Conjugates urease, 71

Figure 5.8 depicts a typical immunoassay-based LAPS. The reaction of an antibody to its antigen will immobilise the species and after washing, only those species that have been successfully bound will be left onto the LAPS surface. Here, a conjugated enzyme, e.g., urease, will change the pH value of the test sample by enzymatic catalysis after injection of urea. 

Ammonium carbamate is formed in citrate or Tris buffer. It is a 480-kDa protein with a pH optimum of 6.0. The enzyme is very specific for urea or hydroxyurea. It is inhibited by heavy metals, while it is stabilized by 1 X 10 M EDTA. The specific activity of urease is very high (10 000 U/mg), but the potential detection sensitivity is lost because the reaction products are difficult to detect. Urease conjugates because of free thiols are not particularly stable. Urease is important in noninstrumented assays because activity can be visualized via pH-sensitive dyes. 

Electrode-Based Enzyme Immunoassays Using Urease Conjugates... 

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. 

Preparation of Urease-BSA Conjugate. The method of glutaralde-hyde conjugation used was based on the previously reported techniques of Avrameas for preparation of enzyme-protein conjugates. Two milligrams of BSA and 2 mg of urease (40,000 U/gram) were dissolved in 1.0 ml of 0.1 M phosphate buffer, pH 6.8, and 0.1 ml of 1% aqueous glu-taraldehyde was added slowly to the stirred solution. The reaction mix-... 

Urease-cyclic nucleotide conjugates were not prepared by the carbodiimide method because of high loss of urease activity during the course... 

Contrary to other previously published mixed anhydride procedures using other proteins, it is an absolute requirement in the case of urease that all oi ganic solvent be removed prior to final conjugation, since it was found that exposure of the urease to even a slight amount of hydrophobic solvent renders it irreversibly inactive. Using the above method excellent final yields of enzyme activity are obtained (approximately 80%). 

Conjugation of the nucleotides to urease was confirmed by ultraviolet (UV) spectra between 300 and 240 nm. Based on the change in the urease spectra before and after conjugation and the known molar extinction coefficients of the cyclic nucleotides, an estimate as to the degree of conjugation can be made. Typically, a conjugation between 2 and 7 mol of nucleotide per mole of enzyme is obtained based on a urease molecular weight of 480,000. 

For BSA assay, to 1.5 ml of buffer-BSA solution, typically 40 fi of a 1 100 dilution of rabbit anti-BSA (2.4 mg/ml) and 40 /aI of a 1 10 dilution of urease-BSA conjugate were added. For cAMP assay, 30 ju.1 of a 1 10 dilution of the (NH4)2S04 fraction of cAMP antiserum (0.5 mg/ml) and 30/al of a 1 10 dilution of enzyme-cyclic nucleotide conjugate were added to 1.0 ml of buffer or buffer-nucleotide standard. 

Fig. 4. Calibration curve obtained for bovine serum albumin (BSA), by monitoring amount of urease-BSA conjugate bound to anti-BSA antibody. Data were obtained with tubes containing 1.5 ml of BSA standard, 40 fil of 1 100 anti-BSA serum, 40 /al of 1 10 urease-BSA conjugate, and 300 1 of insolubilized goat anti-rabbit y-globulin suspension, all prepared in 10 mAf Tris-HCl-EDTA, pH 7.5.

Fig. 5. Titration of various amounts of a 1 10 dilution of urease-bovine serum albumin (BSA) conjugate with rabbit antibody to BSA O, 25 fil A, 80 /il , 200 /u.1. Conditions as in Fig. 4, except that 1.5 ml of buffer replaces standards in all tubes.

Figure 6 shows a typical calibration curve obtained for the inhibition of binding of a urease-cAMP conjugate to anti-cAMP antibody as determined by the ammonia electrode. One hundred percent of activity refers to blank tubes, which had rates of 11-12 mV/min in the absence of cAMP. Selectivity of the assay over structurally similar cGMP is also shown in Fig. 6. It takes approximately 1000 times more cGMP than...

Fig. 6. Calibration curves obtained for cAMP and cGMP using a urease-cAMP conjugate and cAMP antibody. Data obtained in 0.1 M Tris-HCl-EDTA, pH 7.5, using I.O ml of nucleotide standard, 30 ju.1 of 1 10 rabbit anti-cAMP antibody, 30 /itl of 1 10 urtjfise-cAMP coiyugate, and 300 1 of second antibody suspension.

Fig. 7. Calibration curves obtained from inhibition response to cAMP, cGMP, AMP, and GMP, using a urease-cGMP conjugate and cAMP antibody. Data were obtained as for Fig. 6.

Meyerhoff, M.E. Rechnitz, G.A. Electrode-based enzyme immunoassays using urease conjugates. Anal. Biochem. 1979, 95, 483-493. 

Numerous studies done on conjugated proteins were oriented toward the differentiation of the material without performing special separations. For example, direct pyrolysis of several enzymes showed a significant difference in the chromatographic profile of the pyrolysate. The enzymes analyzed by this procedure included a-chymotrypsin, creatine kinase, lactate dehydrogenase, catalase, acetylcholinesterase, and urease [19,20]. 

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