Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures

Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit-deficient mice should be ideal tools for testing the specificity of subunit-directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the alpha3-, alpha4-, alpha7-, beta2-, and beta4-nicotinic acetylcholine receptor subunits on brain tissues of the respective knock-out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild-type and knock-out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.     

鸡Toll样受体21单克隆抗体的研制及其初步应用

【目的】研制鸡Toll样受体21(chTLR21)单克隆抗体,考察禽致病性大肠杆菌(APEC)及高致病性禽流感病毒(HPAIV)感染鸡组织中的chTLR21蛋白的表达情况。【方法】以人工合成的多肽免疫原chTLR21(203–225aa)与KLH偶联蛋白免疫6周龄BALB/c小鼠,以多肽免疫原chTLR21 (203–225 aa)与BSA偶联蛋白作为检测抗原,通过间接ELISA技术筛选阳性杂交瘤细胞株,同时,采用间接免疫荧光技术(IFA)检测单克隆抗体的荧光反应性;上述单克隆抗体用于chTLR21在鸡巨噬细胞(HD11)中的定位;在APEC O1血清型E516菌株、HPAIV H5N6亚型毒株感染35日龄SPF鸡模型中,应用获得的单克隆抗体以免疫印迹(Western blotting)和免疫组化(IHC)方法检测感染鸡组织中chTLR21的表达情况。【结果】建立了间接ELISA检测方法,确定最适抗原包被浓度为2.5μg/mL,最适血清稀释度为1:6400。经ELISA、IFA筛选,获得4株阳性杂交瘤细胞株,分别命名为1G3、2C10、3B6和4F11。上述单克隆抗体亚类分别为IgG2...     

Development of monoclonal antibodies for simple identification of Vibrio alginolyticus

AIMS: The present study was aimed to produce monoclonal antibodies (MAbs) for simple and specific identification of Vibrio alginolyticus infection in shrimp. METHODS AND RESULTS: Mice were immunized with heat killed V. alginolyticus four times at 2-week intervals. The best response mouse was used for spleen donor in hybridoma production. Screening of hybridoma clones producing desired antibodies was performed by dot blotting against V. alginolyticus and other bacterial species, Western blotting and immunohistochemistry of infected shrimp tissues. Four groups of MAbs were obtained; the first group of MAbs demonstrated their limited specificity only to V. alginolyticus used for immunization, while the second and the third groups recognized all three isolates of V. alginolyticus used for testing. The fourth group of MAbs bound to all three isolates of V. alginolyticus and also recognized Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis and Vibrio vulnificus but did not bind to Vibrio mimicus, Vibrio cholerae, Vibrio penaeicida and other bacterial species tested. MAbs in groups 1, 2 and 3 were able to use for the detection of bacterial infection in the tissues by means of immunohistochemistry. CONCLUSIONS: MAbs specific to V. alginolyticus was produced. These MAbs can be used for specific identification of the bacteria by simple 'dot blotting' method and immunohistochemistry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated an immunological tool that can be used for simple and accurate identification of V. alginolyticus as well as for the diagnosis of V. alginolyticus infection in animals. This immunological tool can replace costly and laborious biochemical tests.     

成年大脑神经细胞特异性ADAM10基因敲除小鼠模型的建立与鉴定

去整合素和金属蛋白酶10(ADAM10)是一种能够水解30余种跨膜蛋白质的“脱落酶”(sheddase), 参与诸多生理过程和致病机制, 如胚胎发育、细胞粘附、信号转导、免疫反应、癌症和阿尔茨海默病。迄今, 已报道的ADAM10完全基因敲除小鼠和大脑神经前体细胞特异性ADAM10基因敲除小鼠分别于胚胎期或围产期死亡, 致使无法研究成年小鼠大脑神经细胞ADAM10基因的功能。文章利用本研究小组建立的CaMKIIα-Cre转基因小鼠与ADAM10^loxP/loxP转基因小鼠杂交, 获得了CaMKIIα-Cre/ADAM10^loxP/loxP小鼠, 并对其进行鉴定。利用PCR方法检测成年ADAM10 cKO小鼠大脑基因组DNA表明, ADAM10基因缺失主要发生在前脑皮层和海马中。荧光定量PCR检测结果显示, ADAM10 mRNA的表达水平在前脑皮层和海马中分别降低55.7%和60.8% ; 使用Western blotting方法研究发现, ADAM10成熟蛋白质的含量在前脑皮层和海马中分别减少63%和84.8% 。采用免疫组织化学方法检测表明, 成年ADAM10 cKO小鼠与野生型小鼠相比, 其大脑皮层和海马神经细胞的ADAM10免疫染色明显减弱, 而其它细胞如胶质细胞的免疫染色基本一致。总之, 文章成功制备了首个存活至成年的大脑神经细胞特异性ADAM10基因敲除(cKO)小鼠, 克服了小鼠因ADAM10缺失在胚胎期或围产期死亡的弊端, 为研究成年小鼠大脑神经细胞ADAM10基因的功能奠定了坚实的基础。     

