Comparison of recombinant human immunodeficiency virus gag precursor and gag/env fusion proteins and a synthetic env peptide as diagnostic reagents

Diagnostic reagents for detection of human immunodeficiency virus (HIV) exposure with improved reliability may be provided by viral encoded proteins produced by recombinant DNA techniques or by synthetic peptides corresponding to appropriate viral epitopes. We have expressed at high levels in E. coli a gag gene segment corresponding to approximately 97% of the p55 gag precursor protein, as well as a novel gag/env fusion protein that contains antigenic determinants in common with gag p24, env gp41, and env gp120. The gag and gag/env proteins were purified from insoluble inclusion bodies by sequential extraction with increasing concentrations of urea. These components were tested for reactivity with antisera to HIV proteins and peptides. We have also chemically synthesized a peptide corresponding to env residues 578-608, representing a portion of env gp41. The final preparation of gag and gag/env proteins in 8 M urea reacted with sheep anti-HTLV-III p24 gag antibodies and acquired immune deficiency syndrome (AIDS) patient sera. The gag/env fusion protein also reacted with rabbit anti-HIV env 500-511 peptide antibody. Both recombinant proteins and the env peptide were suitable as reagents for evaluation of serum samples by enzyme-linked immunosorbent assay (ELISA). Results of ELISA assays utilizing the recombinant viral proteins and synthetic peptide were in good agreement with results obtained using disrupted virus as antigen in ELISA assays and immunoblotting.     

Specific phosphorylation of yeast hexokinase induced by xylose and ATPMg. Properties of the phosphorylated form of the enzyme.

Rabbit antibodies to partially purified nicotinamide mononucleotide adenylyltransferase precipitated the enzyme, which remained fully active in the insoluble complexes. Precipitation of antigen-excess soluble complexes with sheep anti-rabbit γ globulin increased the sensitivity of the immunoassay. With this double-antibody assay, the enzymes from chicken erythrocytes, liver, kidney, and thymus showed nearly identical reactivity. Goose, pheasant, and turkey enzymes were highly cross-reactive with the chicken form; pigeon liver enzyme was markedly less reactive. There was no cross-reactivity with fish, amphibian, or mammalian enzymes. The specificity of the antiserum was increased by absorption of antibodies to nonenzyme proteins. The absorbed serum still precipitated the enzyme; in complement fixation assays, it reacted with an antigen that behaved like the enzyme. This antigen was detectable in whole chromatin and in the proteins extracted from chromatin by high salt or urea concentrations. Its immunological reactivity survived exposure to 0.5 m urea, but was reduced by exposure to 6.0 m urea plus 0.4 m guanidine. The enzyme was present as an inactive, partially denatured protein in nonhistone chromatin proteins prepared with these reagents.     

Ionic liquids as solvents for biopolymers: Acylation of starch and zein protein

Biopolymers such as starch and zein protein were found to be soluble at 80 °C in ionic liquids such as 1-butyl-3-methylimidazolium chloride (BMIMCl) and 1-butyl-3-methylimidazolium dicyanamide (BMIMdca) in concentrations up to 10% (w/w). Higher concentrations of biopolymers in these novel solvents resulted in solutions with too high viscosity to stir. Solutions of both starch and zein in BMIMCl were acylated with anhydrides in presence of pyridine to give acetyl starch and benzoyl zein with various degrees of substitution. Without pyridine the acylation reaction did not proceed. ¹H NMR and IR spectroscopies were used to determine the degree of substitution of starch. Viscosity studies indicated that the starch underwent slight reduction in molecular weight during the course of acylation. Starch was also soluble in other non-conventional solvents such as choline chloride/oxalic acid and choline chloride/ZnCl₂. However, zein was insoluble in these solvents.     

Enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to human cytomegalovirus

A solid-phase enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to cytomegalovirus (CMV) is described. The assay used purified CMV and extracts of CMV infected cells as antigen. Antigens were desiccated onto the bottom surface of polystyrene microcuvettes. The antibodies bound to the antigens were assayed by anti-IgG-alkaline phosphate conjugate followed by addition of the enzyme substrate. Titration curves have been obtained from the sera of 35 blood donors and of 23 patients. Comparison of results obtained by ELISA with those obtained by complement fixation (CF) shows that there is agreement between the tests. Both purified CMV and extracts of CMV infected cells were found to be suitable antigens. Purified CMV was of value particularly in those sera which show high reactivity against control antigen. The ELISA technique described is approximately 412 to 548 times more sensitive than the CF test when purified CMV or extracts of CMV infected cells, respectively, are used as antigens. No significant heterotypic rise to CMV was observed by ELISA in three sets of sera with seroconversion to herpes simplex virus. The ELISA technique gives objective results, is easily performed, and may be adaptable as a routine test both for serological diagnosis of CMV infection and for screening of the general population.     

