Fluorescent-labeled antibodies: Balancing functionality and degree of labeling

A critical assumption in using labeled antibodies is that the conjugation reaction has no deleterious effects on antibody avidity. This study demonstrates that this assumption need not hold true and presents a methodology to quantitatively determine the degree of inactivation and/or changes in antibody-antigen binding that can occur with conjugation. Fluorescein isothiocyanate (FITC) was conjugated to a mouse monoclonal antibody, Fc125, against hemagglutinin (HA) using varying fluorophore/protein (F:P) labeling ratios. Antibody binding, as a function of the F:P labeling ratio, was evaluated using a kinetic enzyme-linked immunosorbent assay (ELISA) and analyzed using global fitting. A two-parameter adjustment of the antibody concentration and the maximum rate was sufficient to describe the rate changes. The concentration parameter dominated the rate changes, consistent with the hypothesis that the coupling reaction inactivated an increasing fraction of the antibody population with a smaller change (∼15% at the highest F:P ratio) in antibody-antigen binding. An optimal F:P ratio that minimized both inactivation and unlabeled antibody was calculated. This procedure can be used to prepare functional, labeled antibody reagents with defined activity and can aid in quantitative applications where the stoichiometry and functionality of the labeled antibody are critical.     

The protective effect of immunisation against diphtheria, pertussis and tetanus (DPT) in relation to sudden infant death syndrome

Epidemiological evidence indicates infants immunised against diphtheria, pertussis and tetanus (DPT) are at decreased risk of sudden infant death syndrome (SIDS). Asymptomatic whooping cough and pyrogenic toxins of Staphylococcus aureus have been implicated in the aetiology of SIDS. The objectives of the present study were: (1) to determine if the DPT vaccine induced antibodies cross-reactive with the staphylococcal toxins; (2) to determine if antibodies to the pertussis toxin (PT) and the staphylococcal toxins were present in the sera of women during late pregnancy; (3) to examine the effects of infant immunisation on levels of antibodies to PT and the staphylococcal toxins; (4) to assess the effects of changes in immunisation schedules in the UK on the incidence and age distribution of SIDS. Enzyme-linked immunosorbent assays (ELISA) were used to measure binding of rabbit or human IgG to the DPT vaccine, PT, toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins A (SEA), B (SEB) and C (SEC). Neutralisation activity of anti-DPT serum was assessed by a bioassay for induction of nitric oxide from human monocytes by the staphylococcal toxins. Anti-DPT serum bound to the DPT vaccine, PT and each of the staphylococcal toxins. It also reduced the ability of the four toxins to induce nitric oxide from monocytes. In pregnant women, levels of IgG to PT, SEC and TSST-1 decreased significantly in relation to increasing weeks of gestation while antibodies to SEA and SEB increased. In infants' sera there were significant correlations between levels of IgG bound to DPT and IgG bound to PT, TSST-1 and SEC but not SEA or SEB. Antibody levels to the toxins in infants declined with age; sera from infants < or = 2 months of age had higher levels of IgG bound to the toxins than those older than 2 months. This pattern was observed for infants whose immunisation schedules began at 2 months of age or 3 months of age. The decrease in IgG bound to the toxins was, however, less for those immunised at 2 months. The decrease in SIDS deaths after the change in immunisation schedules was greatest in the 4-6-month age range. While DPT immunisation might prevent some unexplained infant deaths due to asymptomatic whooping cough, these data indicate that immunisation with DPT also induces antibodies cross-reactive with pyrogenic staphylococcal toxins implicated in many cases of SIDS. Passive immunisation of infants who have low levels of these antibodies might reduce further the numbers of these infant deaths.     

Analysis of the effect of immunization in rabies time series

The authors analysed time series of rabies cases diagnosed in Hungary between 1967 and 2001. In Transdanubia (West Hungary), an oral immunization program started in 1992 and in East Hungary in 2001. Both long term and seasonal trends were identified in the time series of rabies cases. In order to characterize the underlying processes governing the behaviour of the epidemic, the fluctuations around the trend were analysed before and after immunization separately. It turned out that the tail of the complementary cumulative distribution functions differ. The tail of the distribution follows an inverse power law (IPL) function and describes the distribution of extreme events. The significant difference in the IPL exponents before and after immunization can be explained by the theory of Highly Optimized Tolerance (HOT).     

