RSV. We conclude that neurotrophic factors and receptors are overexpressed in human airways infected by RSV, and may contribute to airway inflammation and hyperreactivity.

Protein profiling utilizing SELDI technology may detect ductal carcinoma in situ (DCIS) with high reliability in patient's serum

9654 Background: Several methods are suited to classify different cancer types by testing RNA or protein expression. There is no method available to screen for DCIS. This study was performed to differentiate normal, premalignant, and malignant cases by protein profiling of human serum samples. In addition, the diagnostic value of SELDI-MS (surface-enhanced laser desorption ionisation -mass spectroscopy) for early diagnosis of DCIS was investigated. Methods: SELDI-MS potentiates quantitative measurement of complex protein mixtures such as blood serum. According to their isoelectric point, proteins of 5 to 200 kDa were separated on a mass spectroscopy sample carrier, and then analysed. Based on the obtained protein profiles, algorithms to predict the sample type were developed. Blood samples from about thirty women were analysed including cases without any breast-related lesion, with lesions of premalignant, and malignant phenotype, respectively. Results: SELDI analysis revealed a good differentiation between premalignant lesions and normal controls. For DCIS, the prediction strength was more than 80%; 12 different proteins were differentially expressed, four of them were used for the set-up of a decision tree. In contrast, due to their heterogeneity the protein expression spectra of patients with invasive breast cancer did not reach a significant prediction strength. Conclusion: Utilizing SELDI technique, the identification of differentially expressed proteins in serum samples -related to women with tumors of different malignant progression -turned out to be possible. In addition, the method may become available to predict the existence of DCIS, or to detect other pre-invasive lesions. No significant financial relationships to disclose.

Identification of patients with head and neck cancer using serum protein profiles.

New and more consistent biomarkers of head and neck squamous cell carcinoma (HNSCC) are needed to improve early detection of disease and to monitor successful patient management. To determine if a new proteomic technology can correctly identify protein expression profiles for cancer in patient serum samples as well as detect the presence of a known tumor marker. Direct proteomic analysis and comparison. The surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF) ProteinChip system was used to screen for differentially expressed proteins in serum samples from 99 patients with HNSCC, 25 "healthy" smokers, and 102 healthy (normal) controls. Protein peak clustering and classification analyses of the SELDI spectral data were performed. Several proteins, with masses ranging from 2778 to 20,800 Da, were differentially expressed between patients with HNSCC and the normal controls. The serum protein expression profiles were used to develop a classification tree algorithm, which achieved a sensitivity of 83.3% and a specificity of 90% in discriminating HNSCC from normal and healthy smoker controls. The positive and negative predictive values were 80% and 92%, respectively. A peak with an average mass of 10,068 Da was detected in sera from HNSCC patients and identified as the known biomarker metallopanstimulin-1 (MPS-1), based on mass. Peak relative intensity of the 10,068-Da protein correlated consistently with MPS-1 levels detected by radioimmunoassay in serum samples of HNSCC patients and controls. The 10,068-Da peak was provisionally identified as MPS-1 by SELDI immunoassay. We propose that this technique may allow for the development of a reliable screening test for the early detection and diagnosis of HNSCC, as well as the potential identification of tumor biomarkers.

Effect of Feeding Transgenic Cottonseed vis-a-vis Non-transgenic Cottonseed on Haematobiochemical Constituents in Lactating Murrah Buffaloes

