Isolation, Production, and Characterization of Serum Immunoglobulin M (IgM) of Indian Major Carp Cirrhinus mrigal (MRIGAL)

A method for the isolation of immunoglobulin M (IgM) in Indian major carp Cirrhinus mrigal (mrigal) serum to produce polyclonal antibodies is described in the present study. The purified immunoglobulins (IgM) were isolated from the serum of mrigal (Cirrhinus mrigal) by the bovine serum albumin (BSA)-CL affinity column purification method, and the IgM was used to produce a polyclonal rabbit anti-mrigal IgM antiserum. The IgM preparations were employed in the characterization of mrigal serum immunoglobulin. Reduced mrigal IgM on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was shown to consist of two subunits, compatible with heavy and light chains. A single heavy chain at approximately 90 kDa and variant of light chain 30 kDa were found. The dominant form of nonreduced IgM had a MW of approximately 900 kDa, suggesting a tetrameric structure based on estimated molecular weights, the relative protein content, and the reactivity with anti-mrigal IgM antisera, was obtained. The antisera were characterized as to specificity and reactivity by means of the enzyme linked immuno-sorbent assay (ELISA) and western blotting method. The information on the structure and character of immunoglobulin of fishes is essential in health management. The study described here investigates the possibility of using the serological techniques to assess the reactivity of antibody with the anti-mrigal IgM antisera.


Introduction
Fish immunoglobulins share various characteristics with mammalian immunoglobulin; however, some diferences exist; those partly arise from the process of evolution from common ancestors and partly refect the efect of fsh adaptation to a changed life environment. Te fsh immune system contains fve types of immunoglobulins: IgM [1], IgD [2][3][4], IgZ [5], IgT [6], and IgH [7]. Te major immunoglobulin of fsh blood serum is tetrameric IgM-like molecule [7] consisting of eight light and eight heavy chains. Te teleost fsh IgM shares some structural and functional characteristics with mammalian IgM; hence, we understand the ability of various teleost fshes to produce heterogeneous mixtures of IgM polymers, monomers [2,8,9], and halfmer subunits [10].
Wllson et al. [11] had reported severe abdominal dropsy in carps from Meghalaya. Parasitic diseases like myxosporidiosis and mixed infection with saprolegniasis and myxosporidiosis [12] have been accounted in IMC. Numerous occurrences of diseases epidemic of A. hydrophila have been reported. [13] Noncommunicable diseases like cataract, tail deformities (lordosis), cholelithiasis, bleeding in eyes, and gas bubble disease have also been reported in IMC. Meticulous knowledge of the molecular structure of the serum IgM heavy chain and the quantifcation of antibody response in cyprinids are an economically signifcant component of aquaculture in our country. Tis will be advantageous for further immunological studies in the feld of aqua culture and thereby its application in the feld of immunology and infammation. Majority of the carp's immunoglobulin bear tetrameric structures.
Tis study presents methods for processing and purifcation of Indian major carp Cirrhinus mrigal immunoglobulin and its reactivity with anti-mrigal IgM antisera. Te antisera were characterized by specifcity and reactivity by means of the enzyme linked immuno-sorbent assay (ELISA) and western blotting method.

Isolation of Fish Serum.
Five mrigal fshes weighing about 500 g each, maintained in a 25 m [2] cement cistern at the freshwater fsh farm of the College of Fisheries, Mangaluru, were injected intraperitoneally (ip) each with 1 mg bovine serum albumin (BSA, Merck, India) dissolved in 250 μl of phosphate bufered saline (PBS, pH 7.4) emulsifed with an equal volume of Freund's complete adjuvant (FCA). Similar doses of BSA emulsifed with an equal volume of Freund's incomplete adjuvant (FIA) were given 15 and 22 days later. Two mrigal fshes injected with PBS (pH 7.4) alone were maintained as control for the collection of unimmunized serum. On the 30th day, fshes were anaesthetized with 10 ppm benzocaine for 3 min followed by the collection of blood by puncturing the caudal vein, which was allowed to clot for 1 h at room temperature and stored overnight at 4°C. Serum was collected after centrifuging at 6000 rpm for 10 min and pooled and stored at −20°C for further use [2].