Conjugate formation in urea: coupling of insoluble peptides to alkaline phosphatase for ELISA and in situ detection of antibody-forming cells

We report here a new method to produce synthetic peptide/alkaline phosphatase (AP) conjugates in the presence of urea. The method allows the use of peptides that are not soluble to a sufficient degree in aqueous buffers. The presence of 8 M urea during the construction of the synthetic peptide/AP conjugates does not influence enzyme activity nor the affinity of the anti-peptide antibodies for the conjugated peptide. We demonstrate that these synthetic peptide/AP conjugates can be used for detection of specific antipeptide antibody-forming cells (AFC) in vivo. This method for constructing enzyme conjugates with insoluble proteins or peptides suggest not only new possibilities for detection of specific AFC in vivo but also for applications in receptor-ligand studies, ELISA (enzyme-linked immunosorbent assay), and spot ELISA for detection of antibody-secreting cells in vitro.     

非洲猪瘟病毒p35蛋白作为诊断抗原的抗原性比较

为了筛选出酶联免疫吸附测定 (Enzyme linked immunosorbent assay,ELISA) 反应性最佳的非洲猪瘟病毒 (African swine fever virus,ASFV) 诊断抗原,通过建立ELISA方法,以杆状病毒昆虫细胞表达系统表达的ASFV p30蛋白诊断抗原为参照,首次探讨原核表达系统表达的ASFV p35蛋白作为诊断抗原的抗原性和潜力。免疫印迹和免疫荧光结果表明,获得了40 kDa的重组p35蛋白和30 kDa的p30蛋白,两种蛋白与ASFV阳性血清均具有较好的免疫反应原性。采用重组p30和p35蛋白作为诊断抗原分别建立ELISA方法,并验证其敏感性、稳定性以及与进口试剂盒的符合率。结果显示,尽管p35-ELISA方法的检测敏感性稍低于p30-ELISA方法,但其敏感性仍可达95.8%,且p35-ELISA方法和p30-ELISA方法的批内和批间变异系数均小于10%。p35-ELISA方法与进口试剂盒比较,符合率达97.2%。结果表明建立的p35-ELISA方法敏感性高且稳定性好,可应用于ASFV感染血清的检测。     

Application of Supercritical Anti-Solvent Technologies for the Synthesis of Delivery Systems of Bioactive Food Components

To develop edible delivery systems suitable for food applications, regulations require that solvents and ingredients are either generally recognized as safe or listed by the Food and Drug Administration as processing aids. In this work, we studied a food grade polymer-corn zein, a category of alcohol-soluble proteins, as the carrier material for microencapsulating bioactives. Zein is insoluble in aqueous solutions; zein-based delivery systems may thus maintain the integrity in aqueous food products during processing and storage. Three alcohols, i.e., ethanol, methanol, and isopropanol, with an appropriate amount of water were used to dissolve zein. A supercritical anti-solvent process was applied to synthesize micro- and nanoparticles of zein for edible delivery systems of bioactive compounds. We studied critical variables during the particle formation: polymer concentration, CO₂ flow rate, and co-solvent chemistry. Particles were produced only when mass transfer was fast enough that the co-solvent in the atomized droplets could be extracted by the reservoir CO₂ and polymers could nucleate and grow into particles. Manipulation of the above variables enabled the production of micro- and nanoparticles, which can be used as bases for microencapsulating bioactives. Our results demonstrated promising applications of the supercritical anti-solvent technology to synthesize food grade delivery systems of bioactive food ingredients that can enhance the healthfulness, safety, and quality of food products.     

