抗CD81抗体对星形胶质细胞增殖的抑制作用

目的 研究抗CD81抗体在星形胶质细胞增殖中的作用.方法 将纯化的星形胶质细胞分为6组,加入不同浓度抗CD81抗体,其浓度依次为0、0.1mg/L、0.5mg/L、1mg/L、5mg/L、10mg/L,以四甲基噻唑蓝(MTT)比色法检测细胞活性.在此检测结果的基础上,选出3个有意义的浓度组,加入浓度分别为0、0.5mg/L、5mg/L的抗CD81抗体,采用流式细胞术观察抗CD81抗体对星形胶质细胞周期的影响并进行统计学分析.结果 抗CD81抗体对星形胶质细胞的增殖有抑制作用,并呈一定的剂量依赖性.加入抗CD81抗体培养24 h后,实验组的星形胶质细胞处于G0/G1期的细胞指数减少,S期细胞指数增多.结论 抗CD81抗体抑制了星形胶质细胞的增殖,使星形胶质细胞的细胞周期受阻,阻滞于S期.     

ICOS/GL50信号在T细胞依赖性体液免疫应答中的作用

研究ICOS/GL5 0信号在T细胞体外增殖、细胞因子分泌以及B细胞抗体分泌中的作用 ,进一步探讨该信号途径在机体体液免疫应答中的作用机制。采用3 H TdR检测GL5 0 L92 9转染细胞和特异性鼠抗人GL5 0单抗对T细胞体外增殖的作用 ;ELISA法检测GL5 0 L92 9转染细胞和抗人GL5 0单抗对T细胞分泌IL 2、IL 10以及ICOS L92 9转染细胞和特异性鼠抗人GL5 0单抗对PWM介导的B细胞分泌抗体的效应。结果提示GL5 0 L92 9转染细胞能够促进经抗CD3单抗活化的T细胞增值和分泌IL 10 ,而抗GL5 0单抗可以阻断这些效应。ICOS分子与B细胞上GL5 0分子的结合上调PWM介导的B细胞分泌抗体而抗GL5 0单抗则下调这一效应。这些表明 ,ICOS/GL5 0信号途径在机体T细胞依赖的体液免疫应答反应中发挥着重要的调节作用     

腺苷和抗IgE抗体对哮喘患者BALF中肥大细胞分泌组胺的影响

目的 :研究支气管哮喘 (哮喘 )患者支气管肺泡灌洗液 (BALF)中肥大细胞的功能特性。方法 :2 9例轻度哮喘患者BALF中的细胞经冲洗后 ,加入含抗IgE抗体、腺苷、缓冲液或腺苷 +抗IgE抗体的LP4试管中进行激发试验。检测BALF肥大细胞中的组胺释放量。结果 :腺苷浓度达 10 0 μmol/L时 ,仍无明显刺激组胺释放的作用 ,仅在浓度高达 10 0 0 μmol/L时 ,方可诱导哮喘患者BALF中肥大细胞释放约 17%的组胺。抗IgE抗体的浓度大于 1× 10 -4g/L时对哮喘患者BALF中肥大细胞的释放组胺有明显的促进作用 ;仅 3× 10 -5g/L的抗IgE抗体与腺苷联合应用的实测值 ,明显高于对应的单独作用组的相加值。结论 :哮喘患者BALF中的肥大细胞对腺苷的刺激反应较差 ,但对抗IgE抗体刺激的反应性明显增强。仅低浓度的抗IgE抗体和腺苷具有协同作用。     

人源性抗HBsAg单链抗体在大肠埃希菌中分泌表达条件的研究

目的 研究抗HBsAS单链抗体A-15在大肠埃希菌中的高效表达,增加该抗体在培养基中的产量.方法 改变工程菌的诱导时机、诱导剂浓度和诱导时间,以观察这些因素对单链抗体表达的影响.另外,加入不同浓度的蔗糖、甘氨酸和Triton X-100,以考察三种化学物质对单链抗体在培养基中分泌量的影响.结果 工程菌培养4 h后,加终浓度0.5 mmol/L IPTG诱导表达8 h为抗HBsAS单链抗体A-15较佳的表达条件.终浓度0.3 mol/L蔗糖,2%甘氨酸和1%Triton X-100同时加入诱导时的培养基中,可使培养基中表达的单链抗体亲和活性分别增加16.78倍.经测定抗HBsAg单链抗体A-15在培养基中最终表达量为7.4 mg/L.结论 抗HBsAg单链抗体A-15的分泌表达条件得到优化,表达量明显提高,这为更有效地制备该抗体用于其结构和功能研究提供了一个切实可行的方法.     

