Determination of penicillamine or tiopronin in pharmaceutical preparations by flow injection analysis

Two flow injection analysis (FIA) methods, using spectrophotometric detection, are proposed for the determination of penicillamine or tiopronin [N-(2-mercaptopropionylglycine)]. The procedures are based on the formation of yellow complexes between these thiol-containing drugs and Pd(II), in a 1 M or 0.25 M HCl medium, respectively. With peak height as a quantitative parameter, penicillamine is determined over the range 1.0 x 10(-5)-7.0 x 10(-4) M; for tiopronin the range is 1.0 x 10(-5)-6.0 x 10(-4) M. The methods have been applied to the routine determination of the drugs in pharmaceutical preparations.     

Time course of antibody response to tetanus toxoid and pneumococcal capsular polysaccharides in patients infected with HIV

The methods of measuring the affinity constants of anti-HIV-1 p17 monoclonal antibodies (MAbs) using the double antibody methods in the liquid phase and the biomolecular interaction analysis by BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden) were compared. MAbs, HyHIV1-6, recognizing residues 12-29 (P12-29) of p17 and the naive protein, p17, were used. The kinetic association constants (KAs) obtained using the double antibody method were 2.40 x 10(7) - 1.40 x 10(8)M(-1) for P12-29, and 4.80 x 106 - 1.80 x 10(7) M(-1) for p17. In the BIAcore system where P12-29 or p17 was used as immobilized antigens onto the sensorchip, the KAs were 1.57 x 10(9) - 4.81 x 10(9) M(-1) for P12-29, and 1.52 x 10(9) - 1.21 x 10(10) M(-1) for p17. On the other hand, when MAbs were immobilized onto the sensorchip and P12-29 or rp17 was used as analyte, the KAs for P12-29 and p17 were in the region 3 x10(8) - 3 x 10(9), 1 x 10(8) - 3 x 10(9) M(-1), respectively. These data show that the KAs were higher than those obtained using the double antibody method, however, no significant difference could be observed. Moreover, the KAs obtained for p17 using MAbs as ligand were similar for BIAcore and the double antibody method except for HyHIV2. Therefore, the BIAcore system can be used for the affinity measurement instead of the double antibody method.     

Development of a biosensor for monitoring of glycerol during alcoholic fermentation

A biosensor for the measurement of glycerol in FIA was constructed using covalently immobilized glycerokinase and glycerol-3-phosphate oxidase in conjunction with a Pt based hydrogen peroxide probe. Different immobilization strategies have been studied including random and asymmetric immobilization onto a polymeric support and immobilization onto two different membranes. The latter resulted in the best configuration for batch measurement. The most effective configuration for measurement in FIA was the immobilization of glycerokinase in a glass beads reactor coupled with glycerol-3-phosphate oxidase on a preactivated Immobilon AV membrane kept at the electrode surface. Using a 250-microliter injection loop, 3 mmol ATP(Mg+2) in 0.1 M borate buffer pH 8.5 and a flow rate of 0.5 ml/min, a linear response in the 2 x 10(-6)/10(-3) mol/l range and a detection limit of 5 x 10(-7) mol/l were obtained for glycerol. Lifetime of the glycerol-3-phosphate membrane was extended up to 1 month by storage in the working buffer containing 1% DEAE-dextran and 5% lactitol. More than 350 samples can be assayed with this system. The biosensor was used to monitor off-line glycerol production during alcoholic fermentations carried out at different pHs and temperatures.     

Biosynthesis of the carbohydrate units of immunoglobulins. 1. Purification and properties of galactosyltransferases from swine mesentary lymph nodes

