Griffin:Antibody Protein Coupling (F:P) Ratios
Antibodies from the same manufacturer can exhibit batch to-batch variation. In addition, some control antibodies are not marketed at the same concentration as the test antibody. Available IgG should undergo rigorous testing in an unconjugated form in order to determine direct conjugate binding efficiency versus the free form of IgG (over- conjugation can produce highly charged /acidic/ non-specific binding). Establish the correct working dilutions for every new antibody, including those from different batches, for each detection system.
Optimizing the tag:antibody (flurochrome:protein) ratio determines a maximum number of tags (fluorescent dye) coupled to antibody without affecting antigen binding specifcity. Inactivation and changes in binding properties may occur during any antibody conjugation. Optimized F:P Ratio is more important than total number of tags that can bind to every SH or NH2 groups within an immunoglobulin. For standard tags like FITC, Biotin, HRP, etc. standard coupling procedures are well established. For others, an optimization process may be necessary.
Most antibody conjugations are through amino (NH2) groups (nucleophilic substitution) (i.e., primary amines from lysine). Certain chemistries can utilize thiol (-SH) groups. SH groups are not readily available and need to be produced by reducing disulfide bonds (S-S) with mild reducing agents prior to coupling. This has the potential of disrupting the IgG secondary and tertiary structures and potentially harming its antigen binding activity. There are 16 Disulfide bonds in IgGs, with a potential to form 32 SH groups.
Suitable coupling ratios
- 3 to 8 biotin per IgG molecule (biotin MW: 244); (long arm) N hydroxysuccinimide ester
- 4 to 7 FITC per IgG molecule (FITC MW: 390)
- 1 to 2 HRP per IgG molecule (HRP MW: 40,000)
- 1 APC per IgG molecule (APC MW: 100,000)
- ~1 PE per IgG molecule (PE MW: 240,000)
- AF405: 1-3 moles of dye: mole of IgG
- AF488: 4-9 moles of dye: mole of IgG
- AF647: 3-7 moles of dye: mole of IgG
UV Absorbance Optical Density (OD)
- 230 nm peak ~ peptide bonds + Sodium azide absorbs strongly at 230 nm / phenolic impurities, carbohydrates, EDTA / TRIzol, chaotropic salts.
- 260 nm peak ~ nucleic acid aromatics Purines (thymine, cytosine and uracil) and pyrimidines (adenine and guanine)
- 280 nm peak ~ aromatics(tryptophan, phenylalanine, tyrosine, histidine)
Protein contamination (aromatic protein absorbance @280nm) <acceptable ratio = degree of protein contamination.
- RNA 260/280 ratio 2.1 = acceptable (2.0 = pure RNA)
- DNA 260/280 ratio 1.8 = acceptable (1.7–2.0 = pure DNA)
- PROTEIN 260/280 ratio 0.6.= acceptable (>0.6 = DNA contamination) DNA is a contaminant of whole cell dervied protein isolates
Subtraction of non-nucleic acid absorbance at 320 nm DNA Purity (A260/A280) = (A260 reading – A320 reading) ÷ (A280 reading – A320 reading)
Organic contamination (phenol, TRIzol, chaotropic salts, aromatics).
- A260/230 ~2.0-2.2 = acceptable / pure
- A260/230 <1.8 = contaminated / may disrupt reverse transcription.
Peptide to Solid Phase Coating
- Coating peptides can be prepared as follows:
- Free peptides, 2.5 ug/50 ul solutions (or suspensions for some peptides of low solubility) in either PBS or 0.05 M sodium carbonate buffer, pH 9.6
(SCB). Peptide-BSA conjugates dissolved in 0.05 M NH4HCO3, pH 7.8 (0.3 mg per ml), centrifuged and the supernatants used for plating.
- The following order of steps, each of which succeeded by washing of the wells with PBS (5x each).
- PVC 96-well microtiter plates were coated for 3hr/ 37C with 50 ul/well of antigen solution.
- After coating, wells were blocked for 1 h at 37C with 100 ul of 0.5% casein in PBS to reduce non-specific binding to the wells.
- After washing with PBS, 50 ul of anti-peptide antiserum, prediluted 1:500, can be added to each well / incubated for 2 h at 37C.
- Reilly JT. Use and evaluation of leucocyte monoclonal antibodies in the diagnostic laboratory: a review. Clin Lab Haematol. 1996 Mar;18(1):1-5. DOI:10.1111/j.1365-2257.1996.tb00728.x |