The fate of antibodies and their radiolabels bound to tumor cells in vitro: The effect of cross-linking at the cell surface and of anti-idiotype antibodies

Summary In order to obtain rapid blood clearance of circulating antibodies (Ab) at a desired time, cross-linking reagents such as second Ab are often employed. Such reagents will generally bind to Ab located at the tumor site as well as free Ab, and we therefore investigated whether the cross-linking of Ab bound to the surface of tumor cells affects the processing of those Ab. Cross-linking was induced in various ways: a polyclonal second Ab [rabbit anti-(mouse IgG)], a monoclonal rat anti-(mouse IgG constant region) Ab, and streptavidin used in conjunction with a biotinylated first Ab. Processing was followed for 3 days, to allow nearly all of the bound Ab to reach its ultimate fate. Results depended strongly on the particular first Ab used. Two basic effects were observed. First, the second Ab efficiently prevented the early dissociation of intact Ab from the cell; once the second Ab bound, there was virtually no dissociation of the primary Ab bound to the cells. For most Ab, where only a small proportion of bound Ab dissociated intact, this effect was relatively small. However, for an unusual Ab, where the majority dissociated intact (L6) the effect of a second Ab in prolonging Ab retention by the cell was dramatic. Second, cross-linking sometimes resulted in markedly accelerated internalization and degradation of the bound Ab, coupled with the release of degradation products into the medium. This process resulted in much shorter retention of the radioisotope by the cell. If a residualizing radiolabel was used,¹²⁵I-dilactitoltyramine, which is probably trapped within lysosomes after Ab catabolism, the effect of the second Ab in accelerating loss from the cell was largely prevented. We also tested anti-idiotype Ab as cross-linking reagents. In addition to testing anti-idiotype Ab known to react with the cell-bound primary Ab, we also tested antiidiotype Ab not expected to bind to cell-bound Ab, initially as a negative control. Unexpectedly, all anti-indiotype Ab tested induced rapid release of the primary Ab from the cell. This effect was similar to the effect of a large excess of unlabeled Ab, and we attribute it to the blocking of the free binding site of a wobbling Ab, which prevents its rebinding to a second antigen molecule. We conclude that the use of selected anti-idiotype Ab to clear circulating Ab, while not reacting with cell-bound Ab, must be done cautiously. These effects must be taken into consideration in developing procedures that utilize second Ab or other crosslinking agents.     

Activation of low and null activity isozymes of maize alcohol dehydrogenase by antibodies

Summary Antisera were raised against several purified, high specific acitivity isozymes of maize alcohol dehydrogenase (ADH1). The various antisera had different effects on the activity of immunoprecipitated ADH. One antiserum completely inactivated maize ADH. This inactivation could be blocked by preicubation of the enzyme with NAD⁺, its cofactor, or with NADP. The different antisera were used to analyze variant froms of ADH1. Isozymes having lowered specific activity were activated to wild-type levels by precipitation of the enzymes with noninactivating antisera. Isozymes having no detectable ADH activity (CRM⁺ nulls) were activated by immunoprecipition with noninactivating antisera when preincubated with NAD⁺ or NADP. All of the CRM⁺ nulls were shown to be unable to bind NAD⁺, a flaw which can account for their lack of activity. The results indicate that a conformational equilibrium between active and inactive forms of maize ADH in solution controls the specific activity of the various isozymes. Both controls the specific activity of the various isozymes. Both NAD⁺ and antibodies raised against high specific activity enzymes can interact with low activity isozymes to shift the balance of the equilibrium toward the active form, thus increasing their specific activity.     

Immunohistochemical studies with antibodies to myosins from the cytoplasm and membrane fraction of human blood platelets

Summary Antibodies were raised to myosins extracted from the cytoplasm and solubilized membranes of human blood platelets. Both antibodies had similar titers as shown by enzyme-immunoassay and bound to the same sites as shown by immunohistochemistry. They were specific for cytoplasmic myosins (e.g., in human white blood cells, platelets and fibroblasts and rat endothelial cells). They showed no crossreaction with human or rat smooth muscle.Abbreviations ATPase adenosine triphosphatase EC 3.6.1.3 - FITC fluorescein isothiocyanate     

Binding parameters of antibodies: pseudo-affinity and other misconceptions

In order to evaluate and compare monoclonal antibodies (Abs), functional affinities are generally determined. While the equations that define affinity relate to monovalent interactions, it has been considered that the binding of Abs to multivalent antigens such as the cell surface could be described by an apparent, or functional affinity. We demonstrate here that this concept is incorrect, since the binding interactions that occur cannot be described in terms of a functional affinity, and the values that are obtained serve only to obscure the true interactions. Bivalent Ab binding must be considered to be an irreversible reaction, in most cases. A correct understanding of Ab binding will be useful in the further development of Abs for therapeutic purposes.     