Specificity controls for immunocytochemistry: the antigen preadsorption test can lead to inaccurate assessment of antibody specificity

The biomedical research community relies directly or indirectly on immunocytochemical data. Unfortunately, validation of labeling specificity is difficult. A common specificity test is the preadsorption test. This test was intended for testing crude antisera but is now frequently used to validate monoclonal and affinity purified polyclonal antibodies. Here, the authors assess the power of this test. Nine affinity purified antibodies to different epitopes on 3 proteins (EAAT3, slc1a1; EAAT2, slc1a2; BGT1, slc6a12) were tested on samples (tissue sections and Western blots with or without fixation). The selected antibodies displayed some degree of cross-reactivity as defined by labeling of samples from knockout mice. The authors show that antigen preadsorption blocked all labeling of both wild-type and knockout samples, implying that preadsorption also blocked binding to cross-reactive epitopes. They show how this can give an illusion of specificity and illustrate sensitivity-specificity relationships, the importance of good negative controls, that fixation can create new epitopes, and that cross-reacting epitopes present in sections may not be present on Western blots and vice versa. In conclusion, they argue against uncritical use of the preadsorption test and, in doing so, address a number of other issues related to immunocytochemistry specificity testing.     

Binding Specificity of Hydrolytic Catalytic Antibody: Lessons from the Diverse Immune System

The diverse immune system has the potential to provide numerous antibodies that catalyze a great variety of reactions and exhibit diversity in mechanism and substrate specificity. However, to find such an antibody is not practical, unless we understand the immune response precisely and utilize it. We applied an alternative screening methodology based on the binding specificity of antibodies for the structure of the transition-state analog p-nitrophenyl phosphonate in order to study the built-in diversity of the immune response. This study uncovered the unpredictable binding and substrate specificity of catalytic antibodies derived from the diverse immune response.     

Biochemical Studies of Olfaction: Binding Specificity of Odorants to a Cilia Preparation from Rainbow Trout Olfactory Rosettes

Cilia isolated from the olfactory epithelium (olfactory rosettes) of rainbow trout (Salmo gairdneri) bind amino acids, which are odor stimuli to this species. We demonstrate that L-threonine, L-serine, and L-alanine bind to a common site, TSA, in the cilia preparation. All possible mixtures of two of the amino acids as competitors, with the third as the 3H-labeled ligand, were studied. The effect of two combined (unlabeled) competitors was always substantially less than additive compared with their actions singly. Along with additional inhibition studies using mixtures of inhibitors, the data show that the three odorants must interact with at least one common binding site, TSA. Binding of L-[3H]lysine to site L was unaffected by addition of L-threonine, L-serine, or L-alanine, establishing its independence from site TSA. L-Arginine inhibited binding of L-[3H]lysine, showing that both of these basic amino acids interact with site L. The data establish the presence, in trout olfactory cilia, of at least two separate and noninteracting populations of odorant binding sites, TSA and L.     

Comprehensive behavioural study of GluR4 knockout mice: implication in cognitive function

Fast excitatory transmission in the mammalian central nervous system is mediated by AMPA‐type glutamate receptors. The tetrameric AMPA receptor complexes are composed of four subunits, GluR1–4. The GluR4 subunit is highly expressed in the cerebellum and the early postnatal hippocampus and is thought to be involved in synaptic plasticity and the development of functional neural circuitry through the recruitment of other AMPA receptor subunits. Previously, we reported an association of the human GluR4 gene (GRIA4) with schizophrenia. To examine the role of the GluR4 subunit in the higher brain function, we generated GluR4 knockout mice and conducted electrophysiological and behavioural analyses. The mutant mice showed normal long‐term potentiation (LTP) in the CA1 region of the hippocampus. The GluR4 knockout mice showed mildly improved spatial working memory in the T‐maze test. Although the retention of spatial reference memory was intact in the mutant mice, the acquisition of spatial reference memory was impaired in the Barnes circular maze test. The GluR4 knockout mice showed impaired prepulse inhibition. These results suggest the involvement of the GluR4 subunit in cognitive function.     

A estradiol-17beta receptor in the reproductive system of the female of Octopus vulgaris: characterization and immunolocalization

In this study, for the first time we have identified an estradiol-17beta receptor (ER) in the reproductive system of the female of Octopus vulgaris. Scatchard analysis revealed that one binding component with high affinity and low capacity for the ligand was present in the cytosol, but not in the nuclear extract of the ovary and the oviduct. A steroid specificity competition assay showed that 3H-estradiol-17beta binding activity showed a preference for estradiol-17beta. DNA-cellulose chromatography confirmed the presence of one 3H-estradiol-17beta binding component. By using antibodies anti ER (578-595), we have localized by Western blotting one band of about 70 kDa. ER immunoreactivity has been localized in the nuclei of the follicle cells of the ovary, in the nuclei of the epithelium lining the proximal portion of the oviduct and in the nuclei, and in the cytoplasm of the inner region of the oviducal gland and in the cytoplasm of the outer region of the oviducal gland. These data, taken together, provide evidence that in Octopus vulgaris the ER has biochemical and immunohistochemical characteristics resembling those of ER in vertebrates.     

