Rapid and specific enzyme immunoassay of serotonin.

A new, highly sensitive enzyme immunoassay (EIA) of serotonin (5HT) is described. The assay is based on the competition between N-succinyl-glycinamide-serotonin (N-SGA-5HT, obtained by acylation of the 5HT in the sample to be assayed) and an enzymic tracer, N-succinyl-5HT-acetylcholinesterase, for binding to rabbit polyclonal antibody coated onto the wells of microtiter plates. The antibody is directed against an immunogen obtained by coupling N-succinyl-5HT to glycyl-bovine serum albumin. The EIA permits the accurate measurement of as little serotonin as 0.5 nmol/L (1.8 pg per well) in blood, plasma, serum, cerebrospinal fluid, urine, platelets, and other tissues, with no significant cross-reactivity with other compounds. The results obtained correlate well with those obtained by HPLC after extraction. The assay has the advantage of permitting the measurement of 5HT in up to 500 samples in as little as 3 h.

Immunoanalysis of histamine through a novel chemical derivatization.

The immunoanalysis of histamine was carried out with the powerful approach of chemical derivatization. A novel acylating reagent, succinyl glycinamide N-hydroxysuccinimide ester, was synthesized, and monoclonal antibodies were raised to acylated histamine. The monoclonal antibodies had 5 X 10(5) greater affinity for acylated histamine than for native histamine. In agreement with theoretical prediction, no interference was generated, even with closely related amines. An immunoassay of exceptional selectivity and sensitivity was designed that can be used to quantify histamine in any biologic fluid or cell extract.

Production of monoclonal antibodies complementary to an antibody-antigen complex. Use in an immunoradiometric assay for follitropin.

Monoclonal antibodies were prepared against human follitropin by fusion of the myeloma cell line P3-X63-Ag8-653 with spleen cells of mice immunized with either human follitropin or follitropin bound to an anti-beta hFSH monoclonal antibody. The latter immunization procedure permits shielding of the immunodominant specific site and allows the production of numerous specific monoclonal antibodies to human follitropin which are complementary to the monoclonal antibody used in the immunogen. In this investigation two specific monoclonal antibodies were used in a two site immunoradiometric assay which was highly specific, rapid, with one incubation step and demonstrated a sensitivity level of 0.1 mIU/ml. It was possible to differentiate between prepubertal and adult levels of follitropin and to recognize individuals with hyposecretory states.

Immunization with immune complexes: characterization of monoclonal antibodies against a TSH-antibody complex.

Monoclonal antibodies (MAb) to human thyrotropin (hTSH) were prepared by immunization of mice and rats according to different procedures. We have previously demonstrated that a specific antigenic region on the surface of the hTSH molecule was highly immunogenic; in order to produce specific MAb to weakly immunogenic regions of hTSH, we immunized mice and rats with a complex composed of hTSH and an anti-beta hTSH MAb 27 directed against the highly immunogenic region. Monoclonal antibodies elicited by this immunization procedure were highly specific and a high percentage was found complementary to the MAb 27 used in the immunogen. We did not search for anti-MAb 27 antibodies, however one hybridoma produced antibody that preferentially reacted with the immune complex. This antibody, called 515, is an IgG1 that binds the complex with 100-fold greater affinity than it does to the anti-beta hTSH MAb 27 alone. This enhancement was also observed with the Fab fragment of the MAb suggesting that the epitope recognized by this anti-complex MAb is displayed in a very different way when hTSH is bound to the first MAb.

Radioimmunoassay of cyclic AMP can provide a highly sensitive assay for adenylate cyclase, even at very high ATP concentrations.

Modifications of the cyclic AMP radioimmunoassay of Cailla et al. [in Hormones and Cell Regulation (J. Dumont and J. Nunez, eds.), Vol. 4, pp. 1-24, Elsevier/North-Holland, Amsterdam/New York (1980)] allowed its use in the determination of adenylate cyclase activity, which was otherwise precluded by high blank values. These high values originate mainly from chemically formed cyclic AMP and from ATP cross-reactivity. The simultaneous presence of ATP and magnesium ions generates cyclic AMP under the alkaline conditions used to succinylate the sample; this interference can be dealt with either by chelation of Mg2+ ions with EDTA during succinylation or by periodic acid oxidation of samples prior to succinylation. In addition, ATP itself contributes to blank values by its cross-reactivity, especially when working with high concentrations of this substrate. This interference can be decreased by a batch adsorption of ATP or oxidized ATP on alumina. Detailed procedures were discussed, with the choice of the additional steps to the standard method of Cailla et al. having to be made on the basis of the sensitivity requirements. When preventing ATP cyclization, the radioimmunoassay was as sensitive as methods using [alpha-32P]ATP as substrate. Elimination of ATP can improve the sensitivity by one order of magnitude. This method is especially interesting with high ATP concentrations and/or with low cyclic AMP production.

A single radioimmunological assay for serotonin, N-acetylserotonin, 5-methoxytryptamine, and melatonin.

