Direct kinetic fingerprinting and digital counting of single protein molecules.

The sensitive and accurate quantification of protein biomarkers plays important roles in clinical diagnostics and biomedical research. Sandwich ELISA and its variants accomplish the capture and detection of a target protein via two antibodies that tightly bind at least two distinct epitopes of the same antigen and have been the gold standard for sensitive protein quantitation for decades. However, existing antibody-based assays cannot distinguish between signal arising from specific binding to the protein of interest and nonspecific binding to assay surfaces or matrix components, resulting in significant background signal even in the absence of the analyte. As a result, they generally do not achieve single-molecule sensitivity, and they require two high-affinity antibodies as well as stringent washing to maximize sensitivity and reproducibility. Here, we show that surface capture with a high-affinity antibody combined with kinetic fingerprinting using a dynamically binding, low-affinity fluorescent antibody fragment differentiates between specific and nonspecific binding at the single-molecule level, permitting the direct, digital counting of single protein molecules with femtomolar-to-attomolar limits of detection (LODs). We apply this approach to four exemplary antigens spiked into serum, demonstrating LODs 55- to 383-fold lower than commercially available ELISA. As a real-world application, we establish that endogenous interleukin-6 (IL-6) can be quantified in 2-µL serum samples from chimeric antigen receptor T cell (CAR-T cell) therapy patients without washing away excess serum or detection probes, as is required in ELISA-based approaches. This kinetic fingerprinting thus exhibits great potential for the ultrasensitive, rapid, and streamlined detection of many clinically relevant proteins.

Assessment of serum allergen-specific IgE levels in horses with seasonal allergic dermatitis and recurrent airway obstruction in Spain.

Allergic conditions are prevalent equine diseases that can be diagnosed by clinical examination alone, but definitive diagnosis is more likely with laboratory testing. The ELISA Allercept© test was used to analyse the serum samples of 73 horses with allergic diseases. Sixty-one horses (83.5%) had allergen-specific IgE levels ≥ 150 ELISA Units (EU), the cut-off defined by the assay. Fifty-four horses had allergic dermatitis (AD) with high IgE levels to Tyrophagus putrescentiae (51.9%), Rumex crispus (48.1%), Tabanus (46.3%) and Dermatophagoides farinae/ D. pteronyssinus (40.7%). Seven horses with recurrent airway obstruction (RAO) had a high prevalence of T. putrescentiae (85.7%), followed by that of Acarus siro (57.1%) and D. farinae/D. pteronyssinus (57.1%). Horses affected with RAO had more positive reactions to mites (2.22 ± 0.84) than did horses with AD (1.51 ± 0.61, P < 0.05). A strong correlation of serum allergen-specific IgE level was found between Culex tarsalis and Stomoxys (r = 0.943) and between Dactylis glomerata and both Secale cereale (r = 0.79) and R. crispus (r = 0.696). These results indicate that among horses with allergic diseases in Spain, ELISA tests demonstrated a high prevalence of serum allergen-specific IgE in response to mites. Our study emphasises the importance of laboratory testing and updating allergy panels to improve the likelihood of a definitive diagnosis and the identification of allergens that should be included in allergic disease treatment.

Comparison of serum concentrations of environmental allergen-specific IgE in atopic and healthy (nonatopic) horses.

Allergic responses in humans, horses and other species are mediated by immunoglobulin E (IgE) antibodies. Serum testing to detect allergen-specific IgE antibodies has been developed for dogs, cats and horses; this allows for the identification of allergens and determination of appropriate allergen- specific immunotherapies. This study compared serum allergen-specific IgE concentrations in atopic and healthy horses. The study was performed on Malopolski breed atopic (n=21) and nonatopic (n=21) clinically healthy horses. Allergen-specific IgE serum concentrations were measured in summer seasons of 2008-2015 using a monoclonal anti-IgE antibody. A Northern and Central European allergen panel containing mite, insect, mould and plant pollen allergens, including 15 tests of individual allergens and 5 tests of allergen mixtures was used. The mean allergen-specific IgE concentrations in the atopic and normal horse populations were compared. Among the atopic horses, the strongest positive reactions occurred against the storage mites Tyrophagus putrescentiae and the domestic mite Dermatophagoides farinae. The atopic horses also demonstrated high IgE concentrations against insects, particularly Tabanus sp., the plant pollens colza, cultivated rye and the mould pollen mixture Aspergillus/Penicillium. No horses in the atopic group were IgE-negative. Among all mite, insect, mould and some plant allergen groups the differences in mean specific IgE concentrations between allergic and healthy horses were significant. The mean IgE concentrations for most allergen groups were significantly higher in the atopic horses than in the healthy animals. However, a high incidence of positive reactions was observed in both healthy and allergic horses. Our results showed a high frequency of polysensitization in atopic horses.

