Rapid and sensitive sandwich enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxin B in cheese.

A rapid and sensitive screening sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of staphylococcal enterotoxin B (SEB) in cheese by using a highly avid anti-SEB antibody (Ab) as the capture Ab (CAb) and as the biotinylated Ab conjugate. The glutaraldehyde fixation method for the immobilization of CAb on polystyrene dipsticks was superior to the adsorption fixation and the adsorption-glutaraldehyde fixation methods. The glutaraldehyde fixation method resulted in a higher surface-saturating CAb concentration as evaluated by the peroxidase saturation technique and by the ability of the CAb-coated dipstick to discriminate between positive and negative controls (index of discrimination). Of nine blocking agents used alone or in pairs, lysine-human serum albumin, bovine serum albumin, human serum albumin, and gelatin effectively saturated available sites on the CAb-coated dipsticks without causing interference with the antigen-Ab reactions. The addition of 1% polyethylene glycol to the diluent of the biotinylated anti-SEB Ab conjugate improved the detection of SEB. A concentration of 4% polyethylene glycol allowed a 5-min reaction time for the streptavidin-biotin-horseradish peroxidase conjugate. Cheddar cheese homogenate reduced the sensitivity of the SEB assay; however, the sensitivity was restored when 1.6% (wt/vol) of either a nonionic detergent (Mega-9) or two zwitterionic detergents (Zwittergent 3-10 and 3-12 detergent) was added to the diluent. By using the rapid sandwich ELISA, a minimum of 0.5 to 1.0 ng of SEB per ml was detected within 45 min. The whole procedure for the analysis of the cheddar cheese samples was completed within 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid and sensitive sandwich enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxin B in cheese

A rapid and sensitive screening sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of staphylococcal enterotoxin B (SEB) in cheese by using a highly avid anti-SEB antibody (Ab) as the capture Ab (CAb) and as the biotinylated Ab conjugate. The glutaraldehyde fixation method for the immobilization of CAb on polystyrene dipsticks was superior to the adsorption fixation and the adsorption-glutaraldehyde fixation methods. The glutaraldehyde fixation method resulted in a higher surface-saturating CAb concentration as evaluated by the peroxidase saturation technique and by the ability of the CAb-coated dipstick to discriminate between positive and negative controls (index of discrimination). Of nine blocking agents used alone or in pairs, lysine-human serum albumin, bovine serum albumin, human serum albumin, and gelatin effectively saturated available sites on the CAb-coated dipsticks without causing interference with the antigen-Ab reactions. The addition of 1% polyethylene glycol to the diluent of the biotinylated anti-SEB Ab conjugate improved the detection of SEB. A concentration of 4% polyethylene glycol allowed a 5-min reaction time for the streptavidin-biotin-horseradish peroxidase conjugate. Cheddar cheese homogenate reduced the sensitivity of the SEB assay; however, the sensitivity was restored when 1.6% (wt/vol) of either a nonionic detergent (Mega-9) or two zwitterionic detergents (Zwittergent 3-10 and 3-12 detergent) was added to the diluent. By using the rapid sandwich ELISA, a minimum of 0.5 to 1.0 ng of SEB per ml was detected within 45 min. The whole procedure for the analysis of the cheddar cheese samples was completed within 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assembly of Adenoviruses