Monoclonal antibodies to human brain acetylcholinesterase: properties and applications

1. Acetylcholinesterase (AChE) was purified 20,000-fold in a 43% yield from 90 g of human cerebellum by combined immunoaffinity and ligand affinity chromatography. The purified enzyme migrated as a 68,000-dalton band during polyacrylamide gel electrophoresis under denaturing and reducing conditions. 2. Balb/c mice were immunized with multiple 10-micrograms injections of this material in order to raise monoclonal antibodies to human brain AChE. Three such antibodies were obtained and characterized. 3. Each antibody cross-reacted distinctively with AChEs from other mammals. No antibody recognized human plasma butyrylcholinesterase but all reacted with AChE from human red blood cells. 4. Antibodies HR5 and HR3 performed well in two-site immunoassays for AChE. With these assays we compared autopsy samples of cortical region A9 from six controls (nonneurological cases) and five patients with Alzheimer's disease. The latter showed a highly significant 60% deficit of AChE protein. 5. The present antibodies will permit additional immunochemical studies of cholinergic systems in dementia.     

志贺氏I型痢疾杆菌O-特异性多糖与破伤风类毒素结合疫苗的制备及其在小鼠体内的免疫原性

从志贺氏I型痢疾杆菌LPS中分离纯化出O SP半抗原 ,以ADH为连接剂将其与TT结合形成O SP TT结合疫苗 ,并用此结合疫苗免疫NIH小鼠 ,结果显示单独使用O SP免疫后 ,小鼠血清中没有抗LPS抗体产生 ,而用O SP TT免疫后小鼠血清中产生了抗LPSIgG和IgM抗体 ,且IgG抗体水平高于IgM抗体 ;O SP TT免疫组第二次和第三次免疫后IgG抗体水平均有显著的升高 (P <0 0 1) ,但第二次和第三次免疫后血清IgM抗体虽有升高 ,但与前一次免疫相比均无显著差异 (P >0 0 5 ) ,表明O SP TT结合疫苗具有加强免疫应答效应。补体介导的体外杀菌活性试验结果证明 ,O SP TT免疫血清在 1∶16 0倍稀释后仍对志贺氏I型痢疾杆菌具有特异性杀菌活性。     

A new sensitive and fast peptide immunoassay based on enzyme amplification used in the determination of CGRP and the demonstration of its presence in the thyroid

An enzyme amplified immunoassay for rCGRP based on cofactor cycling has been found to be clearly superior to a comparable radioimmunoassay employing the same antiserum in terms of sensitivity, speed and convenience. Correlation between the two methods was very good. With the enzyme amplified immunoassay we have been able to demonstrate the existence of rCGRP in thyroid extract.     

Measurement of hypocretin/orexin content in the mouse brain using an enzyme immunoassay: the effect of circadian time,age and genetic background

The hypocretins (1 and 2) have emerged as key regulators of sleep and wakefulness. We developed a high-throughput enzyme immunoassay (EIA) to measure total brain hypocretin levels from large numbers of mice. Hypocretin levels were not altered by circadian time or age. However, significant differences in one or both hypocretin peptides were observed between different mouse strains. We studied hypocretin levels in knockout and transgenic mouse models with obesity, circadian gene mutations or monoaminergic defects. Compared to controls, only histamine receptor knockouts had lower hypocretin levels. This was most pronounced in H1 receptor knockouts suggesting the existence of a positive feedback loop between hypocretin and histaminergic neurons.     

