Micro Miscell, ELISA

 

Bacterial Growth Curve

• Lag Phase:
• No increase in number of bacteria.
• Increase in size, enzymes, and metabolites.
• Maximum size is seen at the end of this phase.

• Log Phase:
• Cell division occurs.
• Bacteria are smaller in size.
• Shows maximum metabolic activity.

• Stationary Phase:
• Some bacteria viable, others dead.
• Features:
• Sporulation.
• Toxin production (Exo Toxin).
• Antibiotic production.
• Bacteriocin production.

• Death Phase:
• Logarithmic decline
• Also known as Decline phase.
• Characterised by cell death.
• Involution forms are seen.

Dimension And Diffusion

ㅤ	SINGLE DIMENSION	DOUBLE DIMENSION
SINGLE DIFFUSION	OUDIN	MANCINI
DOUBLE DIFFUSION	OAKLY FULTHROPE	OUCHTERLONY

• Mnemonic 1
• Dimension up, diffusion down
• Oudin - Oakly (odunna oak)
• Man - ouch

• Mnemonic 2
• Single single
• Ou - din
• Double double
• Ou - chterlony (lengthy)
• signle diffusion double DIMENSION
• Mancini
• double DIFFUSION single dimension
• Fulthrope

Enzyme-Linked Immunosorbent Assay (ELISA)

Basic Principle

• 96 wells

• Enzyme: Horseradish peroxidase

• Substrate: H2O2

• Chromogen: Tetramethylbenzidine(TMB)

• Uses antigen–antibody reaction.
• Substrate added → enzyme converts into Blue colored product
• Intensity α Presence of Ag/Ab.

• Detection done by an enzyme-labeled antibody.

Types of ELISA

1. Direct ELISA

• Enzyme-labeled antibody binds directly to antigen.

• Pros: Simple, fast.

• Cons: Less sensitive (no amplification).

2. Indirect ELISA

• Antigen is fixed on plate.

• Primary antibody (from patient’s serum) binds antigen.

• Enzyme-labeled secondary antibody detects primary antibody.
• Secondary antibody binds to Fc portion of primary antibody.

• Pros: More sensitive (signal amplification via multiple secondary Abs per primary Ab).

• Use: Detect antibodies in patient (e.g., screening for HIV).

3. Sandwich ELISA

• Antibody is fixed on plate (capture antibody).

• Antigen (patient’s sample) binds to it.

• Second enzyme-labeled antibody binds antigen → “sandwich”.

• Use: Detect antigen (e.g., HBsAg in Hepatitis B).

• Most specific type.

4. Competitive ELISA

• Patient’s antigen competes with labeled antigen for binding to antibody.

• Signal inversely proportional to antigen concentration.

• Use: Small molecules (e.g., hormones, drugs).

High-Yield Points

• Indirect ELISA → detects antibody (screening test for HIV).

• Sandwich ELISA → detects antigen (HBsAg detection).

• Competitive ELISA → for small antigens, quantification.

• ELISA = sensitive, specific, quantitative.

• Color intensity ∝ concentration of target molecule.

• Common enzymes used: HRP (horseradish peroxidase), alkaline phosphatase.

• Read using spectrophotometer / ELISA reader.

• HIV screening:
• Indirect ELISA
• Confirmatory: Western blot

• HBsAg detection:
• Sandwich ELISA (HBsAg)

• Assays for small molecules: Competitive ELISA examples: insulin, hCG