• Cellular Ratio for Hybridoma Fusion: In the classical Köhler and Milstein hybridoma technology, somatic cell fusion mediated by polyethylene glycol (PEG) utilizes an optimized ratio of 1 immune spleen lymphocyte to 2 myeloma cells (1:2) (or routinely between $1:1$ and $1:2$).
• Methodological Rationale: Maintaining a high proportion of HGPRT-deficient myeloma partner cells (e.g., Sp2/0-Ag14 or NS0) maximizes fusion collisions with antigen-primed splenic B lymphocytes, producing stable proliferating hybrid cells capable of continuous growth in Hypoxanthine-Aminopterin-Thymidine (HAT) selection medium.

Correct option: (2) 1:2

• Role of Formamide in Molecular Hybridization: Formamide ($\text{HCONH}_2$) is an organic denaturing solvent routinely added (usually at $30\text{--}50\%$) to hybridization buffers in Southern blotting, Northern blotting, and in situ hybridization (ISH).
• Thermodynamic Effect: Formamide destabilizes the hydrogen bonding between complementary base pairs, lowering the duplex melting temperature ($T_m$) predictably by approximately $0.6^\circ\text{C}$ to $0.7^\circ\text{C}$ for every $1\%$ of formamide added.
• Experimental Benefit: This enables high-stringency DNA-DNA or DNA-RNA probe hybridization at physiological temperatures ($42^\circ\text{C}$ instead of $65\text{--}70^\circ\text{C}$), protecting nylon/nitrocellulose membranes from thermal degradation.

Correct option: (4) Formamide

• Bifunctional / Bispecific Antibodies: Monoclonal antibody derivatives engineered to display two distinct antigen-binding fragment (Fab) arms with different paratopes—enabling simultaneous binding to two separate, unique antigens or epitopes—are called bifunctional (bispecific) antibodies.
• Production Method: Generated through the fusion of two distinct hybridoma cell lines (creating a quadroma or hybrid-hybridoma) or via recombinant antibody engineering (e.g., BiTEs, Dual-Variable Domain Ig).
• Contrasting Antibody Types:
• Chimeric Antibodies: Engineered antibodies possessing rodent antigen-binding variable domains fused to human/bovine constant domain backbones.
• Biphasic Antibodies: Classical clinical antibodies displaying temperature-dependent phase activities (e.g., Donath-Landsteiner hemolysin).

Correct option: (3) Bifunctional antibodies