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified incomplete particles of adenoviruses type 2 and 3 revealed that core proteins V and VII and capsid proteins VI, VIII, and X were absent in these particles, but they contained polypeptides not present in complete particles. Two types of incomplete particles were observed in the electron microscope, appearing as deoxyribonucleic acid-less particles with single discontinuities in the capsid structure (about 80%), or more amorphous particles resembling hexon aggregates (about 20%). The amount of incomplete and complete particles increased in parallel during the infectious cycle. Detectable amounts were found at 13 h with a maximum rate of synthesis for both particles at 24 h after infection. (3)H-labeled amino acids were incorporated into incomplete particles without a detectable lag period, but the label appeared in complete particles with a 60- to 80-min lag. Early after the pulse in pulse-chase experiments, the radioactivity was higher for incomplete particles than for complete particles and leveled off before the activity of complete particles reached a maximum. In the adenovirus type 2 system, pulse-chase experiments suggested a precursor-product relationship between incomplete and complete particles. After a short pulse, 19 h postinfection, entrance of (3)H-labeled amino acids into the hexon polypeptide of complete particles was delayed for 80 min, but no delay was observed for the labeling of the hexon polypeptide of incomplete particles. The core polypeptides appear in complete particles without a delay, also suggesting that incomplete particles were precursors to complete particles. Incorporation of (3)H-labeled amino acids into the hexon polypeptide of complete and incomplete particles was drastically decreased by inhibition of protein synthesis with emetine. However, the uptake of label into core proteins of complete particles was only decreased to 50% on inhibition of protein synthesis. The results suggest that incomplete particles are intermediates in virus assembly in vivo and that the assembly of capsid polypeptides into incomplete and complete particles is dependent on continuing protein synthesis.     

Antibodies with specificities against a dispase-produced 15-kilodalton hexon fragment neutralize adenovirus type 2 infectivity.

During the entrance of adenovirus type 2 into cells, it has been suggested that the virion undergoes a conformational change. In this investigation, we have further characterized the hypothetical conformational change, which the structural protein hexon undergoes in response to low pH. From pH 5.0 to pH 6.0, the proteolytic enzyme dispase cleaved the hexon into a few distinct fragments with a dominating low-molecular-weight fragment with a molecular weight of 15,000 (15K peptide), whereas between pH 6.5 and pH 8.0, the cleavage of the hexon was negligible. The degradation of the hexon with dispase at low pH was not due to an increased activity or alteration of the active site of dispase at low pH. The 15K fragment was identified as a segment of the N-terminal part of the hexon polypeptide beginning at amino acid residue 5. An immune serum produced in response to acid-treated and glutaraldehyde-fixed hexons contained a small amount of antibodies directed towards the 15K fragment, as judged by Western immunoblotting. An anti-15K antibody fraction was isolated by affinity chromatography by removing antibodies recognizing the hexon in the alkaline configuration. Such antibodies displayed a higher relative titer at pH 5.0 than at pH 7.5 in an enzyme-linked immunosorbent assay. The isolated antibodies showed a specific neutralizing capacity five times higher than that of the corresponding unfractionated polyclonal anti-hexon serum; however, the neutralizing ability was independent of pH. The neutralization of adenovirus type 2 infection by the isolated anti-15K antibodies implies that the N-terminal end of the hexon may play a critical role in the early steps of the virion-cell interaction.     

Increased bronchovascular permeability after allergen exposure in sensitive asthmatics

To investigate the possibility that an increase in bronchovascular permeability is associated with allergen exposure in sensitive asthmatics we evaluated the amounts of serum proteins in bronchoalveolar lavage (BAL) effluents before and after local challenge with allergen. After exposure of sensitive asthmatic airways (n = 15) to allergen significant increases in total protein compared with controls were observed: 0.08 +/- 0.01 mg/ml in control airways and 0.13 +/- 0.02 mg/ml in challenged airways; P less than 0.05. The greatest changes induced by allergen exposure involved small-molecular-weight proteins (less than 345,000) and an inverse correlation was observed between log molecular weight and percent increase in the concentrations of the specific proteins; r = -0.61. BAL-serum distribution coefficients of serum proteins in airway fluids reflected a greater diffusability of low-molecular-weight proteins immediately after allergen exposure. We also evaluated the movement of serum proteins into lung after local allergen exposure using intravenously administered 99mTc-albumin (n = 10) and found an immediate 3.8-fold increase in amounts of radioactive albumin in allergen exposed airways compared with airways exposed to diluent. Most of the radioactivity was recovered in the first 5 ml of aliquot withdrawn, suggesting a marked increase in the permeability of the bronchial (large airway) vascular-epithelial membrane. An increase in serum proteins was also observed in BAL fluid of asthmatics 2-4 h after aerosol challenge (n = 4), including all proteins in the molecular weight range 45,000-900,000. These studies suggest that allergen exposure in sensitive asthmatics causes an acute increase in bronchovascular permeability to serum proteins.     