Galactosyltransferase (UDPgalactose:glycoprotein galactosyltransferase EC 2.4.1.22) was isolated from swine mesentary lymhromatography on Sepharose 4B colums containing covalently bound p-aminophenyl-beta-D-N-acetylglucosamine. The homogenous enzyme showed a single band on disc gel electrophoresis and had a specific activity of 35 nmol min-1 (mg of protein)-1 at 37 degrees C. A molecular weight of 57 000 was obtained by exclusion chromatography, sucrose density centrifugation, and sodium dodecyl sulfate-gel electrophoresis. The same molecular weight was obtained after reduction and alkylation which indicates that the enzyme is composed of only a single polypeptide chain. The enzyme catalyzed the formation of beta1 leads to 4 bonds between galactose and free terminal N-acetylglucosaminyl residues of soluble preparations of porcine IgG immunoglobulin heavy chain, fetuin, ovalbumin, and ovomucoid. An endogenous glycoprotein, present in particulate subcellular preparations, was also a very good substrate for the enzyme, and it was identified as incomplete IgG immunoglobulin heavy chain. The Km of the purified enzyme was 2.9 x 10(-5) M for fetuin, 5.4 x 10(-5) M for ovalbumin, 2.0 x 10(-5) M for IgG immlnoglobulin heavy chain, and 2.2 x 10(-5) M for UDP-galactose. About 20% of the total galactosyltransferase activity in lymph node homogenates was present in the cytosol fraction, and 80% was in the microsomal and Golgi fractions. The kinetic properties of the bound and soluble galactosyltransferases were similar,and both required Mn2+ for maximal activity. However, the bound enzyme required the addition of detergent, lysolecithin, GDP-mannose, and UDP-N-acetylglucosamine for maximum activity. These compounds did not influence the activity of the soluble transferase. The membrane preparations catalyzed the transfer of galactose from UDP-galactose and N-acetylglycosamine from UDP-N-acetylglucosamine to incomplete oligosaccharide chains of endogenous IgG immunoglobulin bound to these particles. The labeled products of these reactions were isolated, and the structures of their oligosaccharide chains were determined and compared with those isolated from the heavy chain of porcine IgG immunoglobulin. The glycopeptide prepared from the endogenous acceptor and the major glycopeptide prepared by proteolytic digestion of the heavy chain of porcine IgG immunoglobulin has identical structures. The following structure for the carbohydrate chains of porcine IgG immunoglobulin was determined by sequential enzymatic hydrolysis and methylation studies.     

Selective inhibition of primary acute myeloid leukaemia cell growth by lovastatin

The insulin receptors from erythrocytes of 50 patients with non-insulin-dependent diabetes mellitus were tested for their ability to autophosphorylate. The assay was performed by a new enzyme-linked immunosorbent assay system that used monoclonal anti-insulin receptor antibodies absorbed to microtiter plates as a first antibody and polyclonal antiphosphotyrosine antibody as a labeled second antibody. By this assay, 3 patients were identified with defects in their insulin receptor kinase, although their defects appeared heterogeneous. Patient 1 had 85% less maximal autophosphorylation with a normal ED50 (1.6 x 10(-9) M insulin). Patient 2, who had polycystic ovary disease, had a 49.2% decrease in maximal autophosphorylation of insulin receptors, and the ED50 was shifted to the right (5.6 x 10(-8) M). Patient 3 with acanthosis nigricans had a normal maximal autophosphorylation, but the ED50 shifted to the right (2.9 x 10(-8) M). The mechanisms for the diversity detected in this assay is not known, but this technique has sufficient specificity and sensitivity to be used to screen for insulin-resistant patients who have a lack of kinase activity.     

In-situ photochemical spectrofluorimetric detection of 9,10-anthraquinone-labeled bovine serum albumin

Bovine serum albumin (BSA) labeled with 9,10-anthraquinone (AQ) shows a greatly enhanced photochemical fluorometric activity compared with that of free AQ. The spectral characteristics of the photoreduction product of conjugated AQ was investigated and large blue shifts in the excitation and emission bands compared with those of free AQ were observed. The enhancement in the photochemical reactivity can be employed for sensitive detection of labeled BSA by a simple in-situ photochemical kinetic fluorimetric method. The kinetic behavior of the photochemical reaction and the effects of some experimental conditions were investigated. The calibration graph was linear over the range 0-1.8 x 10(-7) M BSA. The detection limit was 1.2 x 10(-10) M BSA and the relative standard deviation was 2.24% for the 1.42 x 10(-8) M BSA (n = 7).     

Specific cytotoxic T lymphocytes are involved in in vivo clearance of infectious bronchitis virus

Cytotoxic T-lymphocyte (CTL) responses to infectious bronchitis virus (IBV) were determined at regular intervals between 3 and 30 days postinfection (p.i.). The maximum response with 82% lysis of labeled target cells was detected at 10 days p.i. The specific CTL response did not begin to decline until the amount of virus, which peaked at day 8 p.i. in both the kidneys and lungs, started to decrease. Clinical respiratory signs of illness also correlated with amount of virus. CTL activity was shown to be major histocompatibility complex (MHC) class restricted because the lysis of MHC-mismatched targets was negligible, and lysis was mediated by CD8+ CD4- T cells, as the CTL response could be abolished with removal of CD8+ CD4- but not CD4+ CD8- lymphocytes. In contrast, immunoglobulin M (IgM) antibody was not detected until day 10 p.i., and levels peaked at day 12 p.i.; IgG antibody levels were minimal until day 15 p.i. but continued to increase exponentially until day 30 p.i., the last day examined. In summary, CTL responses correlated with initial decreases in infection and illness.     

