Measurement Of Antibody Concentrations Research Articles

Antibody Concentration Measurement Using Optical Rotation: Toward Process Analytical Technology

The antibody manufacturing process consists of the cell culture process using animal cells and the purification process including several chromatography steps and filtration steps. The purification process is important to obtain high quality bio-pharmaceutical products by removing impurities, and measurement of antibody concentrations is a fundamental and essential factor to control the manufacturing process and to confirm the yield of each purification step. Here we introduce the optical rotation method as a resolution for measuring antibody concentrations in the manufacturing process. Feasibility evaluation showed that optical rotation is applicable for measuring a wide range of antibody concentrations even in cell culture supernatants containing various contaminants. In addition, as examples of application to the process analytical technology, we show the possibility of the optical rotation method in the continuous cell culture and chromatography process, and the tangential flow filtration concentration process.

Automated High-Throughput Flow Cytometry for High-Content Screening in Antibody Development

The tedious sample preparation for flow cytometry limits the throughput and thus its usage as a primary screening method despite its sensitivity and accuracy. With the growing focus on utilizing antibodies as a therapeutic modality in drug discovery, it is critical to develop a high-throughput flow cytometry (HTFC) workflow to cope with the increasing need to support antibody discovery programs. We have developed a seamless HTFC sample preparation and readout workflow using the HighRes modular robotic system and the IntelliCyt iQue Screener PLUS. To fully utilize the advantages offered by flow cytometry, we typically multiplex multiple cell lines of interest in one well to simultaneously quantitate on-target activity and nonspecific activity along with measurement of antibody concentration. The ability to measure multiple parameters coupled with speed and increased accuracy provides gains in productivity and helps speed up antibody lead discovery.

Ostertagia ostertagi antibodies in bulk tank milk from dairy cattle in Italy: A nation-wide survey.

The objective of this study was to investigate the exposure to Ostertagia ostertagi in dairy cattle herds across Italy through measurement of antibody concentration in the bulk tank milk (BTM) and to evaluate the possible effect of regions, seasons and pasture management practices on the level of exposure to the parasite. The O. ostertagi antibody levels in milk were determined using a commercial ELISA kit (SVANOVIR® O. ostertagi-Ab) and expressed as optical density ratio (ODR). From March 2015 to September 2017, BTM samples were collected in 1209 dairy cattle farms located in 15 regions of Northern, Central and Southern Italy. As reported in previous studies, BTM samples were classified in three different categories according to the level of exposure to O. ostertagi as follows: (i) positive with ODR values >0.60; (ii) negative with ODR values <0.30 and (iii) grey zone with ODR values between 0.30 and 0.60. Finally, differences in ODR values between regions, seasons and herd management practices were studied using the one-way analysis of variance. The overall mean ODR revealed a value of 0.49 in Italy; in particular, dairy cattle farms located in Southern Italy showed the highest O. ostertagi BTM ODR values than the other regions (p < 0.05). The ODR values ranged from 0.45 in autumn to 0.53 in spring and the seasonal difference was significant (p < 0.05). Furthermore, a positive association was found between the ODR values and the access to pasture; herds with access to pasture showed significantly higher titres (p < 0.0001) of O. ostertagi antibodies in milk (mean ODR = 0.63) than in those in total-confinement housing (mean ODR = 0.42). This study, the first of its kind in Italy, will provide a quantitative assessment exposure to O. ostertagi of Italian dairy herds and represents a significant step forward in evidence-based medicine for dairy veterinarians, advisors and farmers.

