Urease label

Bovine serum albumin (BSA) and cyclic AMP (cAMP) are determined by a competitive binding enzyme immunoassay (315). With urease as label, an ammonia gas-sensing electrode is used to measure the amount of urease-labeled antigen bound to a double-antibody solid phase by continuously measuring the rate of ammonia produced from urea as substrate. The method yields accurate and sensitive assays for proteins (BSA less than 10 ng/mL) and antigens (cAMP less than 10 nM), with fairly good selectivity over cGMP, AMP, and GMP. 

Activity assays of enzymes bound to solid phases in EIA systems have previously been limited to fixed-time spectrophotometric methods following incubation of substrate and solid phase for extended periods of time. Kinetic assays of enzyme activity have not been used to date because of the difficulty in directly monitoring initial rates of enzyme reactions in a turbid solid phase suspension. With urease as the label, an ammonia gas sensing electrode can be used to directly quantitate the amount of urease-labeled antigen or hapten bound to a double-antibody solid phase by continuously measuring the initial rate of ammonia produced from urea as a substrate. 

The estimation. Label two 250 ml. conical flasks A and B, and into each measure 5 ml. of urine solution (or about o i g. of solid urea, accurately weighed). Add to each about 20 ml. of water and bring the temperature to about 60°. To A add 3 drops of phenolphthalein solution and to B add i ml. of 0-5% mercuric chloride solution. Now to each solution, add 10 ml. of the urease solution and mix well. The mixture A soon turns red. 

Table 2 gives details of some conventional regimes (see British National Formulary, 2008). The efficacy of therapy can be checked by Radiocarbon-labelled urea breath testing (which depends upon release of labelled carbon dioxide by bacterial urease) or by testing gastric biopsy material for persistence of gastric urease, but should only be done after eradication therapy has been discontinued for at least a month, and whilst any anti-secretory treatment has been discontinued (because it tends to suppress but does not eradicate the organism). 

There are two reports that hydroxamic acid inhibition is reversible 59,90) and one that inhibition is irreversible (91). The inhibition appears to be competitive. The rather extensive screening of 36 hydroxamic acids was accomplished with sword bean urease (90), but Proteus urease (92) and jack bean urease (59) also have been found to be inhibited by these specific inhibitors. Using tritium-labeled caprylohydroxamic acid and sword bean urease, Kobashi et al. (94) have shown the formation of an inactive complex containing two moles of inhibitor per mole of enzyme. 

Your lab ran out of 0.05 N HC1. You found a bottle labeled 0.05 N H2S04. Could you use it for your titration If you do, would your calculation of urease activity be different ... 

Urease solution prepare the enzyme solution on the week of the experiment and store at 4°C. Take 1.0 g urease, dissolve in 500 mL Tris buffer. (One can buy urease with 5 to 6 units activity, for example, from Nutritional Biochemicals, Cleveland, Ohio.) The activity of the enzyme printed on the label should be checked by the stockroom personnel or instructor. 

The use of enzyme labels in place of radioisotopes for the measurement of antigens, antibodies, and haptens has stimulated the new and expanding field of enzyme immunoassay (EIA). This technique has been the focus of several recent reviews, - and its merits compared to radioimmunoassay (RIA) have been discussed. In many cases, EIA can match RIA in terms of sensitivity and selectivity, yet has advantages of speed, convenience, and reduced cost. EIA sensitivity and simplicity is, however, dependent on the choice of enzyme label. It is the purpose of this work to introduce urease as a new enzyme label and to demonstrate the... 

Detection limits in EIA are ultimately determined by how low one can measure the label s concentration via an activity assay. Sensitivity in such a kinetic determination is dependent upon the turnover number of the enzyme molecule and the method employed to detect the product of the catalyzed reaction. Purified urease obtained from Sigma Chemical Co. has considerably higher activity on a molar basis (international units per mole of enzyme) than the best available commercial preparations of some other common enzyme labels such as alkaline phosphatase, /8-galactosi-dase, peroxidase, - and glucose oxidase. This is due to the high mo-... 

In this chapter we demonstrate that urease can be used as a label for protein antigen type of molecules by employing a urease-bovine serum albumin (BSA) conjugate to carry out competitive binding EIA for BSA. 

Results here give evidence that urease is a good choice of enzyme label for sensitive and simple assays. We further show that urease can be effectively applied to hapten assays by developing an EIA system for cAMP. 

In order to fully evaluate urease as a potential label for EIA in general, we felt that it was necessary to demonstrate that urease could be covalently coupled to an antigen and still maintain sufficiently high activity for sensitive immunoassay. In addition, the effect of antibody binding to the labeled antigen should be tested to see whether enzyme activity can be homogeneously inhibited by the antibody. 

The urea breath test measures radiolabelled COj in expired air after ingestion of labelled urea, exploiting the fact that the organism produces urease and can convert urea to ammonia. 

Infection by H. pylori is detected by serological markers produced by host immune responses (e.g., antibodies to antigens of H. pylori) and a breath test. The latter, known as the urea breath test, consists of oral administrations of radioactively labeled urea. This is metabolized to labeled CO2 and ammonia by the urease of H. pylori present in the gastric mucosa. The presence of labeled CO2 measured in the exhaled air confirms infection. 

Urea breath test HP urease breaks down ingested labeled C-urea, patient exhales labeled CO2 Tests for active HP infection 95% sensitive and specific results take about 2 days antibiotics, bismuth, PPIs, and H2RAS may cause false-negative results withhold PPIs or H2RAS (1 to 2 weeks) and bismuth or antibiotics (2 to 4 weeks) before testing may be used posttreatment to confirm eradication... 

The double-sandwich ILA utilizes the purified anti-HCP antibodies separately labelled with fluorescein and biotin (Fig. 6) (Briggs et al, 1990). The first stage of the assay consists in the formation of a tripartite complex between HCPs and the two labelled antibodies. After addition of streptavidin, these immune complexes are then captured on a biotinylated nitrocellulose membrane. Detection stage starts with incubation of the membrane with an anti-fluorescein-urease conjugate. The urease hydrolyses added urea into ammonia that alters the pH of the solution and consequently changes the surface potential on the membrane, which is then measured by a silicon sensor. The general detection range reported by Molecular Devices for HCP ILA assays is 2-160 ng/ml for E. coli HCPs and 2.5-200 ng/ml for Chinese Hamster Ovaiy cells HCPs. 

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