An experiment was conducted to investigate the effect of feeding transgenic cottonseed (Bt.) vis-a-vis non-transgenic (non-Bt.) cottonseed on blood biochemical constituents in lactating Murrah buffaloes. Twenty Murrah buffaloes in mid-lactation were divided into 2 groups of 10 each. Animals of group I were fed with 39.5% non-transgenic cottonseed in concentrate mixture while the same percentage of transgenic (Bt.) cottonseed was included in the concentrate mixture fed to the animals of group II. Animals of both groups were fed with concentrate mixture to support their milk production requirements. Each buffalo was also offered 20 kg mixed green fodder (oats and berseem) and wheat straw ad libitum. The experimental feeding trial lasted for 35 days. There was no significant difference in the dry matter intake between the two groups of buffaloes. All the buffaloes gained body weight, however, the differences were non significant. Total erythrocyte count, hemoglobin content and packed cell volume were 9.27±0.70×10 6 /µl, 13.01±0.60 g/dl and 34.87±1.47%, respectively in group I with the corresponding figures of 8.88±0.33, 12.99±0.52 and 31.08±1.52 in group II. The values of total erythrocyte count, haemoglobin content and packed cell volume did not differ significantly between the two groups of buffaloes. The concentration of plasma glucose, serum total proteins, albumin, globulin, triglycerides and high density lipoprotein were non significantly higher in buffaloes fed non-transgenic cottonseed than in buffaloes fed transgenic cottonseed. The cholesterol concentration was significantly (p<0.01) higher in buffaloes of group I (136.84±8.40 mg/dl) than in buffaloes of group II (105.20±1.85 mg/dl). The serum alkaline phosphotase, glutamic-oxaloacetate transaminase and glutamic-pyruate transaminase activities did not differ significantly between two groups of buffaloes. However, serum glutamic-pyruate transaminase activity was considerably high in buffaloes fed non- transgenic cottonseed as compared to buffaloes fed transgenic cottonseed. Bt. proteins in serum samples of animals of group II were not detected after 35 days of feeding trial. It was concluded that transgenic cottonseed and non-transgenic cottonseed have similar nutritional value without any adverse effects on health status of buffaloes as assessed from haematobiochemical constituents. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 12 : 1732-1737)

Antibodies to Toxoplasma gondii in individuals with first-episode schizophrenia.

We employed enzyme immunoassay (EIA) and Western blotting techniques to measure the level of antibodies to Toxoplasma gondii proteins in serum samples from 38 individuals undergoing their first episode of schizophrenia and from a group of matched control subjects. We found that the individuals with first-episode schizophrenia had significantly increased levels of IgG, IgM, and IgA class antibodies to Toxoplasma proteins, as compared with the control subjects.

Implication of cartilage intermediate layer protein in cartilage destruction in subsets of patients with osteoarthritis and rheumatoid arthritis.

To investigate whether cartilage intermediate layer protein (CILP), a protein recently cloned from human articular cartilage, is recognized as an autoantigen in patients with osteoarthritis (OA) and rheumatoid arthritis (RA), and whether the immune response against CILP is involved in disease pathogenesis. Recombinant fusion proteins, which contain the first half (C1), second half (C2), or 3 fragments within the C2 region (designated C2F1, C2F2, and C2F3) of the non-porcine nucleotide pyrophosphohydrolase-homologous region of CILP, were prepared using Escherichia coli. Autoantibodies to these proteins in serum samples from patients with OA or RA and from age-matched healthy individuals were detected by enzyme-linked immunosorbent assay and Western blotting. In addition, mice were immunized with a mixture of the C1 and C2 fusion proteins to assess the arthrogenicity of CILP. Production of antibodies to the C2 region was detected in 10.5% (11 of 105) of the tested OA patients and in 8.0% (7 of 88) of the tested RA patients, although antibodies to the C1 region were rarely detected in either patient group. All C2F1, C2F2, and C2F3 fragments were found to carry autoepitopes. The C2F2 fusion protein was recognized most frequently in the tested OA patients, whereas the C2F3 fusion protein was dominantly recognized in the tested RA patients. All 4 mice strains, DBA/1J, ICR, C57BL/6, and BALB/c, immunized with the CILP fusion proteins developed chronic arthritis; in particular, the ICR mice developed polyarthritis that was characterized by infiltration of mononuclear cells in the synovium and exfoliation of the surface of cartilage. The immune response to CILP may play a role in the pathogenesis of inflammatory joint destruction. Our results support the role of an immune-mediated process in the joint destruction present in chronic arthropathies such as OA and RA. The results suggest that suppression of immune responses to various components of the cartilage, such as CILP, might be therapeutically beneficial in these chronic arthropathies.