霉酚酸衍生物对小鼠T细胞免疫功能的抑制作用

目的研究2种新型霉酚酸衍生物(代号JU95-07B、JU100-07E)在体外对小鼠T细胞免疫功能的影响。方法小鼠脾细胞用抗CD3单克隆抗体(mAb)刺激,同时加入不同浓度(10-4、10-5、10-6、10-7、10-8mol/L)及在不同时间点(刺激细胞后第0、6、12 h)加入同一浓度(10-5mol/L)的霉酚酸衍生物后,用酶联免疫吸附法(ELISA)检测培养上清中IFN-γ和IL-2的水平。同时利用流式细胞术(flow cytometry,FCM)检测霉酚酸衍生物对T细胞IFN-γ和IL-2的分泌、表面分子CD25的表达以及增殖的影响。结果 2种霉酚酸衍生物对抗CD3刺激条件下诱导的小鼠脾细胞产生的IFN-γ和IL-2均具有浓度依赖性抑制作用,并且在抗CD3刺激的不同时间点分别加入2种霉酚酸衍生物均能不同程度地抑制小鼠脾细胞产生IFN-γ和IL-2;同时,与单独使用抗CD3刺激相比,分别加入10-5mol/L浓度的2种霉酚酸衍生物后,均能抑制小鼠T细胞表面分子CD25的表达以及增殖。结论体外实验表明2种霉酚酸衍生物均对小鼠T细胞免疫功能具有明显抑制作用,该研究提示这2种霉酚酸衍生物可以抑制自身免疫性疾病和移植排斥反应。     

小鼠胚胎整体原位杂交实用技术的建立

目的 优化小鼠胚胎整体原位杂交及数据分析方法。方法 小鼠胚胎用4％多聚甲醛4％固定过夜，10mg／L蛋白酶K(PK)室温消化30min，预杂交2h后，加入探针63℃反应16～18h，用盐溶液高温洗涤多次，RNA酶37℃消化1h，封闭4h后加入地高辛抗体4℃过夜。抗体反应后反复洗涤并在水平摇床上振摇，NBT和BCIP显色系统避光显色。提取图像灰度值，扣除背景、对照等假阳性信号。结果 KIAA1708探针在脑、脊髓等8个区域有杂交信号，对图像数据分析结果表明，只有1个区域的信号在95％可信限范围内，为阳性信号。结论 通过控制蛋白酶K消化时间、探针和地高辛抗体浓度，以及洗涤、显色过程，得到了低本底、着色清晰的杂交结果，结合图像的数据分析步骤，消除假阳性结果。建立了一套实用的小鼠胚胎整体原位杂交方法。     

体外诱导人类B细胞分泌HLA抗体的实验研究

目的:探索体外诱导人类B细胞产生特异性HLA抗体的理想培养条件。方法:以EBV转化致敏肾移植患者B淋巴细胞系(BLCL)为模型,采用ELISA和ELISPOT方法,比较3种培养体系:(1)RPMI1640 抗OPTI单克隆抗体(mAb);(2)RPMI1640 0·1mmol/L非必需氨基酸液 1mmol/L丙酮酸钠 40mg/L转铁蛋白;(3)Hybridoma无血清培养液,以及3种培养条件:(1)对照组(仅培养液),(2)培养液 rIL-4 rIL-10 鼠抗人CD40(α-CD40),(3)培养液 rIL-4 rIL-10 重组人CD40配体(CD40L),人类B细胞产生免疫球蛋白(Ig)和特异性抗HLA抗体的能力。结果:BLCL细胞在Hybridoma SFM培养体系所产生的IgG和IgM浓度分别为25·2～28·1mg/L和7·14～7·95mg/L,高于其他两种培养条件(P<0·05)。BLCL细胞在添加了IL-4、IL-10和α-CD40或CD40L的杂交瘤无血清培养液中能产生特异性抗HLA-I抗体,所获得的斑点频数分别为3·06/每104个BLCL细胞和3·55/每104个BLCL细胞,高于对照组(1·77/每104个BLCL细胞)。结论:BLCL细胞在杂交瘤无血清培养液中和添加了IL-4、IL-10和α-CD40或CD40L的培养条件下,提高分泌IgG和IgM的能力和特异性抗HLA-I类分子的抗体。     