Cloning, expression, purification and serodiagnostic evaluation of fourteen Mycobacterium paratuberculosis proteins

Fourteen proteins of potential diagnostic value for bovine paratuberculosis were identified in the culture filtrate of Mycobacterium paratuberculosis JTC303 by immunoblot and mass spectrometry. The goals of the present study were to express these 14 ORFs in Escherichia coli and evaluate their antigenicity. All 14 proteins were expressed in E. coli BL21(DE3) after transformation with the pET-22b(+) vector. Yields of insoluble proteins were higher than those of the soluble proteins. Polyclonal rabbit antibodies directed against culture filtrate of JTC303 strain confirmed that five of the expressed and purified proteins are culture filtrate components: ModD, Antigen 85C, PepA, MAP1693c, and MAP2168c. Evaluation of ModD as an ELISA solid-phase antigen on a set of bovine sera from well-characterized paratuberculosis cases and infection-free controls revealed that there was strong serum antibody reactivity to rModD in many infected cattle. However, the overall rModD ELISA sensitivity and specificity for bovine paratuberculosis was not greater than those of ELISAs using crude antigens such as cellular extract or culture filtrate for plate coating, as judged by area under the curve (AUC) of Receiver-operating curve (ROC) analysis. However, an ELISA using natural ModD as the solid-phase antigen had a higher sensitivity and AUC than did rModD suggesting diminution of antigenicity in rModD. Taken together, our results showed that the natural forms of the identified proteins may be useful for diagnosis of bovine paratuberculosis.     

Interpreting dual ELISA and qPCR data for bacterial kidney disease of salmonids

Although there are a variety of methods available for the detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmon and trout, the enzyme-linked immunosorbent assay (ELISA) is probably the most widely used method. However, ELISA measures bacterial antigen, which does not necessarily reflect the number of cells present. We hypothesized that dual analysis of kidney tissue by ELISA and a quantitative real-time polymerase chain reaction assay (qPCR) would provide complementary information about antigen level and the number of bacterial genomes. We found that DNA extracted from the insoluble fraction of the ELISA tissue preparation produced the same qPCR result as DNA extracted directly from frozen tissue, permitting true dual analysis of the same tissue sample. We examined kidney tissue in this manner from individual free-ranging juvenile Chinook salmon and antibiotic-treated captive subadult Chinook salmon and observed 3 different patterns of results. Among the majority of fish, there was a strong correlation between the ELISA value and the qPCR value. However, subsets of fish exhibited either low ELISA values with elevated qPCR values or higher ELISA values with very low qPCR values. These observations suggest a conceptual model that allows inferences about the state of infection of individual fish based on dual ELISA/qPCR results. Although this model requires further assessment through experimental infections and treatments, it may have utility in broodstock selection programs that currently apply egg-culling practices based on ELISA alone.     

Detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by ELISA using purified recombinant nucleoprotein

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody against lymphocytic choriomeningitis virus (LCMV) in sera of laboratory animals. In this ELISA system, LCMV-nucleoprotein (NP) expressed by recombinant baculovirus and purified with high molar urea was used as the antigen. Sera from laboratory animals experimentally infected with the Armstrong strain or the newly isolated M1 strain of LCMV were examined to detect anti-LCMV antibody by the ELISA system, and the reactivity was compared with that of IFA test. Regardless of LCMV strain, all the sera of adult mice infected with LCMV were positive with very high optical density (OD). Also, the sera from mice neonatally infected with LCMV M1 strain were positive with slightly lower OD than adult mice. In contrast, all the sera of uninfected mice were negative to LCMV-NP antigen. Similarly, anti-LCMV antibodies were detected in all the sera of hamsters, mastomyses, and gerbils infected with the LCMV Armstrong strain. The results of the ELISA were in complete agreement with those of IFA, and indicate the high sensitivity and specificity of the ELISA system in the detection of anti-LCMV antibody. Because this ELISA system does not require handling infectious LCMV in the course of the antigen preparation and serological assay, there is no risk of contamination in the laboratory or nearby animal facility. In addition, by using negative control antigen in parallel with positive antigen in ELISA, we can exactly check the LCMV contamination in laboratory animals.     

