Mal. J. Anim. Sci. 14:61-66 (2011) Malaysian Society of Animal Production Production of polyclonal antibody against tetracycline using KLH as a carrier protein
Siti Noraini, B.* and Nur Azura, M.S.
Biotechnology Research Centre, Malaysian Agricultural Research and Development Institute, P.O. Box 12301, 50747 Kuala Lumpur, Malaysia *Corresponding author Abstract
The production of polyclonal antibody against tetracycline was described using tetracycline - KLH conjugate (Keyhole Limpet Hemocyanin). Tetracycline was conjugated with KLH as a carrier protein because it was small molecule and unable to stimulate an immune response by itself. The UV absorbance reading of the tetracycline-KLH conjugate and KLH alone slightly shifted the reading of UV absorbance. Ammonium sulphate precipitation and Protein A affinity column were used in the antibody purification. Two peaks were obtained from affinity Protein A column. Peak 1 indicated the unbound material from serum sample and peak 2 was bound antibody with protein A which was eluted with elution buffer. Peak 2 was collected for titer antibody determination using ELISA method. Antibody level was higher at the fourth bleed which reached 1.2 absorbance (UV/nm) and equivalent with 1 mgmL-1 concentration. The entire antibody level declined dramatically at dilutions greater than 0.0001 mgmL-1 protein. The optimum and significant antibody concentration was at 0.00001 mgmL-1 for use in ELISA or other assays Key words: Antibody titer, ELISA, Keyhole Limpet Hemocyanin, polyclonal antibody, tetracycline

efficiency and egg hatchability and as a treatment for avian respiratory disease. Tetracycline is a group of broad- The use of these antibiotics has raised spectrum antibiotics used for medical concerns as the presence of tetracycline purposes as well as animal husbandry. The residues in food may increase microbial tetracycline antibiotics have a broad range of resistance in humans which could lead to activity against a variety of both Gram- human health risks due to their carcinogenic potency. The emergence of resistance is Therefore, tetracycline and its derivatives related with the introduction of tetracycline (chlortetracycline & oxytetracycline) are for clinical, veterinary and agricultural uses widely used for prevention and control of (Aga et al., 2003). Consequently, many diseases. Tetracycline antibiotic is also used countries have set the maximum residue as a growth promoting agent to improve levels (MRLs) to protect consumers. In growth performance in animals. Not only Malaysia, the maximum residue levels for tetracycline is inexpensive, tetracycline tetracycline as outlined in Food Act and antibiotic is also easy to administer and Regulations 1983 are set at 100 ug/kg (in effective through oral dosing via water and chicken's muscle), 200 ug/kg in eggs, 300 feed (Kelly et al., 2006). For these reasons, ug/kg in chicken's liver and 600 ug/kg for tetracycline antibiotic is extremely popular as chicken's kidney. Excessive and abuse use of a veterinary antibiotic. For example in antibiotics in poultry have also drawn great chickens, tetracycline and its derivatives are attention among consumers and affects the being used up to 10 – 500 grams/ton of feed acceptance of our livestock products when to obtain optimum rate of gain, improve feed exported to other countries. For an example, upon the detection of excessive chemicals or Mal. J. Anim. Sci. 14:61-66 (2011) Malaysian Society of Animal Production antibiotics in food commodity being exported, separately, then were mixed in 10mL bottle. the food commodity will be rejected and One mL of sodium acetate ( pH 5.5, 3 molL-1) returned. Besides paying the penalty fee and and 37% (w/v) formaldehyde (0.4ml) were added to the reaction mixture and was exporters may also be at risk in being stirred for 2 hours at room temperature blacklisted from entering the market. under light-protected condition. The solution There are several methods used to mixture was then dialyzed for 3 times with detect tetracycline residue. Microbiological 0.01M phosphate buffer saline (PBS) pH 7.4. assays are usually used for the measurement The final mixture was measured with the UV of tetracycline in food. However, these absorbance to determine the bonding of the methods are complicated, time consuming tetracycline-KLH conjugate. and lack specificity (Kurtitu et al., 2000). Immunogen preparation and Immunization chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC) are sensitive and highly specific but A 200µg dried conjugate was dissolved require expensive equipment, derivatizing in 0.5mL PBS. The conjugate suspension was treatment and time consuming sample mixed with 0.5ml of Freund's Complete cleanup process. Therefore, they are not Adjuvant and was injected subcutaneously in suitable to be used for routine screening of New Zealand white rabbits. The rabbits were large quantity of samples and field detection. rested for 3 weeks before injected with similar immunogen in 0.5ml of Freund's (ELISA) is an immunological method known Incomplete Adjuvant. For antibody titer as a rapid, sensitive, specific and cost effective determination first bleed was taken one technique that needs the presence of specific month after the injection. Blood was obtained antibody for residue detection (Zhang et al., by bleeding the central auricular artery of the 2007). Another advanced and rapid immuno- rabbit's ear. A booster injection was given based assay technique which also needs the two weeks after the first bleed. The second presence of specific antibody in tetracycline booster injection was given 2 weeks after the detection is biosensor. But the challenge is to first bleed. These booster injections and develop a high quality of the immunogen in bleeds were repeated at two-week intervals until the fourth bleed. Tetracycline is small molecule that is unable to generate the immune response by itself. Antibody purification Therefore, conjugation to a carrier protein is required for immunization to stimulate and The collected blood was allowed to also producing a higher level of this antibody. coagulate for three hours. The serum was The aim of this study was to produce a then separated by centrifugation at 5000rpm polyclonal antibody against tetracycline using KLH (Keyhole Limpet Hemocyanin) as a tetracycline was diluted with distilled water carrier protein to enhance in vivo immune (1:10) and then precipitated with saturated response in rabbit. ammonium sulphate with continuous slow stirring to precipitate the serum protein. The Materials and Methods
