Mammaglobin is found in breast tissue as a complex with BU101.

The mammaglobin gene has been shown to be preferentially expressed in breast tissue. Few genes match its specificity. Mammaglobin has generated much interest, and studies are ongoing to develop diagnostic tests for breast cancer based on the detection of mammaglobin. While searching the Incyte Genomics Lifeseq database for tissue-specific markers, we observed a second secretoglobin, BU101, also known as lipophilin B. We report here that mammaglobin, in breast tissue, is found as a complex with BU101. The complex was isolated from breast cancer tissue and was characterized as the biologically relevant form of mammaglobin.

Initial clinical study of indium-111-labeled clone 110 anticarcinoembryonic antigen antibody in patients with colorectal cancer.

A murine monoclonal antibody directed against carcinoembryonic antigen (CEA) was labeled with indium-111 (111In) by means of a benzylisothiocyanate derivative of diethylenetriamine penta-acetic acid (DTPA) and used for clinical radioimmunodetection studies. Twenty-one patients having a history of surgically resected colorectal cancer and rising serum CEA levels suggestive of tumor recurrence were studied. Patients were infused over 20 minutes with 5, 10, or 20 mg of the monoclonal antibody labeled with 5 mCi of 111In. The mean radiochemical purity was greater than 96%. No toxicity was seen. The stability of the radiolabel on antibody in patient serum was demonstrated by high-performance liquid chromatography (HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with autoradiography, and immunoprecipitation for up to 96 hours after infusion. Tumor sites were identified in 20 of 21 patients. Sites of antibody accumulation in 20 patients were confirmed as tumor either by resection at laparotomy (16 patients) or fine-needle biopsy (four patients). Nine patients who had the identified lesion resected or irradiated showed return of the serum CEA antigen level to normal or near normal values. In the absence of high levels of circulating CEA (greater than 500 ng/mL), the disappearance of radioactivity from patient serum demonstrated first order elimination kinetics, with a mean half-life of 38 hours. The serum half-life was not affected by the dose of antibody administered or by serum CEA titers below 500 ng/mL. Despite a mean liver uptake of 18% injected dose (ID) 24 hours after administration, hepatic metastases were easily visualized as areas of increased uptake of radioactivity. Radioimmunodetection of recurrent colorectal cancer, not detected by computed tomographic (CT) scans, appears achievable with this agent. This may allow successful clinical intervention in selected patients.

An optimized antibody-chelator conjugate for imaging of carcinoembryonic antigen with indium-111.

A monoclonal antibody to carcinoembryonic antigen showing minimal cross-reactivity with blood cells and normal tissues was derivatized with benzylisothiocyanate derivatives of EDTA and DTPA. Seven chelators per immunoglobulin could be incorporated without loss of immunoreactivity. The resulting conjugates, labeled with indium-111, showed low liver uptake in animals. A cold kit, comprising the DTPA conjugate at a molarity of antibody bound chelator exceeding 1 x 10(-4) M, gave radiochemical yields of indium labeled antibody of greater than or equal to 95% and was stable for 1 yr.

Fluorescence polarization immunoassay IV. Determination of phenytoin and phenobarbital in human serum and plasma.

Fluorescence polarization immunoassays for phenytoin and phenobarbital in human serum or plasma are described and shown to be clinically useful. No sample pretreatment, extraction, or phase separation is required. A determination can be made with less than 20 microL of sample in 15 min, with incubation at ambient temperature. Abnormal concentrations of protein, lipid, hemoglobin, or bilirubin do not interfere. In addition, drug metabolites and commonly co-administered drugs do not affect the assay results at clinically significant concentrations. Analytical recoveries of each of the two anticonvulsant drugs from serum averaged 101%. Between-assay CVs were less than 6.5%, with sensitivities of 0.5 mg/L. Comparison of this method with "high-performance" liquid chromatography, homogeneous enzyme immunoassay, and radioimmunoassay for phenytoin determinations yielded correlations of 0.99, 0.98, and 0.99, respectively. Similar comparison studies for phenobarbital yielded correlation coefficients of 0.99, 0.98, and 0.99, respectively.

Fluorescence polarization immunoassay. iii. an automated system for therapeutic drug determination.

A fully automated system for performing fluorescence polarization immunoassay has been developed. Reagents for each assay are contained in coded reagent packs, and no reagent reconstitution is required. A common buffer is used for all assays, minimizing changeover and set-up times for each assay. A single sample may be assayed in 5 min, or 20 samples in 10 min. A single-tube blank subtraction for each sample results in highly precise polarization values and obviates sample interferences. We have used this method for assays of gentamicin, theophylline, phenytoin, and phenobarbital. CVs are 1-4%, and the results correlate well with those by other methods. Because of the instrument design and the stability of the reagents, daily calibration is not required; samples may therefore be run immediately upon receipt or batched as desired.

