Protein a quantitation by competitive ELISA using penicillinase

Summary A simple, sensitive and specific competitive ELISA method using penicillinase as a marker for quantitation of protein A has been developed. The sensitivity of the assay is 20 ng/ml. This method is useful for estimating the protein A concentration in fermented and extracted samples.     

Control of vesicle fusion by a tyrosine phosphatase

The tyrosine phosphatase PTP-MEG2 is targeted by its amino-terminal Sec14p homology domain to the membrane of secretory vesicles. There it regulates vesicle size by promoting homotypic vesicle fusion by a mechanism that requires its catalytic activity. Here, we identify N-ethylmaleimide-sensitive factor (NSF), a key regulator of vesicle fusion, as a substrate for PTP-MEG2. PTP-MEG2 reduced the phosphotyrosine content of NSF and co-localized with NSF and syntaxin 6 in intact cells. Furthermore, endogenous PTP-MEG2 co-immunoprecipitated with endogenous NSF. Phosphorylation of NSF at Tyr 83, as well as an acidic substitution at the same site, increased its ATPase activity and prevented alphaSNAP binding. Conversely, expression of a Y83F mutant of NSF caused spontaneous fusion events. Our results suggest that the molecular mechanism by which PTP-MEG2 promotes secretory vesicle fusion involves the local release of NSF from a tyrosine-phosphorylated, inactive state. This represents a novel mechanism for localized regulation of NSF and the first demonstrated role for a protein tyrosine phosphatase in the regulated secretory pathway.     

Development of competitive ELISA for neosporosis by employing immunoproteomics

In this study, proteomics was used to explore the antigenic proteins that are involved in cross-reactivity during serodiagnosis between Neospora caninum (N. caninum) and Toxoplasma gondii (T. gondii). Competitive enzyme-linked immunosorbent assay (C-ELISA) developed by proteomics shed a new light on the infection of N. caninum. Cross-reactivity of antigenic proteins between N. caninum and T. gondii tachyzoites was explored by using the conventional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (1-DE) and two-dimensional gel electrophoresis (2-DE) immunoblot. The proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The protein expression patterns in the immunoblot profiles of N. caninum were similar to bovine, chicken, and rabbit anti-N. caninum serum, but they were not similar to rabbit anti-T. gondii serum. Band at 79 kDa, HSP70, and actin on immunoblot profiles reacted, in general, with bovine, chicken, and rabbit anti-N. caninum serum and also with rabbit anti-T. gondii serum, respectively. Whereas the band at 144 kDa, and NCDG-1 were detected on bovine, chicken, and rabbit anti-N. caninum immunoblot profiles, they were not observed on rabbit anti-T. gondii immunoblot profile. These specific antigenic proteins were recorded as species-specific proteins of N. caninum against T. gondii. Based on the proteome analysis, C-ELISA was developed to screen the cattle infected with N. caninum by using N. caninum tachyzoite lysate as a coating antigen and chicken anti-N. caninum immunoglobulin (Ig)Y as a competitor. C-ELISA was able to detect the antibody of N. caninum without cross-reactivity with T. gondii. Furthermore, it achieved a fine diagnostic performance in the cases of 162 bovine sera.     

A competitive inhibition ELISA as a probe for studying the refolding of human serum albumin

The refolding of iodoacetic acid-blocked human serum albumin (HSA) was studied using a modified competitive inhibition ELISA. A maximum of 89% native activity was detected 24 hours after initiating refolding using an albumin concentration of 600 micrograms/mL. The presence of both monomer and polymer HSA was studied using native polyacrylamide gel electrophoresis of thiol-blocked HSA samples. Monomer HSA was not detected until 2.5 hours after initiating refolding. Fractionated polymer and monomer HSA from a sample trapped at 72 hours after initiating refolding was determined to have 40% and 87% native activity respectively. Both polymer and monomer HSA fractions contribute to the overall immunological activity detected by the ELISA, at various times. The ELISA assay was able to detect the changing HSA conformation associated with refolding of totally reduced HSA.     