Peroxisome proliferator-activated receptor γ B cell-specific-deficient mice have an impaired antibody response.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. PPARγ, a ligand-activated transcription factor, has important anti-inflammatory and antiproliferative functions, and it has been associated with diseases including diabetes, scarring, and atherosclerosis, among others. PPARγ is expressed in most bone marrow-derived cells and influences their function. PPARγ ligands can stimulate human B cell differentiation and promote Ab production. A knowledge gap is that the role of PPARγ in B cells under physiological conditions is not known. We developed a new B cell-specific PPARγ (B-PPARγ) knockout mouse and explored the role of PPARγ during both the primary and secondary immune response. In this article, we show that PPARγ deficiency in B cells decreases germinal center B cells and plasma cell development, as well as the levels of circulating Ag-specific Abs during a primary challenge. Inability to generate germinal center B cells and plasma cells is correlated to decreased MHC class II expression and decreased Bcl-6 and Blimp-1 levels. Furthermore, B-PPARγ-deficient mice have an impaired memory response, characterized by low titers of Ag-specific Abs and low numbers of Ag-experienced, Ab-secreting cells. However, B-PPARγ-deficient mice have no differences in B cell population distribution within primary or secondary lymphoid organs during development. This is the first report, to our knowledge, to show that, under physiological conditions, PPARγ expression in B cells is required for an efficient B cell-mediated immune response as it regulates B cell differentiation and Ab production.

Long-term maintenance of polysaccharide-specific antibodies by IgM-secreting cells.

Many bacteria-associated polysaccharides induce long-lived Ab responses that protect against pathogenic microorganisms. The maintenance of polysaccharide-specific Ab titers may be due to long-lived plasma cells or ongoing Ag-driven B cell activation due to polysaccharide persistence. BALB/c and V(H)J558.3 transgenic mice respond to α1→3-dextran (DEX) by generating a peak anti-DEX response at 7 d, followed by maintenance of serum Ab levels for up to 150 d. Analysis of the cellular response to DEX identified a population of short-lived, cyclophosphamide-sensitive DEX-specific plasmablasts in the spleen, and a quiescent, cyclophosphamide-resistant DEX-specific Ab-secreting population in the bone marrow. BrdU pulse-chase experiments demonstrated the longevity of the DEX-specific Ab-secreting population in the bone marrow. Splenic DEX-specific plasmablasts were located in the red pulp with persisting DEX-associated CD11c(+) dendritic cells 90 d after immunization, whereas DEX was not detected in the bone marrow after 28 d. Selective depletion of short-lived DEX-specific plasmablasts and memory B1b B cells using cyclophosphamide and anti-CD20 treatment had a minimal impact on the maintenance of serum anti-DEX Abs. Collectively, these findings demonstrate that the maintenance of serum polysaccharide-specific Abs is the result of continuous Ag-driven formation of short-lived plasmablasts in the spleen and a quiescent population of Ab-secreting cells maintained in the bone marrow for a long duration.

Assessment of serum levels of allergen-specific immunoglobulin E in different seasons and breeds in healthy horses.