Monoclonal antibody binding affinity determined by microchip-based capillary electrophoresis

The affinity constant of a monoclonal antibody to fluorescently labeled bovine serum albumin (BSA) was measured in diluted mouse ascites fluid using a microfluidic chip to perform affinity capillary electrophoresis. Borofloat glass-based devices could be used repeatedly with samples for many months. On-chip separations were performed in less than 60 s, and 30-60 s was required for manual sample exchange. The change in peak height for BSA with increasing BSA/anti-BSA concentration ratio was used to determine concentration changes in bound and free BSA. A Scatchard plot analysis gave an affinity constant (more exactly the intrinsic association constant) of 3.5+/-0.6 x 10(7) M(-1) for a 1:1 stoichiometric ratio. Two affinity complexes were separated. One complex was identified by the Scatchard method as having a 1:1 stoichiometric ratio. The other complex is proposed to have a stoichiometry with an excess of anti-BSA to BSA, most likely (anti-BSA)2-BSA, on the basis of a faster migration time than the 1:1 complex, a decrease in the amount of this complex with increasing [BSA], and predictions of theoretical models for multi-valent antigens. Potential applications of microchip-based devices in affinity measurements are discussed.     

Serological diagnosis of experimental Enterococcus faecalis endocarditis

A modified rat model of endocarditis with catheterization for 2 days was established in female Lewis rats using different inocula of Enterococcus faecalis (strain no. EF 19) in order to measure IgG antibodies in serum during the course of infection. Increasing the inocula intravenously resulted in an increase in the CFU/g vegetation and the CFU/g spleen, the ID50 being about 10 CFU/ml and the ID90 about 1x10(2) CFU/ml. The lowest bacterial inoculum infecting 100% of the rats was 3x10(3) CFU/ml, and for further investigations we used this inoculum size. Rats were sacrificed on day 2, 5, 7, 9, 11 and 28 after infection. The CFU/g vegetation and the CFU/g spleen increased until day 7 and then decreased. Serum samples were collected from 129 rats at different times after challenge. Three different ELISA systems were established to measure the IgG antibody responses: E. faecalis sonicate ELISA (a pool of four sonicates of strain no. EF 10, EF 11, EF 19 and EF 48), E. faecalis whole cell ELISA (strain no. EF 19) and E. faecalis purified cell wall ELISA (strain no. EF 19). An IgG antibody response was detected already on day 2, and except for a minor decrease on day 6/7 the antibody response continued to increase until day 14 (whole cell ELISA and sonicate ELISA) and day 21 (purified cell wall ELISA) when a plateau was reached. Significant increases in IgG antibody responses (p<0.05) were found between groups of rats from days 0-2, 2-8/9 and 8/9-14 in the E. faecalis whole cell and sonicate ELISAs and from days 0-2, 2-10/11 and 10/11-21 in the E. faecalis purified cell wall ELISA. In conclusion, we established a model of endocarditis in rats with catheterization for 2 days and were able to demonstrate an increase in IgG antibodies during the course of infection.     

Antibody binding to a functionalized supported lipid layer: a direct acoustic immunosensor

A direct immunosensor has been developed using an acoustic wave device as a transducer. The device is based on an acoustic waveguide geometry that supports a Love wave. The biorecognition surface, formed on a gold layer, consisted of a biotinylated supported lipid layer which specifically bound streptavidin and, subsequently, biotinylated goat IgG. The modified surface was used as a model immunosensor and successfully detected rabbit anti-goat IgG in the concentration range 3 x 10(-8) - 10(-6) M. Using the anti-goat IgG binding isotherm and the time-resolved measurements of antibody binding, both the binding and rate constants of the reaction were determined. The specificity of each binding step was studied with the acoustic wave device, and it was concluded that the phospholipid bilayer showed a good suppression of nonspecific binding. Comparative measurements using surface plasmon resonance allowed the response of the immunosensor to be quantitatively correlated with mass binding to the surface.     