Paper-based ELISA diagnosis technology for human brucellosis based on a multiepitope fusion protein

BackgroundBrucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable.Methodology/Principal findingsIn this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen.ConclusionsB cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations.     

Guanine nucleotide exchange factors operate by a simple allosteric competitive mechanism

Guanine nucleotide exchange factors are essential components of the mode of action of GTPases involved in signal transduction. Their fundamental mechanism is generally accepted to derive from stabilization of the nucleotide-free form of GTPases, which is reflected in an increase in the rate of GDP dissociation when such an exchange factor is bound to a GTPase. The known kinetic properties of exchange factors can be explained on the basis of this simple allosteric competitive mechanism. Here, we describe experiments designed to distinguish this mechanism from a newer model, which invokes an active role for the incoming (i.e., displacing) nucleotide, implying the transient formation of a quaternary complex consisting of an exchange factor, a GTPase, and two nucleotides, one which is being displaced while the other stimulates this displacement. We show that for a well-known system (the small GTPase Ras and its exchange factor Cdc25) there is no evidence for an effect of the concentration or the nature (i.e., GDP or GTP) of the displacing nucleotide on the rate constant of GDP release from the Cdc25.Ras.GDP complex, consistent with the simple allosteric competitive model, and in disagreement with the newer suggestion. In addition, we present arguments, which demonstrate how the erroneous conclusions leading to the alternative model were derived.     

Folic acid-decorated nanocomposites prepared by a simple solvent displacement method

A biodegradable nanocarrier system based on PLGA applicable for FR targeting is described. PEI-based conjugates with covalently coupled folic acid are synthesized, characterized with regard to their composition and used for DNA complexation. The preparation of composites is performed by a solvent displacement technique, assuming an electrostatic interaction of PEI-based polyplexes with PLGA. The synthesis of a folic acid-PEG3kDa-PEI25kDa conjugate is achieved. Blending of PLGA with polyplexes results in spherical nanoparticles with sizes ≤ 250 nm. Incorporation of polyplexes and the localization of folic acid on the particle surface, performed by antibody binding, is confirmed. The method is suitable for the preparation of nanosized, folic-acid-decorated nanoparticles.     

Use of oligonucleotide-alkaline phosphatase conjugates as non-radioactive probes for rapid analysis of a proteinase inhibitor gene fromZea mays

Conjugates of oligonucleotides and alkaline phosphatase have been prepared and used as nonradioactive hybridization probes for the study ofPis3 (=MPI) a gene encoding a proteinase inhibitor fromZea mays. Attachment of the alkaline phosphatase was carried out either at the 5′ or 3′ end of two 25-bp oligonucleotides. Sensitivity of each alkaline phosphatase-oligonucleotide probe was assessed using a chemiluminescent substrate for detection of alkaline phosphatase activity. This sensitive method allows the rapid analysis of genomic clones isolated from aZea mays library and the subsequent characterization of the completePis3 gene without the need for construction of restriction maps for the cloned DNA fragments. This general strategy may be valuable for the identification of any gene for which a limited sequence is known and for location of specific DNA sequences that represent a small region within a larger DNA fragment.     

Secretion of human interferon-alpha induced by using secretion vectors containing a promoter and signal sequence of alkaline phosphatase gene of Escherichia coli

We constructed a new vector containing the promoter and the signal sequence of E. coli phoA gene, the structural gene for the periplasmic alkaline phosphatase. One of the most useful characteristics of this vector is the unique HindIII restriction site located just at the end of the phoA signal sequence. This restriction site was generated by oligonucleotide-directed site-specific mutagenesis without changing the amino acid sequence of the signal peptide. Any kind of foreign structural gene can be easily inserted into the HindIII site by using synthetic oligonucleotides to construct a hybrid gene which has neither an extra sequence nor a deletion between the phoA signal sequence and the foreign structural gene. Human alpha-interferon gene was inserted into this HindIII site. When this hybrid gene was expressed under the control of the phoA promoter region, a low but significant activity was recovered in the cold water wash of the cells after an osmotic shock procedure.     