A radioimmunoassay of melatonin using a new iodinated derivative has been developed. Simple chemical treatments have then been designed to convert serotonin, N-acetylserotonin, and 5-methoxytryptamine to melatonin. Thus these four molecules, belonging to the same metabolic pathway, were separately assayed in the same radioimmunological system at the same sensitivity level (0.01 pmol). Some biological results on the circadian variations of melatonin and serotonin in blood, pineal, and miscellaneous brain structures are presented.

Radioimmunoassay of insect juvenile hormones and of their diol derivatives.

We have developed a radioimmunoassay for insect juvenile hormones. The C18 hormone (JH I) was first converted into diol by opening the 10, 11-epoxy ring. Diol was then succinylated and coupled to human serum albumin to make it immunogenic. High titer antisera were obtained from immunized rabbits. Succinyl juvenile hormone was also coupled to the peptide glycyltyrosine and iodinated so as to form a water-soluble 125I-labelled analogue well recognized by antibodies (60% bound by the 1/2000 000 dilution of antiserum routinely used in radioimmunoassay). Standards and biological samples were treated with acidic dioxane in order to convert each hormone into its corresponding diol. In this way, the sensitivity threshold of the radioimmunoassay was under 0.015 pmol. All three diols were equally recognized by the antibodies. Hormones JH I, JH II and JH III could be assayed separately as diols after thin-layer chromatography or high-pressure liquid chromatography purification of the biological samples. This method was used to determine physiological levels of juvenile hormones in the haemolymph of several insects at different development stages including embryos and in corpora allata cultures.

Radioimmunoassay of 5-hydroxyindole acetic acid using an iodinated derivative.

A radioimmunoassay for the main catabolite of serotonin, 5-hydroxyindole acetic acid (5-HIAA), was developed by using specific antibodies and iodinated derivative. The synthesis of a 125I-iodinated analog was performed by coupling 5-HIAA to [125I-]glycyl-tyrosine without any contact between 5-HIAA and iodine or chloramine T. It was purified on a G25 Sephadex column and diluted in citrate buffer up to 2.5 X 10(5) cpm/ml. Antibodies were obtained by coupling 5-HIAA to human serum albumin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and tested by equilibrium dialysis. After the third immunogen injection, the four rabbits gave antisera capable of binding 50% of iodinated 5-HIIA-glycyl-tyrosine at 1/2000 final dilution. A chemical conversion of the biological samples gives to the antigen molecules a better resemblance to the immunogen, thus conferring a 100-fold gain in specificity and sensitivity. This assay allows 5-HIAA to be determined in small amounts of tissue, blood, cerebrospinal fluid or perfusate without purification with a sensitivity threshold below 0.1 ng. Some applications in cat and rat are presented.

Immunochemical detection of vasopressin precursors: artificial processing and quantification along the hypothalamo-hypophysial axis.

A novel immunological approach to the problem of the detection and molar evaluation of vasopressin precursors was taken. First, the specificity of anti-vasopressin antibodies was studied and the hormone antigenic determinant was identified as the sequence Cys-Pro-Arg-Gly-NH2. Then this antigenic determinant, not originally shared by the precursors, was reconstituted by tryptic cleavage followed by chemical fixation of glycinamide. This treatment made quantification of precursors by radioimmunoassay possible at a fmol level in various tissues. In normal rat, precursors were found only in the supraoptic nucleus (192 pmol/mg protein), paraventricular nucleus, median eminence and posterior lobe of the hypophysis. The maturation process was followed by the decrease of the ratio of precursor to hormone from 4-5 to 0.02 along the hypothalamo-hypophysial axis. In Brattleboro rats, genetically deficient in vasopressin, no precursor could be detected over the background level; that ensures the specificity and reliability of this approach.

Radioimmunoassays for serotonin and 5-hydroxyindole acetic acid.

Radioimmunoassays for serotonin and 5-hydroxyindole acetic acid were developed. 1. High titer antibodies, having a well-defined high specificity, have been raised by coupling the side-chain of both molecules to human serum albumin. Serotonin is first converted into N-hemisuccinate, and then treated like 5-HIAA, namely, conjugated with HSA for the immunogen. 2. Synthesis of [125I] iodinated analogues was performed by coupling 5-HIAA or N-succinyl serotonin to glycyltyrosine without any contact between both molecules and the oxidizing reagents. 3. Chemical conversions of biological samples (by succinylation for 5-HT and amidation for 5-HIAA) were carried out. This critical step makes the antigen molecules resemble the immunogen more closely, thus allowing an appreciable gain in specificity and sensitivity. 4. These assays allow the rapid determination of 5-HT and 5-HIAA in small amounts of tissue, blood, cerebral spinal fluid or perfusate without any purification, with a sensitivity threshold of 50 pg.

Statistical theory of chromatography: new outlooks for affinity chromatography.

We have developed further the statistical approach to chromatography initiated by Giddings and Eyring, and applied it to affinity chromatography. By means of a convenient expression of moments the convergence towards the Laplace-Gauss distribution has been established. The Gaussian character is not preserved if other causes of dispersion are taken into account, but expressions of moments can be obtained in a generalized form. A simple procedure is deduced for expressing the fundamental constants of the model in terms of purely experimental quantities. Thus, affinity chromatography can be used to determine rate constants of association and dissociation in a range considered as the domain of the stopped-flow methods.