The present study was designed to asses specific IgE towards environment allergens in 42 healthy horses. Determination of this immunoglobulin in serum serve as diagnostic tools in allergic diseases to improve efficacy of the treatment and proper allergen selection to specific immunotherapy. Serum levels of allergen specific IgE were measured with equine monoclonal antibody, using 15 individual and 5 mix allergens in North European Panel. The study revealed season dependent increased levels of allergen specific IgE in normal horses. It is noteworthy that healthy horses show high percentage of positive reactions, most commonly towards to domestic mites D. farinae (80%), D. pteronyssinus (35.71%) and storage mites T. putrenscentiae (42.86%), Acarus siro (40.48%). These allergens play an important role in equine, canine and feline atopic dermatitis. We also demonstrated high IgE levels in the group of horse specific insect allergens. Tabanus sp. (35.71%), Culicoides sp. (28.57%) and Simulium sp. (26.19%) were the most frequent insect positive reaction allergens. No positive reactions in all groups of allergens were found in winter season, low and merely detectable levels of antibodies have been found relating to D. farianae and T. putrescentiae allergen. We observed elevated mould-IgE levels in horses that live in stables, while outdoor living horses showed very low levels. Amongst all positive reactions we observed only weak and moderate reactions but no strong positive reactions were found. No significant differences were observed between three breeds of horses with the exception of moulds and D. pteronyssinus allergens.

Variable region identical immunoglobulins differing in isotype express different paratopes.

The finding that the antibody (Ab) constant (C) region can influence fine specificity suggests that isotype switching contributes to the generation of Ab diversity and idiotype restriction. Despite the centrality of this observation for diverse immunological effects such as vaccine responses, isotype-restricted antibody responses, and the origin of primary and secondary responses, the molecular mechanism(s) responsible for this phenomenon are not understood. In this study, we have taken a novel approach to the problem by probing the paratope with (15)N label peptide mimetics followed by NMR spectroscopy and fluorescence emission spectroscopy. Specifically, we have explored the hypothesis that the C region imposes conformational constraints on the variable (V) region to affect paratope structure in a V region identical IgG(1), IgG(2a), IgG(2b), and IgG(3) mAbs. The results reveal isotype-related differences in fluorescence emission spectroscopy and temperature-related differences in binding and cleavage of a peptide mimetic. We conclude that the C region can modify the V region structure to alter the Ab paratope, thus providing an explanation for how isotype can affect Ab specificity.

Induction of immunogenic apoptosis by blockade of epidermal growth factor receptor activation with a specific antibody.

Despite promising results in the use of anti-epidermal growth factor receptor (EGFR) Abs for cancer therapy, several issues remain to be addressed. An increasing emphasis is being placed on immune effector mechanisms. It has become clear for other Abs directed to tumor targets that their effects involve the adaptive immunity, mainly by the contribution of Fc region-mediated mechanisms. Given the relevance of EGFR signaling for tumor biology, we wonder whether the oncogene inhibition could contribute to Ab-induced vaccine effect. In a mouse model in which 7A7 (an anti-murine EGFR Ab) and AG1478 (an EGFR-tyrosine kinase inhibitor) displayed potent antimetastatic activities, depletion experiments revealed that only in the case of the Ab, the effect was dependent on CD4(+) and CD8(+) T cells. Correspondingly, 7A7 administration elicited a remarkable tumor-specific CTL response in hosts. Importantly, experiments using 7A7 F(ab')(2) suggested that in vivo Ab-mediated EGFR blockade may play an important role in the linkage with adaptive immunity. Addressing the possible mechanism involved in this effect, we found quantitative and qualitative differences between 7A7 and AG1478-induced apoptosis. EGFR blocking by 7A7 not only prompted a higher proapoptotic effect on tumor metastases compared with AG1478, but also was able to induce apoptosis with immunogenic potential in an Fc-independent manner. As expected, 7A7 but not AG1478 stimulated exposure of danger signals on tumor cells. Subcutaneous injection of 7A7-treated tumor cells induced an antitumor immune response. This is the first report, to our knowledge, of a tumor-specific CTL response generated by Ab-mediated EGFR inhibition, suggesting an important contribution of immunogenic apoptosis to this effect.

Quantitative analysis of the CD4 T-cell repertoire specific to therapeutic antibodies in healthy donors.