Fragments of Bacterial Endoglycosidase S and Immunoglobulin G Reveal Subdomains of Each That Contribute to Deglycosylation

Endoglycosidase S (EndoS) is a glycoside-hydrolase secreted by the bacterium Streptococcus pyogenes. EndoS preferentially hydrolyzes the N-linked glycans from the Fc region of IgG during infection. This hydrolysis impedes Fc functionality and contributes to the immune evasion strategy of S. pyogenes. Here, we investigate the mechanism of human serum IgG deactivation by EndoS. We expressed fragments of IgG1 and demonstrated that EndoS was catalytically active against all of them including the isolated CH2 domain of the Fc domain. Similarly, we sought to investigate which domains within EndoS could contribute to activity. Bioinformatics analysis of the domain organization of EndoS confirmed the previous predictions of a chitinase domain and leucine-rich repeat but also revealed a putative carbohydrate binding module (CBM) followed by a C-terminal region. Using expressed fragments of EndoS, circular dichroism of the isolated CBM, and a CBM-C-terminal region fusion revealed folded domains dominated by β sheet and α helical structure, respectively. Nuclear magnetic resonance analysis of the CBM with monosaccharides was suggestive of carbohydrate binding functionality. Functional analysis of truncations of EndoS revealed that, whereas the C-terminal of EndoS is dispensable for activity, its deletion impedes the hydrolysis of IgG glycans.     

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.     

儿童家长对预防接种不良反应的知识、态度和行为研究

对儿童家长预防接种不良反应的知识、态度和行为进行调查。家长对预防接种不良反应的认识存在偏差。加强预防接种门诊不良反应相关知识的宣传,提高儿童家长对预防接种不良反应的认知度是做好预防接种不良反应监测工作的基础。     

Monoclonal Antibodies to Substance P: Production, Characterization of Their Fine Specificities, and Use in Immunocytochemistry

Five hybrid clones secreting antibodies to the neuropeptide substance P have been obtained by somatic cell fusion of mouse myeloma cells with splenocytes from immunized mice of the Biozzi strain. To perform rapid and sensitive screening tests as well as to study the fine specificities of each monoclonal antibody, we developed a new enzyme immunoassay of substance P using acetylcholinesterase as label. All five monoclonal antibodies were directed to the C-terminal pentapeptide of substance P, especially to the Phe7 residue. They cross-reacted with neurokinin A and to some extent with neurokinin B but not with other nontachykinin mammalian peptides. One monoclonal antibody (SP 14) was used for immunocytochemical experiments in the rat spinal cord and spinal ganglion, both at the light and electron microscopic levels. A strong specific neurokinin-like immunoreactivity was observed in cell bodies, nerve fibers, and terminals, with a very low background staining. Finally, the affinities of several analogues of substance P for SP 14 monoclonal antibody were shown to be correlated with their biological activities, as measured by their hypotensive effects in vivo. These findings suggested a strong structural resemblance between the combining site of the antibody and that of the physiological substance P receptor.     

Affinity, avidity, and kinetics of target sequence binding to LC8 dynein light chain isoforms

LC8 dynein light chain (DYNLL) is a highly conserved eukaryotic hub protein with dozens of binding partners and various functions beyond being a subunit of dynein and myosin Va motor proteins. Here, we compared the kinetic and thermodynamic parameters of binding of both mammalian isoforms, DYNLL1 and DYNLL2, to two putative consensus binding motifs (KXTQTX and XG(I/V)QVD) and report only subtle differences. Peptides containing either of the above motifs bind to DYNLL2 with micromolar affinity, whereas a myosin Va peptide (lacking the conserved Gln) and the noncanonical Pak1 peptide bind with K(d) values of 9 and 40 μM, respectively. Binding of the KXTQTX motif is enthalpy-driven, although that of all other peptides is both enthalpy- and entropy-driven. Moreover, the KXTQTX motif shows strikingly slower off-rate constant than the other motifs. As most DYNLL partners are homodimeric, we also assessed the binding of bivalent ligands to DYNLL2. Compared with monovalent ligands, a significant avidity effect was found as follows: K(d) values of 37 and 3.5 nM for a dimeric myosin Va fragment and a Leu zipper dimerized KXTQTX motif, respectively. Ligand binding kinetics of DYNLL can best be described by a conformational selection model consisting of a slow isomerization and a rapid binding step. We also studied the binding of the phosphomimetic S88E mutant of DYNLL2 to the dimeric myosin Va fragment, and we found a significantly lower apparent K(d) value (3 μM). We conclude that the thermodynamic and kinetic fine-tuning of binding of various ligands to DYNLL could have physiological relevance in its interaction network.     

