幽门螺杆菌UreB单克隆抗体的制备及尿素酶活性抑制能力的研究

目的制备抗幽门螺杆菌尿素酶B亚单位(UreB)单克隆抗体,并检测其对尿素酶活性的抑制能力。方法以重组UreB蛋白为免疫原,通过杂交瘤技术制备抗UreB单克隆抗体,用ELISA检测mAb的效价、亲和常数;用Westernblot检测mAb的特异性。将尿素酶与单克隆抗体预孵育后加入含有尿素和酚红的缓冲液,于550nm测定单克隆抗体对尿素酶活性的抑制能力。结果得到5株稳定分泌抗UreB的单克隆抗体的杂交瘤细胞1D5、9E1、3A2、1D6、6E6。测定抗体亚型1D5,9E1,3A2为IgG1,1D6、6E6为IgG2a,亲和常数分别为4×108、4.6×108、2.8×108、2×109、3×109L/mol。Westernblot鉴定5种单克隆抗体均能和重组UreB及全菌蛋白中的UreB抗原发生特异性结合,且均不与肠道常见菌发生交叉反应。5株单克隆抗体中只有6E6具有对尿素酶活性的抑制作用,且抑制率与单克隆抗体剂量相关。结论制备了5株稳定分泌抗UreB抗体的杂交瘤细胞株,产生的单克隆抗体效价高且特异性好,其中6E6能抑制尿素酶活性。本研究为探讨尿素酶的作用机制、UreB的纯化及Hp的临床诊断和治疗奠定了基础。     

In vivo imaging of rat lymphocytes with an indium 111-labelled anti-T cell monoclonal antibody: a comparison with indium 111-labelled lymphocytes

An indium 111-labelled mouse anti-rat T cell monoclonal antibody, MRC OX-19, was injected intravenously into rats to establish the usefulness of radiolabelled anti-lymphocyte antibodies in imaging lymphoid tissues. Antibody binding in vivo, measured by immunofluorescence analysis of cell suspensions made from lymphoid tissues, was detectable on lymphocytes in blood, spleen and lymph nodes. The extent of binding was time and antibody-dose dependent. Doses of antibody above 80 g/kg body weight resulted in modulation, i.e. loss of CD 5 (T 1) molecules from the cell surface, although the cells remained in the circulation. Modulation was demonstrable within 2 h and for at least 24 h after a single injection of antibody. Intravenous injection of¹¹¹In-MRC OX-19 resulted in levels of in vivo binding comparable with those seen with unlabelled antibody. Scintillation imaging showed early splenic localisation persisting over 48h, a more gradual localisation in the lymph nodes seen clearly at 24 h and a steady background. Comparison of the in vivo distribution of labelled antibody and¹¹¹In-tropolone-labelled lymphocytes showed that both could be used for external imaging of lymphocytes by scintillation camera.     

Immunohistological study of senile brains by using a monoclonal antibody recognizing β amyloid precursor protein: significance of granular deposits in relation with senile plaques

Summary Immunochemical analyses revealed that a monclonal antibody Am-3 recognized amyloid precursor protein (APP) in senile plaques extracted from Alzheimer's brain, but did not recognize amyloid protein. Immunohistochemically, however, the staining pattern of Am-3 in frozen section of Alzheimer's brain was almost the same with that of rabbit polyclonal antibody to amyloid peptide which could recognize both amyloid protein and APP. In other words, APP was present in senile plaques of various types, cerebrovascular amyloid and granular deposits. The granular deposits were 5–10 m in size and laminarily distributed in the 1st, 3rd and 4th layers of cerebral cortex. They were especially abundant in 1st and 4th layers where senile plaques were usually fewer in number. Although the distribution in the cerebral cortex was different between the senile plaques and the granular deposits, the number of the granular deposits was well correlated with that of senile plaques. The granular deposits were negative in Congo-red birefringence, but contained amyloid protein as well as APP fragment judging from positive staining by both Am-3 and polyclonal antibody to synthetic amyloid peptide. Thus, they could be regarded as pre-amyloid.     

Detection of the organophosphorus nerve agent soman by an ELISA using monoclonal antibodies

The development of a specific and sensitive immunologic ELISA detection system for methylphosphonoflouridic acid. 1,2,2-trimethylpropylester (soman) by the use of monoclonal antibodies (MAbs) is described. The monoclonal antibodies F71D7, F71H10, F71B12 and F71H9 originally produced against the soman derivative methyl phosphonic acid,p-aminophenyl 1,2,2-trimethylpropyldiester (MATP) also reacted with soman in a previously developed, direct competitive ELISA. After optimizing the ELISA system by varying the reaction mixture and the solvents for the organophosphate, 5.0×10^–7 mol/l soman (80% purity), e.g. 2.5 ng or 2 ng pure soman per 25 l test buffer, could be detected after a total test duration of 40 min. A shortening of the incubation time to 10 min resulted in a drop of sensitivity to 1.8×10^–6 mol/l soman. Various alcohols which may be used as extraction media for soman from various materials (isopropanol, ethanol and methanol) were shown to inhibit peroxidase activity and thereby reduce the sensitivity of the test. However, the influence of alcohols decreased with the shortening of incubation time. All monoclonal antibodies showed little cross reactivity to sarin and no cross reactivity to tabun and VX. Judging on the reactivity of the MAbs with MATP and soman oxidazed by 1,2-dihydrobenzol, some reactivity with some other (non-toxic) soman analogues containing the same pinacolyl group can be expected. There was no evidence for stereoselectivity of the MAbs tested. Finally, soman could be detected in different biological samples like human serum, goat serum, rabbit serum, chicken serum, milk, and tap water in concentrations between 1.3×10^–6 and 2.0×10^–6 mol/l.     