抗CD71人-鼠嵌合抗体对活化淋巴细胞的效应

目的　通过体外实验探讨抗CD71人 鼠嵌合抗体对活化淋巴细胞效应的影响 ,并与其鼠源性单克隆抗体进行比较。方法　以丝裂原诱导的人外周血单个核细胞 (PBMC)为靶细胞 ,测定嵌合抗体和鼠源性单抗对其增殖抑制率 ;在新鲜补体存在下 ,测定补体依赖性嵌合抗体介导的细胞毒效应(CDC)。以EBV转化的B细胞为刺激细胞 ,测定两种抗体对其诱导的同种异体PBMC的增殖抑制率 ;以同种异体的PBMC为刺激细胞 ,测定两种抗体在单向、双向混合淋巴细胞培养 (MLC)中的增殖抑制率。结果　嵌合抗体和鼠源性单抗均可明显抑制丝裂原诱导的PBMC的增殖反应 ,且二者抑制率差异无显著性(P >0 .0 5 ) ,其抑制作用随抗体浓度增加而增强 ,PBMC和丝裂原共同孵育 12h后加入抗体的增殖抑制效应最明显 ;在新鲜补体存在下 ,嵌合抗体对丝裂原诱导增殖的PBMC具有CDC作用 ,而鼠源性单抗CDC作用较弱 ;两种抗体对混合淋巴细胞培养反应有明显的抑制作用 ,且嵌合抗体组抑制率明显高于鼠源性单抗组 (P <0 .0 5 )。结论　抗CD71人 鼠嵌合抗体在体外实验中可抑制淋巴细胞的活化及其效应 ,其作用明显强于抗CD71鼠源性单克隆抗体。     

IL-6诱导鼻咽癌CNE-2细胞分泌IL-10的机制研究

目的探讨IL-6诱导鼻咽癌CNE-2细胞分泌IL-10的分子机制。方法 IL-6体外刺激鼻咽癌细胞系CNE-2细胞,或加入抗IL-6受体的抗体和信号通路抑制剂预孵育1 h,IL-6再进行刺激,RT-PCR检测IL-10的mRNA表达水平,酶联免疫吸附试验检测细胞培养上清中IL-10的分泌水平。结果与未刺激组相比,IL-6刺激的CNE-2细胞显著上调IL-10的表达和分泌,差异具有极显著性统计学意义(P0.01),并以剂量和时间依赖的方式增加;加入抗IL-6受体的抗体或NF-κB抑制剂预孵育后,IL-6刺激CNE-2细胞表达和分泌IL-10的水平显著下降,差异具有极显著性统计学意义(P0.01)。而PI3K抑制剂、p38/MAPK抑制剂、JNK抑制剂、STAT3抑制剂和MEK1/2抑制剂对IL-6诱导的IL-10表达和分泌水平则无明显的影响。结论 IL-6经IL-6R/NF-κB信号诱导CNE-2细胞分泌IL-10,抑制IL-6信号有助于鼻咽癌的临床免疫治疗。     

抗人TNF单克隆抗体对TNF诱导的NF-κB核转位的抑制作用

目的 :鉴定 3株抗人TNF单克隆抗体 (mAb)D2、E6和F6对TNF诱导的NF κB核转位的抑制作用。方法 :将不同浓度的 3株mAb及无关mAb分别与TNF溶液孵育后 ,加入到ECV30 4细胞培养液中。孵育 1h后收获细胞 ,从中提取核蛋白并测定其含量 ,用电泳迁移率变动分析 (EMSA)检测核提取液中NF κB的核转位。结果 :3株抗TNFmAb均可抑制TNF诱导的ECV30 4细胞中NF κB的核转位。其中mAbD2的抑制作用最强 ,其次为mAbF6和E6 ,无关对照抗体也有一定的非特异性反应。抑制作用与加入的mAb呈良好的剂量依赖关系 ,在 10mg/L和 0 .1mg/L条件下 ,mAbD2的抑制率分别为 94 .2 %和75 .1% ,mAbE6分别为 6 4 .9%和 2 8.6 % ,mAbF6分别为 70 .3%和 4 9.5 % ,无关对照抗体分别为 2 0 .0 %和 11.1%。结论 :mAbD2、E6和F6可特异性地抑制TNF诱导的NF κB核转位 ,为制备对感染性疾病和自身免疫性疾病等均具有治疗作用的嵌合抗体奠定了基础     

The main neutral protease of rat skin is a mast cell enzyme. Immunohistochemical localization of the enzyme in rat skin with the peroxidase-antiperoxidase (PAP) complex method.