12alpha- and 7alpha-hydroxysteroid dehydrogenase activities from Fusobacterium spp

On screening fecal organisms for hydroxysteroid dehydrogenase activities applicable to bile acid metabolism studies, we have isolated a gram negative "Bacteroides-like" anaerobe which yields both 12alpha- and 7alpha-hydroxysteroid dehydrogenase (HSDH) activities in cell-free preparation. At the optimal harvest time of 36 hours, approximately 4500 units 12alpha-HSDH and 360 units 7alpha-HSDH were produced per 10(10) viable cells. The two enzymes appear to be separate entities in the basis of their stabilities on freezing, and prolonged storage at room temperature and elution volumes on Sephadex G 200. Thin layer chromatography studies on oxidation products confirmed the respective sites of oxidation to be the 12alpha-OH and 7alpha-OH position. No 3alpha-OH oriented activity was measurable. Preliminary kinetic studies of the 12alpha-HSDH revealed a broad pH curve with optimal activity at pH 9.5. Michaelis constants for glycodeoxycholate and NADP were estimated at 1.5 x 10(-4)M and 3.3 x 10(-5)M respectively.     

Characterization of the binding properties of protein LG, an immunoglobulin-binding hybrid protein

Protein LG is a 50-kDa hybrid molecule containing four Ig-light-chain-binding domains from protein L of Peptostreptococcus magnus and two IgG-Fe-binding repeats from streptococcal protein G. Here we analyse the binding of protein LG to Ig from several mammalian species. Protein LG was shown to bind human IgG of all subclasses and other Ig classes that carry kappa chains. The binding to human IgG was only marginally influenced by changes in temperature (4-37 degrees C) or salt concentration (0-1.6 M), and was stable over a wide pH range (pH 4-10). Protein LG bound to Ig from 11 of 12 mammalian species, including those of rabbit, mouse and rat. The affinity constants obtained for the interactions between protein LG and polyclonal IgG from rabbit (4.0 x 10(9) M-1), mouse (1.7 x 10(9) M-1) and rat (1.3 x 10(9) M-1) were similar to the value previously reported for the interaction between the hybrid protein and human polyclonal IgG (5.9 x 10(9) M-1). The interaction between protein LG and a mouse IgG mAb was not influenced by the presence of the specific protein antigen, nor was the binding of this antibody to its ligand affected by protein LG. Inhibition experiments demonstrated that the Ig-binding site of one of the fusion partners retained its ligand-binding capacity when the other component was occupied. Protein LG selectively absorbed 85-90% of the total Ig present in human and rabbit sera and 75-80% of the Ig in sera from mouse and rat. Human serum depleted of C1q, factor D and properdin and preabsorbed by protein LG could be used as a source for other complement factors. These data demonstrate that protein LG is a very versatile Ig-binding protein.     

Recent management of pituitary adenomas

Eight 2-day-old SPF chickens were each inoculated orally with a single dose of 5 x 10(5) oocysts of Cryptosporidium baileyi, and immunoglobulin G (IgG) antibody responses were chronologically measured by indirect immunofluorescent antibody (IFA) assay. Anti-C. baileyi IgG antibody levels remained high (1:106.67 to 1:512.00) for at least 4 months with 330 days of a detectable period. Ten days after the negative conversion, each chicken was re-challenged with 1 x 10(7) oocysts of the same species. Subsequent infection in 340-day-old individuals caused sudden elevated IgG antibody levels and the titer peaked on day 28 postchallenge inoculation (PCI), at 1:1.024 with a 65 days of detection period. Chickens in primary infection showed oocyst shedding profiles, but did not exhibit any oocyst shedding before or after experimental reinfection.     

A study of reversibly inactivated lipases for use in a morphine-triggered naltrexone delivery system

A key component of an implant that can be triggered by external morphine to release naltrexone is an inactivated enzyme that can be activated by morphine and which can then rapidly remove a protective coating surrounding a bioerodible polymer containing dispersed naltrexone. In this article we describe a lipase that has been conjugated with O3-carboxymethylmorphine, morphine-beta-3-glucuronide and O3-carboxypropylmorphine. The enzyme conjugate was then inactivated by complexation with affinity-purified goat polyclonal antimorphine antibodies. Antibody lipase interactions were measured by pH Stat and ELISA techniques. Affinity constants of the antibodies determined by radioimmunoassay using tritium-labeled morphine were 4.10 x 10(6), 3.18 x 10(6) and 3.38 x 10(7), respectively. While a concentration of 10(-5)M morphine was required to restore lipase activity, it is likely that a combination of correct morphine tether and correct affinity-purified antibody can increase sensitivity to the desired 10(-8)10(-9)M morphine level. Thus, a functioning device can almost certainly be constructed. However, it is unlikely that reactivation times of 1-2 h necessary for clinical usefulness in treatment of narcotic addiction can be achieved.     

