25.1 Introduction
25.1.1 Background
Immunoassays are very sensitive and efficient tests that are commonly used to identify a specific protein. Examples of applications in the food industry include identification of proteins expressed in genetically modified foods, allergens, or proteins associated with a disease, including celiac disease. This genetic disease is associated with up to 1 % of the world’s population and more than two million Americans. These individuals react immunologically to wheat proteins, and consequently their own immune systems attack and damage their intestines. This disease can be managed if gluten is avoided in foods. Gluten consists of alcohol-insoluble glutenins and alcohol-soluble prolamins, which are mainly found in wheat and also in oat, barley, rye, and other grain flours and related starch derivatives. Rice and corn are two common grains that do not contain significant gluten and are well tolerated by those with celiac disease. Wheat protein makes up 7–15 % of a wheat grain. Prolamins in wheat are called gliadin. About 40 % of the wheat proteins are various forms of gliadin protein.
The immune system of animals can respond to many foreign substances by the development of specific antibodies. Antibodies bind strongly to and assist in the removal of a foreign substance in the body. Animals make antibodies against many different “antigens,” defined as foreign substance that will elicit a specific immune response in the host. These include foreign proteins, peptides, carbohydrates, nucleic acids, lipids, and many other naturally occurring or synthetic compounds.
Immunoassays are tests that take advantage of the remarkably specific and strong binding of antibodies to antigens. Immunoassays can be used to determine the presence and quantity of either antibody or antigen. Antibodies that identify a specific protein (antigen) can be developed by injection of a laboratory animal with this protein, much as humans are vaccinated against a disease. These antigen-specific antibodies can be used to identify the antigen in a food (e.g., detection of gliadin in food products) through the appropriate use of a label, such as an enzyme or fluorescent molecules linked covalently to either the antibody or a reference antigen. This type of immunoassay concept also can be used to determine the presence of specific antibodies in blood. For example, by analyzing for the presence of gliadin-specific antibodies in an individual’s blood, one can determine if the individual has celiac disease.
25.1.2 Reading Assignment
Hsieh, Y-H.P. and Rao, Q. 2017. Immunoassays. Ch.27, in Food Analysis, 5th ed. S.S. Nielsen (Ed.), Springer, New York.
25.1.3 Objective
Determine the presence of gliadin in various food products using a rabbit anti-gliadin antibody horseradish peroxidase conjugate in a dot blot immunoassay.
25.1.4 Principle of Method
A simple dot blot immunoassay will be used in this lab to detect gliadin in food samples. Dot blot assays use nitrocellulose (NC) paper for a solid phase. Initially, gliadin proteins are isolated by differential centrifugation, in which most of the non-gliadin proteins are washed away with water and sodium chloride (NaCl) solutions, and then the gliadin is extracted with a detergent solution. A drop of the food sample extract or standard antigen (gliadin) is applied to the NC paper, where it adheres nonspecifically. The remaining binding sites on the NC paper are then “blocked” using a protein unrelated to gliadin, such as bovine serum albumin (BSA) to minimize the nonspecific binding. The bound gliadin antigen in the food spot then can be reacted with an antigen-specific antibody-enzyme conjugate. Theoretically, this antibody probe then will bind only to gliadin antigen bound already to the NC paper. Next, the strip is washed free of unbound antibody-enzyme conjugate and then placed in a substrate solution in which an enzymatically catalyzed precipitation reaction can occur. Brown-colored “dots” indicate the presence of gliadin-specific antibody and hence gliadin antigen. The color intensity indicates the amount of antigen present in the food sample. The stronger the color, the higher amount of the antigen (gliadin) in the food sample extract.
25.1.5 Chemicals
|
CAS no. |
Hazards |
|
|---|---|---|
|
Bovine serum albumin (BSA) |
9048-46-8 |
|
|
Chicken egg albumin (CEA) |
9006-59-1 |
|
|
3,3’-Diaminobenzidine tetrahydrochloride (DAB) |
7411-49-6 |
|
|
Gliadin standard protein |
9007-90-3 |
|
|
Hydrogen peroxide, 30 % (H2O2) |
7722-84-1 |
Oxidizing, corrosive |
|
Rabbit anti-gliadin immunoglobulin conjugated to horseradish peroxidase (RAGIg-HRP, Sigma, A1052) |
||
|
Sodium chloride (NaCl) |
7647-14-5 |
Irritant |
|
Sodium dodecyl sulfate (SDS) |
151-21-3 |
Harmful |
|
Sodium phosphate, monobasic (NaH2PO4 · H2O) |
7558-80-7 |
Irritant |
|
Tris(hydroxymethyl)aminomethane (TRIS) |
77-86-1 |
Irritant |
|
Tween-20 detergent |
9005-64-5 |
25.1.6 Reagents
-
Blocking solution **
3 % (g/mL) BSA in PBST; 5–10 mL per student
-
DAB substrate**
60 mg DAB dissolved in 100 mL 50 mM TRIS (pH 7.6) and then filtered through filter paper Whatman #1. (Note: DAB may not completely dissolve in this buffer if it is the free base form instead of the acid form. Just filter out the undissolved DAB and it will still work well.) Just 5 min prior to use, add 100 μL 30 % H2O2, 5–10 mL per student.
