Proteolytic Enzymes

The proteolytic enzymes used in the blood bank and their sources are: 1) papain (papaya), 2) ficin (figs), 3) bromelin (pineapples), 4) trypsin (lining of a hog's stomach). Their order of effectiveness in detecting IgG antibodies is ficin > papain > bromelin > trypsin.

Proteolytic enzymes enhance agglutination of IgG antibodies. Mechanisms proposed to explain how include these:

1. Enzymes strip off negatively charged NeuNac (NANA) from the cell membrane, thus reducing zeta potential and allowing the cells to come closer.

2. Enzymes decrease steric hindrance by removing protein adjacent to antigens, thus allowing antibody to have better access to antigens.

3. Enzymes

Enzyme tests can be one-stage or two stage:

One-Stage Test:

Two-Stage Test:

Antibodies detected by enzyme tests include the following:

1. IgG antibodies in the Rh system. Note: enzymes are the best method for detecting of Rh antibodies.
2. Some IgM cold antibodies that are clinically insignificant such as anti-Le^a, anti-Le^b, autoanti-I, and anti-P₁.
3. Some Kidd antibodies, especially if an antiglobulin test is also done.

Advantages

Advantages of the enzyme method include

• best method for detecting Rh antibodies
• sensitive method for Kidd antibodies if an AHG test is done
• rapid method

Disadvantages

• One-stage method is relatively insensitive and over-incubation can destroy the antibody enzymes denature MNSFy^aFy^bCh^aRg^a antigens, therefore cannot detect the corresponding antibodies.

• Enzymes enhance some cold, clinically insignificant antibodies (listed above) that we do not want to enhance.

• Although the two-stage method is sensitive, it requires extensive quality control. Each cell that is enzyme treated requires a positive and negative control to ensure that it has not been under- or over-treated, respectively. The positive control consists of incubating the enzyme-treated cells with a weak IgG antibody such as a diluted anti-D. The negative control consists of incubating the enzyme-treated cells with AB serum (antibody-free serum).

Applications

Proteolytic Enzymes