An Interdisciplinary Enrichment Laboratory on Lectin Production

INTRODUCTION

Lectins are proteins that bind to carbohydrates and can be isolated from diverse plant and animal sources.^1⇓⇓–4 Seed lectins can be bivalent or tetravalent, allowing a single lectin to bind to carbohydrates on the surfaces of several different cells, causing agglutination. If the target carbohydrate is on a red blood cell (RBC) membrane, the lectin mixed with the RBCs will demonstrate hemagglutination.^5⇓–7

In 1888, Stillmark obtained the first lectin to demonstrate hemagglutinating properties from the seeds of the castor oil plant Ricinus cummunis.¹ Plant-derived lectins, or phytohemagglutinins,^8–9 have been used to detect various RBC antigens in the immunohematology laboratory for many years. For example, Dolichos biflorus is a commonly used lectin to differentiate A₁ from non-A₁ RBCs, and Ulex europaeus lectin has specificity for the H antigen and can differentiate group O RBCs from the rare Bombay phenotype.^5⇓–7 A panel of lectins can be used to presumptively identify polyagglutinable RBCs, and often includes lectins from seeds of Glycine max (soy), Arachis hypogaea (peanut), D biflorus (horse gram), Salvia sclarea (clary sage), Salvia horminum (blue clary sage), and Griffonia (Bandeiraea) simplicifolia, an African woody shrub.⁷

Students enrolled in our medical laboratory science (MLS) program’s Principles of Immunohematology course are provided U europaeus and D biflorus seeds to grind and prepare their own lectins for use in the student immunohematology laboratory. In conjunction with this laboratory activity, the MLS students attended an enrichment laboratory in the university’s botany department to learn how lectins are isolated and purified. The demonstration illustrated how lectins are purified from Pisum sativum (pea) seeds by affinity chromatography. Pisum lectin P sativum agglutinin (PSA) binds to glucose residues. Sephadex, a beaded glucose polymer used for separating proteins by size, can be used as an affinity matrix for glucose-binding lectins like those from P sativum and Lens culinaris (lentil).

For this activity, a crude extract of supermarket split peas was passed through a Sephadex column. Following washing of the column to remove nonbinding proteins, the column was connected to a fraction collector, and PSA was eluted with a glucose solution. Students took turns reading the A₂₈₀ of the fractions to detect protein and thus find the fractions containing the eluted lectin.

MATERIALS AND METHODS

To prepare the crude extract, 10 g of dry pea seeds (P sativum) were imbibed by soaking in water at 4 °C overnight. The imbibed seeds were homogenized in 50 mL of 50 mM phosphate buffer, pH 7.2, with a blender for 30–60 seconds until the puree was smooth. The puree was centrifuged at 10 000 × g for 10 minutes. After centrifugation, the still-cloudy supernatant solution was decanted through 2–4 layers of Kimwipes into a beaker with a stirring bar. The pH of the solution was lowered to 5 by slowly adding 2 M citric acid dropwise while the supernatant was being stirred on a stir plate, monitoring the acidity with a pH meter. The solution was again centrifuged at 10 000 × g for 10 minutes. The now-clear supernatant was decanted into a beaker with a stirring bar, returned to the pH meter, and 2 M tris(hydroxymethyl)aminomethane (Tris) was added dropwise while stirring until the pH increased to 7.

The neutralized supernatant was loaded onto a 50-mL (2.5 × 10.5 cm) Sephadex G-100 column. Once all of the sample had entered the column, the column was washed with 4 column volumes (200 mL) of phosphate-buffered saline (PBS) containing 10 mM sodium phosphate, 150 mM NaCl, and a pH of 7.4 to flush out the proteins that did not bind to the column. The column outlet was then connected to a fraction collector. PBS with 0.1 M glucose was then passed through the column to elute Pisum lectin while collecting 7-mL fractions.

The students in the class then took turns reading the A₂₈₀ of the fractions, using a NanoDrop Lite Spectrophotometer to determine which fractions contained lectin. The absorbance readings were recorded on the classroom whiteboard, enabling students to see the rise and fall in the readings as the lectin eluted from the column.

In a follow-up to the enrichment activity and after approval from the university’s institutional review board, the students were surveyed anonymously approximately 6 months later. The survey instrument collected feedback on the activity (Table 1) and assessed the students’ retention of their knowledge of how lectins are extracted, purified, and used in the immunohematology laboratory (Table 2).

RESULTS

Responses to the survey questions (Table 1) indicated that the students were interested to see where lectins come from, that they enjoyed the activity, that they would recommend the activity for future students, and that they were interested in seeing applications for analytic methods outside the MLS profession. One respondent recommended inviting botany students into the immunohematology laboratory to experience how lectins are used in blood typing, and one respondent recommended hosting the botany students in the immunohematology laboratory. The quiz average was 83% (Table 2), indicating good retention of the material learned approximately 6 months after the survey was conducted.

DISCUSSION

It is important to provide students the opportunity to learn the background of the techniques they will use in their professions. The following techniques were employed in this enrichment activity: affinity chromatography/column chromatography, micropipetting, use of the fraction collector, centrifugation, pH meter, and spectrophotometry (NanoDrop). An interdisciplinary approach was applied because of its ability to enrich the students’ overall educational experience.¹⁰

Interdisciplinary education refers to the collaboration of two or more disciplines in relation to research or instruction.^10–⇓12 This type of learning allows students to apply the knowledge they have gained within their own discipline to another field of study, to form connections between concepts across disciplinary boundaries, and to develop transferrable skills.¹⁰ In addition, interdisciplinary collaborations are increasingly used in both research and medicine because they are recognized for their ability to find solutions to complex questions and to determine the optimal treatment regimens for individual patients.¹²

We concluded that this is an excellent opportunity to emphasize to students that discoveries are ongoing, that new lectins continue to be found in diverse plant and animal sources, and that their use is not restricted to blood typing in the immunohematology laboratory. In future planned activities, an electrophoresis demonstration will be added to show the purity of the lectin compared with the mixture of proteins in the crude extract.