Detection, Quantitation, and Identification of Residual Aminopenicillins by High-Performance Liquid Chromatography after Fluorescamine Derivation

A high-performance liquid chromatographic (HPLC) method with fluorescence detection after precolumn fluorescamine derivation was developed to detect residues of two aminopenicillins, amoxicillin (AMPC) and ampicillin (ABPC), in bovine serum. Proteins in serum samples spiked with each of these penicillins were precipitated with sodium tungstate and sulfuric acid, centrifuged, and removed by passage through a C18 solid-phase extraction cartridge. After precolumn treatment of the extraction products of AMPC and ABPC with fluorescamine solution, HPLC analysis with fluorescence spectrophotometric detection at an excitation wavelength of 390nm and an emission wavelength of 485nm was performed to identify these products. Two mobile phases were used for residual analysis by the isocratic HPLC system. An ODP column (polyvinyl alcohol bonded with an octadecyl functional group) that can be used with strongly alkaline mobile phases (pH 2.0 to 13) was selected, and the column temperature was set at 40°C. A mobile phase comprising 100-mM K2HPO4 solution and acetonitrile (72:28, vol/vol), which yielded AMPC and ABPC retention times of 4.1 and 7.9 min, respectively, was suitable for detection of residual ABPC but not for residual AMPC because interference was caused by peaks of other extracted substances. When a mobile phase comprising a different ratio of 100-mM K2HPO4 solution and acetonitrile (78:22, vol/vol) was used, the retention times of AMPC and ABPC were 7.3 and 26.3 min, respectively, and both penicillins could be analyzed using this system. The calculated standard curves of the reaction products with both mobile phases were linear, and the correlation coefficients were greater than 0.999. The lower limit of detection was 10 ng/ml for both penicillins. Analysis of extracts from bovine serum spiked with AMPC and ABPC at a concentration of 1 μg/ml yielded recovery rates of 102.2 ± 5.5% and 79.0 ± 5.2%, respectively. This detection method may be useful for routine laboratory testing of AMPC and ABPC.

Levels of C-reactive protein in serum samples from healthy children and adults in São Paulo, Brazil.

C-reactive protein (CRP) was measured by ELISA in the sera of 165 healthy blood donors and 125 normal children 1 to 14 years old. The serum levels of blood donors ranged from 0.05 to 57.6 mg/l with median and mean values of 1.8 mg/l and 4.86 mg/l, respectively. CRP levels ranged from 0.02 to 14.4 mg/l in the children's sera, the median being 0.45 mg/l and the mean 1.65 mg/l. No individual lacking CRP was detected. The high CRP levels observed in the present study suggest that the population of the State of São Paulo may usually be exposed to subacute infections and/or inflammation without presenting clinical symptoms.

Cross-Reactivity to Borrelia burgdorferi Proteins in Serum Samples from Residents of a Tropical Country Nonendemic for Lyme Disease

Reports of Lyme disease from areas where the disease is not endemic have increased. Eighty-six human serum samples from Papua New Guinea (nonendemic for Lyme disease) were examined for the presence of IgG antibodies that recognize Borrelia burgdorferi antigens, using the currently recommended two-tiered system of analysis (sensitive ELISA with Western blot). The percentage of positive tests dropped from 50% to 10% when individual negative controls were included in the two-tiered analysis. Positive serum samples failed to inhibit the growth of B. burgdorferi in culture and did not yield positive reactions in the fluorescent treponemal antibody-absorption test. These characteristics, together with atypical Western blot antigen recognition patterns and the absence of known vectors, provide evidence that seropositive results for these persons are not the result of exposure to B. burgdorferi. Individual negative controls may minimize false-positive results for serologic tests for Lyme disease, and these tests must be interpreted in the context of clinical and epidemiologic data.