A sandwich enzyme-linked immunosorbent assay for the quantification of insoluble membrane and scaffold proteins

Enzyme-linked immunosorbent assays (ELISAs) are applied for the quantification of a vast diversity of small molecules. However, ELISAs require that the antigen is present in a soluble form in the sample. Accordingly, the few ELISAs described so far targeting insoluble proteins such as integral membrane and scaffold proteins have been restricted by limited extraction efficiencies and the need to establish an individual solubilization protocol for each protein. Here we describe a sandwich ELISA that allows the quantification of a diverse array of synaptic membrane and scaffold proteins such as munc13-1, gephyrin, NMDA R1 (N-methyl-d-aspartate receptor subunit 1), synaptic vesicle membrane proteins, and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). The assay is based on initial solubilization by the denaturing detergent sodium dodecyl sulfate (SDS), followed by partial SDS removal using the detergent Triton X-100, which restores antigenicity while keeping the proteins in solution. Using recombinant standard proteins, we determined assay sensitivities of 78 ng/ml to 77 pg/ml (or 74-0.1 fmol). Calibration of the assay using both immunoblotting and mass spectroscopy revealed that in some cases correction factors need to be included for absolute quantification. The assay is versatile, allows parallel processing and automation, and should be applicable to a wide range of hitherto inaccessible proteins.     

Molecular weight of an extremely hydrophobic protein, zein, in dimethylformamide and in formamide.

Both alpha zein purified from a commericial preparation and beta zein prepared fresh from corn are soluble in the nonaqueous solvents formamide and dimethylformamide; in this regard zein resembles water soluble proteins such as insulin, ribonuclease, and lysozyme. On the basis of osmotic pressure measurements made in both formamide and dimethylformamide, alpha zein has a number average moleular weight of 21000-24000 daltons and shows no tendency to aggregate or dissociate. Beta zein exists in an aggregated state (dimer and higher forms) in dimethylformamide. Formamide dissociates the beta zein dimer into monomer units but aggregation to higher species occurs with increasing protein concentration.     

New Methods for Extraction and Quantitation of Zeins Reveal a High Content of gamma-Zein in Modified opaque-2 Maize

We have developed methods for quantitative extraction and analysis of zeins from maize (Zea mays L.) flour. Extraction involved solubilization of total endosperm proteins in an alkaline buffer containing SDS and 2-mercaptoethanol with subsequent precipitation of nonzein proteins by the addition of ethanol to 70%. Analysis of these proteins by SDS-PAGE with Coomassie blue staining and by Western blotting and ELISA assay with zein antibodies revealed that this extraction method is more quantitative than the traditional Landry-Moureaux procedure, especially for the β- and γ-zeins. This method was used to extract and analyze the zein content of several `Quality Protein Maize' (QPM) varieties developed by the International Maize and Wheat Improvement Center. QPM varieties contain `modifier genes' that confer a vitreous phenotype on opaque-2 genotypes, while maintaining the elevated levels of lysine and tryptophan characteristic of this mutant. This analysis revealed that the QPM types contain 2 to 4 times the amount of the γ-zein than unmodified opaque-2 or normal maize varieties. Possible relationships between the high expression of the γ-zein and the modified opaque phenotype are discussed.     

Fractionation techniques in a hydro-organic environment. I. Sulfolane as a solvent for hydrophobic proteins

Sulfolane (thiophene, tethrahydro-1,1-dioxide), at concentrations of 4 M or above, is an efficient solubilizing agent for water-insoluble proteins (e.g., zein or globin chains). In comparison with urea, it appears indefinitely stable in aqueous solutions and does not chemically modify proteins upon storage. Moreover, it favors protein structure, i.e., it increases their alpha-helix content, while urea decreases it. Sulfolane is compatible with electrophoretic techniques (it only slightly reduces polyacrylamide polymerization efficiency and it does not interfere with protein and peptide detection methods) and with chromatographic methods (it has negligible A280 nm). With hydrophilic proteins, sulfolane behaves as a mild denaturant and precipitates them at concentrations between 5 and 7 M.     

Plant-derived protein bodies as delivery vehicles for recombinant proteins into mammalian cells

The encapsulation of biopharmaceuticals into micro- or nanoparticles is a strategy frequently used to prevent degradation or to achieve the slow release of therapeutics and vaccines. Protein bodies (PBs), which occur naturally as storage organelles in seeds, can be used as such carrier vehicles. The fusion of the N-terminal sequence of the maize storage protein, γ-zein, to other proteins is sufficient to induce the formation of PBs, which can be used to bioencapsulate recombinant proteins directly in the plant production host. In addition, the immunostimulatory effects of zein have been reported, which are advantageous for vaccine delivery. However, little is known about the interaction between zein PBs and mammalian cells. To better understand this interaction, fluorescent PBs, resulting from the fusion of the N-terminal portion of zein to a green fluorescent protein, was produced in Nicotiana benthamiana leaves, recovered by a filtration-based downstream procedure, and used to investigate their internalization efficiency into mammalian cells. We show that fluorescent PBs were efficiently internalized into intestinal epithelial cells and antigen-presenting cells (APCs) at a higher rate than polystyrene beads of comparable size. Furthermore, we observed that PBs stimulated cytokine secretion by epithelial cells, a characteristic that may confer vaccine adjuvant activities through the recruitment of APCs. Taken together, these results support the use of zein fusion proteins in developing novel approaches for drug delivery based on controlled protein packaging into plant PBs.     

