Characterization of factors influencing on-chip complement activation to optimize parallel measurement of antibody and complement proteins on antigen microarrays

Binding of immunoglobulins and complement fragments to targets of adaptive immune responses can be monitored using collections of arrayed antigens and is used to generate profiles of antibody binding and function. The collection of reliable data on these reactions on a large scale requires the establishment of criteria from sample collection through reaction conditions to normalization strategies. We characterized the detection of IgG, complement C3 and C4 under conditions that better resemble in vivo events than most serological assays and are also relevant for in vitro diagnostic purposes. Immune complex formation was modeled using nitrocellulose-based protein arrays and the effects of factors like anticoagulant use, serum dilution, time and bivalent cation concentrations were assessed. Blood samples from healthy controls (n=24) and patients with systemic autoimmune disease (n=60) were collected and correlations between classical laboratory tests and chip-based reference proteins were evaluated to optimize normalization schemes. Best signal-to-noise ratio and acceptable masking of IgG by complement C3 fragments was achieved at modest, five to ten-fold serum dilutions. C3 binding to captured human IgG was found to correlate best with serum C3 concentrations and C3 activity and is therefore an ideal reference feature for normalization of biological and methodological variations in complement activity and detection.     

Protein microarrays: a new tool for profiling antibody cross-reactivity

Antibody cross-reactivity can compromise interpretation of experiments and derail therapeutic antibody development. Standard techniques such as immunohistochemistry or Western analysis provide important but often inadequate approaches to assess antibody specificity. Protein microarrays are providing a new approach to rapidly characterize antibody cross-reactivity against 1,000s of proteins simultaneously. This review will focus on reported examples of antibody cross-reactivity, methods used to characterize them, and the recent development and use of protein microarrays for assessing antibody specificity.     

Identification and testing of control peptides for antigen microarrays

Introduction

Peptide microarray slides usually contain positive control spots to gauge for antibody binding. Unlike the good response on earlier prototype microarrays, human immunoglobulin controls do not function consistently on newer generation slides. This may be due to technical problems in high-density printing or degradation. Our objective was to identify and print reliable control peptides that did not suffer from the same problems as proteins.

Methods

Peptide microarray slides containing 10,000-23,000 synthetic peptides spanning proteins involved in M. tuberculosis or Bordatella pertussis were incubated with secondary antibody in sample dilution buffer. After removal of artefacts due to slide architecture, we identified peptides that gave a high mean response and low co-efficient of variation across all replicates. These control peptides were tested for their performance on newly manufactured slides.

Results

We selected three peptides on the TB slides and three peptides on the B. pertussis slides that had a mean response index of at least 5 and a coefficient of variation less than 15%. When used as controls in newly-designed slides, these peptides gave consistently high responses: the median index ranged from 4.5 to 9.5 in the absence of patient serum and was of a somewhat lesser magnitude when incubated with patient serum. We illustrate the use of these control responses to normalize the peptide responses on a set of slides prepared with human serum.

Conclusions

Our work shows that it is possible to identify control peptides that can be used when protein controls do not function consistently. This has important consequences for the storage of peptide-microarrays and their use in the field: protein chips need to be kept at + 4 °C while peptide chips can be kept at room temperature. Although we focused on TB and B. pertussis, our methodology has relevance for any disease or disorder where peptide arrays are used to assess immune response.     

Global analysis of protein function using protein microarrays

Protein microarrays containing thousands of proteins arrayed at high density can be prepared and probed for a wide variety of activities, thereby allowing the large scale analysis of many proteins simultaneously. In addition to identifying the activities of many previously uncharacterized proteins, protein microarrays can reveal new activities of well-characterized proteins, thus providing new insights about the functions of these proteins. Below, we describe the construction and use of protein microarrays and their applications using yeast as a model system.     

