The relationship between the binding of primary antibody to solid-phase antigen in microtiter plates and its detection by the ELISA

Radioiodinated monomeric and dimeric M315 (mM315 and dM315) prepared from BALB/c ascites fluid by gel filtration and affinity chromatography were used to study the relationship between primary antibody binding to solid-phase dinitrophenylated gelatin in microtiter ELISAs and its indirect detection by enzyme-antibody conjugates and complexes. The relationship between the amount of mM315 or dM315 which binds to dinitrophenylated gelatin to the amount added is linear over a nearly 3-log range with a slope of 1; the amount of M315 which binds in this linear range after 24 h represents all of the active antibody in the system. On a binding site basis, mM315 was inhibited by a significantly lower amount of dinitrophenyl-(DNP)glycine than was dM315. The indirect detection of bound M315 over the same 3-log range using ELISA yielded a sigmoidal titration curve which encompassed a short linear region that had a slope of 0.9 or less. Plateauing of the titration plots at high input of both mM315 and dM315 was shown to be progressively exaggerated in direct relationship to the size of the enzyme-antibody conjugate used for their detection. The data show that the upper region of the sigmoidal ELISA titration plot is the result of steric hindrance of the detection system. Through the combined use of an 131I-enzyme-antibody immune complex (EIC) detection system and 125I-M315, it was shown that the deviation from the linear binding of 125I-M315 observed during its indirect detection in the so-called linear region of the ELISA titration curve was the result of changing ratios of bound EIC: bound primary antibody, not altered enzymic activity.     

Altered production of IgE and IgA induced by IL-4 in peripheral blood mononuclear cells from patients with IgA nephropathy.

In order to elucidate the factors responsible for altered immunoglobulin production in patients with IgA nephropathy (IgAN), the in vitro effects of IL-4 and interferon-gamma (IFN-gamma) on the synthesis of IgE and IgA by peripheral blood mononuclear cells (PBMC) were studied. Spontaneous IgE and IgA synthesis by PBMC was significantly increased in patients with IgA nephropathy compared with controls. The maximum amounts of IgA and IgE synthesis by PBMC after stimulation with IL-4 were almost the same both in patients with IgAN and in controls. The enhancement rate of IL-4-induced IgE and IgA synthesis was significantly lower in IgAN than in the controls, suggesting in vivo preactivation of PBMC in IgAN patients. IFN-gamma suppressed IgA and IgE synthesis by PBMC from IgAN patients as well as controls. However, the suppressive effect on IgE synthesis was less prominent in patients with IgAN. These results suggested that altered IL-4 action might be involved in the development of IgA nephropathy.     

Exopolysaccharide from Lacticaseibacillus rhamnosus induces IgA production in airways and alleviates allergic airway inflammation in mouse model

The currently observed high prevalence of allergic diseases has been associated with changes in microbial exposure in industrialized countries. Defined bacterial components represent a new strategy for modulating the allergic immune response. We show that intranasal administration of exopolysaccharide (EPS) isolated from Lacticaseibacillus (L.) rhamnosus LOCK900 induces TGF-β1, IgA, and regulatory FoxP3⁺ T-cells in the lungs of naïve mice. Using the ovalbumin mouse model, we demonstrate that intranasal administration of EPS downregulates the development of allergic airway inflammation and the Th2 cytokine response in sensitized individuals. At the same time, EPS treatment of sensitized mice, similar to EPS-induced responses in naïve mice, significantly increased the level of total, OVA-specific, and also bacteria-specific IgA in bronchoalveolar lavage and the number of IgA-producing B-cells in the lung tissue of these mice. Thus, EPS derived from L. rhamnosus LOCK900 can be considered a safe candidate for preventing the development of allergic symptoms in the lungs of sensitized individuals upon exposure to an allergen.     

Low antibody affinity restricted to the IgA isotype in IgA nephropathy.

Antibody affinity affects the handling and behaviour of immune complexes, and experimental studies have shown that animals which produce predominantly low-affinity antibody are prone to immune complex deposition resulting in glomerulonephritis. In order to investigate the potential role of antibody affinity in the pathogenesis of IgA nephropathy, affinity of both IgA and IgG antibody isotypes during secondary response to systemic immunization with tetanus toxoid was studied in 20 patients with IgA nephropathy. Patients with IgA nephropathy produced IgA antibodies of significantly lower affinity than controls (P < 0.001), whereas IgG antibody affinities were similar. Contrasting with controls, patients' IgA antibody affinity was inversely related to antibody concentration, with higher responders producing large amounts of low-affinity antibody. IgG antibody affinity increased with time, and maturation of IgG antibody affinity was similar in both controls and patients. IgA affinity in controls decreased with time, and this lack of IgA affinity maturation may explain the relative unimportance of IgA in normal systemic immunity. This temporal decrease in IgA affinity was not observed in patients with IgA nephropathy. The production of low-affinity IgA in IgA nephropathy may provide an explanation for the predominant deposition of IgA in this disease.     

