Research Journal of Biology Volume 1: 36 - 39 (Published: 08 August, 2013) ISSN: 2322-0066 36 SHORT COMMUNICATION Isolation and characterization of IgM from Bengal goat blood serum Atanu Koner * , Pallavi S. Rajput, Rajat Dhyani, Nikki Nidhi and Kuljeet Kaur Department of Biotechnology, The University of Burdwan, Burdwan-713104, West Bengal, India *Corresponding author’s E-mail: atanukoner@gmail.com ABSTRACT Isolation of IgM from Bengal goat blood serum was carried out by centrifugation of the collected serum to eliminate the blood corpuscles and purity of serum was affirmed by the absence of pellets. Purified serum was obtained by ammonium sulphate precipitation. The isolated IgM obtained through dialysis, was quantified through silica gel chromatography using phosphate buffer saline (PBS) as a solvent with varied pH and obtained different fractions (namely I, II, III, IV and V). Quantification of protein was carried out by Lowry method and the molecular weight was determined by SDS-PAGE with a standard marker. The presence of IgM was confirmed by Immunodiffusion and Immuno Dot Blot. The results of the experiment suggest that Fractions I, II and III contain more stressed protein which has some similarity with ovalbumin. The resulting colour intensity obtained on performing Immuno Dot Blot using IgM as primary antibody, demonstrates that Fraction II contains maximum concentration of stressed protein. Key Words: Blood serum, IgM, immuno dot blot, immunodiffusion, SDS-PAGE. (Received: 18/07/2013; Accepted: 04/08/2013; Published: 08/08/2013) INTRODUCTION Immunoglobulin M (IgM) is a major class of immunoglobulin. It is the first antibody produced in an immune response and is later replaced by other types of antibodies. IgM is produced by B cells and forms polymers where multiple immunoglobulins are covalently linked together with disulfide bonds, mostly as a pentamer but sometimes also as a hexamer (Cambier et al., 1974.). IgM has a molecular mass of approximately 900 kDa (in its pentamer form). Because each monomer has two antigen binding sites, a pentameric IgM has 10 binding sites. Typically, however, IgM cannot bind 10 antigens at the same time because the large size of most antigens hinders binding to nearby sites. The J chain is found in pentameric IgM but not in the hexameric form. At present, it is still uncertain whether a J chain-containing pentamer contains one or more than one J chain. Because IgM is a large molecule, it cannot diffuse well, and is found in the interstitium only in very low quantities. IgM is primarily found in serum; however, because of the J chain, it is also important as a secretory immunoglobulin. Due to its polymeric nature, IgM possesses high avidity, and is particularly effective at complement activation. By itself, IgM is an ineffective opsonin; however it contributes greatly to opsonization by activating complement and causing C3b to bind to the antigen (Ohta et al., 1990). IgM antibodies appear early in the course of an infection and usually reappear, to a lesser extent, after further exposure. These two biological properties of IgM make it useful in the diagnosis of infectious diseases. Demonstrating IgM antibodies in a patient's serum indicates recent infection, or in a neonate's serum indicates intrauterine infection (e.g. congenital rubella). IgM in normal serum is often found to bind to specific antigens, even in the absence of prior immunization. For this reason IgM has sometimes been called a "natural antibody". This phenomenon is probably due to the high avidity of IgM that allow it to bind detectably even to weakly cross-reacting antigens that are naturally occurring. For example the IgM antibodies that bind to the red blood cell A and B antigens might be formed in early life as a result of exposure to A- and B-like substances that are present on bacteria or perhaps also on plant materials (Richman et al., 1982). IgM antibodies are mainly responsible for the clumping (agglutination) of red blood cells if the recipient of a blood transfusion receives blood that is not compatible with their blood type. IgM is more sensitive to denaturation by 2- mercaptoethanol than IgG. This technique was historically used to distinguish between these isotypes before specific anti- IgG and anti-IgM secondary antibodies for immunoassays became commercially available. Serum samples would be tested for reactivity with an antigen before or after 2-mercaptoethanol treatment to determine whether the activity was due to IgM or IgG (Paster et al., 1989). MATERIALS AND METHODS Isolation and characterization of IgM antibodies from Bengal goat serum: The blood sample of Bengal Goat was collected and was kept in room temperature (upto 37 ° C) for clotting. The collected serum was flocked together in sterile vials and stored at -20 ° C.Latera pool of serum was prepared and centrifuged at 5000 rpm for 5 minutes to remove residual RBC and the collected supernatant was stored at -20 ° C. Ammonium sulfate precipitation: Immunoglobulin was saturated to 50% by adding 3.13g ammonium sulphate to 10ml serum and constantly stirred for 2 h. The serum was then centrifuged at 10000 rpm for 15 mins and the pellet was dissolved in phosphate buffer saline (PBS, pH 7.0). After addition of PBS, again and kept for 2 h, it was centrifuged in the same way and the collected pellet was dissolved it in PBS. Dialysis: The ammonium sulphate precipitated IgM was taken in a dialysis bag and dialysed against several changes of PBS for 2 days at 25 ° C to obtain crude IgM and ammonium sulphate.