serum mixture was then centrifuged at 5000rpm for 30 minutes at 4°C. The pellet Conjugate preparation was resuspended in PBS and dialyzed for 3 times in 0.01M PBS buffer to remove A 0.001g tetracycline (Sigma) and 0.02 g Keyhole Limpet Hemocyanin (mcKLH Pierce, Partially purified antibody was then run USA) was dissolved in 2ml and 1.5ml of water through a protein A affinity column (nProtein Mal. J. Anim. Sci. 14:61-66 (2011) Malaysian Society of Animal Production A SepharoseTM) using AKTA prime Plus for producing anti-tetracycline antibody. To protein purifier instrument. Phosphate buffer make it immunogenic, it must be conjugated (0.01M, pH 7) was used as the binding buffer to a carrier protein before immunization and glycine buffer (0.1M, pH 3) was used as (Zhang et al., 2007). In this study, tetracycline the elution buffer. The column was was conjugated with Keyhole Limpet equilibrated with 5-column volumes before Hemocyanin (KLH) as a carrier protein to applying samples. Then, 3mL of partially generate immune response and produce purified antibody sample was injected into polyclonal antibody. KLH is a complex high the column and run with a flow rate molecular weight protein used as a carrier 2.0mLmin-1 and 0.3Mpa pressure limit. protein in antibody production because of its Fraction tubes giving the highest absorbance excellent immunogenicity and it confers to reading at 280nm were collected. One molar attach antigens (Nuria et al., 2007). To obtain of Tris-HCl pH 9 was used as neutralized evidence of successful conjugation, UV buffer and dropped into the eluted sample. absorbances recorded from 200 to 900 nm Purified antibody was run through dialysis were measured for tetracycline, KLH and again to remove salt. Antibody was then tetracycline-KLH conjugate as shown in freeze-dried and kept at 20 o C for long term Figure 1. It was observed that tetracycline had two peaks at 276 nm and 358 nm, KLH had one peak at 278 nm while tetracycline- KLH Titer determination conjugate peaks were at 275 nm and 349 nm. Conjugation between tetracycline and KLH Titer determination was conducted using had slightly shifted the reading of UV absorbance. According to Lynn et al. (1998), (ELISA). Microtiter plate was coated with UV absorbance for proteins changed during antigen and incubated overnight at 4°C. The conjugation reaction conditions and the UV plate was emptied and washed 3 times with absorbance for hapten also changed when PBS-Tween (250µL per well). The well surface coupled to proteins. was then blocked by treating with 1:10 (v/v) The tetracycline-KLH conjugates were solution of milk diluents in PBS for 30 injected into New Zealand white rabbits with minutes (250µL per well). Then, the plate was the addition of Freund's Adjuvant which was emptied and washed 3 times with PBS-Tween. an inexpensive strategy for polyclonal Purified antibodies were added (100µL per production. Freund's adjuvant, which is well) and the plate was incubated for 2 hours paraffin oil based, had been used for at 37°C. The plate was emptied and washed 3 stimulation of the immune system by Alkaline phosphatase enzyme conjugate Adjuvant to generate high antibody titers (1:1000) which was diluted in PBS was added (Trott et al., 2008). Not only this adjuvant in the well (100µL per well) and incubated for activated the immune system, it also retained 30 minutes. Again, the plate was emptied and the antigen to be released slowly into the washed 3 times with PBS-Tween. Lastly, injection site (Bollen et al., 1996). Serum 100µL solution of P-Nitrophenyl Phosphatase obtained from each bleed was purified using diluents in diethanolamine (1:1000 w/v) was two main steps of antibody purification. First, added and the plate was read at 405nm. serum was precipitated using saturated ammonium sulphate to precipitate antibody. Results and Discussion
Partially purified antibody was then run through Protein A affinity column to obtain Tetracycline is a small molecule with a pure IgG antibody against tetracycline. High molecular size of 444.4 Dalton. Therefore, yields of pure IgG antibody could be obtained tetracycline itself is non-immunogenic and using Protein A because it was very effective not able to elicit immune response in animals aseptically in purification strategies (Page and Mal. J. Anim. Sci. 14:61-66 (2011) Malaysian Society of Animal Production also suggested that the binding and eluted chromatogram of IgG fraction elution from buffer used in this experiment were suitable Protein A affinity column with two peaks to purify the antibody. The purified antibody obtained from the graph. Peak 1 showed that fractions were collected via fraction tubes the antibody had successfully bound with which showed the highest absorbance at protein A while peak 2 showed that the bound antibody had successfully eluted. This finding (A) Tetracycline (B) KLH (C) Tetracycline-KLH conjugate
Figure 1. Synthesis of tetracycline-KLH conjugate when scanned through spectrophotometer wave-
length. Two peaks of tetracycline was observed at 276 nm and 358 nm, (B) Peak of KLH at 278 nm and
(C) Tetracycline- KLH conjugate peaks were at 275 nm and 349 nm.
