Enzyme Immunoassay for the Quantitative Determination of Tetracycline

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FACULTY OF FOOD SCIENCE AND TECHNOLOGY

FST 5603 FOOD DIAGNOSTIC

SEMESTER 2 2018/2019

LABORATORY REPORT

ENZYME IMMUNOASSAY FOR THE QUANTITATIVE DETERMINATION OF TETRACYLINE IN HONEY

DATE                        : 27/04/2019

LECTURER                : DR RASHIDAH BINTI SUKOR

NAME                        : AGATHA ANAK SIBUAR

MATRIC NUMBER        : GS 52147

TITLE

Enzyme Immunoassay for the Quantitative Determination of Tetracycline in Honey

ABSTRACT

ELISA test is a method used to detect antibodies or infectious agents in a sample. This test relies on the interaction between components of the immune system called antigens and antibodies. Tetracycline is an antibiotic that inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site. Antibiotic residue such as tetracylin causes a major problem in global honey trade. The concentration of antibiotic residues in honey can be in alarming situation. Due to its potential health risk, it is must not present in any honey product. RIDASCREEN ® ELISA TETRACYLIN was used as a test kit to detect the presence of tetracyline in all three samples of honey.   Graph semilog B/Bo versus tetracycline concentration which was directly proportional plotted. The concentration of tetracycline was the highest in Sample 2 which was 3.088 ug/L. Sample 1 had 2.675 ug/L and sample 3 had a concentration of 2.803 ug/L. The correlation coefficient (R) value was 0.9667. The concentration of tetracycline in all honey samples were below the permitted level based on the standard compared in European country as Malaysia does not has any limiting limit of tetracycline in honey.

INTRODUCTION

The indirect ELISA method has high sensitivity since more than one labelled secondary antibody can bind the primary antibody; it is more economical than the direct ELISA as fewer labelled antibodies are needed. Indirect ELISA delivers greater flexibility since different primary antibodies can be used with a single labelled secondary antibody. Also, indirect ELISA assays take longer to run than direct ELISAs since an additional incubation step for the secondary antibody is required. The indirect ELISA is most suitable for determining total antibody concentration in sample (Kohl & Ascoli., 2017).

Bee product such as honey is widely consumed by human population. The honey based product can be in term of medicinal or food. It may carry potential health hazard if contained any antibiotic residues.  The antibiotics may be presence in honey product because the might be use in apiculture for the treatment of bacterial diseases. Besides, antibiotic residues mostly come from environment an improper beekeeping practices (Saridaki-Papakonstadinou et al., 2006)

Accumulation of antibiotic residues in honey is due to the extensive use of it which will lead to reduced quality of the honey. The antibiotic may cause toxic effect towards human since antibiotic residues has a relatively long half life. This is the reason why European country banned use of antibiotic n beekeeping practices as it can lead to accumulation of antibiotic residues in honey. Furthermore, it is important to monitoring antibiotic residues in honey, wax and bees so potential risk to human health of these product can be assess (Tillotson et la., 2006).

MATERIAL

Materials:

• microtiter plate spectrophotometer (450 nm)
• centrifuge
• vortex
• shaker
• waterbath (for dairy product samples)
• ultrasonic bath (optional for milk powder and honey samples)
• mixer (Stomacher, ultra turrax; for meat, fish, shrimp and sausage samples)
• pasteur pipettes
• graduated pipettes
• variable 20 μl - 200 μl and 200 - 1000 μl micropipettes

Standard preparation:

Standard 1: 50 μl standard concentrate (0 μg/l) + 450 μl buffer 0 (μg/l)

Standard 2: 50 μl standard concentrate (0.5 μg/l) + 450 μl buffer 0.05 (μg/l)

Standard 3: 50 μl standard concentrate (1.5 μg/l) + 450 μl buffer 0.15 (μg/l)

Standard 4: 50 μl standard concentrate (3 μg/l) + 450 μl buffer 0.3 (μg/l)

Standard 5: 50 μl standard concentrate (6 μg/l) + 450 μl buffer 0.6 (μg/l)

Standard 6: 50 μl standard concentrate (18 μg/l) + 450 μl buffer 1.8 (μg/l)

Method

For the sample preparation, 1g of honey sample was weighed in a screw top glass vial (80ml). Then, the sample was diluted into the ratio of 1:50 (1+49) with 20mM PBS buffer of pH 7.4. the sample was mixed intensively for 2 minutes on a vortex. The vial that contained the sample was turned upside down briefly before use it for the test. Only 50 µl was used per well in the sample. The sample then was duplicated when running the test.

For the test procedure, a sufficient number of wells were inserted into the mircowell holder for all standards and samples to be run duplicate. The standard and sample positions were recorded. 50 µl of each standard or prepared sample was added to separate duplicate wells. Then, 50 µl of anti-tetracycline antibody was added to each well. The plate was gently mixed by shaking it manually and incubated for 1 h at room temperature (20-25 °C/ 68-77 °F). For the washing step, the liquid were poured out of the well and tapped the microholder upside down vigorously (three times in a row) against absorbent paper to ensure complete removal of liquid from the wells. This step was repeated three times. Next, 100 µl of conjugate was added to each well and shaking it gently to mix it. The plate was incubated for 15 minutes at room temperature (20-25 °C/ 68-77 °F). For the next washing step, the liquid were poured out of the well and tapped the microholder upside down vigorously (three times in a row) against absorbent paper to ensure complete removal of liquid from the wells. All the wells were filled with 250 µl For the washing step, the liquid were poured out of the well and tapped the microholder upside down vigorously (three times in a row) against absorbent paper to ensure complete removal of liquid from the wells wash buffer and the liquid was poured pour again. This step was repeated three times. 100 µl of chromogen/substrate was added to each well. The plate was mixed gently by shaking it manually and incubated for 15 minutes at room temperature (20-25 °C/ 68-77 °F). Finally, 100 µl of the stop solution was added to each well. The plate was mixed gently by shaking it manually and the absorbance at 450nm was measured. It was read within 30 minutes after addition of the stop solution