Immunoassay of leucovorin: use of 125I-labeled protein A to detect immunological binding.

An immunoassay method was developed for the quantitative determination of leucovorin. Leucovorin immobilized by covalent linkage to a solid support bound anti-leucovorin antibody produced in rabbits. The amount of ¹²⁵I-labeled Protein A bound specifically to IgG anti-leucovorin-coated beads served as a measure of antibody binding. The ability of increasing amounts of fluid-phase leucovorin to compete with solid-phase drug for specific antibody was reflected in a dose-dependent decrease in the amount of bound ¹²⁵I-labeled Protein A. This competition was used to obtain a standard curve to measure levels of leucovorin in the sera of rabbits treated with the drug. The relative abilities of methotrexate, 5-methyltetrahydrofolic acid, and other structurally related compounds to act as inhibitors demonstrated the serologic specificity of the leucovorin immune system.     

Binding site of novel 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5- triazine herbicides in the D1 protein of Photosystem II

A series of replacement experiments of [¹⁴C]-triazines, [¹⁴C]-atrazine and [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine, bound to thylakoids isolated from wild-type and atrazine-resistant Chenopodium album (lambsquarters) were conducted. Replacement experiments of [¹⁴C]-triazines bound to wild-type Chenopodium thylakoids with non-labeled atrazine and 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine were carried out, to elucidate whether benzylamino-1,3,5-triazines use the same binding niche as atrazine. [¹⁴C]-Atrazine and [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine bound to wild-type thylakoids were replaced by non-labeled 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine and non-labeled atrazine, respectively. The above two replacements showed mutual competition. To clarify further whether benzylamino-1,3,5-triazines bind at the D1-protein to amino acid residue(s) different from atrazine or not, experiments to replace [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazines bound to atrazine-resistant Chenopodium thylakoids by non-labeled atrazine, 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU and DNOC were carried out. Although the bound [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine was difficult to be replaced even with high concentrations of atrazine, [¹⁴C]-labeled 1,3,5-triazine was competitively replaced by non-labeled 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU or DNOC. Thus, 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine herbicides are considered to bind to the same niche at the D1 protein as atrazine, but use amino acid residue(s) different from those involved with atrazine binding. This revised version was published online in August 2006 with corrections to the Cover Date.     

A solid-phase immunoassay for human immunoglobulin E: use of 125I-labeled protein A as the tracer

A solid-phase immunoassay has been developed for human immunoglobulin (Ig) E. The specific binding of ¹²⁵I-labeled protein A (¹²⁵I-PA) to the Fc region of rabbit IgG anti-IgE served as a quantitative measure of specific anti-IgE antibody bound to the IgE beads under optimal assay conditions. Inhibition of antibody binding by known amounts of standard IgE was reflected in a decreased binding of ¹²⁵I-PA. The degree of inhibition of ¹²⁵I-PA binding was related to the amount of fluid-phase IgE present and gave a standard curve which was used to determine the concentration of IgE in test samples. The sensitivity of this method and a double antibody radioimmunoassay (RIA), which was developed using the same IgE preparation and anti-IgE antibody, was approximately the same. These assays gave similar results when used to determine levels of IgE in normal human sera that had been absorbed with protein A—Sepharose to remove components responsible for specific and nonspecific interference in the assays.     

Immunoassay of 5-methyltetrahydrofolate: Use of 125I-labeled Protein A as the tracer molecule for specific antibody

A sensitive and specific solid-phase radioimmunoassay for 5-methyltetrahydrofolate (5-MTHFA) has been developed. ¹²⁵I-Labeled staphylococcal Protein A (¹²⁵I-PA) was used as the tracer molecule for rabbit IgG antibodies bound to 5-MTHFA immobilized on polyacrylamide beads. The dose-dependent inhibition of antibody binding by fluid-phase drug was reflected in decreased binding of ¹²⁵I-PA. This inhibition, determined in the presence of known amounts of 5-MTHFA, served as the basis for quantification of 5-MTHFA in test samples. An early bleeding was relatively specific: 4.5 ng 5-MTHFA inhibited immune binding by 50% compared to 7700 ng folinic acid or 1200 ng tetrahydrofolate. Other folic acid analogs, including methotrexate, failed to inhibit significantly. The assay using a later bleeding was more sensitive since 1.6 ng 5-MTHFA gave 50% inhibition (detection limit 0.2 ng), but folinic acid cross-reacted significantly. Absorption with immobilized folinic acid markedly enhanced the specificity of this antiserum and resulted in a 15–20% increase in maximum inhibition by 5-MTHFA. The assay could be carried out in the presence of 0.025 ml human serum or urine without affecting the standard curve, and was used to determine levels of 5-MTHFA in serum of drug-treated rabbits.     

