Enhancement Mechanisms

A. Hybrid binding is achieved by constructing the IgG molecule from two different antibody Fab portions, one directed against the antigen and one against the tag.

Figure 4. The Fab and Fc fragments of the antibody can be enzymatically separated for hybrid binding or very specific indirect labeling.

B. Protein A or G can be tagged on the Fc (non-reactive portion) of the IgG, leaving both reactive sites free to bind with the antigen.

Figure 5. Protein A schematic.

 

C. Silver enhancement may be done when a gold particle is too small to be seen, as in a preembedment procedure, or light level observation. The silver will enucleate the gold particle.

Figure 6. silver enhancement of gold.

D. Tyramide Signal Amplification

• Boosts signal 1000x
• Easily incorporated into protocol
• Signal to noise ratio
• Sequential multi-color detection
• EM applications
• Tyramide can't diffuse
• Can use with in situ PCR or as alternative to PCR

Figure 7. Tyramide signal amplification schematic

Enzyme Controls

A. A positive control should be employed whenever possible in parallel with the test sections to demonstrate reagent viability.

B. A negative control can be easily made by destroying the enzyme activity in a positive control by immersing in boiling water for 15 minutes, or by a specific chemical method for the enzyme to be demonstrated.

C. A useful control is to incubate one of the test sections in the reaction mixture without the substrate for the same time that the test samples are being run. Both sections are taken through the rest of the technique together.

Immuno Controls

All experiments should be performed with a series of controls conducted simultaneously. Any “positive” result should be looked at skeptically until all controls are analyzed.

A. Adsorption. The primary antibody is reacted with an excess of antigen. This control shows that the antigen and not some other substance is responsible for the localization seen. The absorbed solution should not react with the antigen in localization protocols.

B. Tag or Unlabeled antibody. These are used separately in place of the conjugated antibody. This control determines that the specific properties of the labeled antibody are responsible for the localization.

C. Omission of primary or secondary antibodies. If labeling is seen under these conditions, then it is not the labeling intended as a result of antibody/antigen interaction.

D. Use of pre-immune sera. Collection of sera prior to production of primary antisera used in place of the primary antibody will test “positive” if some component in the immune sera other than the specific IgG is responsible for the binding. Usually it will not be possible to obtain pre-immune serum from the same animal, but it is possible to employ normal serum from the same species.

Specimen Preparation

For Enzymes

Most enzyme activity requires tissues to be as fresh as possible. . Fixatives, if they can be used, should be refrigerator cold to preserve as much enzymatic activity as possible.

Some common reactions that cannot survive fixation are acid and alkaline phosphatases and esterases. Enzymes do not survive at temperatures over 55 degrees Celcius.

For Immunoreactions Pre-embedding vs. Post embedding

A. Preembedding procedures

1. Cell surface labeling prior to fixation

a. Frozen sections
b. Vibratome sections

2. Permeabalization may be necessary for penetration, but usually results in poor morphology.

a. Freeze/thaw
b. Detergents (Saponin, Triton-X)
c. DMSO

Preembedding immunolabeling

1. Time required	2-3 days
2. Sectioning	easy
3. Labeling	possible throughout section
4. Serial sections	easy
5. Storage	blocks and sections easy
6. Equipment	common in EM lab
7. Disadvantages	Loss of structure, translocation with permeabalization

B. Post-embedding immunolabeling enables more control of morphology

1. Fixation allows storage.
2. Embedment media may mask antigens
3. Fixation changes protein stuctures

a. Etching or unmasking may be necessary
b. Fixation choices for Immunoreactions

The goal of fixation is to stabilize the cellular constituent in their native positions.

1. Chemical fixation will insure good morphology, but may compromise antigenicity and/or enzymatic activity

a. A dot immunoreactive test may help to determine antigenicity.(Appendix B)
b. Overfixed tissues may respond to epitope retreival methods. (Appendix E)

2. Physical fixation may preserve antigen integrity but at the expense of morphology. For many enzymatic techniques frozen specimens may be necessary.

a. To minimize ice crystal damage a cryo protectant is used.

• sucrose
• DMSO
• glycerol
• embedment media

C. Other conditions for optimizing reactions

1. pH
2. Concentration of antibody and/or substrate
3. Concentration of trapping reagent.
4. Temperature.(enzymes usually will not survive temperatures over55 degrees celcius)
5. Reaction or fixation times.

D. Blocking reagents can be used to block background staining due to cross-reactivity or multispecficity.

Closely related antigens whose molecular shape and charge distribution may be similar to the original antigen and may fit the same antibody. They are said to cross- react.

Figure 8. Two antibodies cross react with a related antigen (DNP).

Very different antigens have been known also to bind with a single antibody, the close contacts of the edges of the antigen and the amino acid sites that form the walls of the of the combining site of the antibody may cause it to become bound. This feature is termed multispecificity.

Figure 9. The same antibody reacting with two very different antigens (Multispecificity).