Presence of Ostertagia ostertagi antibodies in bulk tank milk from cattle herds in northern Spain

The objective of this survey was to investigate the Ostertagia ostertagi infection status of cattle dairy herds from northern Spain through measurement of antibody concentration in the bulk tank milk (BTM). In addition, management and performance data were collected to determine possible relationships with BTM O. ostertagi antibody levels. BTM samples were collected in 118 dairy cattle farms from five northern Spain Autonomic Communities in the autumn of 2009 and spring of 2010. The O. ostertagi antibody levels in milk were determined using a commercial ELISA kit (Svanovir®) and farm management information included type and farm size, production level, access to pasture, grazing regime and anthelmintic treatments. The overall mean optical density ratio (ODR) values of the two sampling times ranged from 0.60 (0.08–1.32) in autumn to 0.56 (0.05–1.24) in spring and no seasonal differences were detected. Herds with access to pasture showed significantly higher titers of O. ostertagi antibodies in milk (mean ODR=0.69–0.63) than in those without access (mean ODR=0.36–0.33). The association analyses showed significant correlations between the BTM O. ostertagi ODR and location (Navarra showed the highest ODR values), herd size (ODR decreased with increased herd size), milk production level (ODR negatively associated with milk yield), calves access to pasture (higher ODR when calves <12 months had access to pasture), outside access (no access low ODR, access high ODR) and grazing management (ODR increased proportionally to the percentage of time spent grazing each day). This study emphasizes that O. ostertagi-induced production losses should be considered on dairy farms in northern Spain. Additionally, putative risk-factors should be also considered for preventive measures.

Immune response to pneumococcal polysaccharides 4 and 14 in elderly and young adults. I Antibody concentrations, avidity and functional activity

Streptococcus pneumoniae is a serious worldwide pathogen and the focus of numerous vaccine development projects. Currently the most widely accepted surrogate marker for evaluating the efficacy of a given vaccine is to utilize ELISA. Measurement of antibody concentration by ELISA without reduction in cross-reactive antibodies causes an overestimation of antibody concentration and therefore protection, this is most notable in the aged, an at risk group for this infection. We compared the immune response to the pneumococcal polysaccharides (PPS) 4 and 14 of 20 young to 20 elderly adults. Pre-and post-vaccination IgG antibody concentrations and antibody avidity against PPS4 and PPS14 were measured using two different enzyme-linked immunosorbant assay (ELISA) absorption protocols. All sera were pre-absorbed with either cell-wall polysaccharide (CPS), or CPS and serotype 22F polysaccharide.Pre- and post-vaccination IgG antibody concentrations for serotype 4, but not 14, were significantly lowered with the additional absorption with serotype 22F polysaccharide in both age groups. Young and elderly demonstrated a significant increase from pre- to post-immunization antibody concentration, using either absorption method; and opsonophagocytic antibody titers in response to both PPS4 and PPS14. The correlation coefficients between ELISA and opsonophagocytic assays were improved by additional absorption with serotype 22F in response to serotype 4, but not serotype 14 in all age groups. Opsonophagocytic antibody titers in a sub-group of elderly (>77 years of age) were significantly lower than the opsonophagocytic antibody concentrations in young adults.These results suggest the importance of eliminating cross-reactive antibodies from ELISA measurements by absorption of serum and an age-related impairment in the antibody response to pneumococcal polysaccharides.

Valency or wählency: is the epitope diversity of the B-cell response regulated or chemically determined?

For almost a century, the humoral immune response has been monitored principally by the measurement of antibody concentrations, although antibody affinity and isotype have also long been acknowledged as critical to their biological activity. In this report, it is argued that these measures alone may provide a poor measure of the activity of serum antibodies. A B-cell response that is directed against multiple epitopes on a protein can form immune complexes bearing multiple antibody molecules. This is essential for the efficient initiation of processes such as the complement cascade and the activation of leucocytes via Fc receptors. These processes can be dramatically enhanced when B cells target a greater number of epitopes on any antigen. Evidence that the epitope diversity of an immune response may vary between individuals, and that it may vary in an individual over time, is reviewed. This variability is likely to be influenced by a number of host-specific factors in addition to antigen chemistry. The appropriateness of the chemically deterministic term 'antigen valency' to describe the number of epitopes recognized by an individual's B-cell response is discussed, and the term 'wählency' to emphasize the situational nature of B-cell epitopes is introduced.