Purifcation of Serum.
Purifcation of serum was carried out by afnity column chromatography according to Gopalakrishnan et al. [13], using a 5 ml BSA-CL agarose column (Bangalore Genei, India). Te column was equilibrated by passing 5 column volume of PBS (pH 7.2). Two milliliters of serum from immunized fsh, mixed with an equal volume of PBS (pH 7.2), was fltered through a 0.45 μm syringe flter (MDI, India) and loaded onto the column. Te serum was allowed to interact with the BSA for 45 min. Unbound serum protein was washed out with 10 column volumes of elution bufer (0.1 M citric acid bufer, pH 3.0). Approximately, 10 fractions of 0.55 ml each were collected and neutralized with 0.45 ml of 0.5 M Tris bufer (pH11). Absorbance at 280 nm of each fraction was measured spectrophotometrically. Te eluted samples were pooled and subjected to dialysis at 4°C against PBS using a low molecular weight (12 kDa cut of) dialysis membrane (Sigma, USA). Te dialysis membrane was shifted to a tray containing sucrose to concentrate it to 2 ml, and the concentrated IgM was stored at −20°C for further use.

Characterization of Purifed IgM.
Purifed IgM was characterized by nonreducing SDS-PAGE, briefy 10 μl of the 4x native PAGE sample bufer (10% SDS, 0.5 M Tris-HCl, pH 6.8), glycerol 0.8 ml, and 0.01% bromophenol blue (W/ V) was added to 30 μl of the purifed IgM. Te purifed IgM along with a molecular weight marker (Sigma, USA) and native human IgM (Sigma, USA) were loaded onto the gel containing 3% acrylamide and 0.5% agarose and subjected to electrophoresis at 25 mA till the dye front reached the bottom of the gel which was strained with coomassie blue (10% acetic acid, 40% methanol, 0.1% coomassie blue) for 8 h and destained with destaining solution (10% acetic acid, 40% methanol), and the molecular weight of the IgM was estimated. Further characterization of the IgM was carried out by 10% SDS-PAGE according to Laemmli [14]. About 10 μl of the 4X sample bufer (10% SDS, 2 -β-mercapto ethanol. 0.4 ml, 0.5 M tris-HCl, pH 6.8), glycerol 0.8 ml, and 0.01% bromophenol blue (W/V) was added to 30 μl of the purifed IgM and boiled at 100°C for 5 mins. For comparison, crude serum from an immunized fsh was prepared in a similar way. Te samples along with molecular weight markers (Sigma, USA) were loaded onto 4.5% stacking gel and subjected to electrophoresis at 30 mA in 10% separating gel till the dye front reached the bottom of the gel. Te gel was stained with coomassie blue, and the molecular weight of heavy and light chain Ig were determined [2].

Production of Polyclonal Antibody.
A polyclonal antibody-based ELISA method was adapted for the detection of mrigal antibodies. Ten-week old Swiss albino mice were immunized (ip) with purifed mrigal IgM (50 μg in 250 μl PBS) mixed with 1 : 1 with FCA. A similar second dose (ip) was given with the purifed IgM in FIA (1 : 1) on the 15th day. On the 27th day, a booster dose of 10 μg purifed IgM in 0.05 ml PBS was given intravenously. Blood was withdrawn on the 30th day from the tail vein using a hypodermic syringe and collected blood was allowed to clot for 1 h at room temperature. Later, the serum was obtained by centrifuging the blood clot at 6000 rpm for a period of 10 mins, which was used as a polyclonal antibody.

Western Blotting.
Purifed mrigal IgM was solubilized in the sample bufer under reduced conditions. After separation in 10% polyacrylamide gel [16], the proteins were transferred onto the nitrocellulose membrane [14]. After drying, the nitrocellulose strips were incubated with polyclonal antibodies diluted by 1 : 10000. Te reaction was detected using rabbit anti-mouse IgG antibodies labeled with horse radish peroxidase.

Polyclonal Antibody (Pab) Based Immunodot Assay for the Study Reactivity of Mrigal
IgM. An immunodot assay was carried out by dotting BSA onto nitrocellulose (N.C.) papers cut into 02 cm 2 size. Purifed IgM was dotted on the paper as positive control and Aeromonas hydrophila and vibrio sp bacteria were dotted as a negative control, and three such dotted membranes were prepared separately to detect antibodies in the afnity purifed IgM sample, crude immunized, and unimmunized sera. Free sites on the nitrocellulose membrane were blocked with 05% skimmed milk powder in PBS for 02 hours and washed 03 times with PBS (pH 7.4), and one of the membranes was treated over night with 01 ml of purifed IgM and at a concentration of 0.1 mg/ml, and the other with crude immunized fsh serum diluted to 10 times with PBS (01 ml). Te third membrane was treated overnight with unimmunized fsh serum diluted to 10 times with PBS (01 ml). After washing with PBS Tween 20, rabbit anti-mouse IgG horse radish peroxidase (Bangalore genie, India) in 3% BSA in PBS (1 ml, 1 : 1000 dilution) was added and incubated for 90 minutes. After washing with PBS Tween 20 three times, 1 ml of substrate (0.3 mg of 4 chloro-1-naphthol in 10 μl ethanol, 01 μl of 30% H 2 O 2 , and 01 ml of Tris bufer (pH 7.6)) was added, and clear purple dot development on the nitrocellulose paper was considered as positive reaction.