The isolation and partial sequencing of human bone alkaline phosphatase gene

1. A charon 4A human fetal liver genomic library was screened for human alkaline phosphatase sequences using the cloned human bone cDNA as a hybridization probe. 2. A positive clone was obtained and then characterized by restriction endonuclease cleavage analysis, hybridization experiments and partial DNA sequencing.     

Detection of glycosylated and deglycosylated extensin precursors by indirect competitive ELISA

As part of their defense mechanism against herbivores or phytophagous insects, many plant tissues contain lectins. Some of these lectins are potent toxins which kill animal cells by arresting protein synthesis. An attractive strategy for developing specifically cytotoxic chemotherapeutic agents is to link cell type-specific monoclonal antibodies to potent toxins. The plant protein ricin has emerged as the toxin of choice for such constructs.     

The EWS-Oct-4 fusion gene encodes a transforming gene

The t(6;22)(p21;q12) translocation associated with human bone and soft-tissue tumours results in a chimaeric molecule fusing the NTD (N-terminal domain) of the EWS (Ewing's sarcoma) gene to the CTD (C-terminal domain) of the Oct-4 (octamer-4) embryonic gene. Since the N-terminal domains of EWS and Oct-4 are structurally different, in the present study we have assessed the functional consequences of the EWS-Oct-4 fusion. We find that this chimaeric gene encodes a nuclear protein which binds DNA with the same sequence specificity as the parental Oct-4 protein. Comparison of the transactivation properties of EWS-Oct-4 and Oct-4 indicates that the former has higher transactivation activity for a known target reporter gene containing Oct-4 binding. Deletion analysis of the functional domains of EWS-Oct-4 indicates that the EWS (NTD), the POU domain and the CTD of EWS-Oct-4 are necessary for full transactivation potential. EWS-Oct-4 induced the expression of fgf-4 (fibroblast growth factor 4) and nanog, which are potent mitogens as well as Oct-4 downstream target genes whose promoters contain potential Oct-4-binding sites. Finally, ectopic expression of EWS-Oct-4 in Oct-4-null ZHBTc4 ES (embryonic stem) cells resulted in increased tumorigenic growth potential in nude mice. These results suggest that the oncogenic effect of the t(6;22) translocation is due to the EWS-Oct-4 chimaeric protein and that fusion of the EWS NTD to the Oct-4 DNA-binding domain produces a transforming chimaeric product.     

Quantification of human angiotensinogen by a novel sandwich ELISA

The urinary angiotensinogen excretion rates show a clear relationship to kidney angiotensin II content, suggesting that urinary angiotensinogen may serve as an index of angiotensin II-dependent hypertensive rats. However, simple and accurate methods to measure human angiotensinogen are unavailable at this time. We have developed two antibodies and a sensitive and specific quantification ELISA system for human angiotensinogen to be applicable to human subjects. The ELISA is able to detect human angiotensinogen at range of 0.01-1 microg/well (R(2)=0.9945) using standard ELISA plates. This ELISA will be a useful tool to investigate the relationship between urinary angiotensinogen excretion rates and reactivity to antihypertensive drugs in hypertensive human subjects.     

Chemical synthesis and expression in E. coli of a human Val8-calcitonin gene by fusion to a synthetic human interferon-gamma gene

A gene coding for human Val8-calcitonin (Val8-hCT) was synthesized by the solid-phase phosphite approach and fused to a synthetic human immune interferon-gamma (IFN-gamma) gene. The IFN gene was previously shown to be expressed at a very high level in E. coli [(1986) Gene, in press] due to the control of a strong synthetic promoter and strong ribosome binding site. The cells harboring the fused gene produced 100-150 micrograms per l of bacterial suspension of immunoreactive calcitonin in the form of hybrid IFN-gamma-Val8-hCT protein consisting of 140 amino acids. The Val8-hCT can be released from this protein by CNBr treatment.     