Quantitative trait loci (QTL) analysis reveals a close linkage between the hinge region and trimeric IgA dominancy in a high IgA strain (HIGA) of ddY mice

Polymerization of IgA has been suggested as one of the causes of mesangial deposition in IgA nephropathy. HIGA mice are an inbred model of IgA nephropathy, established by selective mating of ddY mice. This strain is characterized by a unique profile of the IgA molecule that is dominantly polymeric and has high serum levels with intense IgA deposition on the mesangium. We carried out quantitative trait loci (QTL) analysis, using F2 generations by crossing HIGA with BALB/c mice. Significant linkage of polymeric IgA in serum samples was identified around D12Mit263, which is close to the gene of the immunoglobulin heavy chain on chromosome 12. The amino acid sequence of the alpha heavy chain revealed marked differences between BALB/c and HIGA mice. Furthermore, most differences were focussed on the hinge region. The DBA/2J strain, which has the same amino acid sequence in the hinge region as the HIGA strain, also showed polymeric IgA dominance but low IgA levels in sera. Size fraction analysis revealed that these polymeric IgA showed trimer dominance in both DBA/2J and HIGA mice. In conclusion, the hinge region plays a key role in trimeric IgA formation in HIGA mice.     

Specific IgG and IgA antibodies to herpes simplex virus (HSV)-induced surface antigen in patients with HSV infections and in healthy adults

Herpes simplex virus (HSV)-specific IgG and IgA antibody response in patients with HSV infection and in healthy adults was studied by the immunoperoxidase antibody-membrane antigen (IPAMA) technique. In all HSV infections in which specific IgM antibodies were detected by enzyme-linked immunosorbent assay (ELISA), a significant rise in the titer of HSV IgG and IgA antibodies was found. In contrast, in patients with recurrent herpes labialis in which no specific HSV IgM antibodies were detected by ELISA, HSV IgG and IgA antibodies were not found to fluctuate significantly during the course of infection. A higher geometric mean titer (GMT) for HSV IgG and for IgA antibodies was found in seropositive individuals with a previous history of recurrent HSV than in seropositive individuals without a previous history of recurrent HSV infection. Nineteen of 26 HSV IgG seropositive healthy medical students without a previous history of recurrent HSV infection had HSV IgA antibodies to membrane antigen. The significance of this finding in understanding the mechanism of latency in healthy seropositive individuals without previous history of HSV recurrent infections is discussed.     

Cancer risk among patients with IgA deficiency or common variable immunodeficiency and their relatives: a combined Danish and Swedish study

The extremely high risk reported for some types of cancer among patients with common variable immunodeficiency (CVID) is based on a limited number of investigations. Therefore, we examined the risks for cancer among 562 Danish and Swedish patients with CVID or IgA deficiency and 2071 relatives in 1958-96. The patients were identified through an Immunodeficiency Register and hospital records, while the relatives were traced through population registers. Cancer incidence was assessed by linkage to the Cancer Registries and compared with that in the general population. Among 386 patients with IgA deficiency, the incidence of cancer was not increased (standardized incidence ratio (SI) = 1.0); but two cases of stomach cancer were found, resulting in a non-significant increase in risk (SIR = 5.4; 95% CI = 0.7-19.5). Among 176 patients with common variable immunodeficiency (CVID), the incidence of cancer at all sites combined was increased (SIR = 1.8; 95% CI = 1.0-2.9), which was due mainly to significant excesses of malignant lymphoma (obs = 4; SIR = 12.1; 95% CI = 3.3-31.0) and of stomach cancer (obs = 3; SIR = 10.3; 95% CI = 2.1-30.2). Among the 626 relatives of patients with CVID, no increase in risk was found for these types of cancer or for cancer overall (obs = 53; SIR = 1.0; 95% CI = 0.8-1.3). Our data show that the risks for malignant lymphoma and stomach cancer among patients with CVID may be lower than reported previously. The absence of an increased risk among relatives suggests that the increased cancer morbidity in patients with CVID is related to the immunodeficiency per se rather than to specific genetic traits shared with their relatives.     

