Effects of the centrally acting cholinesterase inhibitors tetrahydroaminoacridine and E2020 on the basal concentration of extracellular acetylcholine in the hippocampus of freely moving rats

The effects of the centrally acting cholinesterase (ChE) inhibitors, tetrahydroaminoacridine (THA) and E2020 (1-benzyl-4-[(5,6-dimethoxy-l-indanon)-2-yl] methylpiperidine hydrochloride), potential drugs for the treatment of senile dementia, on the basal extracellular acetylcholine (ACh) concentration in the hippocampus of freely moving rats, were determined using a microdialysis technique without the use of a ChE inhibitor in the perfusion fluid and a sensitive RIA. The mean (±SEM) basal ACh content in the perfusate was 103.1 ± 3.6 fmol/sample collected over 30 min when microdialysis probes with a length of 3 mm dialysis membrane were used. The content of ACh decreased to an almost undetectable level upon perfusion of magnesium, suggesting that, in the present study, most of the ACh detected in the perfusates was due to cholinergic neuronal activity. THA (1.65 mg/kg, i.p.) produced an insignificant increase in the extracellular ACh concentration, but a dose of 5 mg/kg, i.p. caused a prolonged and significant 5.5-fold increase from the control value. E2020 (0.65 and 2 mg/kg, i.p.) produced significant, prolonged and dose-dependent increases (4 and 12 times the control value, respectively), the peak effect occurring within 1 h. Perfusion with 10 mol/l physostigmine produced an about 30-fold increase of ACh output, suggesting that the basal extracellular ACh concentration is highly dependent on ChE activity. When ChE was inhibited locally by perfusion with physostigmine, THA (5 mg/kg) produced a transient and, at its maximum, a 1.42-fold increase in extracellular ACh concentration. These results demonstrate that the basal, physiological, extracellular ACh concentration in the hippocampus of freely moving rats can be determined using a microdialysis technique and a sensitive RIA, and suggest that THA and E 2020 increase ACh concentration in the synaptic cleft of the hippocampus in a dose-dependent manner mostly through ChE inhibition. Correspondence to: K. Kawashima at the above address     

放射免疫分析法测定重组人白细胞介素-2含量

目的:建立用双抗体放射免疫分析法测定重组人白细胞介素-2(IL-2)注射剂的含量.方法:以重组人IL-2为抗原与放射物标记的抗体结合,采用放射免疫分析仪测出抗原-抗体-第二抗体免疫复合物的放射强度计数cpm值.建立重组人IL-2药物浓度与复合物放射强度之间的关系曲线.根据此关系曲线得出重组人IL-2的含量.结果:重组人IL-2在0～150 μg·L-1浓度范围内标准曲线较好.注射用粉针剂重组人IL-2的含量以重量浓度单位表示为50 mg·g-1.质量控制符合要求.精密度RSD为4.34%,在误差许可范围内.结论:双抗体放射免疫分析法可用于制剂中重组人IL-2的定量分析,相关辅料对测定结果无影响.     

Production of monoclonal antibodies with preselected submolecular binding specificities to protein antigenic sites: antibodies to sperm whale myoglobin sites

The five synthetic antigenic sites of sperm whale myoglobin were used in their free form (i.e. not coupled to any carrier) to immunize separate groups of BALB/cByJ mice. The synthetic peptides corresponded to: site 1, residues 15-22; site 2, residues 56-62; site 3, residues 94-99; site 4, residues 113-119; site 5, residues 145-151. Serum samples obtained from each group of mice contained antibodies that bound specifically to myoglobin and exclusively to the immunizing antigenic site. Monoclonal antibodies to each of the five antigenic sites were subsequently obtained by hybridizing Fa/O mouse myeloma cells with spleen cells derived from each group of mice. These monoclonal antibodies were either IgM(kappa) or IgGl(kappa). They expressed the same isotypes as the antigen specific serum antibodies produced by the mice whose spleen cells were used for hybridization. Solid phase radioimmunoassay studies also indicated that each monoclonal antibody, like the immune serum of the parent animals, bound specifically to myoglobin and exclusively to the synthetic peptide used as an immunogen. These results suggested that the hybridoma antibodies expressed submolecular binding specificities that were the result of peptide immunization rather than hybrid selection. This strongly supports our previous findings that it is possible to produce monoclonal antibodies with preselected submolecular binding specificities to continuous protein determinants by the techniques of somatic cell hybridization when the corresponding free synthetic determinants are used as immunogens.     

