Production of chicken yolk IgY to sulfamethazine: comparison with rabbit antiserum IgG

Abstract

Sulfamethazine was used as target analyte to produce IgY from chicken aiming to compare the performance of these two antibodies in term of yield, titer, sensitivity, selectivity and matrix effect under parallel conditions. The results showed that the total yield of IgY produced by one chicken during 43 weeks' experimental period was about 15 g. This output is much more than IgG (150 mg from one rabbit). Besides, IgY titers increased during 10 weeks and remained stable over 30 weeks. The peak titer of IgY was 1:2¹⁸. As the antibody titer rose, the sensitivity was increased. For IgG, the maximum titer was observed to be 1:2²³. In addition, both IgY and IgG were highly sensitive. The limits of detection were 0.54 ng/mL for IgY and 0.24 ng/mL for IgG. These results indicated that the IgY potentially provides a practical and ethical alternative to IgG in veterinary drug residue immunoanalysis.

Keywords:

• IgY
• IgG
• sulfamethazine
• ELISA
• comparison

1. Introduction

Immunoassays are widely used bioanalytical method for the detection of veterinary drug residues due to their low cost, specificity and high sensitivity (Dias & Tambourgi, 2010; Reig & Toldra, 2011). These methods are usually based on polyclonal or monoclonal antibodies, which are produced in mammal. However, there are certain limitations in using mammal-produced antibodies, such as complex preparation, high cost, frequent bleeding of animals and low titer against mammalian antigens (Klimentzou et al., 2006; Larsson & Sjoquist, 1990; Tu, Chen, & Chang, 2001). Recently, chicken yolk antibody (IgY) has attracted considerable attention for the prevention and treatment of infectious gastrointestinal diseases (Hirai et al., 2010; Malekshahi, Gargari, Rasooli, & Ebrahimizadeh, 2011; Rahman, Van Nguyen, Icatlo, Umeda, & Kodama, 2013; Sarker, Pant, Juneja, & Hammarstrom, 2007). IgY is isolated from the egg yolk of immunised hens instead of blood or ascitic fluid. It is a non-invasive method other than the generation of polyclonal or monoclonal antibodies. Moreover, this technology is able to continuously produce large quantities of IgY (Meulenaer & Huyghebaert, 2001; Schade et al., 2005; Svendsen, Crowley, Stodulski, & Hau, 1996). For example, one immunised chicken can produce as much as 3 grams of IgY per month, which is much more than the serum antibodies obtained from one rabbit (Tini, Jewell, Camenisch, Chilov, & Gassmann, 2002). Nowadays, IgY is applied in the detection of chemical compounds (Bottari, Oliveri, & Ugo, 2014; Meulenaer, Baert, Lanckriet, Van Hoed, & Huyghebaert, 2002; Sotiropoulou, Pampalakis, Prosnikli, Evangelatos, & Livaniou, 2012; Van Coillie, Block, & Reybroeck, 2004; Wang et al., 2013). However, few immunoassays based on IgY were reported for the detection of veterinary drugs. Probably because there was little report to investigate the advantages and disadvantages of IgY and IgG against veterinary drugs.

Sulfamethazine (SMZ) is a widely used veterinary drug to treat and prevent various diseases, such as gastrointestinal and respiratory tract infections (Agunos, Léger, & Carson, 2012; Clark, Mackey, & Scheel, 1966; O'Connor, Wellman, Rice, & Funk, 2010; Sampson, Bing, Grueter, Ose, & Havens, 1973). As a consequence, the overuse of SMZ has caused many food safety issues (Ding et al., 2006; Ko, Song, & Park, 2000; Shaikh & Chu, 2000). In China and the European Commission, the maximum residue level for SMZ in animal product is set at 100 µg/kg (Chinese Agriculture Ministry, 2002; European Community Council Regulation, 1990). The aim of this study was to produce IgY against SMZ for the first time and evaluate IgY and IgG by systematically comparing their advantages and disadvantages.

2. Materials and methods

2.1. Apparatus

White opaque 96-well polystyrene microtiter plates were purchased from Costar Inc. (Milpitas, CA, USA). Spectra Max M5 micro-plate reader (Molecular Devices, Sunnyvale, CA, USA) was used for this experiment. Centrifugation was performed with a refrigerated centrifuge supplied by Shanghai Anting Scientific Instrument Factory (Shanghai, China). Protein concentration was carried out using the Thermo NanoDrop 2000 (Waltham, MA, USA). Water was purified using a Milli-Q system (Bedford, MA, USA). Other reagents were analytical grade and obtained from Beijing Regent Corp. (Beijing, China).