-
Gliadin antibody probe **
1:500 (mL/mL) diluted RAGIg-HRP using 0.5 % (g/mL) BSA in PBST; 5–10 mL per student
-
Gliadin extraction detergent**
1 % (g/mL) SDS in water; 10 mL per student
-
Gliadin standard protein, 4000 μg/mL, in 1 % SDS**
One vial of 150 μL per student
-
Negative control sample**
3 % CEA (or other non-gliadin protein) in PBST; 100 μL per student
-
Phosphate-buffered saline (PBS)**
0.05 M sodium phosphate, 0.9 % (g/mL) NaCl, pH 7.2
-
Phosphate-buffered saline + Tween 20 (PBST) **
0.05 M sodium phosphate, 0.9 % (g/mL) NaCl, 0.05 % (mL/mL) Tween 20, pH 7.2, 250 mL per student
25.1.7 Hazards, Precautions, and Waste Disposal
Adhere to normal laboratory safety procedures. Wear gloves and safety glasses at all times. Handle the DAB substrate with care. Wipe up spills and wash hands thoroughly. The DAB, SDS, and hydrogen peroxide wastes should be disposed of as hazardous wastes. Other wastes likely may be put down the drain using a water rinse, but follow good laboratory practices outlined by environmental health and safety protocols at your institution.
25.1.8 Supplies
-
1–10 μL, 10–100 μL, and 200–1000 μL positive displacement pipettors
-
Disposable tips, for pipettors
-
Filter paper, Whatman #1
-
Food samples (e.g., flour, crackers, cookies, starch, pharmaceuticals, etc.)
-
Funnels, tapered glass
-
Mechanical, adjustable volume pipettors, for 2 μL, 100 μL, and 1000 μL ranges, with plastic tips (glass capillary pipettes can be substituted for 2 μL pipettors)
-
Microcentrifuge tubes, 1.5 mL, two per sample processed
-
Nitrocellulose paper (Bio-Rad 162–0145) cut into 1.7 × 2.3 cm rectangular strips (NC strips)
-
Petri dishes, 3.5 cm
-
Test tubes, 13 × 100 mm, six per student
-
Test tube rack, one per student
-
Tissue paper
-
Tweezers, one set per student
-
Wash bottles, one per two students for PBST
-
Wash bottles, one per two students for distilled water
25.1.9 Equipment
-
Mechanical platform shaker
-
Microcentrifuge
-
pH meter
-
Vortex mixer
25.2 Procedure
25.2.1 Sample Preparation
- 1.
Weigh accurately (record the mass) about 0.1 g of flour, starch, or a ground processed food and add to a 1.5 mL microcentrifuge tube. Add 1.0 mL distilled water and vortex for 2 min. Place in the microcentrifuge with other samples and centrifuge at 800 × g for 5 min. Discard the supernatant (albumins). Repeat.
- 2.
Add 1.0 mL of 1.5 M NaCl to the pellet from Step 1 and resuspend it by vortexing for 2 min. If the pellet is not resuspending, dislodge it with a spatula. Centrifuge at 800 × g for 5 min. Discard the supernatant (globulins). Repeat.
- 3.
Add 1.0 mL of 1 % SDS detergent to the pellet from Step 2 and resuspend it to extract the gliadins. Vortex for 2 min. Centrifuge at 800 × g for 5 min. Carefully pipette off most of the supernatant and transfer to a clean microcentrifuge tube. Discard the pellet.
25.2.2 Standard Gliadin
The standard pure gliadin is dissolved at a concentration of 4000 μg/mL in 1 % SDS. To provide for a series of standards to compare unknown samples, dilute the standard serially by a factor of 10 in 13 × 100 mm test tubes to make 400 μg/mL, 40 μg/mL, and 4 μg/mL standards in 1 % SDS. Use 100 μL of the highest standard transferred to 900 μL of 1 % SDS detergent for the first tenfold dilution. Repeat this procedure serially to produce the last two standards. As each standard is made, mix it well on a vortex mixer.
25.2.3 Nitrocellulose Dot Blot Immunoassay
- 1.
Mark two NC strips with a pencil into six equal boxes each (see drawing below).
- 2.
Pipette 2 μL each of sample, standard or negative controls onto the NC paper. Lay the NC strips flat onto some tissue.
- (a)
On NC strip A, pipette 2 μL of four different SDS food sample extracts.
- (b)
On NC strip B, pipette 2 μL of four different gliadin standards.
- (c)
On the remaining two squares (5 and 6) on strips A and B, add 2 μL of 1 % SDS and 2 μL of the 3 % CEA protein negative control, respectively.