The highly sensitive PAP immunoperoxidase method was used to localize the main neutral protease of rat skin. The use of the neutral detergent, Triton X-100, in the reagent and washing solutions was observed to effectively decrease the nonspecific staining. The specific staining was localized to the mast cell granules.     

Comparison of indirect peroxidase and avidin‐biotin‐peroxidase complex (ABC) immunohistochemical staining procedures for c‐fos in rat brain

c‐Fos is the product of a gene expressed within neurons in the brain that serves as an anatomical marker of cellular activation. Immunohistochemical staining for c‐fos allows a characterization of the effects of many different types of experimental manipulations on neuronal activity, making it a powerful technique for understanding brain, drug and behavior relationships. This study compared visualization of an anti‐c‐fos primary antibody in 40‐μm‐thick cryostat sections of formaldehyde‐fixed rat brainstem using either a peroxidase enzyme‐conjugated secondary antibody (indirect peroxidase) or the peroxidase‐conjugated avidin‐biotin complex (ABC) method. All sections were treated with H₂O₂ to quench endogenous peroxidase enzyme and sodium borohydride to enhance permeability of the tissue and improve staining quality. Every other section was used to examine either the indirect peroxidase or the ABC method. Sections for the indirect peroxidase method were treated with Triton X‐100 detergent to increase tissue permeability, goat serum to reduce non‐specific binding of the secondary antibody and, in some cases, bovine serum albumin (BSA) to reduce non‐specific binding of the primary antibody. Sections for the ABC method were treated with dilute normal serum, and avidin and biotin solutions and, in some cases BSA. Alternate sections were incubated for 72 h in either rabbit anti‐c‐fos primary antibody (1 : 20 000) or its vehicle (negative control). For the indirect peroxidase protocol, tissues were treated with peroxidase‐conjugated goat anti‐rabbit secondary antibody. For the ABC protocol, tissues were treated with biotinylated goat anti‐rabbit secondary antibody and ABC peroxidase complex. All sections were reacted with 3,3′‐diaminobenzadine (DAB) and H₂O₂, mounted and coverslipped. Both methods produced specific staining of c‐fos‐containing neurons, relative to the negative control sections. The indirect peroxidase protocol produced clear staining of c‐fos‐containing neurons, with very little background in the negative control sections. Staining for c‐fos was enhanced using the ABC method in that c‐fos stained neurons were darker and more clearly visible after shorter treatment with DAB. However, negative control sections showed a greater amount of non‐specific staining with the ABC method. Thus, the ABC method was more sensitive but showed reduced specificity, with BSA treatment slightly reducing the level of non‐specific staining. Overall, the ABC method produced better visualization and contrast of c‐fos‐containing neurons against the background color of the tissue.     

In situ hybridization with novel biotinyl-tyramide: fundamental studies and its utility of the detection of human papilloma virus in tissue sections

We developed an in situ hybridization (ISH) method with a higher sensitivity and less background staining than the generally used catalyzed reporter deposition amplification method of in situ hybridization (CARD-ISH). The characteristics of this method are follows: sections heated in citrate buffer (pH6.0) containing 0.1% Triton X-100 showed the strongest signals, and a well-preserved morphology. The strongest signals were observed when borate buffer of biotinyl-tyramide stock and phosphate buffer of working solution were changed to Tris-HCl buffer. Compared with hematoxylin counter staining, alcian blue counter staining made it easier to identify dot, diffuse, and mixed type signals, respectively. Thus, we were able to clearly detect positive signals in the SiHa cell line with 1-2 copies of the integrated human papilloma virus-16 (HPV-16) gene, and in formalin-fixed, paraffin-embedded cervical lesion specimens for the HPV-16 gene.     