Structure of human prostatic acid phosphatase gene.

Two cDNA clones containing the complete protein-coding sequence of 1,188 nucleotides as well as the 5' and 3' non-coding regions of human prostatic acid phosphatase (PAP) were isolated and sequenced. The size of PAP mRNAs from benign prostate hyperplasia and cancerous prostate was estimated to be 3.2Kb, indicating that the 3' downstream polyadenylation signal was used. Several genomic clones containing parts of the human PAP gene were isolated and the nucleotide sequence of ten exons and their flanking regions was determined. The protein-coding sequence of the human PAP gene was interrupted by nine introns. The positions of all nine introns present in the human PAP gene were homologous to those of the first nine introns in the human lysosomal acid phosphatase (LAP) gene. However, the last (11th) exon of the LAP gene encoding the COOH-terminal domain, which includes a transmembrane segment, was found to be absent in human PAP gene. Southern blot analysis of ten mammalian genomic DNAs gave multiple EcoRI fragments. The data of human genomic DNAs were consistent with the total length of the PAP gene of at least 50 kilobases.     

Immunological characterization of human acid phosphatase gene products

The immunological cross-reactivity of heterogeneous acid phosphatase isozymes from different human tissues has been studied using monospecific antisera prepared against four homogeneous acid phosphatases. The enzyme characterized as tartrate-inhibitable, prostatic acid phosphatase is also found to be present in leukocytes, kidney, spleen, and placenta. The tartrate-inhibitable (liver) lysosomal enzyme is also found in kidney, fibroblasts, brain, placenta, and spleen, but it is not detectable in erythrocytes and prostate. In several tissues, 10–20% of the tartrate-inhibitable enzyme is not precipitated by any of the antisera used; an exceptionally high amount (54%) of such an enzyme is present in human brain. Antiserum against a low molecular weight tartrate-resistant liver enzyme (14 kDa) does not cross-react with the erythrocyte enzyme. (10–20 kDa). All other tissues except placenta, prostate, and fibroblast cells show a cross-reactivity with the 14-kDa acid phosphatase antiserum. Thus, the low molecular weight human liver acid phosphatase is distinct from the erythrocyte enzyme, and there are also at least three different tartrate-inhibitable acid phosphatases in human tissues. Chromosomal assignments have been made for only two of the (at least) five acid phosphatases that are present in adult human tissues.This study was supported by DHHS Research Grant GM 27003 from the U.S. National Institute of General Medical Sciences and by Grant SFB-104 from the Deutsche Forschungsgemeinschaft.     

Detection of trace aflatoxin M1 in human urine using a commercial ELISA followed by HPLC

Aflatoxin is a liver carcinogen, and rapid, inexpensive methods to detect its urinary biomarkers are needed. We used a commercial enzyme-linked immuno-sorbent assay (ELISA) for aflatoxin M1 in urine (Helica Biosystems) to test 52 Haitian samples. Using this ELISA, we detected traces above the limit of detection (0.2?ng/ml urine) but below the limit of quantitation (0.4?ng/ml) in 14 samples. Liquid chromatography of all 52 Haitian urine samples revealed that only 11 had quantifiable AFM1 (mean: 29.5?pg/ml, standard error: 10.8, range: 2.94–96.5?pg/ml). The Helica ELISA may have detected forms of aflatoxin other than AFM1 in the Haitian samples, or matrix enhancement may have affected results at low AFM1 concentrations. This ELISA may serve as an initial, qualitative indicator of aflatoxin exposure for epidemiological purposes. But this method’s utility as a precise and specific indicator of AFM1 concentrations will require additional refinement and validation.