共价连接的多肽酶联免疫测定法的研究

采用紫外线照射及戊二醛活化处理聚苯乙烯酶标板后再包被八肽胆囊收缩素（CCK-8）,以此为基础进行酶联免疫吸附试验（ELISA）,通过不同的洗涤过程对CCK-8与酶标板相互作用的性质进行研究.结果表明,该包被方法可使CCK-8通过共价交联的方式稳定地结合于固相载体上,该方法适用于不同来源的酶标板,处理后的酶标板性质稳定,可储存使用,测定的批内及批间变异系数分别为4.75%和7.80%,方法稳定可靠,重复性好,便于推广应用.     

The N-terminal peptide of the hexon of human adenovirus type 2: its chemical synthesis and antigenic properties]

Peptides corresponding to the N-terminal section of protein of a hexone of the 2nd type human adenovirus have been synthesized. Being conjugated with a carrier of BSA they induced formation of antibodies which reacted both with peptides and with viral proteins. Sera to native proteins were also bound to synthetic peptides. Immunogenicity strengthening in peptides conjugated with synthetic polyelectrolytes is shown. The N-terminal section of hexon is supposed to participate in formation of an antigenic determinant possessing group specificity.     

Role of Adenovirus Structural Proteins in the Cessation of Host-Cell Biosynthetic Functions

Two of the adenovirus capsid proteins, the fiber and the hexon, complexed with either KB cell or type 5 adenovirus deoxyribonucleic acid (DNA). Maximal binding occurred at 0.01 m NaCl; increasing the ionic strength of the reaction mixture to 0.2 m NaCl resulted in a decrease in the association of either antigen to DNA. Variations of pH between 6.3 and 8.4 did not affect the binding of fiber antigen to DNA. Below pH 7.5, however, there was a small decrease in the ability of the hexon to bind nucleic acid. The association between the adenovirus structural proteins and DNA was reversible and was independent of whether the DNA was native or denatured. The fiber or hexon protein inhibited the DNA-dependent ribonucleic acid (RNA) polymerase and the DNA polymerase from KB cells. On a weight basis, the fiber protein inhibited enzymatic activity to a greater extent than the hexon. Increasing the template DNA concentration decreased this inhibition. The inhibition of the DNA-dependent RNA polymerase activity by either antigen could be reversed by increasing the ionic strength of the reaction mixture. After infection of KB cells with type 5 adenovirus, the levels of DNA and RNA polymerases remained unchanged for 15 to 20 hr. Thereafter, the specific activity of both enzymes decreased. By 30 hr postinfection, the polymerase activities were only about 30% of the enzyme activities in uninfected cells.     

Particle-by-particle quantification of protein adsorption onto poly(ethylene glycol) grafted surfaces

The use and advantage of flow cytometry as a particle-by-particle, low sampling volume, high-throughput screening technique for quantitatively examining the non-specific adsorption of proteins onto surfaces is presented. The adsorption of three proteins: bovine serum albumin (BSA), immunoglobulin gamma (IgG) and protein G, incubated at room temperature for 2 h onto organosilica particles modified with poly(ethylene glycol) (PEG) of increasing MW (2000, 3400, 6000, 10,000 and 20,000 g mol(-1)) and grafted amounts (0.14-1.4 mg m(-2)) was investigated as a model system. Each protein exhibited Langmuir-like, high affinity monolayer limited adsorption on unmodified particles with the proteins reaching surface saturation at 1.8, 4.0 and 2.5 mg m(-2) for BSA, IgG and protein G, respectively. Protein adsorption on PEG-modified surfaces was found to decrease with increasing amounts of grafted polymer. PEG grafting amounts >0.6 mg m(-2) effectively prevented the adsorption of the larger two proteins (BSA and IgG) while a PEG grafting amount >1.3 mg m(-2) was required to prevent the adsorption of the smaller protein G.     

