Explain monoclonal antibody production

6.3.6 · Biology › Biotechnology Applications

WHAT is a monoclonal antibody?

WHY do we need the fusion trick?

Toh koi bhi akela parent kaam nahi karta. Hybridoma dono ke best properties inherit karta hai.

HOW is it made — step by step (derive the logic)

Step 1 — Immunise. Mouse ko target antigen inject karo. Yeh step kyun? Isse mouse ka immune system trigger hota hai aur B-cells produce hoti hain jo already us antigen ke against antibodies bana rahi hain. Hum yeh B-cells spleen se harvest karte hain.

Step 2 — Myeloma cells lo. Myeloma (cancerous B-cells) culture karo. Yeh step kyun? Hume "immortality gene package" chahiye. Yeh specially chosen myeloma cells HGPRT⁻ bhi hoti hain (enzyme HGPRT absent hai) — yeh Step 4 ke selection mein crucial ho jaata hai.

Step 3 — Fuse karo. Spleen B-cells + myeloma cells ko ek fusion agent ke saath mix karo (PEG, polyethylene glycol — yeh membranes ko ek saath pighlata hai). Yeh step kyun? Fusion physically do cells ko ek hybrid mein merge karta hai = hybridoma. Lekin fusion random hoti hai, isliye dish mein ab yeh sab hain: unfused B-cells, unfused myeloma, aur hybridomas. Pehle do ko remove karna zaroori hai.

Step 4 — HAT medium mein select karo. Sab kuch HAT medium (Hypoxanthine–Aminopterin–Thymidine) mein grow karo. Yeh step kyun? (clever wali baat)

• Cells DNA do routes se banati hain: de novo pathway aur salvage pathway.
• Aminopterin de novo pathway BLOCK kar deta hai. Toh ek cell sirf salvage pathway use karke survive kar sakti hai, jiske liye enzyme HGPRT chahiye.
• Unfused myeloma cells HGPRT⁻ hain → salvage blocked → mar jaati hain. ✅
• Unfused B-cells normal hain lekin mortal hain → kuch dinon mein waise hi mar jaati hain. ✅
• Hybridomas ko B-cell parent se HGPRT milta hai AUR myeloma parent se immortality → survive karti hain aur divide karti hain. ✅ Toh HAT medium ek filter hai jo sirf hybridomas ko chodta hai.

Step 5 — Screen aur clone karo. Hybridoma cultures test karo taaki woh wala dhundha ja sake jo actually tumhari manpasand antibody bana raha ho, phir itna dilute karo ki har well ek single cell se grow ho (= ek clone). Yeh step kyun? Alag-alag hybridomas alag-alag antibodies banate hain. Hume ek specific epitope ke against clone chahiye — yahi cheez ise monoclonal banati hai.

Step 6 — Mass-produce karo. Chosen clone ko bade bioreactors mein (in vitro) ya mice mein ascites ke roop mein (in vivo) grow karo. Antibody purify karo. Yeh step kyun? Ek immortal clone → unlimited identical antibodies.

Uses (WHY it matters — the 80/20)

Common mistakes

Flashcards

Connections

• Antibody Structure and Epitopes
• Cell Culture and Bioreactors
• Cancer and Myeloma Cells
• ELISA and Diagnostic Kits
• Immune Response - B and T cells
• Recombinant Therapeutics

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