舌下滴入多价细菌疫苗对支气管哮喘合并反复呼吸道感染儿童疗效观察

目的　探讨支气管哮喘合并反复呼吸道感染(RRI)经规律吸入糖皮质激素(ICS)控制不理想的儿童联用多价细菌疫苗(兰菌净)的临床疗效及安全性。方法　采用随机、双盲法将2 0 0 1年6月至2 0 0 2年6月在广州医学院第一附属医院及呼吸病研究所收治的4 8例哮喘合并RRI经系统ICS治疗控制不佳的患儿分为2组,分别给予舌下滴入兰菌净或安慰剂治疗,观察两组患儿RRI及哮喘临床症状、血清IgA及唾液分泌型IgA(sIgA)水平及可能出现的不良反应。血清IgA及唾液sIgA采用单向免疫扩散法检测。结果　与安慰剂组比较,兰菌净组呼吸道感染次数、发热天数、抗生素使用天数、咳嗽及气喘发作天数均减少,差异有显著性(P均<0 0 5 )。兰菌净组治疗后血清IgA及治疗后第1、2、3个月唾液sIgA与治疗前比较均显著升高(均P <0 . 0 5 ) ,而治疗后第6个月唾液sIgA与治疗前比较差异无显著性(P >0 .0 5 )。安慰剂组治疗后血清IgA及唾液sIgA与治疗前比较差异均无显著性(均P >0 . 0 5 )。两组不良事件发生率及脱落率差异均无显著性(均P >0. 0 5 )。结论　哮喘合并RRI经ICS控制不理想儿童联用多价细菌疫苗(兰菌净)能有效预防RRI ,从而减少哮喘发作的次数,减轻哮喘严重程度。多价细菌疫苗(兰菌净)安全性好、使用方便,值得推广应用。     

儿童新月体性IgA肾病临床与病理分析

目的 了解儿童原发性新月体性IgA肾病的临床、病理和免疫病理的特征 方法 分析9例儿童原发 性新月体性IgA肾病患儿的临床、病理和免疫病理资料,进行疗效观察及随访 结果 临床表现为肾病综合征5例, 急进性肾炎3例,无症状性血尿和蛋白尿1例。尿蛋白均>2 g／d,其中>3 g／d者7例:有持续性肉眼血尿8例,其中>2 周者7例;肾功能减退苦8例,其中5例仅内生肌酐清除率(CCr)轻度下降者;伴高血压7例 9例肾病理均有不同程 度的弥漫性系膜增生和小管-间质病变,平均74.5％肾小球有新月体形成,半数病例可见球囊粘连、小球硬化、节段内 皮增生和间质灶状纤维化。免疫荧光无1例单纯IgA型,IgA M和IgA M G型占55.6％,4例呈"满堂亮"。8例经大剂 量甲基泼尼松龙冲击2~4个疗程治疗,肉眼血尿、高血压全部消失,肾功能恢复正常,蛋白尿有不同程度改善 7例随 访3~18个月,3例尿蛋白正常,2例轻度蛋白尿(<1 g／d),尿蛋白>3 g／d者2例 结论 本组儿童原发性新月体性IgA 肾病临床以肾病综合征为主,持续性肉眼血尿和大量蛋白尿突出,肾功能减退程度不一;肾小球、小管和间质均有急、 慢性病理改变,球囊粘连、小球硬化和间质纤维化易见,免疫病理以IgA合并IgM、IgG沉积为主,可见"满堂亮"现象 及时大剂量甲基泼尼松龙冲击治疗,短期疗效好。     

黄芪提取物对IgA肾病小鼠的作用研究

目的:观察黄芪提取物对 IgA肾病小鼠的作用及其机理.方法:复制 IgA肾病小鼠模型,给予模型小鼠黄芪提取物 40 g/kg体重 (以生药计 ),检测实验动物 24 h尿蛋白、血尿、血清尿素氮 (BUN)、肌酐 (Cr)、总蛋白 (TP)、白蛋白 (ALB)等生化指标,作肾脏病理学检查,综合各种数据分析黄芪提取物对 IgA肾病的作用.结果:黄芪提取物可以降低模型小鼠的 24 h尿蛋白,血清尿素氮、肌酐含量,并具有利尿和降低血尿发生率、抑制系膜增生、缓解肾脏病理损伤等作用.结论:黄芪提取物对 IgA肾病有一定的治疗作用.