Selection and Preliminary Characterization of Temperature-Sensitive Mutants of Type 5 Adenovirus

Eight temperature-sensitive (ts) mutants that replicate normally at 32 C but poorly, if at all, at 39.5 C have been isolated from mutagenized stocks of a wild-type strain of type 5 adenovirus. Three mutagens were employed: nitrous acid, hydroxylamine, and nitrosoguanidine. Ts mutants were isolated from mutagenized viral stocks with frequencies between 0.01 and 0.1%. All eight mutants had reversion frequencies of 10(-5) or less. Complementation experiments in doubly infected cultures at the nonpermissive temperature separated the mutants into three nonoverlapping complementation groups. Complementation yields ranged from a 2.3- to a 3,000-fold increase over the sums of the yields from the two singly infected controls. Genetic recombination was also demonstrated; approximate recombination frequencies ranged from 0.1 to 15%. Preliminary biochemical and immunological characterization of the mutants indicated that: (i) the single mutant in complementation group I did not replicate its deoxyribonucleic acid (DNA) or synthesize late proteins at the nonpermissive temperature but did inhibit host DNA synthesis to 25% of an uninfected control; (ii) the four group II mutants replicated viral DNA, shut off host DNA synthesis, synthesized penton base and fiber, but did not synthesize immunologically detectable hexon; the three mutants in complementation group III synthesized viral DNA, shut off host DNA synthesis, and made immunologically reactive capsid proteins (hexon, penton base, and fiber).     

Minimizing ELISA background in the diagnosis of swine trichinellosis.

After confirming that long-term serum storage (frozen at -20 C for greater than 3 mo) causes optical density to drift upward, several modifications of an enzyme-linked immunosorbent assay (ELISA) protocol were evaluated to identify a protocol that would reduce background in porcine sera tested for trichinellosis. Modifications evaluated included blocking the antigen-coated ELISA plate with sample diluent containing 10% bovine serum albumin (BSA) or 10% nonfat milk powder (bovine lacto transfer optimizer or BLOTTO), diluting sera in sample diluent containing 10% BSA or 10% BLOTTO, and preincubating samples in sample diluent containing 10% BSA or 10% BLOTTO. Overnight preincubation (approximately 12 hr at 2 C) of fresh sera diluted (1:10) in sample diluent containing 10% BLOTTO significantly reduced background and improved the detection of experimentally infected pigs by enhancing positive-negative discrimination. When testing stored sera, the modified protocol effectively reduced the effect of storage and the kit revealed specificity of 98.4%; there was no loss in sensitivity. The effect of long-term storage at -20 C must therefore be considered when testing swine sera for trichinellosis by ELISA and possibly also when conducting other immunoglobulin assays. The modification described here may prove useful if there is no alternative to using serum stored for greater than 3 mo at -20 C.     

秦岭大熊猫精液的细管冷冻保存

2008年3月1日至4月27日和2009年3月3日至5月1日,在陕西省珍稀野生动物抢救饲养研究中心对处于繁殖期内的4只雄性秦岭大熊猫(Ailuropoda melanoleuca qinlingensis)精液进行了细管冻精实验。比较组成不同的4种稀释液:葡萄糖-果糖-柠檬酸三钠-卵黄-甘油-双抗(稀释液1)、葡萄糖-蔗糖-柠檬酸三钠-卵黄-甘油-双抗(稀释液2)、葡萄糖-柠檬酸三钠-卵黄-甘油-双抗(稀释液3)和美国进口的TEST(加入3.5%甘油),以及直接降温平衡法(方法 1)与逐级降温平衡法(方法 2)2种冷冻保存操作方法,对秦岭大熊猫精液进行细管冷冻保存后精子活力和顶体完整率的影响。结果表明:稀释液1的精子活力为46.25%±11.67%,顶体完整率为80.75%±7.89%,TEST的精子活力为48.75%±8.54%,顶体完整率为84.50%±7.59%,两者的精子活力和顶体完整率均无明显差异(P0.05),但是都明显高于稀释液2(P﹤0.01)和稀释液3(P﹤0.01);采用方法 1冷冻保存秦岭大熊猫精液,解冻后精子的活力和顶体完整率分别为45.67%±10.54%和81.37%±8.42%,都显著高于方法 2(P﹤0.01);方法 1解冻后畸形率为23.50%±3.51%,明显低于方法 2(P﹤0.01)。经比较确定,方法 1(用稀释液1)是一种较好的细管冷冻保存秦岭大熊猫精液的方法。     

