Fluorescent antibody method

Был разработан спектрофотометрический метод количественного определения белка и 1-диметиламинонафтален-5-сульфоновой кислоты в флюоресцентных антителах. Выяснилось, что после основательного диализа коньюгаты не содержат свободного красителя, который был бы помехой определений. В зависимости от условий коньюгации среднее отношение веса белка к флюоресцентному красителю в меченом γ-глобулине составляло 76–245, что отвечает 3–9 молекулам красителя, связанным с 1 молекулой глобулина.     

Fluorescent antibody method

Гыл синтезирован флюоресцентный краситель 1-диметиламинонафтален-5-суляфонил-хлорид и исследовались условия его коньюгации с белка.ми. Концентрация белков и ионная сила раствора в реакцcии не оказывали суцественного влияния на интенсивность связывания, тогда как деиствие pH было противоположным. При всех опытах приименялся одии вид гамма-глобулина, иммунологические свойства которого в результате химической интеракции с данзилхлоридом не менялися. С 1 молекулой гамма-глобулина связывались—В в зависимости от условий опыта—3—9 молекул флюооресцирующего красителя. При длительном отстаивании комплекса белки-краситель при +4° C на-блюдалась неболящая диссоциация, которая не имела существенного влияния на красяцую способностя кончюгата.     

Fluorescent antibody demonstration ofPasteurella tularensis

The authors studied certain factors determining the possibility of the demonstration ofPasteurella tularensis by the immunofluorescence technique and the sensitivity, specificity and reliability of the reaction under experimental conditions. On the basis of the results they regard this method as suitable for rapid microbiological diagnosis (both for demonstratingPasteurella tularensis in material from patients and infected animals and for detecting antibodies in the serum of convalescents). The next step will be to study in greater detail the nature of the antigens and antibodies participating in the reaction and to verify the method in actual practice.     

Fluorescent probes in membrane studies.

A number of spectroscopic techniques are suitable for studying biological membranes. Of these, fluorescence has the sensitivity and time resolution for following membrane events associated with nerve excitation. In this paper, the nature of the information derived from measurements of the fluorescence properties of externally introduced chromophores in membranes is examined. In particular, the locations of various probes are described on the basis of nuclear magnetic resonance (n.m.r.) experiments in model situations. Then the motional characteristics of the probe molecules (rotation and diffusion) are discussed. Finally experiments designed to relate the detailed observations that can be made in lipid bilayers using n.m.r. and fuorescence measurements to those (more limited in nature) that can be made in membranes are described.     

Fluorescent labeling of antibody fragments using split GFP

Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs) using the split green fluorescent protein (GFP) system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11), is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems.     

Hypomorphic lethal mutations and their implications for the interpretation of lethal complementation studies in Drosophila

In a small region of the X chromosome of Drosophila melanogaster, we have found that a third of the mutations that appear to act as lethals in segmental haploids are viable in homozygous mutant individuals. These viable mutations fall into four complementation groups. The most reasonable explanation of these mutations is that they are a subset of functionally hypomorphic alleles of essential genes: hypomorphic mutations with activity levels above a threshold required for survival, but below twice that level, should behave in this manner. We refer to these mutations as "haplo-specific lethal mutations." In studies of autosomal lethals, haplo-specific lethal mutations can be included in lethal complementation tests without being identified as such. Accidental inclusion of disguised haplo-specific lethals in autosomal complementation tests will generate spurious examples of interallelic complementation.     

Tumour necrosis factor alpha antibody protects against lethal meningococcaemia

Tumour necrosis factor alpha (TNF-alpha) has been shown to be the principal mediator of Gram-negative bacterial endotoxin-induced shock. Nevertheless, evidence suggests that TNF-alpha plays a beneficial role in controlling bacterial infections when multiplication of the microorganism is required to kill the host. Using an infant rat model of Neisseria meningitidis infection, we found that blood TNF-alpha concentration reaches a peak three hours after intraperitoneal injection of 3 x 10(6) bacteria. Thereafter, the level of TNF-alpha decreased and was undetectable six to eight hours after infection. A correlation was observed between the magnitude of initial TNF-alpha response and a fatal outcome. Pretreatment of the animals with polyclonal anti-TNF antiserum significantly reduced mortality relative to animals pretreated with control serum. However, pretreatment of animals with anti-TNF antibody did not alter the bacterial invasion of the cerebrospinal fluid. Injection of heat-killed bacteria did not cause death and induced lower TNF-alpha levels than the same number of live bacteria. This excludes the possibility that the role of TNF-alpha is to mediate a shock induced by the endotoxin component of the bacterial inoculum. These results indicate that TNF-alpha has a deleterious effect in this model of bacteraemia. Identification of the critical factors that determine the action of TNF-alpha during lethal bacteraemia will lead to a better understanding of these diseases and the development of appropriate therapeutic intervention.