Immunoassay using 125I- or enzyme-labeled protein A and antigen-coated tubes

Antigen-coated plastic tubes were used with ¹²⁵I- or enzyme-labeled staphylococcal protein A in a general immunoassay method for antigens and haptens. Protein A reacts with immunoglobulin G (IgG) regardless of antibody specificity at sites distal to the antigen combining site and does not inhibit the immune reaction. It therefore serves as a general tracer and its use eliminates the need to purify and to label individual components for each assay. Macromolecular antigens were bound to polystyrene or polypropylene tubes by direct passive adsorption. Haptens with free carboxyl groups were bound covalently to poly-l-lysine and these conjugates passively adsorbed to the tube surface. Optimal assay conditions were established for the quantitative determination of immunoglobulins and the folate derivatives, methotrexate and 5-methyltetrahydrofolate, using ¹²⁵I-labeled protein A or protein A labeled with alkaline phosphatase. The method has been used to estimate levels of IgG, IgA, IgM, and IgE in serum in volumes up to 1 ml.     

The development of a radioimmunoassay for 12-L-hydroxyeicosatetraenoic acid

Antibodies directed toward 12-L-hydroxyeicosatetraenoic acid (12-L-HETE) were generated in rabbits by immunization with conjugates of 12-L-HETE and human serum albumin. The concentration of antibodies was determined by incubating immune plasma with 12-L-HETE that had been covalently linked to a solid support, washing the 12-L-HETE support, and measuring the quantity of bound antibodies by reaction with [¹²⁵I] Protein A. The addition of 0.5 ng-10 ng of fluid-phase 12-L-HETE to the standard mixture of solid-phase 12-L-HETE and anti-12-L-HETE plasma inhibited by 21–80% the binding of antibodies and consequently of [¹²⁵I]Protein A to the solid support. The 12-OH function positioned between two double bonds was the immunodominant determinant of this antigen-antibody reaction, but the carboxyl function also was recognized. This radioimmunoassay was used to detect and quantitate 12-L-HETE resolved by high pressure liquid chromatography.     

Radioimmunoassay of cytidine 3',5' monophosphate (cCMP). I. Development of the assay.

A method is presented for the production of reagents for a radioimmunoassay for cCMP. cCMP was succinylated at the 2′0 position with [1,4 ¹⁴C] succinic anhydride, and the monosuccinyl cCMP coupled to Keyhole limpet hemocyanin and injected into rabbits. Antibodies to cCMP were produced that showed minimal crossreactivity with other cyclic nucleotides. Monosuccinyl cCMP was coupled to tyrosine methyl ester, then labeled with ¹²⁵I, and used as the radiolabeled ligand in the immunoassay of cCMP. By use of this assay, the concentration of cCMP in various tissues of rat and guinea pig have been determined.     

Radioimmunoassay for pmol-quantities of indole-3-acetic acid for use with highly stable [125I]- and [3H]IAA derivatives as radiotracers

A radioimmunoassay for the detection of as little as 0.5–1 pmol indole-3-acetic acid (IAA) in unpurified or partially purified plant extracts is described. The assay makes use of either IAA[¹²⁵I]tyrosine methyl ester or [³H]IAA methyl ester as radioactive antigens and IAA methyl ester as the assay standard (measuring range: 1–200 pmol). Levels of extractable IAA in a number of biological samples have been estimated.Abbreviations BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxy acetic acid - DMF dimethyl formamide - GC-MS gas chromatography-mass spectroscopy - IAA indole-3-acetic acid - RIA radioimmunoassay - SICM selected ion current monitoring - TLC thin layer chromatography - TME tyrosine methyl ester Part 18 in the series: Use of immunoassay in plant science     

An NMR method for the study of proton transport across phospholipid vesicles

We have identified [1-¹⁴C]-oxindole-3-acetic acid as a catabolic product of [1-¹⁴C]-indole-3-acetic acid metabolism in Zea mays seedlings. The isolation, and chemical and mass spectral characterization of oxindole-3-acetic acid from corn kernel tissue is described together with data suggesting oxindole-3-acetic acid to be a major catabolic product of indole-3-acetic acid.     

