Liver tissue was collected from rohu (Labeo rohita) and kept in deep freezer (−20˚C) till used. Isolation of fish leptin was carried out as per Peinado et al. [6] . In brief, the rohu liver (1 gm) collected was thawed in 0.4 ml of cold Urea/thiourea buffer (7 M urea, 2 M thiourea, 4% CHAPS, 45 mM Tris, pH 7.4, 60 mM DTT) and complete protease inhibitors (one tablet/20 ml, Roche, Barcelona, Spain) supplemented with 0.1 mM NaCl. Hepatocytes were mechanically disrupted and briefly sonicated (Hielscher Ultrasonics GmBH, Germany). Samples were adjusted to 900 μl with lysis buffer (20 mM Tris, pH 7.4; 100 mM NaCl; 1% Triton and protease inhibitors) and incubated for 15 min at 35˚C. After cooling on ice (10 min), 100 μl of 0.1 M Tris, pH 7, and 50 mM MgCl₂ were added to the homogenate, which were then incubated with DNase-1 (30 U Sigma) on ice (10 min). The homogenate was centrifuged for 15 min at 10,000 g (4˚C) and the aqueous phase between the upper lipid phase and the lower cellular debris phase was collected. Finally, the extract was separated by chloroform/methanol precipitation and dissolved in phosphate buffer solution (pH-7.4). The total protein concentration was determined using Lowry’s method [7] .

Isoelectric focusing and 2D-PAGE of proteins obtained from chemically treated fish liver tissue was performed as per Peinado et al. [6] . Hepatocyte protein was treated with 0.2 ml rehydration buffer (8 M urea, 2% CHAPS, 50 mM DTT, 0.2% ampholytes pH 3 - 10 and 0.002% bromophenol blue). Electrophoresis was carried out following the manufacturer’s protocol (Bio-Rad Laboratories, USA). Briefly, non linear pH 3 - 10 IPG strip was rehydrated with 0.25 ml rehydration buffer containing 0.05 ml sample overnight at 25˚C and then subjected to isoelectric focusing. The strip was then equilibrated with reducing buffer (6 M urea, 2% SDS, 30% glycerol, 50 mM Tris-HCl and 25 mM DTT) followed by alkylation buffer (6 M urea, 2% SDS, 30% glycerol, 50 mM Tris- HCl and 135 mM IAA). The strip was then placed on 12.5% SDS polyacrylamide gel using 1% agarose in SDS- PAGE running buffer. After electrophoresis gels were stained by SYPRO Rubi dye (Bio Rad Laboratories, USA). The stained gel was washed with washing solution (10% methanol, 7% glacial acetic acid) and the final washing was done with Milli-Q water for 10 min according to manufacturer’s instructions. Gel was scanned with the help of Fuji scanner in the fluorescence mode to visualize the stained gel. The spot volume was measured and reported as percent volume of the spot (normalized volume of spots, % volume) in relation to the sum of all detected spot and this provided normalized spot volume. The change in % volume is the basis of detection of differential protein identification.

Leptin being glycoprotein, purification of native rohu leptin was attempted using affinity chromatography kit (Genei, India) having Concavalin-A (Con-A) attached with carbon background (cellulose). In the first step, Con- A agarose column was prepared in sodium acetate buffer as per manufacture’s instruction. Then the sample protein was charged on the column and the unbound fraction was eluted with sodium acetate buffer. In next step, bound fraction was eluted with the help of elution buffer. The fractionated proteins (Con-A bound) were pooled and dialyzed against distilled water at 4˚C. The collected protein pool was concentrated by sucrose, sterilized through a membrane filter (0.22 μ), and stored at −20˚C. The protein concentration of the obtained fractionated protein was estimated by Lowry’s method [7] .

Crude and affinity fractionated rohu hepatocyte proteins were analyzed by sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE) as per Laemmli [8] using 12.5% polyacrylamide gel in a vertical slab gel electrophoretic apparatus (Attao, Japan). The samples were mixed with sample buffer in a proportion of 1:1 and subsequently the solution was heated at 100˚C for 3 min. The amount of protein applied was 50 μg per track. Proteins were run at 18 mA for 150 min. The bands were visualized by staining with monochromatic silver staining [9] . Standard medium range molecular weight marker (PMW-M, Genei, India) was run parallel along with sample proteins to determine the molecular mass of the polypeptides.

Hyperimmune serum, against the affinity fractionated rohu hepatocyte protein, was raised in rabbit as per Mishra et al. [10] with some modifications that encompass increased doses. Briefly, two New Zealand White (NZW) male rabbits weighing 1200 g were injected intramuscularly with 5 doses of affinity purified antigen, mixed with equal volume of Freund’s adjuvant (Sigma, USA) at 10 days interval with increased subsequent doses ranging from 600 μg to 1200 μg per injection. First dose was given with Freund’s complete adjuvant (FCA) and subsequent 4 doses with Freund’s incomplete adjuvant (IFA). One rabbit of same breed, sex and weight was also maintained without immunisation to collect normal serum.