Thiophilic adsorption revisited

Specific and efficient selection of serum immunoglobulins, but not other proteins, on T-gel remains difficult. T-gel capacity was determined for different activation conditions and serum loadings. Mass spectrometry analysis was used to identify the proteins found in the flow-through and in the eluted fractions. Alpha-2-macroglobulin and albumin were the major contaminants of the eluates. The influence of the competition between immunoglobulins and the other serum proteins on the adsorption was also studied. Using a serum depleted in immunoglobulins (flow-through of a first chromatography on T-gel), many serum proteins were retained on the T-gel, including albumin. We conclude that T-gel selectivity is less than absolute and may reflect for a large part the experimental conditions of the adsorption.     

Cryoelectron microscopy map of Atadenovirus reveals cross-genus structural differences from human adenovirus

A three-dimensional (3D) cryoelectron microscopy reconstruction of the prototype Atadenovirus (OAdV [an ovine adenovirus isolate]) showing information at a 10.6-A resolution (0.5 Fourier shell correlation) was derived by single-particle analysis. This is the first 3D structure solved for any adenovirus that is not a Mastadenovirus, allowing cross-genus comparisons between structures and the assignment of genus-specific capsid proteins. Viable OAdV mutants that lacked the genus-specific LH3 and p32k proteins in purified virions were also generated. Negatively stained 3D reconstructions of these mutants were used to identify the location of protein LH3 and infer that of p32k within the capsid. The key finding was that LH3 is a critical protein that holds the outer capsid of the virus together. In its absence, the outer viral capsid is unstable. LH3 is located in the same position among the hexon subunits as its protein IX equivalent from mastadenoviruses but sits on top of the hexon trimers, forming prominent "knobs" on the virion surface that visually distinguish OAdV from other known AdVs. Electron density was also assigned to hexon and penton subunits and to proteins IIIa and VIII. There was good correspondence between OAdV density and human AdV hexon structures, which also validated the significant differences that were observed between the penton base protein structures.     

Determination of the nucleotide sequence for the penton-base gene of human adenovirus type 5

The major structural proteins of adenovirus (Ad), which form the external capsid, are hexon, penton base and fiber. The primary structure of the Ad5 penton base has been deduced from the nucleotide sequence of the corresponding gene. It has 98.6% homology with the sequence of the analogous protein from Ad2. This result is in contrast with the significantly lower homology found for the two other major structural proteins, the hexon and the fiber.     

Simple purification of immunoglobulins from whey proteins concentrate

We have developed a new protocol with only two steps for purification of immunoglobulins (Ig) from a protein concentrate of whey. Following this protocol, we have an 80% recovery of immunoglobulins, fairly pure. The purification was achieved by eliminating the BSA, via a strong adsorption on DEAE-agarose. Full desoprtion of the other serum proteins could be achieved without contamination with BSA. Thus, a protein solution containing only Ig and very small proteins (e.g., beta-lactoglobulins and alpha-lactalbumin) was obtained. Offering this protein mixture to a lowly activated aminated support, only Ig adsorbed on the support. It has been shown that BSA is able to interact with other proteins (including Ig and lactalbumins). This ability to form complexes with other proteins prevented the success of the direct adsorption of Ig on this mildly activated support, even although Ig should be the largest protein presented in dairy whey.     

Circulating levels of insulin-like growth factors increase and molecular forms of their serum binding proteins change with human pregnancy.