Fluorescence polarization immunoassay. I. Monitoring aminoglycoside antibiotics in serum and plasma.

Fluorescence polarization immunoassays of the aminoglycoside antibiotics gentamicin, tobramycin, and amikacin in plasma and serum are described and shown to be clinically useful. The aminoglycoside tracers were prepared by reacting the parent compounds with 5-[(4,6-dichlorotriazin-2-yl)-amino] fluorescein. Antisera specific for the compounds were raised in rabbits by conventional procedures. Tracer, sample, and diluted antiserum are combined and, after a 15-min incubation at ambient temperature, the polarization of the fluorescence of the tracer is determined in a specially designed fluorometer. The assays are designed to give accurate trough (i.e., minimum during therapy) values and to be free of matrix effects. Severely icteric samples may interfere, but this can be overcome by blank subtraction. The performance of the assays with clinical specimens compared favorably with that of some commercially available assays.

Kinetic analysis of calcium binding to concanavalin A.

The kinetics of calcium binding to concanavalin A was studied utilizing ultraviolet difference spectral measurements. The results show that calcium binds to the lectin in a biphasic process: a rapid and reversible phase, followed by a relaxation phase with a kobs of 0.012 sec-1. Kinetic measurements were used to calculate the association constant, Ka, for calcium binding to concanavalin A of 2.7 x 10(4) M-1, in reasonable agreement with values obtained by equilibrium methods.

Kinetics and mechanism of bilirubin binding to human serum albumin.

The kinetics of bilirubin binding to human serum albumin at pH 7.40, 4 degrees C, was studied by monitoring changes in bilirubin absorbance. The time course of the absorbance change at 380 nm was complex: at least three kinetic events were detected including the bimolecular association (k1 = 3.8 +/- 2.0 X 10(7) M-1 S-1) and two relaxation steps (52 = 40.2 +/- 9.4 s-1 and k3 = 3.8 +/- 0.5 s-1). The presence of the two slow relaxations was confirmed under pseudo-first order conditions with excess albumin. Curve-fitting procedures allowed the assignment of absorption coefficients to the intermediate species. When the bilirubin-albumin binding kinetics was observed at 420 nm, only the two relaxations were seen; apparently the second order association step was isosbestic at this wavelength. The rate of albumin-bound bilirubin dissociation was measured by mixing the pre-equilibrated human albumin-bilirubin complex with bovine albumin. The rate constant for bilirubin dissociation measured at 485 nm was k-3 = 0.01 s-1 at 4 degrees C. A minimum value of the equilibrium constant for bilirubin binding to human albumin determined from the ratio k1/k-3 is therefore approximately 4 X 10(9) M-1.

Mechanism of steroid binding to serum proteins.

Association and dissociation rate constants of steroid complexes with progesterone-binding globulin (PBG) and with corticosteroid-binding globulin have been determined, utilizing the fluorescence quenching phenomenon observed on steroid binding to protein. Stopped-flow techniques were used in most cases. The dissociation rates of the complexes with steroid-binding proteins of serum are much greater than those of steroid-receptor complexes, in accordance with the biological functions of these two types of proteins. Association of steroids with PBG is accompanied by conformational changes in both components of the complexes. Chemical modification of tryptophan, lysine, and tyrosine in PBG results in inactivation of the binding site; complex formation with progesterone protects against this inactivation. A comparison of the affinity constants of PBG complexes with steroids of different structures leads to a conceptual image of the binding site and to localization of the various forces of interaction over the binding site area.

Alterations in the ultraviolet absorption spectra of steroids upon binding to serum proteins.

Difference spectra of progesterone-binding globulin (PBG) complexes with progesterone and testosterone were measured. The contributions of steroid and protein to the difference spectra were resolved by use of 5alpha-pregane-3,20-dione and dihydrotestosterone to compensate for the perturbation of PBG. The absorption spectra of seven bound steroids all showed increased extinction coefficients, sharpened absorption bands, a small blue shift, and an increased area implying an enhanced transition moment. This is in contrast to the steroid complexes with the low affinity binders, human serum albumin, and alpha 1-acid glycoprotein, which exhibit decreased extinction coefficients and reduced transition moments.

Kinetic and equilibrium studies on steroid interaction with human corticosteroid-binding globulin.