A double antibody radioimmunoassay for mouse hemoglobins: use of polyethylene glycol in conjunction with the second antibody.

A double antibody radioimmunoassay (RIA) system is described for detection of small quantities of hemoglobins. Mouse (C57BL/6) hemoglobin and horse anti-mouse hemoglobin antiserum were used to develop the system.The first phase of the RIA, i.e., the initial reaction between the antigen and the antibody, was found to be complete within 24 h. The reaction proceeded better at 4°C than at 25°C. The second phase, i.e., separation of bound from unbound antigen, was achieved by precipitation with a second antibody (goat anti-horse IgG) and polyethylene glycol (PEG). A 50 g/l concentration of PEG was found to be best suited for the assay. Mixing of all the reagents together was found to decrease the binding as compared to the system in which second antibody and PEG were added after completion of the first phase. Maximum precipitation of the complex took place within 30 min after the addition of the second antibody and 1 h after the addition of 50 g/l PEG.The RIA system described here combines the conventional double antibody RIA with the PEG method. This method has decreased the amount of time necessary for precipitation from 24 h (or longer) to 1 h. Large molecular weight antigens could not be estimated in the conventional PEG method because of their insolubility in 200 g/l PEG utilized in the assay. The use of a low concentration of PEG along with the second antibody in the method described here allows the estimation of large molecular weight antigens. This double antibody-PEG method has a general applicability for small as well as large molecular weight antigens.     

A rapid, sensitive two-site immunometric assay for TSH using monoclonal antibodies: investigation of factors affecting optimisation

Twenty-three monoclonal antibodies specific for human thyrotropin (TSH) have been prepared and characterised. Four different epitopes have been identified, 3 of which are sufficiently distinct to allow simultaneous binding of the corresponding antibodies. Only 1 epitope is expressed on free TSH beta-subunit. The best antibodies have affinities for TSH approaching 10(11) M-1 and cross-reactions with lutropin and chorionic gonadotropin of 0.2% or less. A 2-site immunoradiometric assay for TSH was established using 2 monoclonal antibodies, one of which was absorbed to plastic tubes and the other labelled with 125I. Several parameters affecting assay performance were investigated, including conditions for adsorption of antibodies to various plastics, selection of antibody combinations, concentration of labelled antibody and incubation protocol. The optimised assay covered a working range of 1-60 mU/1 (0.17-10 ng/ml) TSH in a 4 h, single incubation protocol, with no significant interference from other glycoprotein hormones at their maximum physiological or pathological concentrations.     

Detection of IgE in the sera of rodents: comparison of the applicability of ELISA and RIA

RIA and ELISA were compared for their ability to detect IgE in different rodent species. With a sheep anti-rat IgE antibody good correlation (P less than 0.001) between the 2 assay methods for IgE was found in rats. RIA failed to detect the IgE of Mastomys natalensis while ELISA proved to be a suitable test. However, both tests failed to measure IgE in sera of Nile rats.     