Insulin-like growth factors (IGF) and binding proteins were measured in serum from pregnant and nonpregnant women. IGF-I determined by immunoassay after acid-ethanol extraction was increased by pregnancy (p less than 0.005) and was highest in the third trimester (p less than 0.01). Size exclusion chromatography of serum in acid before assay (i) gave a very similar IGF-I pattern, (ii) showed that IGF-II was much higher than IGF-I and (iii) revealed less serum IGF-binding protein activity in pregnancy and lactation. All IGF-binding proteins except binding protein-1 were markedly reduced by pregnancy. This indicates a major change in the main carrier protein for IGFs in the circulation and suggests that tissue targetting of IGFs may be altered during pregnancy.     

Adsorption and decolorization of dyes using solid residues from Pleurotus ostreatus mushroom production

We investigated the use of solid residues from Pleurotus ostreatus mushroom production in adsorbing and decolorizing different dyes. The solid residue used in this study was composed of hemicellulose and cellulose (52.81 %), acid-insoluble lignin (25.42%), chitin (6.5%), and water extractives (14.82%). After incubating 14% (wt/vol) solid residue in distilled water for 4 h, laccase and manganese peroxidase (MnP) activities were 0.5 U/g and 12 mU/g, respectively. Enzymatic decolorization percentages were up to 100 for azure B (heterocyclic dye) and indigo carmine (indigoid dye), 74.5 for malachite green (MG) (triphenylmethane dye), and zero for xylidine (azoic dye). The optimum temperature for decolorization was in the range of 26 ∼ 36°C for all dyes. Data obtained on adsorption (enzymatic decolorization was prevented with sodium azide) at different dye concentrations and in a pH range of 3 ∼ 7 were used to plot Freundlich isotherms. The spent fungal substrate (SFS) displayed large differences in adsorption capacity, depending on the dye tested. The highest adsorption capacity was observed at pH 3 for MG, while xylidine was slightly adsorbed at pH 3 and 4 and not adsorbed at higher pH values. Laccase and MnP production were affected by the presence of the dyes. The highest enzyme levels were observed in the presence of MG, when laccase and MnP increased 1.39- and 2.13-fold, respectively. Decolorization and adsorption to SFS are both important processes in removing dyes from aqueous solutions. The application of this spent substrate for wastewater treatment will be able to take advantage of both of these dye removal processes. An important problem in bioremediation processes involving microorganisms is the amount of time required for their growth. In this report, we used the spent substrates from mushroom cultivation in wastewater treatment, thus solving the problem of waiting for microorganisms to grow.     

Use of nonfat dry milk to block nonspecific nuclear and membrane staining by avidin conjugates

The use of fluorescein-avidin or rhodamine-avidin conjugates in conjunction with biotinylated secondary antibodies for indirect immunohistology frequently results in pronounced nonspecific nuclear staining in kidney sections. This nonspecific nuclear staining can be effectively blocked by using 5% (w/v) nonfat dry milk in buffered saline as the diluent for the avidin conjugates. In contrast, 5% (w/v) bovine serum albumin, a commonly used blocking agent, has only a modest effect. Nonfat dry milk is also effective as a blocking agent and carrier when used in fluorescence-activated cell sorter (FACS) analysis. These results emphasize the broad usefulness of milk-based blocking reagents.     

Chimeric hexon HVRs protein reflects partial function of adenovirus

Adenovirus is widely used in gene therapy and vaccination as a viral vector, and its hypervariable regions (HVRs) on hexon are the main antigen recognition sites of adenovirus. The modification of this area by genetic engineering will change the antigenic specificity of the virus. In addition, recent studies have demonstrated the importance of coagulation factor X (FX) in adenovirus serotype 5-mediated liver transduction in vivo. The binding site of adenovirus to FX is the HVRs on hexon. By constructing five proteins containing chimeric HVRs from different adenovirus serotypes, we focused on the antigenic specificity and the affinity for FX of these proteins compared with the corresponding viruses. Our data showed that HVR5 and HVR7 had only a part of hexon activity to neutralizing antibodies (NAbs) compared with the complete activity of HVR1-7. Results also demonstrated a differential high-affinity interaction of the HVRs proteins with FX and indicated that HVRs protein had a similar binding ability with corresponding adenovirus serotype. These results highlighted some properties of chimeric HVRs proteins and revealed the influence on the structure and function of hexon proteins and adenovirus resulting from the HVRs.