2.2. Reagents and buffers

SMZ, sulfamerazine, sulfadimethoxine, sulfamethoxazole, sulfabenzamide, sulfapyridine, sulfaquinoxaline, sulfamethoxypyridazine, sulfamonomethoxine, sulfachlorpyrazine, sulfadoxine, sulfadiazine, sulfamethoxydiazine, sulfaclozine, sulfathiazole, sulfisoxazole, sulfabenzamine, sulfanilamide, sulfaguanidine, sulfamethizole, sulfaphenazolum, sulfaethoxypyridazine, bovine serum albumin (BSA), ovalbumin (OVA), Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), 3,3′,5,5′-tetramethylbenzidine, urea hydrogen peroxide and casein were purchased from Sigma-Aldrich (St. Louis, MO, USA). The peroxidase-conjugated rabbit anti-chicken IgY (HRP-IgY) and peroxidase-conjugated sheep anti-rabbit IgG (HRP-IgG) were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Proclin-300 was purchased from Zymed Laboratories, Inc. (South San Francisco, CA, USA). Other reagents used were analytical grade from Beijing Reagent Corp. (Beijing, China).

The coating buffer was 0.05 M carbonate solution (pH 9.6), containing 0.015 M Na₂CO₃ and 0.035 M NaHCO₃. The blocking buffer consisted of 0.01 M phosphate buffered saline (PBS) (pH 7.4), 5% bovine serum, 0.5% casein (w/v) and 0.01% proclin 300. Assay buffer was 0.01 M PBS containing 0.15 M NaCl and 2.7 mM KCl. PBS with 0.05% Tween 20 (v/v) was used as washing solution. PBS with 5% foetal bovine serum was used for the dilution of peroxidase-conjugated rabbit anti-chicken IgY and sheep anti-rabbit IgG. The substrate was 0.1% 3,3′,5,5′-tetramethylbenzidine and 0.05 M citrate buffer, pH 4.5. The stop solution was 2 M H₂SO₄. The stock solutions of SMZ and structure-related sulfonamides (including sulfamerazine, sulfadimethoxine, sulfamethoxazole, sulfabenzamide, sulfapyridine, sulfaquinoxaline, sulfamethoxypyridazine, sulfamonomethoxine, sulfachlorpyrazine, sulfadoxine, sulfadiazine, sulfamethoxydiazine, sulfaclozine, sulfathiazole, sulfisoxazole, sulfabenzamine, sulfanilamide, sulfaguanidine, sulfamethizole, sulfaphenazolum, sulfaethoxypyridazine) in methanol (2 mg/mL) were stored at −20°C in amber glass bottles.

2.3. Synthesis of immunogen and coating antigen

Glutaraldehyde was used for the coupling of SMZ with OVA or BSA according to the previous articles (Haasnoot et al., 2000; He, Shen, Suo, Jiang, & Hou, 2005). Briefly, 63.6 mg of SMZ and 10 mg of BSA were dissolved in 2.5 mL of PBS (0.01 M, pH 7.4). About 150 µL of 1% glutaraldehyde was added and incubated for 3 h at room temperature. Then this mixture was dialysed for three days in PBS. The obtained immunogen, SMZ-G-BSA, was stored at −20°C for further use. The coating antigen (SMZ-G-OVA) was synthesised by using 30 mg of SMZ and 5 mg of OVA in the same strategy as SMZ-G-BSA.

2.4. Immunisation

The use of experimental animal in this study was approved by the Animal Care Committee of Beijing. The animal house in China Agricultural University has been certificated in 2004 and evaluated twice each year by the same committee. Three leghorn chickens and four New Zealand white rabbits were used to raise specific antibodies. The chickens were housed in 0.5 m × 0.5 m floor pens with nest boxes in the standard animal room (20°C ± 4°C). They were fed laying hen diet, and water ad libitum. The rabbits were kept in single cages in rabbit standard animal room (20°C ± 4°C) and fed rabbit diet, water ad libitum. Care of the animals was in accordance with animal welfare act.

For the first immunisation, three chickens were immunised intramuscularly in the breast muscle with 200 µg immunogen (SMZ-G-BSA) in PBS, which was emulsified in FCA (1:1, v/v). Each of the four rabbits was subcutaneously injected on the backs, using the same antigen and dose as chicken immunisation treatment. Booster immunisations for both chickens and rabbits were carried out at weeks 2, 4, 6, 8, 11, 13 and 16 with 250 µg immunogen in PBS with FIA (1:1, v/v). Finally, the immunisation procedure was stopped.

2.5. Antibody isolation and quantity determination

The eggs were collected from each immunised chicken every day for further use. IgY was isolated from chicken yolks according to the method of Akita and Nakai (1992) with slight modification. Egg yolk was separated from egg white and dried with filter paper, and then it was diluted 6-fold with water. The pH was adjusted between 5.0 and 5.2 by 1 M HCl. The mixture was incubated overnight at 4°C and then centrifuged (10,000 rpm, 30 min, 4°C). The supernatant was filtered through filter paper. Subsequently, saturated ammonium sulfate was added with stirring in order to achieve a 40% saturated solution. After overnight incubation, this solution was centrifuged (5000 rpm, 20 min, room temperature). The residue was dissolved in 5 mL PBS and stored in small aliquots at −20°C for further use. The concentration of IgY was evaluated by the NanoDrop assay according to the instructions.