Differential chemical modification of substrate binding areas in porcine-pancreatic alpha-amylase by three regioisomeric photolabile ligands

Binding studies of metallothionein and rat spermatozoa were performed using 125I-Tyr-metallothionein (125I-MT). Reactions between 125I-MT and spermatozoa indicated that MT bound in two forms, namely, middle affinity (Kd1 = 2 x 10(-9) M) binding and low affinity (Kd2 = 1 x 10(-8) M) binding. Labeled MT binding to spermatozoa was inhibited by adding anti-MT antibody. Total binding reactions of MT were temperature and incubation time dependent. By transmission electron microscopy using a gold conjugate (indirect method), gold particles bound to MT were shown to bind to the cell membrane of the head and the proximal portion of the tail. By optical autoradiography, grains of labeled MT were localized mainly in the head and the proximal portion of the tail. By electron microscopical autoradiography, grains of labeled MT were identified mainly in the cell membrane of the head and tail and partly in the nucleus. These results suggest that MT has both specific and non-specific binding sites on the spermatozoal membrane.     

Automated homogeneous liposome-based assay system for total complement activity

We developed an automated homogeneous immunoassay, based on immune lysis of dinitrophenyl (DNP)-labeled liposomes, for measuring total complement activity. Liposome lysis caused by complement activity was detected spectrophotometrically from entrapped glucose-6-phosphate dehydrogenase activity. Complement activity in human sera was quantified by comparison with a calibration curve. For ease of application to fully automated routine clinical analyzers, we adopted a two-reagent system, one reagent containing a homogeneous population of small DNP-labeled liposomes and one containing antibody/substrate. This system required calibration only once a week. Within-run and between-run CVs were 0.4-1.3% (n = 10) and 1.8-4.7% (n = 10), respectively. Serum results were linear upon dilution (with saline) over a twofold range. Bilirubin, hemoglobin, Intrafat, and serum proteins such as rheumatoid factor, M protein, IgG, and IgA did not affect the assay results. The results (y) correlated well with those from a hemolytic complement activity test (x): y = 1.05x - 1.14, r = 0.92, on 66 samples in the range < 10- > 50 kU/L. This method should therefore be of great use for the determination of complement activity.     

Analysis of autoantibody epitopes on steroid 21-hydroxylase using a panel of monoclonal antibodies

A panel of five mouse monoclonal antibodies (MAbs) to human recombinant steroid 21-hydroxylase (21-OH) were produced, characterized, and used to study the interaction of 21-OH autoantibodies (AAbs) with different epitopes on human 21-OH. AAbs in patients with isolated autoimmune Addison's disease, autoimmune polyglandular syndromes types I and II, and 21-OH antibody-positive patients without overt Addison's disease (25 patients in total) were studied. Four MAbs were IgG1 subclass, one was IgG2a, and all had kappa light chains. The affinities of four of the antibodies were in the range 2.0 x 10(8) M(-1) to 7.0 x 10(8) M(-1), and the affinity of the other was 2.3 x 10(7) M(-1) 21-OH MAbs did not cross-react with 17alpha-hydroxylase (17alpha-OH)) or P450 side chain cleavage enzyme. Studies using a series of 21-OH fragments allowed the identification of short stretches of amino acids (AA) that were involved in forming the MAb binding sites. AA 391-405, defined as epitope region (ER) 1, were found to be important for binding of M21-OH1 and M21-OH2, AA 406-411 (ER2) were important for M21-OH3 and M21-OH4 binding, and AA 335-339 (ER3) for M21-OH5 binding. In addition, MAb Fab or F(ab')2 fragments were used to study 21-OH AAb epitopes in competition experiments. These investigations demonstrated that 21-OH AAbs recognize similar epitopes to the MAbs, with ER2 and ER3 being part of two distinct major epitopes, and ER 1 being part of a minor epitope. Mixtures of M21-OH antibody Fab or F(ab')2 fragments caused almost complete inhibition (80%-95%) of AAb binding in 24 out of 25 sera, and in the case of the remaining serum, the effect was marked but incomplete (67% inhibition). There were no major differences between the binding characteristics of AAbs from patients with different forms of autoimmune adrenal disease. All five 21-OH MAbs reacted with human adrenal tissue in an immunofluorescence test, but only M21-OH1 and M21-OH2 reacted with bovine adrenal tissue in these experiments. None of the MAbs reacted with human ovarian tissue in an immunofluorescence test. Overall, these studies indicate that 21-OH AAbs bind to at least three different epitopes in the C-terminal part of 21-OH, and two of these epitopes appear to be human 21-OH specific.     

