Purification Of Specific Antibodies Research Articles

Development of the ELISA Test System for Quantitative Determination of IgG to the Varizella zoster virus in Human Serum and Assessment of its Diagnostic Efficiency

Relevance. The introduction of Varicella vaccine prophylaxis explains the need to develop a methodology for monitoring the vaccination effectiveness and the intensity of population immunity. This problem can be solved using quantitative immunoassay methods. Aim. Development of an enzyme-linked immunosorbent assay for the concentration of class G immunoglobulins (AB) to Varicella zoster virus (VZV) determining and assessing its functional characteristics and diagnostic efficiency. Materials and methods. Recombinant antigen GE VZV. WHO International Standard for Antibodies to VZV W1044. Blood serum samples from healthy people and patients with Chickenpox and Herpes zoster, blood serum samples containing IgG antibodies to herpes simplex viruses of the first and second types, cytomegalovirus, Epstein-Barr virus. Anti-VZV ELISA (IgG) reagent kit (Euroimmun, Germany). Indirect enzyme-linked immunosorbent assay. Immunization of animals with recombinant antigen GE, isolation, and purification of specific antibodies. Conjugation of monoclonal antibodies to human IgG with antibodies to antigen GE and with horseradish peroxidase. Results. An enzyme-linked immunosorbent assay in «an indirect» format has been developed to determine the specific antibodies to VZV concentration (IU/ml) in human serum/plasma. An artificial calibrator for determining the concentration of AB-VZV had been synthesized and standardized according to the International WHO-standard W1044. The main functional characteristics of the developed enzyme-linked immunosorbent assay are determined in accordance with GOST 51352-2013. The diagnostic kit was tested on blood serum samples from children with chickenpox (n = 43), adults with Herpes zoster (n = 158), healthy individuals (n = 781). The diagnostic sensitivity of the test system was 85%, the diagnostic specificity was 87% according to the ROC analysis. The absence of cross-reactivity of the test system was shown on samples with serological markers of other herpesvirus infections (n = 94). Comparative trials of the developed test system and its commercial analog, the Anti-VZV ELISA (IgG) reagent kit, did not reveal statistically significant differences between their functional characteristics. Conclusions. The developed test system for determining of the AB-VZV concentration in human serum/plasma in terms of its functional characteristics meets the GOST requirements, is characterized by high diagnostic efficiency, can be used to monitor the effectiveness of vaccine prophylaxis and strength of population immunity, as well as to assess the immune response in chickenpox and Herpes zoster.

Immunohistochemical Localization of Proteins in the Phloem: Problems and Solutions.

Immunolocalization of proteins in differentiated phloem cells is a challenging task given their special anatomy, organellar infrastructure, and the phloem tissue's heterogeneity. Incorporation of specific wall components in the thickened cell walls of phloem cells is often the source of unspecific labeling, leading to erroneous localization. Therefore, special care is required regarding generation and purification of specific antibodies. In addition, tissue preservation of phloem cells, which contain a high osmotic pressure in their functional state, is a very challenging task prone to various pitfalls. This chapter provides practical advice for cautious tissue preparation and antibody purification. Furthermore, methods that can be used to verify immunohistochemical localization data, such as promoter-reporter studies or activity tests, are discussed. Such confirmation experiments are essential for unambiguous determination of protein location in cells of the phloem.

Selection of antibody probes to correlate protein sequence domains with their structural distribution.

We propose a new approach that permits correlation of specific domains defined by their primary sequence with their location in the structure of complex macromolecular aggregates. It is based on the combination of well-established structural analysis methods that incorporate the use of overlapping peptides on cellulose membranes for the isolation and purification of specific antibodies from a polyclonal antiserum. Monospecific antibodies to the connector protein of bacteriophage phi29 were isolated from polyclonal antisera using a new development of the spotscan method. These antibodies can be purified in quantities that allow antigenicity testing in enzyme-linked immunosorbent assays, Western blotting and immunoprecipitations, demonstrating the specificity of this isolation procedure. This approach has allowed us to generate direct antibody probes for immunoelectron microscopy mapping of different connector protein domains in a low resolution three-dimensional epitope map.

Purification of specific antibody against aspartate and immunocytochemical localization of aspartergic neurons in the rat brain

The distribution of l-aspartate known as a putative excitatory neurotransmitter in the central nervous system was investigated immunocytochemically in the rat brain. Anti-aspartate antiserum was raised in rabbits using l-aspartate covalently conjugated to rabbit serum albumin with glutaraldehyde as the immunogen and was found to be cross-reactive with an l-glutamate conjugate. Monospecific anti- l-aspartate antibody was successfully purified using affinity gels coupled with several amino acids including l-aspartate and l-glutamate and with the l-glutamate conjugate. Putative aspartergic neurons were generally immunoreactive to the purified antibody, but epithelia of the choroid plexus were also stained. These results show that the antibody is a useful tool for the immunocytochemical demonstration of possible aspartergic neurons in the central nervous system, although the immunochemical expression of l-aspartate not used as a neurotransmitter must be taken into consideration.

Letter: Acrylamide affinity chromatography for immunohistochemistry. Purification of specific antibodies.

Free accessAbstractFirst published February 1975Letter: Acrylamide affinity chromatography for immunohistochemistry. Purification of specific antibodies.A Martinez-Hernandez, D A Merrill, […], M A Naughton, and C Geczy+1-1View all authors and affiliationsVolume 23, Issue 2https://doi.org/10.1177/23.2.46878

The purification of specific antibody as F(ab′)2 by the pepsin digestion of antigen-antibody precipitates, and its application to immunoglobulin and complement antigens

The purification of specific antibody as F(ab′)2 by the pepsin digestion of antigen-antibody precipitates, and its application to immunoglobulin and complement antigens

The purification of specific antibody as F(ab′)2 by the pepsin digestion of antigen-antibody precipitates, and its application to immunoglobulin and complement antigens

Pepsin treatment of antigen antibody precipitates has been shown to be an efficient way to obtain purified antibody as F(ab′)2 to pepsin labile protein antigens. A method of so doing is described which has been used to make class specific (and sub-class specific) anti-immune globulin antibody and antibodies to some complement components.