A Simplified Method for Phenotyping Alpha-2-HS-glycoprotein

Alpha-2-HS-glycoprotein is considered a reliable genetic marker for paternity testing and forensic investigation. We describe a simple, semiautomated method for phenotyping this protein in serum samples, bloodstains and dental pulp using isoelectric focusing methods followed by immunofixation techniques. This method produces clear results even with very old biological evidence.

Antibody response to the M2 protein of influenza A virus expressed in insect cells.

A recombinant baculovirus expressing the M2 protein from influenza A/Ann Arbor/6/60 (H2N2) virus (AA60 virus) was constructed. The expressed M2 protein was recognized by a monoclonal antibody specific for the M2 protein and comigrated with the M2 protein from cells infected with AA60 virus on SDS-polyacrylamide gels. Immunofluorescence studies indicated that the expressed M2 protein was present on the surface of Spodoptera frugiperda (Sf9) cells infected with the recombinant baculovirus. Immunoassays using the expressed M2 protein were able to detect antibodies to the M2 protein in serum samples from humans and ferrets infected with influenza A viruses.

Differential-pulse polarography of amino acids in acetaldehyde-borax medium

A differential-pulse polarographic method for the determination of amino acids is reported, based on the formation of Schiff's base compounds in borax buffer solution (pH 10.10) containing acetaldehyde. The compounds are reduced at a mercury electrode with peak potentials of about −1.5 V (vs. SCE) and well defined polarographic waves are obtained which can be used to determine amino acids in borax medium. The differential-pulse polarographic method was found to be the most sensitive and suitable for the determination of amino acids in the concentration range 1 × 10 −6–8 × 10 −4 M (lysine) and 2.8 × 10 −6–1 × 10 −3 M (arginine). The polarographic characteristics of these waves were studied by differential-pulse polarographic and cyclic voltammetric methods. The waves are ascribed to the reduction of the imido group in the Schiff's base compounds. The procedure was applied to the determination of lysine and arginine in foodstuffs and the total proteins in serum samples.

Changes in concanavalin A-reactive proteins in inflammatory disorders.

Quantitative changes of concanavalin A (Con A)-reactive proteins in serum samples obtained from rats with induced inflammation and from patients with inflammatory and autoimmune diseases were examined by use of lectin blots. Treatment of rats with a single dose of fermented yeast to induce inflammation caused an extensive increase in Con A-reactivity. These changes were time dependent and were similar in both sexes of the animals. When we examined serum samples obtained from patients with various inflammatory disorders for their Con A-reactive proteins as compared with normal donors, we noted that the Con A-reactivity increased in patients with rheumatoid arthritis and systemic lupus erythematosus. Among all the glycoproteins examined by lectin blots with use of Con A, a set of five proteins was selected for detailed analysis by densitometric scanning. These included alpha 2-macroglobulin, P-150, P-95, P-40, and P-35, of Mr 180,000, 150,000, 95,000, 40,000, and 35,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Densitometric scanning analysis of the lectin blots revealed that the Con A-reactivity of these proteins increased during inflammation. Because alpha 2-macroglobulin is not an acute-phase protein in humans, an increase in Con A staining of this protein suggested that altered glycation is associated with autoimmune diseases. Thus, study of changes in Con A-reactive proteins in human sera may facilitate our understanding of the etiology and pathophysiology of autoimmune diseases.