Comparative biodistribution of indium- and yttrium-labeled B3 monoclonal antibody conjugated to either 2-(p-SCN-Bz)-6-methyl-DTPA (1 B4M-DTPA) or 2-(p-SCN-Bz)-1,4,7,10-tetraazacyclododecane tetraacetic acid (2B-DOTA)

The biodistribution of indium-111/yttrium-88-labeled B3 monoclonal antibody, a murine IgG1k, was evaluated in non-tumor-bearing mice. B3 was conjugated to either 2-(p-SCN-Bz)-6-methyl-DTPA (1B4M) or 2-(p-SCN-Bz)-1,4,7,10 tetraazacyclododecane tetra-acetic acid (2B-DOTA) and labeled with ¹¹¹In at 1.4–2.4 mCi/mg and ⁸⁸Y at 0.1–0.3 mCi/mg. Non-tumor-bearing nude mice were co-injected i.v. with 5–10 Ci/4–10 g of ¹¹¹In/⁸⁸Y-labeled B3 conjugates and sacrificed at 6 h and daily up to 168 h post-injection. Mice injected with ¹¹¹In/⁸⁸Y (IB4M)-B3 showed a similar biodistribution of the two radiolabels in all tissues except the bones, where significantly higher accretion of ⁸⁸Y than ¹¹¹In was observed, with 2.8% ± 0.2% vs 1.3% ± 0.16% ID/g in the femur at 168 h, respectively (P<0.0001). In contrast, mice receiving the ¹¹¹In/⁸⁸Y-(DOTA)-B3 conjugate showed significantly higher accumulation of ¹¹¹In than ⁸⁸Y in most tissues, including the bones, with 2.0% ± 0.1% vs 1.2% ± 0.09% ID/g in the femur at 168 h, respectively (P<0.0001). Whereas the ratios of the areas underneath the curve (%ID × h/g) in the blood, liver, kidney and bone were 0.96, 1.12, 1.13, and 0.74 for ¹¹¹In/⁸⁸Y-(IB4M)-B3 and 0.84, 1.23, 1.56, and 1.31 for ¹¹¹In/⁸⁸Y (DOTA)-B3, respectively, ratios 1 were observed between ¹¹¹In-(IB4M)-B3 and ⁸⁸Y-(DOTA)-B3. In summary, while neither IB4M nor DOTA was equally stable for ¹¹¹In and ⁸⁸Y, the fate of ⁸⁸Y- (DOTA)-B3 could be closely traced by that of ¹¹¹ In-(IB4M)-B3.     

Comparison of non-invasive approaches to red marrow dosimetry for radiolabelled monoclonal antibodies

Red marrow is usually the dose-limiting organ during radioimmunotherapy. Several non-invasive approaches to calculate the red marrow dose have been proposed. We compared four approaches to analyse the differences in calculated red marrow doses. The data were obtained from immunoscintigraphy of two antibodies with different red marrow kinetics [iodine-131-16.88 IgM and indium- 111-OV-TL-3 F(ab)₂]. The approaches are based on, respectively, homogeneously distributed activity in the body, a red marrow-blood activity concentration ratio of 0.3, scintigraphic quantification, and a combination of the second and third approaches. This fourth approach may be more adequate because of its independence from the chosen antibody. In addition, the influence of activity accumulation in liver, kidneys or cancellous bone on red marrow dose was studied. The calculated red marrow dose varied between 0.14 and 0.42 mGy/MBq for ¹¹¹ In-OV TL-3 and between 0.13 and 0.68 mGy/MBq for ¹³¹I-16-88. If the radiopharmaceutical shows high affinity for cancellous bone or another organ situated near the red marrow, the activity in these organs must be included in dose calculations. This study shows a large variation in calculated red marrow dose and selection of the definitive non-invasive approach awaits validation. Correspondence to: M.A.B.D. Plaizier     

Growth suppression of human colorectal carcinoma in nude mice by monoclonal antibody C27-abrin A chain conjugate