The effects of reverse micelles on the reaction mechanism of alpha-chymotrypsin

Reverse micelles were employed to test the accuracy of the widely accepted mechanism for alpha-chymotrypsin in a highly structured aqueous system similar to intracellular conditions. Results yielded from spectrophotometrical assays of the alpha-chymotrypsin catalyzed hydrolysis of both p-nitrophenyl acetate (p-NPA) and p-nitrophenyl trimethylacetate (p-NPTA) were kinetically analyzed to determine constants typical of the proposed mechanistic model. This was accomplished through the establishment of a control, i.e. the well studied buffer system, for comparison between the reverse micellular environment and a bulk aqueous solution. Control group results yielded kinetic constants in favor of the proposed mechanism (Km = 1.55 x 10(-5) +/- 1.40 x 10(-6) M for p-NPA and a Km = 4.97 x 10(-6) +/- 2.29 x 10(-7) M, Km(app) = 4.92 x 10(-6) +/- 2.33 x 10(-8) M, k2 = 4.34 x 10(-3) +/- 1.31 x 10(-3), k(cat) = 1.96 x 10(-3) +/- 2.47 x 10(-4), and Ks = 1.60 x 10(-5) +/- 4.61 x 10(-6) M for p-NPTA). In contrast, similar reactions of the enzyme in a reverse micellular system produced kinetic constants atypical to that representative of the textbook mechanism. (Km = 1.59 x 10(-4) +/- 2.70 x 10(-5) M, Ks = -8.67 x 10(-5) +/- 4.46 x 10(-5) M and Km(app) = -4.80 x 10(-5) +/- 7.05 x 10(-5) M for p-NPA and Km = 1.95 x 10(-4) +/- 9.28 x 10(-5) M, Km(app) = -1.79 x 10(-4) +/- 2.36 x 10(-5) M, and Ks = -3.95 x 10(-4) +/- 1.18 x 10(-4) M for p-NPTA). In addition to negative kinetic constants, alpha-chymotrypsin seemed to display characteristics indicative of super-activity and a hysteretic response. Overall, the widely accepted mechanism for alpha-chymotrypsin appeared to fail within the confines of reverse micelles, due to the direct influence of the system's highly structured form.     

Genetic analysis of the humoral immune response of White Leghorn chicks

Total agglutinin antibody titers, 2-mercapto ethanol (2-ME) sensitive and 2-ME resistant antibody titers were determined in 598 White Leghorn chicks after intramuscular injection with sheep red blood cells. Antibody titers were determined on day 0 and on days 3, 7, 10, 13 postinjection. Mean total tier (5.2, log2 value) was highest on day 7. Females showed a significantly higher response to injection with sheep red blood cells than males. Also, significant hatch effects were noted. Heritability estimates generally varied from 0 to .5 for all parameters. In the earlier stages of the immune response the sire estimate of heritability for total and 2-ME sensitive antibody titer was higher than the dam estimate. Additive genetic correlations between 2-ME sensitive (days 3 to 13) and resistant (days 7 to 13) antibody titers were negative, varying from -.30 to -.93. The response to selection for total antibody titer is, therefore, not easily predicted.     

Rat liver transcript profiling in normal and disease states using a kinetic polymerase chain reaction assay

The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porcine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp32-NPY peptide at Lys4 was investigated using SK-N-MC neuroblastoma cell membranes containing the Y1 receptor. The release rate of the spin labeled peptides was monitored by electron spin resonance and the KD was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr1N epsilon 4-proxyl]-NPY, the KD was 8 x 10(-10) M and koff was 2.7 x 10(-4) sec-1 yielding a value for kon of 3.3 x 10(5) sec-1 M-1. The [Ac-Tyr1, N epsilon 4-proxyl,-D-Trp32]-NPY antagonist ligand had a value of KD equal to 1.35 x 10(-7) M and koff was 1.7 x 10(-4) sec-1 leading to a value for kon of 1.2 x 10(3) sec-1 M-1. The difference in the kon rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-N epsilon 4 proxyl-D-Trp32-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.