Acquiring genome-wide gene expression profiles in Guthrie card blood spots using microarrays

Standard Guthrie cards have been widely used to collect blood samples from essentially all USA and Japanese neonates for newborn screening programs. Thus, archival blood spot samples are a unique and comprehensive resource for molecular pathology studies. However, the challenge in using these samples is the presumed low quantity and degraded quality of nucleic acids that can be isolated from these samples, particularly the RNA. Here, we report a new assay using Agilent 4x44K microarrays for acquiring genome-wide gene expression profiles from blood spots on Guthrie cards. Due to the small amount of RNA obtained from each sample, major modifications, such as concentrating and amplifying the RNA and using a different labeling procedure, were performed. Approximately 9000 expressed genes can be detected after normalization of data, an increment of 260% in detection power compared with previously reported cDNA microarrays made in-house with standard procedures. The correlation coefficients in technical and biological replicates were 0.92 and 0.85, respectively, confirming the reproducibility of this study. This new and comprehensive assay will add value to the utility of archival Guthrie cards (e.g. neonatal blood spot cards) and open new opportunities to molecular epidemiology, pathology, genomic, and diagnostic studies of perinatal diseases.     

Algorithm of determination of circadian gene expression profiles analysed with DNA microarrays

DNA microarrays allow to simultaneously determine the expression level of thousands of genes. A nycthemeral study must enable to conclude which ones show a circadian rhythm. Two aspects prove this to be quite difficult: firstly, what does "circadian" exactly mean and how to quantify this qualification, and secondly which genes pertain to this definition. Our method, derived from linear optimisation procedures, consists in determining a cost function, depending from magnitudes characterising the notion of circadian rhythm. Given number of genes present on the microarray are known to be expressed rhythmically; their time series are considered as reference series. We have further constructed random series having the same temporal structure as the circadian gene series. We then carried out an optimisation procedure to determine the weighting coefficients in order to obtain a cost function value which orders the time series as follows: the reference series are in the first rows and the random series have low scores. We have tested this method on over 6000 genes expressed in mouse liver. We obtained a circadian gene detection probability of 100% with a false positive rate inferior to 1%.     

HLA antigen profiles in Thai tuberculosis patients

HLA antigens were studied in 35 Thai patients suffering from active pulmonary tuberculosis. An increase in the frequency of HLA-Bw46 and -DR4 and a decrease in the frequency of HLA-B12 were found when compared with the matched controls. These findings suggest that the pathogenetic role of HLA-B12 is to confer resistance and that HLA-Bw46 and -DR4 are associated with susceptibility. In addition it provided further information on HLA antigen profiles in pulmonary tuberculosis patients in another ethnic group (viz. Thais). Conclusions regarding genetic control over therapeutic efficacy must await further study.     

Effect of antibody affinity on the isotherm of antibody binding to surface-immobilized antigen

The binding of monoclonal antibodies to surface-adsorbed antigen was studied. Mouse IgG antibodies directed against dinitrophenyl groups (DNP) and O6-ethyl-2'-deoxyguanosine with known affinity for the antigen were used. The hapten was coupled to a protein, bovine serum albumin (BSA) or keyhole limpet hemocyanin, and adsorbed to polystyrene or silicone surfaces. Four different DNP-BSA epitope densities were used. Antibodies were incubated with the antigen-coated surface overnight. The bound antibodies were detected either optically by ellipsometry or by enzyme-conjugated anti-mouse IgG antibodies in the common ELISA technique. Absorbance values from ELISA measurements were transformed to surface density through calibration by ellipsometry. The experimental data showed that the binding of a high affinity antibody (Ka = 2.0 X 10(10] was diffusion rate limited after 24 h incubation time. Identical binding isotherms were found for high and low affinity clones of anti-DNP antibodies (Ka = 4.1 X 10(7) and 3.5 X 10(5] when antigen of high epitope density was used. At low epitope density the amount of bound low affinity antibodies decreased. Electron microscopy was used for studies of the distribution of colloidal gold-antibody complexes bound to surface-immobilized antigen. The results of the experiment showed that low affinity antibodies were bound in clusters whereas high affinity antibodies bound as single particles. These findings were related to the ELISA measurements. The results indicate that the binding isotherm of antibody to surface adsorbed antigen is not merely a reflection of the intrinsic antibody affinity measured in solution. Other macromolecular properties of antibodies, e.g., lateral intermolecular interactions and phase separation, affect the heterogeneous binding reaction.     

