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The United States of America As Represented by the Secretary of

The United States of America As Represented by the Administrator of the

The United States of America As Represented by the Department of Health

Grace L Picciolo

Emmett W Chappelle

David S Kaplan

James C Fletcher

Michael J Barza

Louis Weinstein

Jody W Deming

Hillar Vellend

Robert A Frosch

Richard R Thomas

Eldon L Jeffers

Edward P Mueller

Stephanie A Tuttle

Christian G Shrock

A chemiluminescence method for continuously monitoring in real time the generation of oxidative products such as hydrogen peroxide and superoxide from in vitro cell-biomaterial interactions using cell lines such as Human Leukemic cells (HL-60); tumor cell line hybridomas; cells lacking the respiratory burst such as Chronic Granulomatous Disease cells as controls; Monocytic cell lines; Primary Human cells such as monocytes, pmns, fibroblasts, endothelial cells; and whole and isolated blood cells. The oxidative products have the potential for the degradation of the biomaterial and thus this information can be used to aid in predicting the functional lifetime of the biomaterial when it is used to fabricate an implanted medical device. Further, this method may be used to determine the amount of activation of biological cells which is inferred from the amount of oxidative products. This activation level aids in the determination of the degree of the inflammatory response and thus influences the degree of acceptance of the biomaterial. Thus, toxicity and biocompatability tests could be supported by these measurements.

Real-time monitoring of oxidative products from in vitro

The present invention discloses a device and a process for quantitation of Toxoplasma gondii and Treponema pallidum antibody titer in a biological sample by immunofluorescent photometric microscopy. The process comprises:

Device for quantitative endpoint determination in immunofluorescence

The present invention discloses a device and a process for quantitation of end point in the formation of fluorescent reaction product in microfluorophotometry. The process comprises:

Method and device for quantitative end point determination in

A bioluminescent assay for ATP in water borne bacteria is made by adding nitric acid to a water sample with concentrated bacteria to rupture the bacterial cells. The sample is diluted with sterile, deionized water, then mixed with a luciferase-luciferin mixture and the resulting light output of the bioluminescent reaction is measured and correlated with bacteria present. A standard and a blank also are processed so that the light output can be correlated to bacteria in the sample and system 'noise' can be substracted from the readings. A chemiluminescent assay for iron porphyrins in water borne bacteria is made by adding luminol reagent to a water sample with concentrated bacteria and measuring the resulting light output of the chemiluminescent reaction. The light output is correlated with bacteria present. A standard and a blank are also processed so that the light output can be correlated to bacteria in the sample and system 'noise' can be subtracted from the readings. Modifications may be made in the methodology to differentiate between live and dead bacteria. An automatic system automatically performs a biolumenscent ATP assay on a concentrated bacterial sample. Reservoirs are provided for the sample, standard and blank. These are sequentially mixed with nitric acid from a reservoir by using two channels of a peristaltic pump. This acid mixture is then mixed with sterile, dionized water from another reservoir using two additional channels of a peristaltic pump and the resulting mixture is then mixed with a luciferase-luciferin mixture from an additional reservoir by employing two more channels of a peristaltic pump. The resulting solution flows through a photometer which indicates the level of the bioluminescent light reaction.

Rapid, quantitative determination of bacteria in water

Method for the quick determination of the susceptibilities of various unidentified bacteria contained in an aqueous physiological fluid sample, particularly urine, to one or more antibiotics. A bacterial adenosine triphosphate (ATP) assay is carried out after the elimination of non-bacterial ATP to determine whether an infection exists. If an infection does exist, a portion of the sample is further processed, including subjecting parts of the portion to one or more antibiotics. Growth of the bacteria in the parts are determined, again by an ATP assay, to determine whether the unidentified bacteria in the sample are susceptible to the antibiotic or antibiotics under test.

Determination of antimicrobial susceptibilities on infected urines

The susceptibility of bacteria, particularly those derived from body fluids, to antimicrobial agents is determined in terms of an ATP index measured by culturing a bacterium in a growth medium, assaying the amount of ATP in a sample of the cultured bacterium by measuring the amount of luminescent light emitted when the bacterial ATP is reacted with a luciferase-luciferin mixture, subjecting the sample of the cultured bacterium to an antibiotic agent and assaying the amount of bacterial adenosine triphosphate after treatment with the antibiotic by measuring the luminescent light resulting from the reaction, whereby the ATP index is determined from the values obtained from the assay procedures.

Application of luciferase assay for ATP to antimicrobial drug

An improved method is provided for determining bacterial levels, especially in samples of aqueous physiological fluids, which method depends on the quantitative determination of bacterial adenosine triphosphate (ATP) in the presence of non-bacterial ATP. The bacterial ATP is released by cell rupture and is measured by an enzymatic bioluminescent assay. A concentration technique is included to make the method more sensitive. It is particularly useful where the fluid to be measured contains an unknown or low bacteria count.

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Fort Washington, MD, United States of America

Grace L Picciolo