Indirect ELISA with recombinant GP5 for detecting antibodies to porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome is caused by the PRRS virus (PRRSV), which has six structural proteins (GP2, GP3, GP4, GP5, M and N). GP5 and N protein are important targets for serological detection by enzyme-linked immunosorbent assay (ELISA) and other methods. Toward this goal, we developed an indirect ELISA with recombinant GP5 antigens and this method was validated by comparison to the LSI PRRSV-Ab ELISA kit. The results indicated that the optimal concentration of coated recombinant antigen was 0.2 μg/well for a serum dilution of 1:40. The rate of agreement with the LSI PRRSV-Ab kit was 88.7% (266/300). These results support the potential use of recombinant GP5 as an antigen for indirect ELISA to detect PRRSV antibodies in pigs.     

A quantitative enzyme-linked immunosorbent assay for Dermatophagoides pteronyssinus-specific immunoglobulin G

A monoclonal mouse anti-human IgG was used to develop an enzyme-linked immunosorbent assay (ELISA) for the measurement of Dermatophagoides pteronyssinus (DP)-specific IgG in human sera. This monoclonal antibody (HG2-25) binds to all subclasses of IgG but not to IgA, IgM, or IgE. For the assay, the DP antigen is coated firmly on polystyrene beads through physical adsorption and any leakable antigen is washed off. The assay gives satisfactory reproducibility and parallelism of the dilution curves. Using 0.1% human serum albumin as a substitute for the DP-specific IgG preabsorbed diluent gave extremely low backgrounds and high sensitivity. Horseradish peroxidase-labeled HG2-25 prepared with the optimum degree of conjugation and free of polymerized conjugates gave responses fairly proportional to the doses. This ELISA gives a satisfactory recovery and is not affected by nonspecific IgG levels in human sera.     

Indirect ELISA with Recombinant GP5 for Detecting Antibodies to Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome is caused by the PRRS virus (PRRSV),which has six structural proteins (GP2,GP3,GP4,GP5,M and N). GP5 and N protein are important targets for serological detection by enzyme-linked immunosorbent assay (ELISA) and other methods. Toward this goal,we developed an indirect ELISA with recombinant GP5 antigens and this method was validated by comparison to the LSI PRRSV-Ab ELISA kit. The results indicated that the optimal concentration of coated recombinant antigen was...     

High cytoplasmic expression in E. coli, purification, and in vitro refolding of a single chain Fv antibody fragment against the hepatitis B surface antigen.

A single-chain Fv (scFv) antibody fragment against the hepatitis B surface antigen (HBsAg) was expressed in Escherichia coli in the form of two independent fusion proteins, with either 60 ('long') or 27 ('short') amino acid N-terminal encoding sequences related to human interleukin-2. Both fusion proteins were expressed insolubly and at high levels in the bacterial cytoplasm (approximately 30% of total bacterial protein in MM294 cells at a laboratory scale). When recombinant cells were cultured in 5-1 fermentors, expression and optical density increased 2- and 4-fold, respectively, compared to a previous periplasmic insoluble version of the same anti HBsAg scFv. After extraction and solubilization in urea, the cytoplasmic scFvs were purified using immobilized metal ion affinity chromatography, followed by DTT treatment, and refolding by dialysis against a basic pH buffer containing EDTA. The refolded scFvs recognized the recombinant HBsAg in ELISA. Results of an ELISA where antigen affinity chromatography repurified scFvs were used as standards, indicated that refolding efficiencies were high: 56.2% for the 'short' fusion scFv, and 50.6% for the 'long' fusion scFv. Corrected final yields of active scFv were 30.3 and 27.3 mg l-1, respectively, for the aforementioned fusion proteins, 5-6 times better than those reported for the periplasmic scFv variant.