Localization of glycylproline dipeptidyl aminopeptidase in the liver tissue and possible mechanism of its release into blood flow

We examined the localization of glycylproline dipeptidyl aminopeptidase (GPDAP) in the liver tissue and the mechanism of its release into blood flow. An immunohistochemical study using rabbit polyclonal antibody against the purified GPDAP from human liver obtained at autopsy was performed in liver biopsy samples with the peroxidase-antiperoxidase stain technique. GPDAP staining was detected on the liver cell membrane around bile canaliculi. After treatment with deoxycholic acid (DOC) or Triton X-100, this enzyme disappeared. GPDAP was solubilized rapidly from microsome of human liver by treatment with either DOC or Triton X-100. DOC or Triton X-100 solubilized GPDAP coincided with the isozyme that was the specific isozyme in sera of patients with acute hepatitis or obstructive jaundice. These results suggest that GPDAP localizes on the liver cell membrane around bile canaliculi and that this enzyme is released by the detergent action of bile acids.     

Triton detergents and the frog neuromuscular end-plate: An electrophysiological and ultrastructural study

The effects of the nonionic detergents Triton X-45 and Triton X-100 were studied in the frog muscle end-plate, by intracellular recordings of spontaneous miniature end-plate potentials (m.e.p.p.'s) and the potential changes produced by iontophoretic application of acetylcholine (ACh-potentials). In addition, the ultra-structural changes produced by Triton X-100 were studied by transmission electron microscopic and freeze-fracture techniques. It was found that Triton X-45 and Triton X-100 caused a rapidly developing reduction of the amplitude of the m.e.p.p.'s. The response lo iontophoretic application of acetylcholine was reduced by Triton X-100. Following return to normal Ringer solution the ACh-potentials recovered, although not completely. The dissociation constant calculated from the rate constants for onset and offset of the reaction (K_D= k₂/k_l) was 5–50 μM depending on the type of stoichiometric reaction presumed to occur between Triton X-100 and the cholinergic receptor. The ultrastructural changes observed indicate that the nerve terminal plasma membrane and mitochondria are affected by Triton X-100. Leakage of Ca²⁺ from the latter may therefore be the cause of the increase in m.e.p.p. frequency. It is concluded that the influence on the amplitude of the m.e.p.p.'s and the ACh-potentials can be attributed to a direct effect of the detergent upon the acetylcholine receptor protein.     

Differential detergent treatment allows immunofluorescent localization of the Newcastle disease virus matrix protein within the nucleus of infected cells

Paramyxoviruses are cytoplasmic viruses and presumably do not require any nuclear function for their replication. However, recent studies using monoclonal antibodies directed against the Newcastle disease virus matrix (M) protein have found a large portion of the M protein apparently associated with the nucleus of infected cells. Whether the M protein is associated with the cytoplasmic surface of the nucleus or whether the M protein is actually located within the nucleus has not been clearly determined. To examine this question, conditions for selectively permeabilizing the cytoplasmic membrane were sought. After treating fixed cells with a low concentration (0.02%) of the nonionic detergent Triton X-100, the cytoplasmic antigen vimentin was stained with a monoclonal antibody, but nuclear antigens were not. Apparently, 0.02% Triton permeabilizes the plasma membrane while leaving the nuclear membrane intact. Under these conditions, monoclonal antibodies directed against the NDV phosphoprotein and hemagglutinin/neuraminidase glycoprotein stained infected cells, but a monoclonal antibody to the M protein did not. The inability of the anti-M monoclonal antibody to stain the nucleus, even though the outer nuclear membrane is accessible under these conditions, indicates that the M protein is not associated with the outer membrane of the nucleus. The nuclei of infected cells treated with a higher concentration (0.05%) of Triton X-100 were stained both with antibodies to nuclear antigens and with the anti-M monoclonal antibody.     

Sendai virus glycoproteins are T cell-dependent B cell mitogens

UV-inactivated Sendai virus is mitogenic for murine splenocytes, whereas infectious Sendai virus kills spleen cells in vitro. The isolated hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of Sendai virus are also mitogenic for cultured mouse spleen cells. A mixture of these glycoproteins (1 microgram/well) gives maximum stimulation 96 h after culture initiation. Viral proteins remaining insoluble after Triton X-100 extraction are also mitogenic for mouse spleen cells, with maximum stimulation occurring at 72 h after culture initiation with 1 to 5 microgram/well. On the basis of protein concentration, the HN and F glycoproteins are approximately three times more mitogenic than the Triton X-100-insoluble material. The mitogenic response of the HN and F glycoproteins has two components, a T cell-independent B cell proliferation, which is less than one-half of the total stimulation observed, and a T cell-dependent B cell proliferation. In contrast, the Triton X-100-insoluble material is a T cell-dependent B cell mitogen. Purified T lymphocytes do not respond to the mitogenic signal of either HN-F or Triton X-100-insoluble material.     