The use of homologous and hetebologous 125 I-radioligands in the radioimmunoassay of progesterone

Eight homologous and heterologous¹²⁵ I-radioligand systems for the radioimmunoassay of progesterone were examined. Using an antiserum raised to 11α-hydroxyprogesterone 11-succinyl-bovine serum albumin, standard curves were set up with the homologous radioligands, 11α-hydroxyprogesterone 11-succinyl-[¹²⁵I]-iodotyramine, -[¹²⁵I]-iodohistamine and -[¹²⁵I]-iodotyrosine methyl ester. Heterologous bridge systems were represented by progesterone-11α-oxycarbonyl-[¹²⁵I]-iodotyrosine methyl ester and 11α-hydroxyprogesterone 11-phthalyl-[¹²⁵I]-iodotyrosine methyl ester, and heterologous site systems by progesterone-3-(O-carboxymethyl)oxime-[¹²⁵I]-iodotyramine, progesterone-12-(O-carboxymethyl) oxime-[¹²⁵ I]-iodotyramine, and progesterone-20-(O-carboxymethyl) oxime-[¹²⁵I]-iodohistamine. The preparation of the steroid derivatives and iodination by a two-phase method are described. The curves obtained from the homologous radioligands were relatively insensitive compared with a tritiated system, with the tyrosine methyl ester derivative providing a more sensitive assay than the corresponding tyramine or histamine analogues. The heterologous bridge systems gave more sensitive curves than the homologous tracers whilst the 3- and 12-(O-carboxymethyl) oxime derivatives of progesterone furnished curves as sensitive as the tritiated reference. Progesterone-20-(O-carboxymethyl)oxime-[¹²⁵I]-iodohistamine was not bound by the antibody.     

Streptavidin-biotinylated IgG conjugates: a simple procedure for reducing polymer formation

Disulfide links of the IgG2ak anti-ovarian carcinoma antibody, 5G6.4, were site-specifically biotinylated [≈2 biotins/ IgG2a] using a novel crosslinking procedure using the biotin derivatized ETAC (equilibrium transfer alkylation crosslink reagent) 1a. Complexation of ETAC 1a biotinylated 5G6.4 on a column of immobilized protein A at high dilution, followed by passage of [¹²⁵I]streptavidin, washing and pH change leads to elution of a streptavidin-free product with a molecular mass in the 200–300 kDa range. By contrast, direct mixing with [¹²⁵I]streptavidin rapidly gave larger oligomers of ⪢669 and ≈440–669 kDa molecular mass, respectively. The biodistribution of the 200–300 kDa complex showed significantly diminished liver, kidney and spleen uptake as well as higher blood activity than the 440–669 kDa complex. The methodology represent the first application of ETAC chemistry to disulfide-bond directed biotinylation of antibodies and the synthesis of streptavidin antibody conjugates which minimizes their polymerization.     

A radioimmunoassay for chicken avidin. Comparison with a [14C]biotin-binding method.

A double-antibody solid-phase radioimmunoassay for chicken avidin is reported. Avidin was labelled with 125I by the chloramine-T method. The bound and free avidin were separated with a second antibody bound to a solid matrix. In the logit-log scale the standard curve was linear from 1-2 to 100-200ng of avidin/ml. Cross-reaction of ovalbumin was less than 0.015%. Saturation of biotin-binding sites of avidin with an excess of biotin decreased radioimmunoassay values by about 15%. Recovery studies indicated that avidin can be assayed from all chicken tissues studied with radioimmunoassay, whereas the [14C]biotin/bentonite method gave poor recoveries for avidin in the liver and kidney. Radioimmunoassay and the [14C]biotin/bentonite method gave similar concentrations for oviduct avidin.     

The biosynthesis of the thiazole moiety of thiamine in Salmonella typhimurium

The mechanism of biosynthesis of 4-methyl-5-β-hydroxyethyl thiazole, the thiazole moiety of thiamine was studied in Salmonella typhimurium. Using the adenosine derepression technique the incorporation of various ¹⁴C-labeled precursors was determined. We found that [Me-¹⁴C]methionine, [2-¹⁴C]methionine, [U-¹⁴C]alanine, and [2-¹⁴C]glycine were not incorporated whereas [2-¹⁴C]-tyrosine was incorporated. Degradation of the 4-methyl-5-β-hydroxyethyl thiazole obtained after [2-¹⁴C]tyrosine incorporation revealed that all of the activity was located on carbon-2. These findings are discussed and compared with previous findings concerning 4-methyl-5-β-hydroxyethyl thiazole biosynthesis.     