The seroreactivity of affinity fractionated hepatocyte protein and titre of the antiserum were assessed by AGPT as per Ouchterlony [11] .

Affinity fractionated leptin protein was separated on a 12.5% gel (SDS-PAGE), and electro transferred onto nitrocellulose membrane using a semi dry blotting unit (Atto, Japan) as per Towbin et al. [12] . The blotted proteins were subjected to immunoblot analysis using hyper immune serum raised in rabbit (polyclonal antibodies) and anti-rabbit horse radish peroxidase (HRPO) immunoconjugate (Genei, India). Standard molecular weight marker (Fermentas) was run parallel along with sample proteins to determine the relative molecular weights of the polypeptides.

The molecular mass of affinity fractionated rohu hepatocyte protein was determined by Voyager DE Pro^TM mass spectrometer equipped with 337 nm N₂ laser (Applied Biosystems, USA) as per Mandal et al. [13] . The sample protein (2 μl) was mixed with MALDI matrix, synapinic acid (8 μl), then 2 μl of mixture solution was spotted onto the MALDI 100 well stainless steel sample plate and allowed to air dry (for 5 hr) prior to the MALDI analysis. The spectra were recorded in the positive ion linear mode in accelerating voltage 20 kV. Peptide mono- isotopic mass was obtained in linear mode with external calibration. It was performed using calibration mixture 1 (Applied Biosystems, USA) having Arg1-Bradykinin (m/z, 904.468), angiotensin I (m/z, 1296.685), Glu1-fi- brinopeptide B (m/z, 1570.677) and ACTH (18 - 39) (m/z, 2465.199). Reproducibility of the spectrum was checked 5 times from separately spotted samples.

Dip-stick ELISA was performed with the affinity fractionated protein following the procedure of Jiahao et al. [14] with some modifications in substrate buffer composition. Coating of two sticks was done by affinity fractionated protein @ 2 μg/srip. After blocking and subsequent washing of the sticks, hyperimmune and normal serum were added in the sticks, respectively. In the next step, after proper washing, anti-rabbit HRPO conjugate (Genei, India) was added to the sticks. The colour development step was carried out by dipping the sticks into substrate solution (40 ml H₂O₂, 0.025 gm diaminobenzydine in 10 ml Tris HCl, pH 7.5).

Using known concentration of affinity fractionated protein, a plate ELISA was standardized based on sandwich ELISA principle. Blood was collected from apparently healthy rohu (n = 24), serum separated and used as unknown sample for detection of leptin concentration using the standardized protocol developed. The concentration (ng/ml) of each unknown sample was extrapolated by putting the O.D. values in the standard graph prepared from the known concentrations (5 ng/ml through 160 ng/ml).

The concentration of the crude protein from hepatocyte tissue of rohu was observed as 6.2 mg/ml and affinity fractionated protein was found to be 0.15 mg/ml

Two Dimensional Gel electrophoresis of crude hepatocyte protein after processing showed several proteins in the range of molecular weight 3 - 97.4 kDa. The proteins were mainly present in 4 - 8 pI range. In the sample, proteins of low and high molecular weight were less abundant. Proteins were mainly visible in the range of 6 - 70 kDa range (Figure 1).

When the crude protein obtained from liver tissue was subjected to SDS-PAGE analysis, 25 (twenty-five) polypeptides in the molecular weight range 3 to 130 kDa were obtained. The affinity bound fraction yielded only four polypeptides of which one (16 kDa) was major and three (4.7 and 54 kDa) were minor (Figure 2).

The specificity between fractionated protein and hyperimmune serum was indicated by clear band between the wells. The titre of the hyperimmune serum was found as 4. (Result not shown)

When western blot analysis was performed with the affinity fractionated protein using polyclonal antibodies, a band corresponding to 16 kDa was observed indicating the presence of 16 kDa leptin protein in the affinity fractionated sample (Figure 3).

The affinity fractionated protein when subjected to MALDI-TOF mass spectrometry to determine the molecular mass more precisely, one of the constituent proteins revealed as 16283.38 Da (Figure 4).

Affinity fractionated rohu hepatocyte protein was further used in dip-stick ELISA to ascertain its sero-diagnostic potentiality. The difference in intensity of developed colour between hyperimmune and normal serum is shown in Figure 5. Out of the two dip-sticks used, the first stick showed strong seroreactivity.

The O.D. values of unknown serum samples ranged from 0.672 to 0.903. When the individual values (n = 24) were used for determining leptin concentration from the standard graph (Figure 6), the concentration varied between 38 to 72 ng/ml.