The influence of natural antibody specificity on antigen immunogenicity

The natural antibody repertoire in humans, apes and Old World primates is distinct from the repertoire of all other placental mammals, and encodes antibodies specific for the carbohydrate epitope Galalpha1-3Galbeta1-4GlcNAc-R (alphaGal). Here, we examined whether conjugating antigens to the alphaGal epitope can augment their immunogenicity in alpha(1,3)galactosyltransferase knockout mice (GT0 mice) which, like humans, produce alphaGal-specific antibodies. Immunization of GT0 mice with BSA conjugated to alphaGal (alphaGal-BSA) led to significant production of anti-BSA IgG antibodies without the need for adjuvant. This response was dependent on the presence of alphaGal-reactive antibodies. Immunization of wild-type mice with alphaGal-BSA failed to induce an anti-BSA response. The presence of alphaGal-reactive antibodies also led to an increase in the T cell response to BSA following immunization with alphaGal-BSA when compared with mice that received BSA alone, resulting in an increased frequency of IFN-gamma- and IL-4-producing BSA-specific T cells. In addition, the ability to produce alphaGal-reactive antibodies enhanced the cytotoxic T lymphocyte anti-viral antigen response following vaccination with murine leukemia virus transformed cell lines that express alphaGal on their cell surface. Natural antibodies that bind alphaGal therefore play a key role in increasing the efficiency of priming to antigens decorated with alphaGal epitopes.     

Human neutrophil antigen and antibody studies: a Taiwanese experience

Neutrophil alloantibodies are clinically known to cause neonatal neutropenia and transfusion-related lung injuries. Furthermore, neutrophil autoantibodies are associated with autoimmune neutropenia. This article is a review on neutrophil alloantigen in the Taiwanese population, and case investigations related to allo/auto neutrophil antibodies over a period of 20 years in the immunohaematology reference laboratory of Mackay Memorial Hospital, Taiwan. In Taiwanese populations, HNA-1a is more frequent than HNA-1b (90% and 50% respectively) and HNA-1c is not seen. Furthermore, the FcgRIIIb-deficient individual homozygous for FCGR3B gene deletion is estimated to be approximately 1 in 180 and is not as rare as previously thought. Although mothers homozygous for either HNA-1a or HNA-1b are commonly found in Taiwan, the number of patients with neonatal neutropenia caused by HNA-1 alloantibodies is seldom seen. In a search among our previous laboratory requests of neonatal cases for granulocyte antibodies testing over a 10-year period, only four cases showed neutrophil alloantibodies in the mother and baby’s sera. Lastly, primary autoimmune neutropenia among 51 children was retrospectively investigated. The study revealed that the specificity of the autoantibody was most commonly directed against HNA-1a. In addition, 31 cases were genotyped for HLA-DRB1 and -DQB1 genes. The HLA allele HLA-DQB1*05:03 was found to be associated with primary autoimmune neutropenia in Taiwan.     

Effects of circulating antigen on monoclonal antibody localization.

The potential effects of free circulating antigen on the ability of monoclonal antibodies to target tumors in vivo were investigated. Tumor models consisted of HCC, NuE and PLC cell lines producing AFP xenografted in nude mice, and the NuE-treated mouse designated as the NuE-bearing mouse injected with AFP prior to the administration of antibody. Immunoscintigraphy and biodistribution were evaluated by using 125I-labeled monoclonal antibody 19F12 raised against AFP. Gel chromatography analysis of plasma from the PLC-bearing mouse which excreted 400 ng AFP/ml in blood injected with 125I-19F12 indicated that all injected antibody 19F12 formed an immune complex in plasma. No immune complex was present in plasma from the NuE-bearing mice, where blood AFP levels were 7 ng/ml, while the intact antibody was found to remain partly in plasma from the NuE-treated mouse. Radioactivities in the whole body of NuE-bearing and NuE-treated mice eventually cleared at the same rate. Our experimental results indicated that the endogeneous circulating antigen retained the antibody in the whole body for a longer period. The ability of monoclonal antibodies to target tumors was influenced not only by how much antigen was present but also by how rapid the antigen was cleared in the blood.     

Chloramphenicol-specific antibody: III. Differential antibody decay rates to separate determinants of one antigen

In rabbits, peak titres to repeated immunization with chloramphenicol bound to bovine γ-globulin (CAP—BGG) appear on the 4th to 7th day for the BGG part of the antigen, but not until the 9th to 12th day for CAP. Similarly the antibody to CAP declines in titre much more rapidly than the antibody to the BGG and 4–6 weeks later anti-CAP antibody is no longer detectable while the anti-BGG antibody is still found in high titre.