Comparison of separation techniques in radioimmunoassays for 17-hydroxyprogesterone

Separation techniques have been studied in the development of a direct radioimmunoassay to determine levels of 17-hydroxyprogesterone in serum. The same highly specific sheep antiserum was used throughout, together with the same amount of 125I-labelled 17-hydroxyprogesterone to which was added sodium salicylate to eliminate interference by endogenous binding proteins in serum samples. In one approach, dextran-coated charcoal was employed to adsorb the free fraction and, in another, the antibodies were covalently coupled to magnetisable particles. The antiserum was also adsorbed to assay tubes either directly or indirectly through second (double) antibodies. Analytical recovery and specificity were similar irrespective of the separation technique as was the correlation with results obtained by a reference assay. Levels of 17-hydroxyprogesterone in sera from normal adults and from treated and untreated patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency were also similar. However, the assay employing dextran-coated charcoal for separation showed the best precision and resulted in the greatest sensitivity, while the use of antibodies adsorbed indirectly to assay tubes was superior in terms of practicability.

Effects of dextran on five biuret-based procedures for total protein in serum.

We evaluated the effect of dextran on values for total protein in serum as measured by the biuret method with five widely used automated instruments: the American Monitor Parallel; the Du Pont aca II; the Roche Cobas-Bio; the Kodak Ektachem 400; and the Beckman Astra 8. Dextran concentrations as great as 25 or 30 g/L had relatively little or no influence on total protein measurements by the latter three instruments. Dextran concentrations exceeding 6 g/L caused falsely low results with the aca, whereas the Parallel gave falsely high results when the dextran concentration exceeded 2 g/L. The aca total protein procedure could be protected from the interference by dextran concentrations up to 30 g/L by injecting 0.4-0.8 mL of ethylene glycol directly into the reagent pack before sampling. However, we could not eliminate the interference with the Parallel procedure by any simple means; we thus recommend that it not be used for measuring total protein in serum samples from patients who are being treated with dextran.

Determination of retinol-binding protein in serum by kinetic immunonephelometry with polyethylene glycol pretreatment.

This work describes the use of polyethylene glycol as a pretreatment reagent to remove endogenous light scattering material from serum samples prior to automated immunonephelometric analysis on a centrifugal analyser. An assay system for retinol-binding protein is described, which allows rapid (10 minutes) quantitation of retinol-binding protein in serum samples with a detection limit of 5 mg/L and between-assay coefficients of variation ranging from 2.9% to 4.0%. The assay range is 5-80 mg/L and accuracy comparisons with a Mancini single radial immunodiffusion method yield a regression line y = 0.89 x + 0.52 (r = 0.98, n = 22). The problem of analyte precipitation associated with use of pretreatment regimes is discussed.

Identification of alpha-fetoprotein polymers. Artifacts of the isolation procedure

By combining polyacrylamide gel electrophoresis and radioimmunoassay, a method has been developed to detect polymeric forms of alpha-fetoprotein (AFP) during the isolation procedure. The method is capable of detecting AFP polymers in the presence of other serum proteins in serum samples that contain as little as 100 ng of AFP. Using this method, we have found that AFP polymers do not exist in cord serum; if encountered, we feel they should be considered to be artifacts of the isolation procedure. AFP polymers appear in 7.5% polyacrylamide gel as multiple bands. We believe the multiple bands to be AFP polymers and not serum protein contaminants for the following reasons: (1) the electrophoretic pattern is very similar to that of serum albumin; (2) the polymers do exhibit AFP activity, although less than the monomeric form does; (3) the molecular weight of the dimer is exactly twice that of the monomer; (4) in the presence of sodium dodecyl sulfate and mercaptoethanol, the polymers move to the same position as do the monomers in polyacrylamide gel electrophoresis; (5) the polymers cannot be removed by repeated use of an immunoadsorbent affinity column designed to remove contaminant serum proteins.

Quick two-dimensional electrophoresis for simultaneous analysis of several serum protein samples. Study of various species serum

A two-dimensional agar hydrolyzed-starch electrophoresis technique facilitating simultaneous analysis of several serum protein samples in 5 to 6 hr is described. Results obtained with human, rabbit, dog, rat, mouse, and guinea pig serum proteins are shown.