PURPOSE: The aim of this study was to assess an immunotoxin, monoclonal antibody C27-abrin A chain conjugate (MAAC), that might be effective in the treatment of colorectal carcinoma. METHODS: The immunotoxin was prepared by a specific monoclonal antibody against carcinoembryonic antigen (CEA), monoclonal antibody C27, linked toN-succinimidyl-3-(2-pyridyldithio)propionate and then coupled covalently to the toxic abrin-A chain to synthesize MAAC. The therapeutic role of this immunotoxin in suppressing thein vitro andin vivo growth of CEA-secreting human colorectal cancer cells (LS174T) was assayed by methods of protein biosynthesis inhibition, cell colony proliferation, and treatment of tumor cells before and after inoculation in nude mice. RESULTS: We found that MAAC effectively suppressed the growth of LS174T in culture medium and completely eradicated cells in inoculated nude mice. In contrast, irrelevant immunotoxin antiferritin-abrin A chain conjugate and isotype-matched monoclonal immunoglobin (MOPC21IgG1)-abrin A chain conjugate did not cause such effects. Thein vitro toxicity was highly specific because the conjugate (MAAC) inhibitedde novo protein biosynthesis, impeded growth, and caused death of cells possessing surface CEA determinants. The 50 percent inhibition dose values of the conjugate for colonogenic survival and for protein biosynthesis in LS174T cells were 0.09 g/ml and 0.06 g/ml, respectively. Colony survival was inhibited 96.3 percent after prolonged MAAC treatment. MAAC showed selective cytotoxicity; the inhibitory effect of MAAC to the CEA-secreting LS174T cells over the CEA-nonsecreting human embryonic kidney cells was 16-fold. CONCLUSION: These results indicate that MAAC may be of benefit in therapy during or soon after resection of colorectal carcinoma or in patients who have micrometastasis.Supported by a grant from the National Science Council and the Veterans General Hospital-Taipei, Taipei, Taiwan.     

Monitoring pathological assembly of tau and β-amyloid proteins in Alzheimer's disease

This double-labelling confocal microscopy study of the neuropathology of Alzheimer's disease (AD) reports the use of a fluorescent dye, thiazin red, which has staining properties similar to thioflavin-S. Thiazin red fluorescence can be visualised selectively in the red channel, and we have used this property to compare it with the labelling seen using monoclonal antibody (mAb) 423, which detects tau protein C-terminally truncated at Glu-391, and mAb 4G8, which detects -amyloid protein. Thiazin red is shown to recognized the typical histopathological deposits associated with both proteins. However, not all deposits containing these proteins are stained. Specifically, diffuse -amyloid plaques and severely degraded extracellular tangles are unlabelled. Likewise a characteristic mAb 423-reactive granular plaque-like structure, typically present in cases with abundant extracellular tangels, is unlabelled by thiazin red. Such plaques can be shown to be continuous with the basal dendrites of degraded tanglebearing pyramidal cells. These findings suggest that paired helical filaments (PHFs) continue to undergo degradation in the extracellular space, which is associated with loss of thiazin red binding sites, but preservation of mAb 423 immunoreactivity. This epitope appears to be characteristic of a stable core element of the PHF which is highly resistant to proteolysis. Compounds such as thiazin red with high affinity for -pleated protein structures can be used to monitor the state of pathological assembly of amyloidogenic protein species found in AD.Supported in part by CONACyT grant #1624-N9208 (to R.M.), the Medical Research Council (U.K.), Zeneca Pharmaceuticals and the Alzheimer Disease Research Fund and the Leopold Muller Estate     

直接还原法标记^99mTc与抗人膀胱癌单克隆抗体BDI—1

通过２ － 巯基乙醇直接还原法将抗人膀胱癌单克隆抗体 Ｂ Ｄ Ｉ－ １ 与核素９９ｍ Ｔｃ 进行标记, 制备９９ ｍ Ｔｃ － Ｂ Ｄ Ｉ－ １ ． 优化条件后进行标记并进行一系列检测． 结果： 标记率为７２ ％ , 经 Ｓｅｐｈａｄｅｘ 柱纯化后标记抗体的放化纯度高于９５ ％ ; 标记抗体的免疫活性分数为８７ ％ , 与 Ｅ－ Ｊ细胞的结合率为９０ ％ ; 无毒、无菌、无热原． 结果表明： ２ － 巯基乙醇直接还原法将抗人膀胱癌单克隆抗体 Ｂ Ｄ Ｉ－ １ 与核素９９ ｍ Ｔｃ 进行标记为进一步进行膀胱癌的放射免疫显像诊断奠定了基础．     

单克隆抗体在形态学难以分型的急性白血病分型中的应用

目的：探讨单克隆抗体在形态学难以分型的急性白血病分中的价值。方法采用ＡＰＡＡＰ法检测１４例形态学难以分型的急性白血病患者的免疫表型。结果：１４例患者均有某个系列特异性（或相关性）标志表达,免疫学分型为急性髓性白血病７例,急性淋巴细胞白血病４例,急性混合细胞白血病２刀性未分化白血病１例。结论：单克隆抗体有助于形态学难以分型的急性白血病的分型。