Binding Specificity and Possible Analytical Applications of the Cytokinin-binding Antibody, Anti-N-Benzyladenosine

Antibodies elicited in rabbits by immunization with an N⁶-benzyladenosine-bovine serum albumin conjugate bound N⁶-benzyladenosine specifically. The affinity constants and specific binding site concentrations for a number of cytokinins and related compounds were estimated by nonlinear least squares analysis of direct or competitive ultrafiltration data. The antisera contained 230 to 330 nanomoles of cytokinin binding sites per gram protein. Affinity constants were 8.8 × 10⁸ molar⁻¹ for 6-benzylaminopurine, 8.4 × 10⁷ molar⁻¹ for kinetin, 9.1 × 10⁷ molar⁻¹ for 6-(3-methyl-2-butenylamino)purine, 6.6 × 10⁶ molar⁻¹ for 6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine, and 2.0 × 10⁴ molar⁻¹ for 6-methylaminopurine. Affinity constants were below the limit of detectability (<10⁴ molar⁻¹) for benzylamine, adenine, and other adenine derivatives without an N⁶-side chain. The N⁶-substituent was thus immunodominant, but the purine moiety was also necessary for binding affinity. The antibodies were immobilized on cyanogen bromide-activated Sepharose with 95% retention of binding capacity and without apparent change in affinity constants. Columns of the immobilized antibody retained 64% of the [³H]6-(3-methyl-2-butenylam-ino)purine from 2 nanomolar solutions and readily trapped [¹⁴C]6-benzylaminopurine that had been added to crude extracts of cabbage. Aqueous 10% pyridine adjusted to pH 7.3 with formic acid effectively eluted bound cytokinins from gel columns without loss of binding capacity of the immobilized antibody.     

Inhibition of insulin receptor phosphorylation by indomethacin

Insulin stimulated phosphorylation of tyrosine residues by the insulin receptor kinase may be part of a signalling mechanism associated with insulin's action. We report that indomethacin inhibited the phosphorylation of the -subunit of the solubilized adipocyte insulin receptor. Indomethacin also inhibited several insulin-sensitive processes in intact rat adipocytes. Indomethacin (1 mM) inhibited basal phosphorylation of the -subunit of the solubilized insulin receptor by 6007o and insulin-stimulated phosphorylation by 30%. In adipocytes, indomethacin inhibited basal 3-0-[methyl-¹⁴C]-methyl-D glucose transport by 50070 (P < 0.01), D-[6-¹⁴C]-glucose oxidation by 5007o (P < 0.01), D-[6-¹⁴C]-glucose conversion to lipid by 30010 (P < 0.01), and D-[1-¹⁴C]-glucose conversion to lipid by 6007o (P<0.01). Similarly, indomethacin inhibited insulin-stimulated 3-0-[methyl-¹⁴C]-methyl-D-glucose transport by 75070 (P<0.01), D-[6-¹⁴C]-glucose oxidation by 20% (P<0.05), D-[1-¹⁴C]-glucose oxidation by 35070 (P<0.01), D-[6-¹⁴C] glucose conversion to lipid by 25010 (P<0.01), and D-[1-¹⁴C] glucose conversion to lipid by 4501o (P<0.01). In contrast, insulin binding to its receptor, basal D-[1-¹⁴C]-glucose oxidation and both basal and insulin-stimulated activation of glycogen synthase were unaffected by indomethacin. Thus, indomethacin partially inhibited autophosphorylation of the solubilized insulin receptor on tyrosine and partially inhibited some but not all of insulin's actions. This supports the hypothesis that insulin's metabolic effects are linked to activation of the insulin receptor protein kinase and indicates that there may be heterogeneity in the mechanisms of intracellular metabolic control by insulin.     