Identification of urotensin II-related peptide as the urotensin II-immunoreactive molecule in the rat brain

Urotensin II (UII) has been reported as the most potent known vasoconstrictor. While rat and mouse orthologs of UII precursor protein have been reported, only the tentative structures of UII peptides of these animals have been demonstrated, since prepro-UII proteins lack typical processing sites for their mature peptides. In the present study, we isolated a novel peptide, UII-related peptide (URP), from the extract of the rat brain as the sole immunoreactive substance to anti-UII antibody; the amino acid sequence of the peptide was determined as ACFWKYCV. cDNAs encoding rat, mouse, and human precursor proteins for URP were cloned and revealed that the sequences of mouse and human URP peptides are the same as that for rat URP. Prepro-URP gene is expressed in several rat tissues such as those of the thymus, spleen, testis, and spinal cord, although with lower levels than the prepro-UII gene. In the human, the prepro-URP gene is expressed comparably to prepro-UII in several tissues except the spinal cord. URP was found to bind and activate the human or rat UII receptors (GPR14) and showed a hypotensive effect when administered to anesthetized rats. These results suggest that URP is the endogenous and functional ligand for UII receptor in the rat and mouse, and possibly in the human. We also describe the preparation of specific monoclonal antibodies raised against UII peptide and the establishment of a highly sensitive enzyme immunoassay system for UII peptides.     

Plasmodium berghei and Eperythrozoon coccoides: Antibody and immunoglobulin synthesis in germfree and and conventional mice simultaneously infected

The immune response to Eperythrozoon coccoides and the malaria parasite Plasmodium berghei was evaluated in germfree (GF) and conventionally reared (CV) mice infected with both parasites. Following infection, the mice showed significant changes in the levels of the immunoglobulins IgM, 7Sγin1, 7Sγ2a, and 7Sγ2b, but no detectable changes in IgA. Increases in immunoglobulin levels were first observed in GF mice, but by the twelth day both GF and CV mice had comparable levels. 7Sγ2a globulin had a bimodal distribution in both groups of mice which probably was due to heterogeneity in the allotype of this immunoglobulin. IgM levels closely paralleled the antibody responses to P. berghei suggesting that most of the antibody to this parasite was IgM. Relatively low levels of antibody to both parasites, in comparison to the large immunoglobulin response, were detected in GF and CV mice. The possible causes for the low titers are discussed.     

广西2017年免疫规划疫苗分配及管理策略

目的 分析广西壮族自治区(广西)2017年免疫规划疫苗分配状况,探讨免疫规划疫苗管理策略。方法 整理2017年广西各级免疫规划疫苗使用进度报表数据,对免疫规划疫苗分配情况进行统计学分析。结果 2017年共分配免疫规划疫苗2292.59万剂次,使用2187.26万剂次,各类疫苗损耗系数以市为单位达到国家要求;部分疫苗分配显示,广西各级分配疫苗基本平衡(SD<20),疫苗供应短缺和疫苗分配平衡呈负相关(Pearson=-0.984,P=0.016),单人份疫苗比多人份疫苗分配平衡性好;疫苗供应不足和多人份疫苗在一定程度上影响疫苗分配平衡性。结论 2017年广西免疫规划疫苗使用规范,广西各级分配疫苗基本平衡,但有个别地级市向下级分配疫苗不平衡。需从补充完善冷链设备、建设疫苗管理信息化系统、强化业务督导考核和人员培训等方面加强对免疫规划疫苗的分配管理工作。     

Interaction between granulocytes and antibodies in the enhancement of lung defenses against Staphylococcus aureus after intranasal immunization of mice with live-attenuated bacteria

Abstract Immunization with live-attenuated Staphylococcus aureus induced measurable levels of specific IgG and IgA in the lungs, but the pulmonary clearance of S. aureus in immunized mice did not differ from that of control mice. Aerosol exposure of mice to Pseudomonas aeruginosa induced a significant recruitment of polymorphonuclear leukocytes (PMNL) to the lungs in both immunized and control mice, whereas S. aureus challenge did not. However, challenge with a mixture of P. aeruginosa-S. aureus or exposure to an aerosol of Escherichia coli lipopolysaccharide (LPS) before S. aureus challenge induced PMNL migration and a significant enhancement of pulmonary clearance of S. aureus in immunized mice. The presence of both antibodies and PMNL was required for enhancement of S. aureus pulmonary clearance.