Association of surrogate molecular classification with PD-L1 negative and positive expression

1. Two types of antibodies are used:
 Polyclonal antibodies
 Monoclonal antibodies
Polyclonal antibodies
 They are a heterogenous mixture of antibodies directed against various epitopes of same
antigen
 Generated by different B-cell clones of the animals that are immunochemically dissimilar
Monoclonal antibodies
 They are a homogenous population of lg directed against a single epitope.
 Generated by a single B-cell clone from one animal that are immunochemically similar

2. Preparation of Antigen Retrieval Buffer
1. TRIS-EDTA Buffer or tris(hydroxymethyl)aminomethane-EDTA Buffer (pH 9.0)
 Tris 12.1 gm
 EDTA 3.7 gm
 Distilled water 1.0 liter
 Above prepared buffer solution is 10X (10 times concentrated) so usable buffer
solution is prepared by diluting it to 1X with distilled water.
 Adjust pH to 9.0 with NaOH or HCL
2. EDTA Buffer (pH 8.0)
 EDTA 3.7gm
 Distilled water 1.0 liter
 Above prepared buffer solution is 10X (10 times concentrated) so usable buffer
solution is prepared by diluting it to 1X with distilled water.
 Adjust pH to 8.0 with NaOH or HCL

3. 3. Citrate Buffer (pH 6.0)
 Anhydrous Citric acid 29.4 gm
 Distilled water 1.0 liter
 Above prepared buffer solution is 10X (10 times concentrated) so usable buffer
solution is prepared by diluting it to 1X with distilled water.
 Adjust pH to 6.0 with NaOH or HCL
TBS (Tris buffered saline) solution – wash buffer (7.4)
 Tris 60.0 gm
 NaCl 40.4 gm
 Distilled water 1.0 liter
 Above prepared buffer solution is 10X (10 times concentrated) so usable buffer
solution is prepared by diluting it to 1X with distilled water.
 Adjust pH to 7.4 with NaOH or HCL

4. Advantages
 consistency of staining
 Fast and accurate results
 Decreased use of reagents
 Less use of man power
Disadvantages
 one
 Two
 Three