Results
Fish serum was obtained after immunization with bovine serum albumin (BSA), the sera obtained was purifed by afnity column chromatography method eluted fractions and characterized by poly acrylamide gel electrophoresis (PAGE) under reducing and nonreducing conditions (Figures 1 and 2). Polyacrylamide gel electrophoresis (PAGE) under reduced condition revealed that the immunoglobulin contained heavy and light chains with molecular weights of 90 and 30 kDa, respectively, and under nonreducing conditions in a 3% gel revealed a single protein having a molecular weight of 900 kDa. Te method was further employed for production of polyclonal antibodies against anti-mrigal serum in the mouse, and thus, polyclonal antibodies against mrigal anti serum was obtained. Te sensitivity and the titer were determined using ELISA. Te antibody titer was found to be 1 : 12400. Te antibody sensitivity is signifcant in purifed mrigal IgM while compared with crude mrigal antiserum, unimmunized negative control serum did not react. In dot immuno blot assay also, mrigal polyclonal antibodies reacted signifcantly with the pure mrigal IgM while compared to crude mrigal antiserum, and unimmunized negative control sera did not react. Similarly, in western blot analysis of mrigal IgM, two distinctive bands showing heavy and light chain was observed, the molecular weight of heavy chain was 90 kDa and light chain was 30 kD, which reacted signifcantly towards pure mrigal IgM and crude mrigal antiserum, and unimmunized negative control sera had not reacted (Figures 3-5).

Discussion
In the present study, we tried to purify the immunoglobulin of mrigal by a single step afnity purifcation by the BSAlinked column, which was suitable to get adequate amount of immunoglobulin (Ig); moreover, the same column was found to be useful to purify more than once without reduction in the binding capacity of the antibody adapting established immunization protocol and the purifcation technique, and an average yield of immunoglobulin (Ig) was 1.25 mg/ml of serum. Ig levels in diferent fshes after immunization vary within the range of 0.25-23.5 mg/ml [11].
Te immuno dot test clearly showed specifc reaction of the antibodies in the afnity-purifed IgM and crude antisera with BSA. In the NC membrane treated with afnity-purifed IgM ( Figure 5) and the membrane treated with crude immunized serum, the BSA spots were clearly stained indicating the specifc reactivity of immunized crude antiserum and purifed IgM. Te membrane treated with unimmunized mrigal serum did not develop purple blue dot In the present study, only one form of IgM-like molecule was eluted in the afnity purifcation. In the earlier studies on Cirrhinus mrigal (mrigal), Catla catla (Catla), and Lobeo rohita (rohu) with nonreducing SDS-PAGE, diferent populations of Ig molecules such as tetrameric and dimeric ano-monomeric forms have been reported, but we have observed a single band of protein in nonreducing SDS-PAGE indicating that protein is pure and was eluted in its native form. Te molecular weight of C. mrigal IgM was 900 kDa in nonreducing SDS-PAGE, which is similar to the molecular weight of IgM of the fshes such as Turbot (835 kDa African catfsh 840 kDa Cod 851 kDa).
SDS-PAGE analysis revealed two clear bands of polypeptides having molecular weights of 90 and 30 kDa which are considered as the heavy and light chains of the purifed IgM molecule, respectively. Te immunized crude serum also shows the corresponding heavy and light chain bands. Te molecular weights of heavy and light chains can be compared with those from other species of fshes like Tilapia   [16]. Te present paper describes the attempts made and strategy employed to obtain specifc polyclonal antibody against mrigal antiserum to develop a strategy to obtain a specifc polyclonal antibody against mrigal antiserum and to develop a sensitive, rapid, and specifc ELISA to employ in routine screening in disease management. In the present study, BSA antigens were selected to raise the antibodies in mrigal because of the accessibility of afnity columns for antibody purifcation. BSA has been used as an immunogen for elucidating antibody production in diferent fshes by many investigators [16][17][18].
Eluted fractions of immunoglobulin were analyzed afterwards by the antiserum which was found extremely immunogenic as assessed by the titration of immune sera. Te ELISA developed was shown to be very sensitive with titer of 1 : 12400. Te antibody reacted more strongly with pure mrigal IgM than with crude mrigal sera as ELISA, immmunodot, and western blot has confrmed the results [13,19].

Data Availability
Te data used to support the study are included in the paper.

Conflicts of Interest
Te authors declare that they have no conficts of interest. Advances in Pharmacological and Pharmaceutical Sciences 5