Therapeutic antibodies are generally partially to fully humanized, yet they can show unwanted immunogenicity and lead to antibody response and adverse effects when administered to humans. As immunogenicity relies on a T-cell-dependent mechanism, we have evaluated in vitro the size of the preexisting CD4 T-cell repertoire specific to therapeutic antibodies in healthy donors. Specific CD4 T cells of individuals with different HLA-DR allotypes were amplified by in vitro stimulation and quantified. Well-known immunogenic proteins, KLH and a murine antibody, exhibited a strong in vitro T-cell response characterized by a mean of preexisting T cells >1 cell/10(6) cells. In contrast, the preexisting CD4 T-cell repertoires specific to 2 chimeric, 2 humanized, and 2 fully human antibodies remained generally inferior to this value, confirming the role of species-specific sequences in their shaping. Mean values ranged from 0.01 to 0.3 cell/10(6) cells and varied not necessarily in relationship with the humanization level of the therapeutic antibodies. Relationship with their known immunogenicity is discussed. These results contribute to a better understanding and prediction of immunogenicity of therapeutic antibodies in humans.

Distinct region-specific alpha-synuclein oligomers in A53T transgenic mice: implications for neurodegeneration.

Aggregation of alpha-synuclein (alpha-syn), a process that generates oligomeric intermediates, is a common pathological feature of several neurodegenerative disorders. Despite the potential importance of the oligomeric alpha-syn intermediates in neuron function, their biochemical properties and pathobiological functions in vivo remain vastly unknown. Here we used two-dimensional analytical separation and an array of biochemical and cell-based assays to characterize alpha-syn oligomers that are present in the nervous system of A53T alpha-syn transgenic mice. The most prominent species identified were 53 A detergent-soluble oligomers, which preceded neurological symptom onset, and were found at equivalent amounts in regions containing alpha-syn inclusions as well as histologically unaffected regions. These oligomers were resistant to SDS, heat, and urea but were sensitive to proteinase-K digestion. Although the oligomers shared similar basic biochemical properties, those obtained from inclusion-bearing regions were prominently reactive to antibodies that recognize oxidized alpha-syn oligomers, significantly accelerated aggregation of alpha-syn in vitro, and caused primary cortical neuron degeneration. In contrast, oligomers obtained from non-inclusion-bearing regions were not toxic and delayed the in vitro formation of alpha-syn fibrils. These data indicate that specific conformations of alpha-syn oligomers are present in distinct brain regions of A53T alpha-syn transgenic mice. The contribution of these oligomers to the development of neuron dysfunction appears to be independent of their absolute quantities and basic biochemical properties but is dictated by the composition and conformation of the intermediates as well as unrecognized brain-region-specific intrinsic factors.

LL-37 complexation with glycosaminoglycans in cystic fibrosis lungs inhibits antimicrobial activity, which can be restored by hypertonic saline.

There is an abundance of antimicrobial peptides in cystic fibrosis (CF) lungs. Despite this, individuals with CF are susceptible to microbial colonization and infection. In this study, we investigated the antimicrobial response within the CF lung, focusing on the human cathelicidin LL-37. We demonstrate the presence of the LL-37 precursor, human cathelicidin precursor protein designated 18-kDa cationic antimicrobial protein, in the CF lung along with evidence that it is processed to active LL-37 by proteinase-3. We demonstrate that despite supranormal levels of LL-37, the lung fluid from CF patients exhibits no demonstrable antimicrobial activity. Furthermore Pseudomonas killing by physiological concentrations of exogenous LL-37 is inhibited by CF bronchoalveolar lavage (BAL) fluid due to proteolytic degradation of LL-37 by neutrophil elastase and cathepsin D. The endogenous LL-37 in CF BAL fluid is protected from this proteolysis by interactions with glycosaminoglycans, but while this protects LL-37 from proteolysis it results in inactivation of LL-37 antimicrobial activity. By digesting glycosaminoglycans in CF BAL fluid, endogenous LL-37 is liberated and the antimicrobial properties of CF BAL fluid restored. High sodium concentrations also liberate LL-37 in CF BAL fluid in vitro. This is also seen in vivo in CF sputum where LL-37 is complexed to glycosaminoglycans but is liberated following nebulized hypertonic saline resulting in increased antimicrobial effect. These data suggest glycosaminoglycan-LL-37 complexes to be potential therapeutic targets. Factors that disrupt glycosaminoglycan-LL-37 aggregates promote the antimicrobial effects of LL-37 with the caveat that concomitant administration of antiproteases may be needed to protect the now liberated LL-37 from proteolytic cleavage.

