Antibody purification: ammonium sulfate fractionation or gel filtration.
ABSTRACT Antibodies can be purified by a variety of methods based on their unique physical and chemical properties such as size, solubility, charge, hydrophobicity and binding affinity. This chapter focuses on ammonium sulfate precipitation as a convenient first step in antibody purification in that, it allows the concentration of the starting material and the precipitation of the desired protein. The principle of ammonium sulfate precipitation lies in "salting out" proteins from the solution. The proteins are prevented to form hydrogen bonds with water and the salt facilitates their interaction with each other forming aggregates that afterward precipitate out of solution. Gel filtration or size- exclusion chromatography is also discussed in this chapter. Gel filtration is based on the relative size of protein molecules and it is of great value to separate IgMs, exchange buffers and/or desalt solutions. The columns designed to separate the proteins are composed of porous beads and the proteins will flow through the packed column inside and around the beads, depending on its size.
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ABSTRACT: The first milk, colostrum, is an important source of nutrients and an exclusive source of immunoglobulins (Ig), essential for the growth and protection from infection of newborn pigs. Colostrum intake has also been shown to affect the vitality and behaviour of neonatal pigs. The objective of this study was to evaluate the effects of feeding colostrum and plasma immunoglobulin on brain development in neonatal pigs. Positive correlations were found between growth, levels of total protein and IgG in blood plasma and hippocampus development in sow-reared piglets during the first 3 postnatal days. In piglets fed an elemental diet (ED) for 24h, a reduced body weight, a lower plasma protein level and a decreased level of astrocyte specific protein in the hippocampus was observed, as compared to those that were sow-reared. The latter was coincident with a reduced microgliogenesis and an essentially diminished number of neurons in the CA1 area of the hippocampus after 72h. Supplementation of the ED with purified plasma Ig, improved the gliogenesis and supported the trophic and immune status of the hippocampus. The data obtained indicate that the development of the hippocampus structure is improved by colostrum or an Ig-supplemented elemental diet in order to stimulate brain protein synthesis and its development during the early postnatal period.International journal of developmental neuroscience: the official journal of the International Society for Developmental Neuroscience 03/2014; DOI:10.1016/j.ijdevneu.2014.03.003 · 2.92 Impact Factor
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ABSTRACT: Background & objectives: Despite major control efforts, malaria remains a major public health problem that still causes high mortality rate worldwide especially in Africa and Asia. Accurate and confirmatory diagnosis before treatment initiation is the only way to control the disease. The present study was undertaken to develop reagents using sandwich ELISA for simultaneous detection of PfHRP2 (Plasmodium falciparum histidine rich protein) and PfLDH (P. falciparum lactate dehydrogenase) antigens in the proven malaria cases. Methods: The antibodies were raised against two epitopes of PfHRP2 protein and three unique and unexplored epitopes of PfLDH protein. These antibodies were able to detect PfHRP2 and PfLDH antigens in culture supernatant and parasitized RBC lysate of P. falciparum, respectively up to 50 parasites/μl. The in-house reagents were tested in 200 P. falciparum positive patients residing in Baghpat district of Uttar Pradesh in northern India. Results: Microsphere (PLGA) with CpG ODN were used to generate high titre and high affinity antibodies against selected peptides of PfHRP-2 and pLDH antigen in mice and rabbit. The peptide specific peak titre varied from 12,800 - 102,400 with an affinity ranging 0.73 - 3.0 mM. The indigenously developed reagents are able to detect PfHRP2 and PfLDH antigens as low as 75 parasites/μl of blood with a very high sensitivity (96-100%) and specificity (100%). Interpretation & conclusions: The study highlight the identification of unique epitopes of PfHRP2 and PfLDH, and the generated antibodies against these antigens were used for quantitative estimation of these two antigens using sandwich ELISA. No corresreactivity with P. vivax infected patients was observed with the sera.The Indian Journal of Medical Research 12/2013; 138(6):977-87. · 1.66 Impact Factor
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ABSTRACT: Gelatin from bighead carp (Hypophthalmichthys nobilis) scale was extracted with the gel strength of 415.7 ± 9.9 g. Ammonium sulfate fractional precipitation (ASFP) with different ammonium sulfate saturations (20%, 25%, 30%, 35% and 40%, respectively) separated fish gelatin components with different gel strengths (469.7 ± 12.0 g, 419.7 ± 9.9 g, 181.9 ± 17.7 g, 83.9 ± 9.9 g and 15.3 ± 2.1 g, respectively). ASFP did not destroy characteristic FTIR peaks (amide A, amide I, amide II and amide III) and X-ray diffraction peaks (around 7° and 20–21° represented the triple-helix structure and the single left-hand helix chain, respectively) in the fish gelatin. In addition, lower ammonium sulfate saturation precipitated the fish gelatin component with stronger hydrogen bond, more triple-helix structures, higher molecular weight distribution and denser microstructure. Prominently, fish gelatin with higher gel strength had greater amount of high molecular weight components and denser microstructure.Food Hydrocolloids 05/2014; 36:173–180. DOI:10.1016/j.foodhyd.2013.09.024 · 4.28 Impact Factor