Measurement of antibody concentrations by hemagglutination.

During every stage of development and production of diagnostic or therapeutic antibodies, it is necessary to have an assay to measure antibody concentration. Several techniques are routine in virtually all antibody laboratories. High-performance liquid chromatograpy (HPLC) using an affinity matrix (protein A or protein G) is rapid and quantitative and measures most types of antibody, though the equipment is quite costly. Enzyme-linked immunosorbent assay (ELISA) is widely used and extremely versatile. By judicious choice of anti-Ig reagents, specific for heavy chain, light chains, or particular domains, it is possible to screen for almost any desired Ig molecule or fragment. This is specially useful when analyzing a complex mixture (1). Native gel electrophoresis is a useful technique for screening relatively concentrated samples (e.g., from fermentors) since different antibodies can be readily distinguished by their characteristic mobilities. However, none of these methods are really suited to the rapid semiquantitative testing of huge numbers of samples that is often necessary early in a project (when screening for a rare hybridoma or transfectant, or for somatic mutants) and for routine analysis of process samples during cell culture. Instead we have found that red cell agglutination as originally developed by Coombs (2,3), is convenient, quick, and very cheap.

No influence of cadmium on the production of specific antibodies in mice

The in vivo effect of cadmium on specific antibody production of the immune system was investigated in mice. Female Balb/c mice were given 1, 10 or 100 ppm cadmium as cadmium chloride in the drinking water for 7, 14, 21, 28, 60 or 90 days. Immunization of the mice was carried out twice with a specific antigen (dinitrophenyl-bovine serum albumin) at an interval of 14 days. The antibodies (IgM and IgG) produced by this antigen were determined jointly with the aid of ELISA on days 0, 7, 14, and 21 after the first sensitization. The leucocyte count in the blood of the animals exposed to cadmium was unchanged. In spite of marked spleen and thymus atrophy in the 100-ppm group, the measurement of antibody concentrations in the blood, also, did, not show any significant differences from controls. Our results show that orally administered cadmium at the doses selected does not have any effect on the specific antibody production and thus on the most important function of the humoral immune system. The marked loss of weight of the thymus, however, does suggest there were effects on the cellular immune system.

Passage of host immunoglobulin across the mid-gut epithelium into the haemolymph of blood-fed buffalo flies Haematobia irritans exigua

Host immunoglobulin G was detected in the haemolymph of blood-fed buffalo flies. Immunoblot analysis of this haemolymph showed that the immunoglobulin was intact by comparison of its molecular weight to that of an affinity purified immunoglobulin G control. The concentrations of total immunoglobulin and anti-myoglobin antibody in the haemolymph were measured using enzyme linked immunosorbent assays (ELISAs). The concentration of total immunoglobulin in pooled haemolymph from flies which had ad libitum access to blood remained relatively constant over the sampling period (range: 2.1–7.3 μg/ml). Ninety-four per cent of flies known to have taken a blood meal had detectable levels of immunoglobulin in the haemolymph. Immunoglobulin could be detected in the haemolymph 24 h after flies had fed on a single blood meal. An ingested specific antbody (anti-myoglobin) which passed into the haemolymph retained the capacity to bind antigen. Measurement of antibody concentration in haemolymph is a valuable guide to the feasibility of targeting specific antigens in a vaccine against the buffalo fly.

Forssman Antigen and Antibody: Preparation of Water Soluble Antigen and Measurement of Antibody Concentration by Precipitin Analysis, by C′1a Fixation and by Hemolytic Activity

Summary Purified fractions of rabbit antisera containing 19 S Forssman antibody were analyzed for antibody content on a molecular basis by precipitin analysis, by C′1a fixation and transfer and by direct hemolysis with complement. We have found that the C′1a fixation and transfer test detects all antibody molecules which react with water soluble Forssman antigen, while direct hemolytic assay detects about one third of these molecules. Possible reasons for this paradox are discussed.