BRCA1 Antibodies Matter.

Breast cancer susceptibility gene 1 (BRCA1) encodes a tumor suppressor that is frequently mutated in familial breast and ovarian cancer patients. BRCA1 functions in multiple important cellular processes including DNA damage repair, cell cycle checkpoint activation, protein ubiquitination, chromatin remodeling, transcriptional regulation, as well as R-loop formation and apoptosis. A large number of BRCA1 antibodies have been generated and become commercially available over the past three decades, however, many commercial antibodies are poorly characterized and, when widely used, led to unreliable data. In search of reliable and specific BRCA1 antibodies (Abs), particularly antibodies recognizing mouse BRCA1, we performed a rigorous validation of a number of commercially available anti-BRCA1 antibodies, using proper controls in a panel of validation applications, including Western blot (WB), immunoprecipitation (IP), immunoprecipitation-mass spectrometry (IP-MS), chromatin immunoprecipitation (ChIP) and immunofluorescence (IF). Furthermore, we assessed the specificity of these antibodies to detect mouse BRCA1 protein through the use of testis tissue and mouse embryonic fibroblasts (MEFs) from Brca1+/+ and Brca1Δ11/Δ11 mice. We find that Ab1, D-9, 07-434 (for recognizing human BRCA1) and 287.17, 440621, BR-64 (for recognizing mouse BRCA1) are specific with high quality performance in the indicated assays. We share these results here with the goal of helping the community combat the common challenges associated with anti-BRCA1 antibody specificity and reproducibility and, hopefully, better understanding BRCA1 functions at cellular and tissue levels.

Relationship of donor HLA antibody strength to the development of transfusion-related acute lung injury.

BACKGROUND: Antibodies against the human leukocyte antigen (HLA) in donors' blood are implicated in the development of transfusion-related acute lung injury (TRALI). Screening of female donors for HLA antibodies has been introduced to prevent TRALI; however, the relationship of HLA antibody strength in the transfused components to the development of TRALI has not been evaluated in detail. STUDY DESIGN AND METHODS: Donors involved in 1038 cases of nonhemolytic transfusion reactions (NHTRs) including 283 cases of TRALI were screened for HLA antibodies by the fluorescence beads method. HLA antibody specificity and strength were analyzed in detail. The usefulness of enzyme-linked immunosorbent assay (ELISA) for screening HLA antibodies was also evaluated. RESULT: Among 21 cases of TRALI, four cases of possible TRALI, and five cases of other NHTRs, the sum of mean fluorescence intensity (MFI) of donors' HLA antibodies to patients' cognate antigen(s) was determined in 18, four, and three cases, respectively. The sum of MFI in TRALI cases was significantly higher than that in other NHTR cases (p<0.05). When HLA antibody-positive samples were reevaluated by ELISA, the ELISA optical density ratio was significantly higher in donors' samples associated with TRALI than in those associated with other NHTRs (p<0.01) CONCLUSIONS: A correlation between the HLA antibody strength and development of TRALI was indicated. The antibody strength measured by ELISA could be used as the basis for the screening of HLA antibodies in place of the fluorescence beads method. This study provided clues to the establishment of a cutoff value for HLA antibody screening in an evidence-based manner for the prevention of TRALI.

Characterization of antisperm antibody binding patterns in relation to sperm phenotypic attributes and field fertility in dairy bulls.

