A novel approach to enhance antibody sensitivity and specificity by peptide cross-linking

Most current techniques employed to improve antigen-antibody signals in Western blotting and in immunohistochemistry rely on sample processing prior to staining (e.g., microwaving) or using a more robust reporter (e.g., a secondary antibody with biotin-streptavidin). We have developed and optimized a new approach intended to stabilize the complexes formed between antigens and their respective primary antibodies by cupric ions at high pH. This technique improves the affinity and lowers cross-reactivity with nonspecific bands of approximately 20% of antibodies tested (5/25). Here we report that this method can enhance antigen-antibody specificity and can improve the utility of some poorly reactive primary antibodies.     

A thermodynamic approach to PCR primer design

We developed a primer design method, Pythia, in which state of the art DNA binding affinity computations are directly integrated into the primer design process. We use chemical reaction equilibrium analysis to integrate multiple binding energy calculations into a conservative measure of polymerase chain reaction (PCR) efficiency, and a precomputed index on genomic sequences to evaluate primer specificity. We show that Pythia can design primers with success rates comparable with those of current methods, but yields much higher coverage in difficult genomic regions. For example, in RepeatMasked sequences in the human genome, Pythia achieved a median coverage of 89% as compared with a median coverage of 51% for Primer3. For parameter settings yielding sensitivities of 81%, our method has a recall of 97%, compared with the Primer3 recall of 48%. Because our primer design approach is based on the chemistry of DNA interactions, it has fewer and more physically meaningful parameters than current methods, and is therefore easier to adjust to specific experimental requirements. Our software is freely available at http://pythia.sourceforge.net.     

A new approach to apple proliferation detection: a highly sensitive real-time PCR assay

The present paper describes a new approach for diagnosis of apple proliferation (AP) phytoplasma in plant material using a multiplex real-time PCR assay simultaneously amplifying a fragment of the pathogen 16S rRNA gene and the host, Malus domestica, chloroplast gene coding for tRNA leucine. For the first time, such an approach, with an internal analytical control, is described in a diagnostic procedure for plant pathogenic phytoplasmas enabling distinction between uninfected plant material and false-negative results caused by PCR inhibition. Pathogen detection is based on the highly conserved 16S rRNA gene to ensure amplification of different AP phytoplasma strains. The newly designed primer/probe set allows specific detection of all examined AP strains, without amplifying other fruit tree phytoplasmas or more distantly related phytoplasma strains. Apart from its specificity, real-time PCR with serial dilutions of initial template DNA ranging over almost five orders of magnitude (undiluted to 80,000-fold diluted) demonstrated linear amplification over the whole range, while conventional PCR showed a reliable detection only up to 500-fold or 10,000-fold dilutions, respectively. Compared to existing analytical diagnostic procedures for phytoplasmas, a rapid, highly specific and highly sensitive diagnostic method becomes now available.     

A new approach to primer design for the control of PCR bias in methylation studies

Background

Nerve growth factor (NGF) helps in the healing and survival of ganglion cells, photoreceptors, and optic nerve after injury and has been implicated to have a role in pathophysiology of glaucoma. So far, in animal studies, injury to iris in vitro has revealed an increase in NGF levels in aqueous. There is a great interest in investigating the levels of NGF in human aqueous in glaucomatous eyes, as suggested by animal studies, to gain a better understanding of the pathophysiology of glaucoma.

Findings

In this study, we examined the presence of NGF levels in aqueous humor collected from human eyes and the limitations in determining the NGF levels in human samples. NGF was assessed by ELISA immunoassay in undiluted aqueous samples collected from 32 consecutive patients undergoing surgery for cataract (control) or primary open angle glaucoma (POAG). Recombinant NGF was used as positive control. NGF levels were below undetectable levels in aqueous humor from eyes with POAG and controls by immunoassay. Less than 10% of samples had detectable NGF levels and these were considered outliers.

Conclusion

Our result highlights the undetectable levels of NGF in human aqueous samples.     

A quantum-theoretic approach to enzyme specificity: Proposals for a new type of biochemical control mechanism

A quantum-theoretic approach to the problem of enzymic specificity is presented. The concept of a “measuring system” analogized with the enzyme is utilized. Along these lines a quantum mechanical hypothesis for the mechanism of enzyme reactions was advanced (Enzymologia,35, 117–130, 1968). In the measuring process an ℐ-observable, linked to the proper values of the substrate'sA ₁...A _m observables will indicate the state of the measuring apparatus. On the corresponding Hilbert space of the enzymesubstrate complexH _ES , through the respective statistical operatorU _ES we get a “state” vector [s, a]. Theng:Γ _S xΓ _E →Γ _ES , that is, to an oriented pair 〈s, a〉 ∈Γ _S xΓ _E will correspond a “state” vector [s, a], and to a proper valuei _k of ℐ will correspond, throughg, the respective equivalence classes of Г-spaces. Introducing the concept of enzyme-substrate complex entropyL _ES , a theorem is demonstrated asserting thatL _S =L _ES -L _E + 1/2kn wheren is the number of the degrees of freedom which may fluctuate. The values ofL _ES are denoted “specific values,” and it is demonstrated that a microphysical systemS may be a substrate specific forE, if and only if it can realize one of theL _ES specific values. Along these lines a model of a stochastic process for the enzymic reaction is constructed, and the set of Kolmogorov equations for the respective probabilities is derived. When a “perturbation” is induced in our model, an interesting prediction concerning the fluctuations in the kinetics of the corresponding enzyme is obtained. A relevant experimental proof ensued from these theoretical considerations. When a “gentle perturbation” was induced in a substrate by mild X-ray or UV-irradiations, an interesting oscillatory behavior of enzymic activity was recorded. A biochemical control mechanism is constructed (a simple “flip-flop” type) utilizing nonoverlapping oscillations in the activity of two enzymes at a key metabolic intermediate level. Eventually, a relevant experimental proof for the respective control model is presented.     