Kinetic and equilibrium studies on the interaction of steroids with human corticosteroid-binding globulin (CBG, transcortin) were performed with pH, temperature, and steroid structure as variables. Dissociation rate constants were determined fluorometrically; the values for cortisol, corticosterone, deoxycorticosterone, and progesterone are 0.031, 0.047, 0.10, and 0.16 s-1, respectively, at 20 degrees C, pH 7.4. The pH dependence of the dissociation rate constant for the corticosterone complex below pH 10.5 at 20 degrees C is given by koff = 0.043 (1 + [H+]/10(-6.50)) s-1; above pH 11, koff = 0.030 (1 + 10(-12.15/[H+] s-1. A temperature-dependence study of koff for the cortisol and progesterone complexes gave values of 0.0028 s-1 and 0.012 s-1 at 4 degrees C, respectively, and 0.88 s-1 and 4.5 s-1 at 37 degrees C, with progesterone dissociating about four to five times faster over the entire temperature range. The affinity constants, determined by equilibrium dialysis, for the binding of cortisol, corticosterone, and progesterone at 4 degrees C were 7.9, 7.2, and 7.0 X 10(8) M-1; values of 0.40 and 0.26 X 10(8) M-1 were determined at 37 degrees C for cortisol and progesterone. The close similarity of the affinity constants of the three steroids combined with differing dissociation rates implies that the association rate changes with steroid structure, in contrast to our earlier findings with progesterone-binding globulin.

pH dependence of progesterone interaction with progesterone-binding globulin. Kinetic and equilibrium studies.

The kinetics of binding and dissociation for the progesterone-binding globulin (PBG)-progesterone complex have been measured as a function of pH. The association rate constant appears to be independent of pH from pH to 10 with an average value of kon = 8.5 X 10(7)M-1 S-1. The dissociation rate constant is strongly pH dependent with the dependency defined by: koff = k0 (1 + [H+]/K1 + K2/[H+])(1 + K3*/[H+])/(1 + K3/[H+]). The best values for the various parameters were k0 = 0.0785 s-1, pK1 = 5.30, pK2 = 10.54, pK3* = 7.41, and pK3 = 7.21. Simpler expressions were inadequate to fit the data, and it was concluded that at least three ionizing residues are responsible for the stability of the PBG-progesterone complex. The affinity constant was determined by equilibrium dialysis over the range of pH 3 to 12. The ratio of the association and dissociation rate constants is in agreement with the affinity constant from pH 6.5 to 10.5. The influence of pH on the conformation and binding activity of PBG was also investigated. Denaturation by acid, base, or guanidine hydrochloride leads to a reversible loss of binding activity. Regain of binding activity in all cases is slow with half-times of 0.5 to 2.7 h, depending on conditions. The rate of acid denaturation was found to be incompletely protonated at pH 1.4, suggesting a buried carboxylic acid residue. The slow renaturation of PBG might be due to the difficulty of burying a charged residue in the protein's interior coupled with steric hindrance by the large carbohydrate moiety of PBG.

Steroid-protein interactions. Stopped flow fluorescence studies of the interaction between steroid hormones and progesterone-binding globulin.

Stopped flow fluorometry, measuring changes in the intrinsic fluorescence of progesterone-binding globulin (PBG), was used to determine the association and dissociation rates of the interaction of PBG with seven delta4-3-ketosteroids. The rates of formation and dissociation of the PBG-progesterone complex were measured as a function of concentration and temperature. At 20 degrees, kon = 8.7 X 10(7) M-1 S-1 and koff = 0.060 S-1. The association rate constants for progesterone, deoxycorticosterone, testosterone, testosterone acetate, and medrogestone were found to be the same within experimental error. The different affinities of PBG for these steroids result from the dissociation rate constants of the steroids which ranged from 0.43 S-1 for testosterone to 0.024 S-1 for medrogestone. Two corticosteroids, corticosterone and cortisol, were both bound somewhat more slowly (approximately 5 X 10(7) M-1 S-1). Reflecting their very low affinity for PBG both steroids dissociate very rapidly: corticosterone at 1.4 S-1 and cortisol at 90 S-1. The ratio of association to dissociation rate constants gave affinity constants in agreement with independently determined constants.

Conformational changes in the progesterone binding globulin-progesterone complex.

An improved purification procedure for the progesterone-binding globulin (PBG) of the pregnant guinea pig has been developed utilizing sulfopropyl Sephadex, a strong cation exchanger, in the first step. The method exploits the low pI (2.8) and favorable acid stability of the glycoprotein. Subsequent chromatographies on DEAE-cellulose and Sephadex G-200 afford a highly purified PBG that exhibits the previously observed polydispersity (R.M. Burton et al. (1974), Biochemistry 13, 3554-3561). Circular dichroism, optical rotatory dispersion, and difference uv spectra all indicate the purified protein to undergo a conformational transition upon forming a complex with a steroid ligand. The CD and ORD spectra cannot be interpreted in terms of tertiary structure probably due to carbohydrate contributions. However, the difference spectra indicate strong perturbation of both a tryptophan residue and the steroid chromophore in the complex.