Mls 308: Medical Laboratory Science Plant Lectins

Plant lectins are extracts from plants which have red cell agglutinating properties. They are naturally-occurring proteins found in a wide variety of plants, especially in seeds, legumes, and grains. They can also be found in animals, fungi, bacteria, and viruses, but are found more in plants.

These agglutinins appear to be globulins, but are not as complex as the antisera produced in animals.

They have the unique ability to bind specifically and reversibly to carbohydrates present on the surface of cells, particularly red blood cells. Because they have these unique properties, they are widely used in haematology and immunohaematology, especially in blood grouping and identification of red cell antigens.

Lectins are known to combine with a simple sugar or part of a simple sugar on the red cell membrane. Seven components of the red cell membrane are known to interact with lectins. These are:

• Galactose

Mannose

• Fucos

• Glucose

N-acetyl-glucosamine

N-acetyl-galactosamine

N-acetylneuraminic acid

Known specificities of lectins include: Anti-A.1, anti-B, anti-A+B, anti-H, anti-M, anti-N, anti-A+N, anti-T, and anti-Tn.

Characteristics of Lectins

1. Lectins are non-immune proteins (unlike antibodies).

2. They possess specific carbohydrate sites.

3. They can cause agglutination of R.B.C's.

4. Their reactions are highly specific but not Ag-Ab (Antigen-Antibody) based.

5. They are usually resistant to moderate heat and pH changes depending on the type.

Sources of Plant Lectins

Lectins are extracted from various plants, particularly legumes. Common examples include:

1. Dolichos biflorus – Anti-A.1 lectin. This lectin is extracted from Lima beans. It is specific for A.1 and can be used to differentiate subgroups of A. It is N-acetylgalactosamine specific with high avidity.

2. Ulex europaeus and Lotus tetragonolobus – Sources of anti-H. They are extremely useful because they react better with the subgroups of A, and also distinguish between secretor and non-secretor. They are specific for L-fucose.

3. Vicia graminea – Anti-N. They possess anti-N activity. The extracts also cross-react weakly with M cells, but some other antisera show this fault which can be corrected by dilution.

4. Marasmius oreades – This is an anti-B lectin extracted from mushroom. It is present in many fungi.

5. Arachis hypogaea (peanut) – Anti-T lectin. They show anti-T activity.

6. Iberis amara – Has been shown to be anti-M in activity. It can distinguish M from N.

7. Salvia sclarea – Has anti-Tn activity.

8. Bandeiraea simplicifolia – They are anti-B lectins.

9. Extracts from snails – Anti-A. These lectins are used exactly as the one obtained commercially and they give reactions expected for their specificity, but the optimum concentration for use may be obtained by dilution.

Functions and Applications of Lectins

1. Blood Grouping: Lectins are very useful in distinguishing blood group subgroups. For example, Dolichos biflorus lectin agglutinates A.1 cells but not A.2 cells.

2. Identification of Red Cell Antigens: They help detect specific carbohydrate antigens on R.B.C membranes.

3. Research and Diagnostics: Lectins are used in cell biology, cancer research (altered glycosylation patterns), and immunology studies.

Mechanism of Action

Lectins bind to specific sugar residues on the red cell membrane, and this binding leads to cross-linking of cells and visible agglutination. Unlike antibodies, lectins do not require prior sensitization.

Advantages of Lectins

• High specificity for certain carbohydrate antigens.

- Useful in differentiating blood group subgroups.

• Readily available from plant sources.

Limitations

• Not as versatile as antibodies.

• Some lectins may show non-specific reactions.

- Activity may vary depending on preparation.

Preparation and Standardization of Antisera from Lectins (e. g., Dolichos biflorus)

Lectin antisera are prepared by extracting active lectin proteins from plant seeds. These extracts act similarly to antisera in blood grouping because they agglutinate specific red cells.

A. Preparation

Materials: Dried Dolichos biflorus seeds, mortar and pestle, Normal saline (0.85% N-A-C-L), Centrifuge, filter paper or muslin cloth, Sodium azide (preservative).

1. Grinding: Weigh an appropriate quantity of seeds (e.g., 10–20g). Grind into a fine powder using a mortar and pestle.

2. Extraction: Add normal saline in a ratio such as 1:10 (Weight/Volume). For example, 10g powder + 100ml saline. Mix thoroughly to allow extraction of lectins.

3. Incubation: Allow the mixture to stand for several hours (or overnight) at 4 superscript circle C or room temperature. This enhances protein extraction.

4. Filtration: Filter using muslin cloth or filter paper to remove debris.

5. Centrifugation: Centrifuge filtrate at about 3000 rpm for 10 to 15 mins. Collect the clear supernatant (this is the crude lectin extract).

6. Preservation: Add Sodium azide (0.1%) to prevent microbial growth. Store at 2 to 8 degrees Celsius.

B. Standardization of Lectin Antisera

Standardization ensures that the lectin extract has the appropriate strength and specificity for laboratory use.

1. Preparation of Red Cell Suspension:

• Prepare 2 to 5% suspension of known R.B.C's:

- A.1 cells (Positive control)

- A.2 cells (Negative control)

2. Titration Procedure:

• Label a series of test tubes.

• Perform serial doubling dilutions of the lectin extract: 1:2, 1:4, 1:8, 1:16 etcetera

• Add equal volumes of: Lectin dilution and Red cell suspension.

• Mix and incubate at room temperature (15 to 30 mins).

3. Reading Results: Observe for agglutination.

4. Determination of Titre: The titre is the highest dilution showing visible agglutination. For example, if agglutination is seen up to 1:32 dilution, the titre is 32.

5. Specificity Testing: Test against A.1 cells (should agglutinate) and A.2 cells (should not agglutinate). This confirms the specificity of Dolichos biflorus lectin.

6. Quality Control – Ensure: Clear agglutination with A.1 cells. No reaction with A.2 or O cells. Reject preparation if non-specific reactions occur.

C. Storage and Stability

Store at 2 degrees to 8 degrees Celsius.

• Avoid contamination.

Do not freeze unless specified.

• Check activity periodically.

D. Safety Precautions

• Handle biological samples carefully.

- Use gloves and laboratory coat.

• Sodium azide is toxic. Handle with caution.

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