To test our hypothesis that antisperm antibodies (ASA) might alter sperm phenotypic attributes thus leading to sub-fertility/infertility in bulls, ASA were generated in crossbred male calves by immunizing with sperm two times. Cryopreserved spermatozoa from crossbred bulls (n = 24) with different field fertility ratings were incubated with ASA and different patterns of ASA immunolocalization were studied. In addition, sperm membrane integrity, acrosomal integrity and cryo-capacitation status were also assessed. Immunolocalization of sperm antigens using antisperm antibody revealed three major patterns (Acrosomal-AR, apical-AP and, acrosome and tail-AT). The proportion of ASA reactive spermatozoa was significantly (P < 0.05) higher in low- and medium-fertile bulls compared to high-fertile bulls. Among the three patterns, the proportion of spermatozoa with AR pattern was significantly (P < 0.05) higher in low- and medium-fertile bulls compared to high-fertile bulls. The proportion of membrane and acrosome intact spermatozoa was significantly (P < 0.05) higher in high-fertile bulls compared to medium- and low-fertile bulls. There were no significant differences in the proportion of cryo-capacitated spermatozoa among high-, medium- and low-fertile bulls. The relationship between ASA reactive spermatozoa and conception rates (CR) of bulls was highly (P < 0.01) significant and negative. Similarly, AR and AT pattern were also significantly (P < 0.01) and negatively related to CR of bulls. The reactivity of spermatozoa with ASA was also significantly (P < 0.01) and negatively related to the membrane and acrosome integrity of spermatozoa. It was concluded that the proportion of spermatozoa responding to ASA was higher in low-compared to high-fertile bulls and ASA localization in sperm acrosomal area was negatively related to sperm membrane and acrosomal integrity and bull fertility.

Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity.

Although expanded polyglutamine (polyQ) repeats are inherently toxic, causing at least nine neurodegenerative diseases, the protein context determines which neurons are affected. The polyQ expansion that causes Huntington's disease (HD) is in the first exon (HDx-1) of huntingtin (Htt). However, other parts of the protein, including the 17 N-terminal amino acids and two proline (polyP) repeat domains, regulate the toxicity of mutant Htt. The role of the P-rich domain that is flanked by the polyP domains has not been explored. Using highly specific intracellular antibodies (intrabodies), we tested various epitopes for their roles in HDx-1 toxicity, aggregation, localization, and turnover. Three domains in the P-rich region (PRR) of HDx-1 are defined by intrabodies: MW7 binds the two polyP domains, and Happ1 and Happ3, two new intrabodies, bind the unique, P-rich epitope located between the two polyP epitopes. We find that the PRR-binding intrabodies, as well as V(L)12.3, which binds the N-terminal 17 aa, decrease the toxicity and aggregation of HDx-1, but they do so by different mechanisms. The PRR-binding intrabodies have no effect on Htt localization, but they cause a significant increase in the turnover rate of mutant Htt, which V(L)12.3 does not change. In contrast, expression of V(L)12.3 increases nuclear Htt. We propose that the PRR of mutant Htt regulates its stability, and that compromising this pathogenic epitope by intrabody binding represents a novel therapeutic strategy for treating HD. We also note that intrabody binding represents a powerful tool for determining the function of protein epitopes in living cells.

Immunohistochemical validation of multiple phospho-specific epitopes for estrogen receptor alpha (ERalpha) in tissue microarrays of ERalpha positive human breast carcinomas.

Estrogen receptor alpha (ERalpha) activity is regulated by phosphorylation at several sites. Recently several antibodies specific for individual phosphorylated sites within ERalpha have became available. Such antibodies potentially provide invaluable tools to gain insight into the relevance in vivo of phosphorylated ERalpha in human breast tumors. However, validation of these antibodies for immunohistochemistry in particular is necessary in the first instance. In this study we have investigated the usefulness of several antibodies generated to specific phosphorylated sites within ERalpha for immunohistochemistry of formalin-fixed, paraffin-embedded human breast cancer biopsy samples. As well, these data demonstrate for the first time, the detection of multiple phosphorylated ERalpha forms in breast cancer (P-S104/106-ERalpha, P-S118-ERalpha, P-S167-ERalpha, P-S282-ERalpha, P-S294-ERalpha, P-T311-ERalpha, and P-S559-ERalpha) suggesting the possibility that profiling of phosphorylated ERalpha isoforms might be useful in selecting subgroups of breast cancer patients that would benefit from endocrine therapy.

