Tsunehiro Kitagawa

Novel preparation method of immunogen for hydrophobic hapten, enzyme immunoassay for daunomycin and adriamycin.

The present study was undertaken to develop a novel method of preparing a hydrophobic hapten as immunogen. The anticancer drug, daunomycin (DM) was used as prototype and coupled to mercaptosuccinylated bovine serum albumin (MS, BSA) with N-(gamma-maleimidobutyryloxy)succinimide (GMBS). Injection of rabbits with a conjugate (DM-GMBS-MS.BSA) which contained 7.3 DM per BSA molecule produced good levels of anti-DM antibody which was detected by the reaction of diluted antiserum with beta-D-galactosidase-labeled DM. beta-D-galactosidase-labeled DM was used to develop a double antibody enzyme immunoassay for DM and for the DM homologue adriamycin (AM) which reproducibly detected as little as 1 pmole of either drug. A variety of commonly used other anticancer drugs were tested and had little reactivity in this immunoassay. These studies indicate that the anti-DM serum produced is highly specific.

Enzyme immunoassay of bradykinin using β-d-galactosidase as a labeling enzyme☆

Abstract An enzyme immunoassay of bradykinin, using β- d -galactosidase from Escherichia coli as a labeling enzyme, is described. Bradykinin, conjugated to the enzyme with a hetero bifunctional type of coupling agent, N-(m-maleimidobenzoyloxy)succinimide, was prepared as a labeled antigen, Antisera against bradykinin were obtained from male rabbits immunized with bradykinin linked to albumins (ovalbumin or bovine serum albumin) with toluene 2,4-diisocyanate or l-ethyl-3-(3-dimethylaminopropyl) carbodiimide. These antisera were tested for their abilities to bind the labeled antigen and for their sensitivities. The antigen-antibody reaction was performed in an ice bath for 18 hr; this was incubated for another 4 hr, after addition of anti-rabbit IgG antiserum from goat (double antibody method) to separate the bound antigen from free antigen; the enzyme activity in the precipitate was measured with a fluorogenic substrate. Some of the antisera showed good sensitivity when assayed by this method, the sensitivity having been comparable to that of radioimmunoassays of bradykinin. With this method, 30 pg/tube (0.2 ml) of bradykinin could be measured, and the standard curve was obtained in the range of 30 pg to 10 ng of bradykinin. The kininogen level in human plasma was determined by conversion of kininogen to bradykinin by trypsin after heating plasma at 60°. Kininogen levels obtained from six human subjects were in good agreement with those obtained by bioassay.

Preparation of polyamine antibody and its use in enzyme immunoassay of spermine and spermidine with β-D-galactosidase as a label

An enzyme immunoassay for polyamines is described which uses beta-galactosidase labeled spermine and antiserum raised in rabbits against spermine-bovine serum albumin synthesized by coupling spermine to mercaptosuccinylated bovine serum albumin with a bifunctional cross-linker, N-(gamma-maleimidobutyryloxy)-succinimide. The lower limit of detection by this assay, which involves a double antibody technique for the separation of antibody-bound and free antigen, was 1 ng of spermine per tube. The anti-spermine serum showed 88% cross-reaction with spermidine but only 0.13% with putrescine, 0.08% with 1,3-diaminopropane, and 0.04% with cadaverine. The method has been used to measure serum polyamine levels in healthy subjects and cancer patients, in whom mean concentrations of 58.1 ng/ml and 94.8 ng/ml (as spermine), were respectively noted. This enzyme immunoassay is specific, accurate and easy to perform, and appears suitable for routine clinical use.

Immunoelectron Microscopic Study for Polyamines

The polyamines (PAs) are ubiquitous polycationic metabolites in eukaryotic and prokaryotic cells and are believed to be intimately involved in the regulation of DNA, RNA, and protein biosynthesis, the exact function of which remains unclear, mainly because of a lack of knowledge of PA subcellular localization. In this study, using immunoelectron microscopy, we have demonstrated that PAs are predominantly located on free and attached ribosomes of the rough endoplasmic reticulum in the neurons of the lateral reticular nucleus of rat medulla oblongata. The nuclei, axons, and nerve endings were devoid of PA. This suggests that PAs are one of the components of biologically active ribosomes, being closely involved in the translation processes of protein biosynthesis.

A sandwich enzyme immunoassay of rabbit immunoglobulin G with an enzyme labeling method and a new solid support.

A method was devised for enzyme labeling goat antibody to rabbit immunoglobulin G with beta-D-galactosidase (EC 3.2.1.23) using a heterobifunctional cross-linker, N-(gamma-maleimidobutyryloxy)-succinimide. Labeling of the purified antibody was by a continuous 2-step process and including a chromatographic purification procedure could be completed within one day. The partially purified anti-rabbit IgG was coated on Amino-Dylark cylinders, a new solid support, using glutaraldehyde as the coupling reagent. With enzyme-labeled antibody and the solid-phase anti-rabbit IgG, a sandwich enzyme immunoassay for rabbit IgG was developed with a lower limit of detection at 3.5 pM (0.1 ng/tube). The specificity of the assay was excellent and all 4 types of IgG tested showed 0.0001% or less cross-reactivity with rabbit IgG.