Phytochrome in green tissue: Spectral and immunochemical evidence for two distinct molecular species of phytochrome in light-grown Avena sativa L.

A method is described for the extraction of phytochrome from chlorophyllous shoots of Avena sativa L. Poly(ethyleneimine) and salt fractionation are used to reduce chlorophyll and to increase the phytochrome concentration sufficiently to permit spectral and immunochemical analyses. The phototransformation difference spectrum of this phytochrome is distinct from that of phytochrome from etiolated shoots in that the maximum in the red region of the difference spectrum is shifted about 15 nm to a shorter wavelength. Immunochemical probing of electroblotted proteins (Western blotting), using a method sensitive to 50 pg, demonstrates the presence of two polypeptides in green tissue that bind antiphytochrome antibodies: a predominant species with a relative molecular mass (M_r) of 118000 and a lesser-abundant 124000-M_r polypeptide. Under nondenaturing conditions all of the 124000-M_r species is immunoprecipitable, but the 118000-M_r species remains in the supernatant. Peptide mapping and immunochemical analysis with monoclonal antibodies show that the 118000-M_r species has structural features that differ from etiolated-oat phytochrome. Mixing experiments show that these structural differences are intrinsic to the molecular species from these two tissues rather than being the result of post-homogenization modifications or interfering substances in the green-tissue extracts. Together the data indicate that the phytochrome that predominates in green-tissue has a polypeptide distinct from the well-characterized molecule from etiolated tissue.Abbreviations and symbols Ig immunoglobulin - M_r relative molecular mass - Pfr, Pr far-red-absorbing and red-absorbing forms of phytochrome respectively - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - _max ^R , _max ^FR maxima of the phototransformation difference spectrum in the red and far-red region     

Danon disease: a focus on processing of the novel LAMP2 mutation and comments on the beneficial use of peripheral white blood cells in the diagnosis of LAMP2 deficiency

Danon disease (DD) is a monogenic X-linked disorder characterized by cardiomyopathy, skeletal myopathy and variable degrees of intellectual disability. DD develops due to mutations in the gene encoding lysosomal-associated membrane protein 2 (LAMP2). We report on a family exhibiting the clinical phenotype comprising of hypertrophic cardiomyopathy and ventricular pre-excitation, myopia and mild myopathy in two male patients and cardiomyopathy and myopia in a female patient. The diagnosis of DD in this family was based on the assessment of the clinical phenotypes and the absence of LAMP2 in skeletal and/or cardiac muscle biopsy specimens. Sequence analysis of the LAMP2 gene and its mRNA revealed a novel LAMP2 mutation (c.940delG) in all three patients. Approximately 25% of the female patient's cardiomyocytes were LAMP2 positive apparently due to the unfavorable skewing of X chromosome inactivation. We further performed qualitative LAMP2 immunohistochemistry on peripheral white blood cells using the smear technique and revealed the absence of LAMP2 in the male patients. LAMP2 expression was further assessed in granulocytes, CD4+ and CD8+ T lymphocytes, CD20+ B lymphocytes, CD14+ monocytes and CD56+ natural killer cells by quantitative polychromatic flow cytometry. Whereas the male DD patients lacked LAMP2 in all WBC populations, the female patient expressed LAMP2 in 15.1% and 12.8% of monocytes and granulocytes, respectively. LAMP2 expression ratiometrics of highly vs. weakly expressing WBC populations discriminated the DD patients from the healthy controls. WBCs are thus suitable for initial LAMP2 expression testing when DD is a differential diagnostic option. Moreover, flow cytometry represents a quantitative method to assess the skewing of LAMP2 expression in female heterozygotes. Because LAMP2 is a major protein constituent of the membranes of a number of lysosome-related organelles, we also tested the exocytic capacity of the lytic granules from CD8+ T lymphocytes in the patient samples. The degranulation triggered by a specific stimulus (anti-CD3 antibody) was normal. Therefore, this process can be considered LAMP2 independent in human T cells. The c.940delG mutation results in a putatively truncated protein (p.A314QfsX32), which lacks the transmembrane domain and the cytosolic tail of the wild-type LAMP2. We tested whether this variant becomes exocytosed because of a failure in targeting to late endosomes/lysosomes. Western blotting of cardiac muscle, WBCs and cultured skin fibroblasts (and their culture media) showed no intra- or extracellular truncated LAMP2. By comparing the expression pattern and intracellular targeting in cultured skin fibroblasts of normal LAMP2 isoforms (A, B and C) tagged with green fluorescent protein (GFP) and the A314Qfs32-GFP fusion, we found that the A314Qfs32-GFP protein is not even expressed. These observations suggest that the truncated protein is unstable and is co-translationally or early post-translationally degraded.