Tyrosine-phosphorylated extracellular signal--regulated kinase associates with the Golgi complex during G2/M phase of the cell cycle: evidence for regulation of Golgi structure.

Phosphorylation of the extracellular signal-regulated kinases (ERKs) on tyrosine and threonine residues within the TEY tripeptide motif induces ERK activation and targeting of substrates. Although it is recognized that phosphorylation of both residues is required for ERK activation, it is not known if a single phosphorylation of either residue regulates physiological functions. In light of recent evidence indicating that ERK proteins regulate substrate function in the absence of ERK enzymatic activity, we have begun to examine functional roles for partially phosphorylated forms of ERK. Using phosphorylation site--specific ERK antibodies and immunofluorescence, we demonstrate that ERK phosphorylated on the tyrosine residue (pY ERK) within the TEY activation sequence is found constitutively in the nucleus, and localizes to the Golgi complex of cells that are in late G2 or early mitosis of the cell cycle. As cells progress through metaphase and anaphase, pY ERK localization to Golgi vesicles is most evident around the mitotic spindle poles. During telophase, pY ERK associates with newly formed Golgi vesicles but is not found on there after cytokinesis and entry into G1. Increased ERK phosphorylation causes punctate distribution of several Golgi proteins, indicating disruption of the Golgi structure. This observation is reversible by overexpression of a tyrosine phosphorylation--defective ERK mutant, but not by a kinase-inactive ERK2 mutant that is tyrosine phosphorylated. These data provide the first evidence that pY ERK and not ERK kinase activity regulates Golgi structure and may be involved in mitotic Golgi fragmentation and reformation.

Preparation of immunogens and production of antibodies.

The quality of reagents greatly affects the interpretation of serological tests. Methods used in conventional viral purification and molecular cloning and expression of target viral proteins to obtain antigens for immunization are presented. Immunization of rabbits, mice and chickens and isolation of immunoglobulin from immunized animals also are described.

Parallelized Microscale Expression of Soluble scFv.

Antibody phage display is a key technology to generate recombinant, mainly human, antibodies for diagnostic and therapy, but also as tools for basic research. After antibody selection by "panning," a crucial step is the screening of monoclonal binders to isolate those which show antigen specificity. For this screening procedure, a highly parallelized approach to produce soluble antibody fragments in microtiter plates is essential. In this chapter, we give the protocol for the parallelized microscale production of scFvs for the screening procedure or further assays.

Antibody-based approaches in Alzheimer's research: safety, pharmacokinetics, metabolism, and analytical tools.

High morbidity, enormous socioeconomic costs, and lack of specific treatments emphasize the importance of research on protective therapies against Alzheimer's disease. The efficacy of anti-amyloid immunization strategies has been demonstrated preclinically, prompting the design of clinical studies. However, the detailed mechanisms of action of therapeutic antibodies, especially their influence on the complex amyloid beta peptide (Abeta) metabolism and various Abeta-equilibria present both within and outside the CNS, are far from being clear. Furthermore, physiological Abeta metabolism is poorly understood and the analytical tools to characterize and quantify treatment effects on Abeta metabolism are suboptimal. Thus, the design of immunization strategies with optimized benefit-to-risk ratios for patients is subjected to significant obstacles. Indeed, an active immunization trial with Abeta was discontinued because of severe adverse effects. Anti-Abeta immunization protocols designed to attain high blood levels of antibodies bear the potential to induce brain inflammation and/or hemorrhage, thus directing the biomedical research towards development of more predictable therapies for minimizing the risk of adverse effects. The focus of this review is to summarize current knowledge of Abeta metabolism under physiological and antibody-based therapeutic conditions and to introduce a promising approach, namely the passive immunization using antibody fragments, which are characterized by entirely different pharmacokinetic and pharmacodynamic properties compared with conventional monoclonal antibodies.