Immunocytochemical localization of polyamines in the gastrointestinal tracts of rats and mice

An immunocytochemical method using a recently produced monoclonal antibody (ASPM-29) with an antibody specificity to spermine (Spm) and spermidine (Spd) fixed in situ, was used to demonstrate an immunocytochemical localization of polyamine (PA) pools in the gastrointestinal tracts of rats and mice. High PA immunoreactivity was always found in the cytoplasm of cells not only at the cell proliferative zone or the precursor cell zone but also at the neighboring non-proliferative premature cell zone of the epithelium, and a gradient of decreasing PA levels was noticed from these cells to the fully mature differentiated gastric surface mucous cells and absorptive cells of the small and large intestines. Also, strong staining for PAs was seen in the cytoplasm of fully differentiated gastric chief cells and neurons of both the myenteric and submucous plexuses, whereas the nuclei of the cells remained virtually unstained. These results may suggest that PAs are closely associated with the high biosynthetic activity in the cells of the gastrointestinal mucosa of normal rats and mice. This seems to be consistent with the PA imunocytochemical results previously obtained for neoplastic cells and active protein- or peptide-secreting cells, including exocrine or endocrine cell types.

Polyamine-like immunoreactivity in rat neurons.

The localization of polyamine (PA) pools in motor, sensory, and autonomic neurons and in the nerve cells of the hypothalamo-hypophysial system of rats was examined by immunocytochemical method using the monoclonal antibody ASPM-29 specific to spermine (Spm) and spermidine (Spd) fixed in situ. Strong PA immunoreactivity was found in the cytoplasm and dendrites of the large perikaryon of motor neurons in the anterior spinal column, in the Purkinje cells of the cerebellum, in the pyramidal cells of the cerebrum, in the nerve cells of the paraventricular and supraoptic nuclei in the hypothalamus, and in the nerve cells of the spinal and autonomic ganglions. No PA immunoreactivity was seen in the nucleus and nerve terminals of the neurons. The PA immunoreactivities in the motor and sensory neurons were characterized by clustered masses and blocks of immunoreactive cells. Irrespective of location, small and medium-sized neurons were weakly PA-positive. The glia cells, some stellite cells, and Schwann cells were almost completely PA-negative. These results may suggest that in neurons PAs are not transported axonally, but are located in conjunction with Nissl bodies (the rough endoplasmic reticulum), specified as sites for protein synthesis within cells.

Sandwich enzyme immunoassay of tumor-associated antigen sialosylated Lewisx using β-d-galactosidase coupled to a monoclonal antibody of IgM isotype

Enzyme conjugates with antibody of IgG type have been used extensively in immunohistochemistry, but conjugates with antibody of IgM type have not been reported. This paper describes the beta-D-galactosidase (Gal) labeling of a monoclonal IgM antibody designated CSLEX1 (for cytotoxic sialosylated Lewisx), which is directed against a tumor-associated antigen sialosylated Lewisx (S-Lex). The antibody was first acylated with a heterobifunctional agent N-(gamma-maleimidobutyryloxy)succinimide (GMBS) to introduce the maleimide groups into the molecule; excess reagent was removed by gel filtration and then the activated antibodies were crosslinked to the thiol groups of Gal. The conjugates were partially purified of free Gal by DEAE-Toyopearl column chromatography with an increasing linear concentration of NaCl. The conjugates thus prepared retained almost full enzyme activity and were demonstrated to be free of CSLEX1 by affinity chromatography using anti-galactosidase antibody bound to Sepharose 4B. The conjugates were used as a label in a sandwich enzyme immunoassay (SEIA) to detect the antigen at concentrations as low as 0.2 U/well. The SEIA was used to measure serum S-Lex levels in both healthy subjects and lung cancer patients and mean concentrations of 70 U/ml and 198.6 U/ml were detected respectively.

Inactivation of pyroglutamyl aminopeptidase by L-pyroglutamyl chloromethyl ketone.

A chloromethyl ketone derivative of pyroglutamic acid was newly synthesized and its reactivity with bacterial pyroglutamyl aminopeptidase (L-pyroglutamyl-peptide hydrolas, EC 3.4.11.8) as an affinity labelling reagent was examined. The compound was found to inactivate the enzyme markedly and rapidly at very low concentrations, though the enzyme was resistant to N-tosyl-phenylalanyl chloromethyl ketone. The rate of the enzyme inactivation by pyroglutamyl chloromethyl ketone was retarded in the presence of a poor substrate, pyroglutamyl valine. The enzyme inactivated by treating with p-chloromercuribenzoate failed to react with pyroglutamyl chloromethyl ketone. These results strongly suggest an active site-directed mechanism for the enzyme inactivation by pyroglutamyl chloromethyl ketone. This compound was shown to be useful as a titrant for the catalytically active protein of pyroglutamyl aminopeptidase.

Monoclonal antibodies against glutaraldehyde-conjugated histamine: application to immunocytochemistry

Abstractu2002We have developed mouse monoclonal antibodies (AHA-1–5, all IgG1 sub-isotype mAbs) against histamine (HA) conjugated to bovine serum albumin using glutaraldehyde-NaBH4. Among these, AHA-1 mAb was found to be the most useful for HA immunocytochemistry (ICC) in terms of specificity and sensitivity without non-specific immunobinding. AHA-1 was demonstrated to be specific to HA with an enzyme-linked immunosorbent assay (ELISA) binding test, simulating the ICC of tissue sections, and not reactive to any of the other amino acids and peptides with N-terminal histidine tested. By use of this antibody, indirect immunoperoxidase staining was observed in rat stomach fixed with glutaraldehyde (GA) in combination with NaBH4 reduction. In contrast, no immunoreactivity was seen in tissue fixed only with GA. Absorption controls indicated that the immunostaining could be completely inhibited by GA-conjugated HA, which was consistent with the results of an ELISA inhibition test. No cross-reactivity occurred with other GA-conjugated amino acids. ICC staining was dense in the cytoplasm of gastric enterochromaffin-like cells and very weak in mast cells. A new finding was that staining was noticed in some cell bands of the intermediate layer between the stratum lucidum and the stratum corneum of the stratified squamous epithelium of the gastric cardia, esophagus, tongue, and skin in rats. The results strongly suggest that the monoclonal antibody allowed highly specific detection of HA in animal tis- sues.