- (d)
Let the spots air-dry on the tissue.
Below are diagrams of the NC strips marked off in boxes and numbered by pencil. The circles are not penciled in, but rather they represent where the 2 μL sample or standard spots will be applied.
A series (food sample):
B series (gliadin standards):
1 = food sample 1 @ 1× dilution
1 = gliadin standard @ 4000 μg/mL
2 = food sample 2 @ 1× dilution
2 = gliadin standard @ 400 μg/mL
3 = food sample 3 @ 1× dilution
3 = gliadin standard @ 40 μg/mL
4 = food sample 4 @ 1× dilution
4 = gliadin standard @ 4 μg/mL
5 = 1 % SDS control
5 = 1 % SDS control
6 = 3 % protein negative control (CEA)
6 = 3 % protein negative control (CEA)
- (a)
- 3.
Place both NC strips into a petri dish containing 5 mL of blocking solution (3 % BSA in PBST) and let incubate for 20 min on the mechanical shaker so that the NC strips are moving around slightly in the solution.
- 4.
Rinse the strips well with PBST using a wash bottle over a sink, holding the strips by the tips of their corners with tweezers.
- 5.
Place the NC strips into a petri dish containing about 5 mL of 1:500 diluted RAGIg-HRP conjugate, and incubate for 60 min on the mechanical shaker.
- 6.
Wash the NC strips with PBST using a wash bottle and then incubate them for 5 min in a clean petri dish half full with PBST. Rinse again well with PBST, and rinse one last time with distilled water.
- 7.
Add NC strips to a petri dish containing the 5 mL of DAB/H2O2 substrate, and watch for the development of a brown stain. (Note: Handle the substrate with care. Wipe up spills, wash hands thoroughly, and wear gloves. Although there is no specific evidence that DAB is a carcinogenic compound, it should be treated as if it were.) Stop the reaction in 10–15 min, or when the background nitrocellulose color is becoming noticeably brown, by rinsing each strip in distilled water.
- 8.
Let the NC strips air-dry on tissue paper.
25.3 Data and Calculations
Make any observations you feel are pertinent to this laboratory. Attach the developed NC strips to your lab report with a transparent tape.
Describe the results based on observations of the degree of brown-colored stain in standards and samples relative to negative controls. You can use a crude quantitative rating system like +++, ++, +, ±, and − to describe and report the relative intensities of the dot reactions (note: the brown dot images will fade in several days).
Make very crude approximations of the quantity of gliadin in each substance relative to (more or less than) the standard gliadin dots. Comment on this crude estimate relative to the food product’s gluten status [i.e., gluten free or not; Codex Alimentarius (http://www.codexalimentarius.net/) defines less than 20 mg of gluten/kg in total, based on the food as sold or distributed to the consumer to be classified as “gluten-free”].
Tabulate your results in a manner that is easy to interpret.
To make the gluten status estimation, you must know the values of both the concentration of the food sample (g food/mL extraction solution) extracted and the concentration of the gliadin standards (mg gliadin/mL extraction solution) to which you are making a comparison.
Example calculation:
If the food sample has a concentration of 100 mg/mL and reacts equivalently to a 4 μg/mL gliadin standard, it can be estimated that 4 μg gliadin is in 100 mg food sample, because both are applied at equal volumes so the two concentrations can be related fractionally (i.e., 4 μg gliadin/100 mg or 40 mg gliadin/kg food sample). The gliadin content of gluten is generally taken as 50 %. Since this sample has a gliadin concentration higher than the limit set by Codex Alimentarius (10 mg gliadin/kg or 20 mg gluten/kg food), the food cannot be considered as “gluten-free.”
25.4 Questions
- 1.
Draw a set of symbolic pictures representing the stages of the dot blot assay used in this laboratory, including the major active molecular substances being employed (i.e., nitrocellulose solid matrix, antigen, BSA blocking reagent, antibody-enzyme conjugate, substrate, product).
- 2.
Why should you block unbound sites on nitrocellulose with 3 % BSA in a special blocking step after applying samples to the membrane?
- 3.
Why is a spot of 1 % SDS (gliadin extraction detergent) used in the dot blot?
- 4.
Why is a protein negative control spot (3 % CEA) used in the dot blot?
- 5.
Describe the basic role of horseradish peroxidase enzyme (i.e., why is it attached to the rabbit antibody), and what roles do DAB and H2O2 play in the development of the colored dot reaction in this immunoassay? Do not describe the actual chemical reaction mechanisms, but rather explain why a color reaction can ultimately infer a gliadin antigen that is present on the nitrocellulose paper.
Acknowledgment
This laboratory exercise was initially developed for the first edition of the laboratory manual by Mr Gordon Grant and Dr Peter Sporns, Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada. Dr. Hsieh, who has now updated the laboratory for two editions, gratefully acknowledges the original contribution.