Calmodulin loss in vascular smooth muscle following Triton X-100 or saponin skinning

The calmodulin (CaM) content of intact and chemically skinned strips of rat caudal artery was measured using a¹²⁵I-CaM radioimmunoassay. The total CaM measured following homogenization of arterial tissue with EGTA and EGTA/Triton X-100 was 2.58 mol/kg wet tissue. Based on a smooth muscle volume of 40%, this value corresponds to a cellular CaM concentration of 6.5 M. Approximately 97% of total CaM was soluble and approximately 3% was EGTA-nonextractable. Permeabilization of the plasmalemma with 0.15 mg/ml saponin or 0.5% Triton X-100 caused significant detergent-dependent loss of CaM. At the end of a 1 h skinning period, tissues exposed to saponin lost 30% of total CaM. By comparison, tissues skinned under the same conditions with Triton X-100 lost 50%. During a subsequent 4h exposure to relaxing solution, total tissue CaM continued to decline. The exponential loss over the 5h period was described by a first order model having diffusible and nondiffusible CaM components. The diffusible CaM component of saponin skinned tissue (59%) was significantly less than the diffusible component of those skinned with Triton X-100 (88%); however, the rate coefficients for CaM diffusion (0.78 h^–1 and 0.91 h^–1, respectively) did not statistically differ. The nondiffusible component of CaM was significantly larger in saponin treated strips (42%) than in Triton X-100 permeabilized tissue (12%). Arterial strips skinned with Triton X-100, which were subsequently exposed to relaxing solution for up to 22 h, lost significantly more CaM than those retained in Triton X-100 skinning solution for a comparable duration These studies demonstrate the diffusion of CaM from detergent skinned arterial strips and characterize the time course of that loss.     

Analysis of three variables in sampling solutions used to assay bacteria of hands: type of solution, use of antiseptic neutralizers, and solution temperature.

Tests were performed using the sterile bag technique to determine the effects of type of sampling solution, use of antiseptic neutralizers, and solution temperature on the detection and quantitation of bacteria on hands. Using paired hand cultures, three sampling solutions were compared: quarter-strength Ringer solution, a phosphate buffer containing Triton X-100, and the same buffer containing antiseptic neutralizers. The phosphate buffer containing Triton X-100 was significantly better than quarter-strength Ringer solution in mean bacterial yield; the neutralizer-containing sampling solution was slightly better than Triton X-100-containing solution, although differences were not significant at the P = 0.05 level. Temperature (6 or 23 degrees C) of the sampling solution showed no consistent effect on bacterial yield from hands tested with the fluid containing neutralizers.     

Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis.

Crossed immunoelectrophoresis was used to study two complex antigenic preparations from Neisseria gonorrhoeae, one of cytoplasmic origin and the other derived by Triton X-100 extraction of isolated washed gonococcal envelopes, with the aim of developing suitable reference antigen-antibody systems that could be subsequently used to investigate the immune response to gonococcal infection and to monitor envelope preparations for cytoplasmic contamination. A number of parameters were investigated to optimized and standardize antigen preparation, e.g., harvesting and washing of gonococci, methods of bacterial disruption, and washing of envelopes. The effects of Triton X-100 concentration, initial total envelope protein concentration, and the composition, pH, and concentration of buffer on cell envelope extractability were studied to obviate the need to concentrate material before use in crossed immunoelectrophoresis. The electroendoosmotic properties of agarose were a major determining factor in resolving envelope antigens. From 25 to 30 immunoprecipitates were revealed in the envelope antigen-antibody system; 75 to 80 were revealed in the cytoplasmic sytem. Envelope immunoprecipitates with reduced nicotinamide adenine dinucleotide and lactate dehydrogenase activities were identified. Crossed immunoelectrophoresis with intermediate gels revealed the presence of antibodies in a preimmune rabbit antiserum pool to a distinctive fact-moving component in both the envelope and cytoplasmic antigen preparations. The intermediate gel technique also demonstrated that extensive washing of envelope preparations with buffer did not remove cytoplasmic ontamination completely. The method provides a much more sensitive means of monitoring the purity of envelope fractions than the use of single enzy,e markers as indexes of such contamination. The use of rabbit antisera raised to formolized gonococci in intermediate gels indicated that both reference antigen-antibody systems were of potential use in screening immune responses to N. gonorrhoeae.