Metabolism of [14C]indole-3-acetic acid by the cortical and stelar tissues of Zea mays L. roots

Reverse-phase high-performance liquid chromatography was used to analyse ¹⁴C-labelled metabolites of indole-3-acetic acid (IAA) formed in the cortical and stelar tissues of Zea mays roots. After a 2-h incubation in [¹⁴C]IAA, stelar segments had metabolised between 1–6% of the methanol-extractable radioactivity compared with 91–92% by the cortical segments. The pattern of metabolites produced by cortical segments was similar to that produced by intact segments bathed in aqueous solutions of [¹⁴C]IAA. In contrast, when IAA was supplied in agar blocks to stelar tissue protruding from the basal ends of segments, negligible metabolism was evident. On the basis of its retention characteristics both before and after methylation, the major metabolite of [¹⁴C]IAA in Zea mays root segments was tentatively identified by high-performance liquid chromatography as oxindole-3-acetic acid.Abbreviations HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid     

Androgenesis in Citrus aurantifolia (Christm.) swingle

By means of gas chromatography-selected ion monitoring-mass spectrometry using an isotope-dilution assay with 4,5,6,7-tetradeutero-indole-3-acetic acid as the internal standard, indole-3-acetic acid has been estimated to be present in aseptically cultured gametophytes of wild-type Physcomitrella patens (Hedw.) B.S.G. at a level of 0.075 g g^–1 dry weight or 2.1 ng g^–1 fresh weight.Abbreviations IAA indole-3-acetic acid - d₄IAA 4,5,6,7-tetra-deutero-indole-3-acetic acid - [¹⁴C]IAA indole-3-[2-¹⁴C]-acetic acid - GC-SIM-MS gas chromatography-selected ion monitoring-mass spectrometry     

Biosynthesis and metabolism of indole-3-ethanol and indole-3-acetic acid by Pinus sylvestris L. needles

Combined gas chromatography-mass spectrometry has been used to identify indole-3-ethanol (IEt) in a purified extract from needles of Pinus sylvestris L. Quantitative estimates obtained by high-performance liquid chromatography with fluorescence detection, corrected for samples losses occurring during purification, indicate that Pinus needles contain 46±4 ng g^-1 IEt. This compares with 24.5±6.5 ng g^-1 indole-3-acetic acid (IAA) and 2.3±0.4 ng g^-1 indole-3-carboxylic acid (ICA) (Sandberg et al. 1984, Phytochemistry, 23, 99–102). Metabolism studies with needles incubated in a culture medium in darkness revealed that both [3-¹⁴C]-tryptophan and [2-¹⁴C]tryptamine mine are converted to [¹⁴C]IEt. It was also shown that [3-¹⁴C]IEt acted as a precursor of [¹⁴C]IAA. The observed metabolism appears to be enzymic in nature. The [2-¹⁴C]IAA was not catabolised to [¹⁴C]ICA in detectable quantities implying that, at best, only a minor portion of the endogenous ICA pool in the Pinus needles originates from IAA.Abbreviations DEAE diethylaminoethyl - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - ICA indole-3-carboxylic acid - IEt indole-3-ethanol - PVP polyvinylpyrrolidone     

Catabolism of indole-3-acetic acid in chloroplast fractions from light-grown Pisum sativum L. seedlings

Abstract The catabolism of indole-3-acetic acid was investigated in chloroplast preparations and a crude enzyme fraction derived from chloroplasts of Pisum sativum seedlings. Data obtained with both systems indicate that indole-3-acetic acid undergoes decarboxylative oxidation in pea chloroplast preparations. An enhanced rate of decarboxylation of [1′-¹C]indole-3-acetic acid was obtained when chloroplast preparations were incubated in the light rather than in darkness. Results from control experiments discounted the possibility of this being due to light-induced breakdown of indole-3-acetic acid. High performance liquid chromatography analysis of [2′-¹⁴C]indole-3-acetic acid-fed incubates showed that indole-3-methanol was the major catabolite in both the chloroplast and the crude enzyme preparations. The identification of this reaction product was confirmed by gas chromatography-mass spectrometry when [²H₅]indole-3-methanol was detected in a purified extract derived from the incubation of an enzyme preparation with ³²H₅]indole-3-acetic acid.     

Changes in the Level of [C]Indole-3-Acetic Acid and [C]Indoleacetylaspartic Acid during Root Formation in Mung Bean Cuttings

Changes in the levels of [¹⁴C]indole-3-acetic acid (IAA) and [¹⁴C]indole-acetylaspartic acid (IAAsp) were examined during adventitious root formation in mung bean (Vigna radiata [L.] R. Wilcz. `Berken') stem cuttings. IAAsp was identified by GC-MS as the primary conjugate in IAA-treated cuttings. During root formation in IAA-treated cuttings, the level of [¹⁴C]IAAsp increased rapidly the first day and then declined; [¹⁴C]IAA was rapidly metabolized and not detected after 12 hours.