SOD固相-抗的RIA

放射免疫分析大大提高SOD的灵敏度，用SOD抗血清固相聚苯乙烯微球，使微球具有一抗的功能，又代替二抗及PEG等分离剂，因而分析简单，减少干扰；使用Bolton-Hunter试剂联合碘标记法标记SOD获得高的比活性。     

Monoclonal antibody against a "Pan-T-cell" antigen expressed by thymocytes, peripheral T-lymphocytes and T-cell leukemias

A monoclonal antibody, LAU-A1, which selectively reacts with all cells of the T-lineage, was derived from a fusion between spleen cells of a mouse immunized with paediatric thymocytes and mouse myeloma P X 63/Ag8 cells. As shown by an antibody-binding radioimmunoassay and analysis by flow microfluorometry of cells labelled by indirect immunofluorescence, the LAU-A1 antibody reacted with all six T-cell lines but not with any of the B-cell lines or myeloid cell lines tested from a panel of 17 human hematopoietic cell lines. The LAU-A1 antibody was also shown to react with the majority of thymocytes and E-rosette-enriched peripheral blood lymphocytes. Among the malignant cell populations tested, the blasts from all 20 patients with acute T-cell lymphoblastic leukemia (T-ALL) were found to react with the LAU-A1 antibody, whereas blasts from 85 patients with common ALL and 63 patients with acute myeloid leukemias were entirely negative. Examination of frozen tissue sections from fetal and adult thymuses stained by an indirect immunoperoxidase method revealed that cells expressing the LAU-A1 antigen were localized in both the cortex and the medulla. From the very broad reactivity spectrum of LAU-A1 antibody, we conclude that this antibody is directed against a T-cell antigen expressed throughout the T-cell differentiation lineage. SDS-PAGE analysis of immunoprecipitates formed by LAU-A1 antibody with detergent lysates of radiolabeled T-cells showed that the LAU-A1 antigen had an apparent mol. wt of 76,000 under non-reducing conditions. Under reducing conditions a single band with an apparent mol. wt of 40,000 was observed. Two-dimensional SDS-PAGE analysis confirmed that the 76,000 mol. wt component consisted of an S-S-linked dimeric complex. The surface membrane expression of LAU-A1 antigen on HSB-2 T-cells was modulated when these cells were cultured in the presence of LAU-A1 antibody. Re-expression of LAU-A1 antigen occurred within 24 hr after transfer of the modulated cells into antibody-free medium.     

Efficient selection of human tumor growth-inhibiting monoclonal antibodies

A method is described for selection early after fusion for hybridomas that secrete IgG2a monoclonal antibodies (MAbs) with binding specificity for antigens on the human tumor cells used to immunize mice. By combining this preselection method with antibody-dependent macrophage-mediated cytotoxicity assays, it was possible to select those MAbs mediating a tumoricidal effect against tumor cells in culture and inhibiting the growth of tumor xenografts in nude mice. Two such MAbs, GA733 and CO441, inhibited the growth of colorectal carcinoma cells, and one of them (GA733) was effective even when administered 6 days after implantation of tumor cells. These results suggest the potential usefulness of the 2 MAbs in immunodiagnosis and immunotherapy of human tumors.     

Immunochemical characterization of a high molecular weight basic allergen (HMBA) of rye grass (Lolium perenne) pollen

A high molecular weight basic allergen (HMBA) was isolated from the mixture of non-dialysable components of the aqueous extract of defatted rye grass pollen by a combination of gel filtration and isoelectrofocussing. HMBA, a glycoprotein of mol. wt 56,800 (17% carbohydrate) contained all naturally occurring amino acids. A hyperimmune rabbit anti-HMBA serum gave only a single precipitin band with the crude extract of the rye grass pollen in crossed immunoelectrophoresis. Thus. it was concluded that HMBA was a unique and highly purified antigen. The allergenicity of HMBA was revealed by its ability to elicit immediate skin reactions in grass allergic patients. Moreover, all patients' sera tested had IgE antibodies to HMBA detectable by direct RAST with HMBA allergosorbent discs, These observations indicated that HMBA was a major allergenic constituent of rye grass pollen. Treatment of HMBA by 6 M guanidine HC1 led to a significant reduction in its ability to combine with human IgE antibodies. The treatment also resulted in the complete loss of allergenicity (i.e. inability to elicit PCA reactions with a murine reaginic antiserum to HMBA) and antigenicity (inability to form precipitins with rabbit anti-HMBA): hence, it would appear that the allergenic and antigenic determinants of HMBA are ‘conformational’.