AT2010Jul09no004:10_UV Figure 2: Chromatogram of IgG elution from Protein A affinity column using AKTAprime protein purifier Mal. J. Anim. Sci. 14:61-66 (2011) Malaysian Society of Animal Production In the present study antibody titer booster injections given to the rabbits during determination was conducted through ELISA immunization period had stimulated the body method. The higher antibody titer indicated to produce more specific IgG against the good quality of antibody produced. The tetracycline, as previously reported by results as shown in Figure 3 indicated that the Faridah (2010). Antibody production was antibody production against tetracycline was highest at the fourth bleed reaching an successful compared to the preimmune absorbance of 1.2 at 1 mgmL-1 concentration. antibody. Preimmune was the serum taken All antibody bleeds declined dramatically at before the rabbits were exposed to antigen. dilutions greater than 0.0001 mgmL-1 protein. This suggested that the immunization and The optimum and economic concentration of purification method used were successful. antibody was about 0.00001 mg/ml. Based on Figure 3 also shows the whole pattern of the antibody titer result, KLH can be antibodies level in rabbits for all bleeds. considered as a good carrier protein for a Absorbance reading and antibody titer small haptein molecule such as tetracycline.
increased from first to fourth bleed. The Antibody concentration, mg/ml
Figure 3: Pattern of antibody production against tetracycline in rabbit for all bleeds Conclusion
successfully purified using Protein A affinity column and the fourth bleed showed the best The result of our study revealed that antibody titer sensitivity. Polyclonal antibody tetracycline was successfully conjugated with produced against tetracycline in this study KLH as the carrier protein and was able to has the potential to be used as biomaterial in generate immune response when immunized the detection of tetracycline residue where in white rabbits with the addition of Freund's the optimum and economic concentration of antibody is 0.00001mg/ml. Mal. J. Anim. Sci. 14:61-66 (2011) Malaysian Society of Animal Production Lynn, L.J., Daniel, A. J. and Bruce, D.H. 1998. This work was supported by MARDI via WRM Immunosorbent Assay for atrazine fund (JP-RB-0224). mercapturic acid in human urine. Chem. Res. Toxic. 11: 342-352. References
Nuria, P.N., Sergi, M., Angel, M. and Rosa, P. Aga, D.S., Goldfish, R. and Kulshrestha, P. 2003. development of a sensitive immunoassay Application of ELISA in determining the for tetracycline residues application to fate of tetracyclines in land-applied honey samples. Analytica Chimica Acta. livestock waste. Analyst. 128: 658-662. Bollen, L.S., Crowley, A., Stodulski, G. and Hau, Page, M. and Thorpe, R. 2008. IgG purification J. 1996. Antibody production in rabbits in immunochemical protocols, Second and chickens immunized with human IgG. Edition, Ed. John D. Pound. New York: A comparison of titre and avidity Humana Press. Pp: 95-111. development in rabbit serum, chicken Trott, D.L., Hellested, E.M., Yang, M. and Cook, serum and egg yolk using three different M.E. 2008. Addition of killed whole cell adjuvants. J. Immuno. Meth. 191: 113- 120. Complete Adjuvant alter laying hen antibody response to soluble protein typhimurium. Ph.D. Thesis. Cranfield antigen. Poult. Sci. 87: 912-917. Zhang, Y., Shengxin, L., Wei, L., Zhao, C. and Kelly, M., Tarbin, J. A., Ashwin, H. and Rimo, X. 2007. Preparation of anti- tetracycline antibodies and development compliance with organic meat production of an indirect heterologous competitive standards by detection of permitted and enzyme linked immunosorbent assay to nonpermitted uses of veterinary medicines detect residues of tetracycline in milk. J. (tetracyclines antibiotics). J. Agric. Food Agric. Food Chem. 55: 211-218. Chem. 54: 1523-1529. Kurtittu, J., Lonnberg, S., Virta, M. and Karp, M. A. 2000. A group specific microbiological test for the detection of tetracyclines residues in raw milk. J. Agric. Food Chem. 48: 3372-3377.


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