A new approach to microhomogenization

A versatile microhomogenizer is described which is capable of handling sample volumes ranging from 1 μl to several millititers. The sample and homogenization buffer are sealed within a segment of flexible plastic tubing. The sample is disrupted by rubbing or rolling a metal rod back and forth over the length of tubing. Centrifugation of the homogenate is performed in the same sealed tubing segment. This method can be used to homogenize single cells.     

A new approach to extending the wheat marker pool by anchored PCR amplification of compound SSRs

A study was undertaken to determine the utility in bread wheat of anchored PCR for the development of single locus SSR markers targeted at compound repeat motifs. In anchored PCR, microsatellite amplification is achieved using a single primer complementary to the flanking sequence, and one which anchors to the repeat junction of the compound SSR. The recovery rate of useable markers was found to be similar (43%) to that reported for conventionally generated SSRs. Thus, anchored PCR can be used to reduce the costs of marker development, since it requires that only half the number of primers be synthesised. Where fluorescence-based platforms are used, marker deployment costs are lower, since only the anchoring primers need to be labelled. In addition, anchored PCR improves the recovery of useful markers, as it allows assays to be generated from microsatellite clones with repeat sequences located close to their ends, a situation where conventional PCR amplification fails as two flanking primers cannot be designed. Strategies to permit the large-scale development of compound SSR markers amplified by anchored PCR are discussed.Communicated by P. Langridge     

A new approach to enhance reproductive performance in sheep using royal jelly in comparison with equine chorionic gonadotropin

The objective was to compare the effects of royal jelly (RJ) and eCG treatments on reproductive performance of ewes synchronized using intravaginal progesterone-releasing devices. Forty-two cycling Awassi ewes were treated intramuscularly (i.m.) with 15 mg PGF2alpha. On the following day, all ewes were administered with CIDR-G for 12 days and were randomly allocated to three (RJ, eCG and control) groups of 14 ewes each. Ewes in the RJ-treated group received daily i.m. treatments of 400mg RJ during the period of CIDR-treatment. Each ewe in the eCG-treated group received an i.m. treatment of 500 IU eCG at the time of CIDR-G removal (day 0) and no further treatment was given to ewes in the control group. Ewes were exposed to four fertile rams for 72 h, from the time of CIDR-G removal, and checked for breeding marks at 6-h intervals. Blood samples were collected from day -13 until day 0 and thereafter until day 19 for progesterone analysis. Royal jelly treatment resulted in a greater rate of decline and lower (P<0.02) progesterone concentrations between days -10 and 0 than eCG-treated and control ewes. Expression of estrus was similar among the three groups and intervals to onset of estrus were shorter (P<0.01) in RJ-treated (31.3h) and eCG-treated (29.8h) than control (41.3h) ewes. First-cycle pregnancy and lambing rates were greater (P<0.05) in RJ-treated (71.4 and 71.4%) and eCG-treated (85.7 and 78.6%) than in control (42.9 and 35.7%) ewes, respectively. Results demonstrate that the treatments of RJ and eCG in conjunction with CIDR-G were similarly effective in induction of estrus and improvement of pregnancy and lambing rates.     

A new approach to sympatric speciation

Sympatric speciation has always been a controversial issue. Recently, the debate has centred around the possibility that host race formation in phytophagous insects occurs as a first stage in this process. Now a new approach to host race formation - examining the population genetics of performance on alternative hosts - is challenging a major underlying assumption of this model. The evidence suggests that improved performance on one host is not generally accompanied by reduced performance on others.     

A new approach to microtubule depolymerization

Microtubule thermal denaturation is treated in the framework of the Ising model. The partition function is calculated with a new iterative procedure which allows us to calculate values of the denaturation curves and enthalpy changes in excellent agreement with the experimental data, We also obtain the excess heat capacity due to polymerization, OCp.     

A new approach to immunoFET operation

A new method is presented for the detection of an immunological reaction taking place in a membrane, which covers the gate area of an ISFET. By stepwise changing the electrolyte concentration of the sample solution, a transient diffusion of ions through the membrane-protein layer occurs, resulting in a transient membrane potential, which is measured by the ISFET. The diffusion rate is determined by the immobile charge density in the amphoteric protein layer, which changes upon formation of antibody-antigen complexes. No membrane potential is induced at zero fixed charge density as occurs at a protein characteristic pH. Isoelectric points of embedded proteins can be determined by detecting the zero potential response. Up to now, the authors have studied the membrane adsorption of lysozyme, human serum albumin (HSA) and the immune reaction of HSA with the antibody anti-human serum albumin (alpha HSA). The influence of protein parameters on the amplitude of the transient can be described with an empirical equation. Assuming Langmuir behaviour, the protein concentration in the solution can well be correlated with the concentration in the membrane. This new detection method is unique concerning direct measurements of charge densities and isoelectric points of amphoteric macromolecules adsorbed in the membrane. The simple procedure of one incubation stage followed by one detection stage, without separate washing and labelling techniques, gives direct information about specific charge properties of the macromolecules to be studied.     

A new approach to biogeographical subdivision

The authors propose a new approach permitting subdivision of a water area into sectors, each including species with a similar biogeographical status. This approach relies on computer methods for construction of maps presenting the spatial distribution of continuous fields. The method is illustrated by data on the nekton from the north-western Sea of Japan. Different modifications of the method and prospects of its further development are discussed.