In order to monitor the presence of IgG in rabbit, blood samples (1 mL) were collected from the marginal ear vein one week after each booster immunisation. Finally, each rabbit was exsanguinated by heart punctures one week after the final booster immunisation and the blood samples were collected. Then all the rabbits were euthanised by lethal injection. Blood samples were allowed to incubate at 4°C overnight, followed by centrifuging to remove particulate material. The serum was kept at −20°C before use.

2.6. Antibody titer determination

To study the response of the immunised chickens and rabbits, noncompetitive ELISA was adopted to test the titers of antibodies. Microplates were previously coated with coating antigen and blocked. Then 100 µL of IgY or IgG (at appropriate dilution) was added into each well. After incubating for 30 min at 37°C, the microplates were washed. After that, rabbit anti-chicken IgY-HRP or sheep anti-rabbit IgG-HRP (1/5000, 100 µL/well) was added, followed by another incubation. The plates were washed and substrate solution was added (100 µL/well). Finally, the enzyme reaction was stopped by sulfuric acid. The absorbance was measured by microplate absorbance reader (450 nm as test wavelength and 630 nm as reference wavelength). Preimmune IgY preparation or rabbit serum was used as negative control, and antibody titer was defined as the maximum dilution that gave an absorbance of 2.2 times that of the preimmune chickens or rabbits.

2.7. Antibody sensitivity determination

Indirect competitive ELISA was employed to test the sensitivity of IgY and IgG. The procedure was similar to the noncompetitive ELISA described above. Checkerboard titration was carried out to select the optimal concentration of coating antigen and antibody dilution. Fifty microlitres of SMZ and 50 µL of antibody were added to each well. Antibodies with relatively low IC₅₀ values would be used in the further experiments. In this study, the IC₅₀ value, dynamic range and limit of detection (LOD) were served as the analyte concentrations obtained at 50%, 20%–80% and 90%, respectively. Standard curves were obtained by plotting inhibition rate against the value of analyte concentration and fitted to the four parameters function. The equation was:

where A = response at high asymptote, B = slope factor, C = concentration corresponding to 50% specific binding (IC₅₀), D = response at low asymptote and X = calibration concentration.

2.8. Antibody selectivity

The selectivity of the antibodies was tested using 22 structure-related sulfonamides. The test compounds were SMZ, sulfamerazine, sulfadimethoxine, sulfamethoxazole, sulfabenzamide, sulfapyridine, sulfaquinoxaline, sulfamethoxypyridazine, sulfamonomethoxine, sulfachlorpyrazine, sulfadoxine, sulfadiazine, sulfamethoxydiazine, sulfaclozine, sulfathiazole, sulfisoxazole, sulfabenzamine, sulfanilamide, sulfaguanidine, sulfamethizole, sulfaphenazolum and sulfaethoxypyridazine. The ELISA was performed as described above. The selectivity of antibody was revealed by the cross-reactivity (%) for each interfering compound, and it was calculated as the following equation:

2.9. Matrix effect

Dairy products are frequently contaminated with SMZ. A preliminary study was carried out to test the matrix effect of milk. Milk samples bought in a local shop were centrifuged at 4°C (10,000 rpm, 10 min), and the upper fat layer was removed. Artificially contaminated milk samples were used to obtain standard curves. These samples were diluted with PBS at different spiked levels. Finally they were compared to the curves prepared in buffer. The dilutions of spiked milk were 1:2, 1:5 and 1:10, respectively.

2.10. Recovery test

To confirm the precision and trueness of the developed ELISA, recovery test was performed. Blank milk samples were fortified with SMZ at the levels of 1/2, 1 and 2 × MRLs. Samples were thoroughly mixed and centrifuged for 10 min at 10,000 rpm to remove the upper fat layer. Then they were diluted with PBS. Fifty microlitres of these diluted milk samples were used in the indirect competitive ELISA assay.

3. Results and discussion

3.1. Antibody isolation and quantity determination

In this study, IgY was separated from egg yolk using acidified water dilution method due to its cheap cost, high purity and facility of the process (Akita & Nakai, 1992, 1993). The quantity of specific antibody was determined by NanoDrop according to the instructions. During the 43-weeks experimental period, each hen laid about 200 eggs (except one hen that barely laid eggs after immunisation). About three-fourths of those eggs were obtained during the highest activity period. And nearly 0.1 g of IgY can be recovered per yolk after treatment. These results implied that the total quantity of IgY with high titer and good sensitivity produced by one chicken was about 15 g (0.1 × 150). In the case of rabbit, considering that multiple bleeding would seriously hurt the rabbits, antiserum was collected by heart puncture. And about 150 mg of IgG were obtained from 30 mL of antiserum per rabbit. These data indicated that the IgY output per hen was significantly higher than IgG output per rabbit.

3.2. Antibody titer and sensitivity determination

Indirect competitive ELISA was introduced to detect the changes of IgY and IgG titers. Since the concentration of coating antigen may have significant effect on the antibody titer, the same concentration of coating antigen was employed in the comparison of IgY and IgG. Because one of the three chickens stopped laying eggs 30 days after the first immunisation, the IgY produced by this chicken was excluded from consideration. The remaining two chickens immunised with SMZ-G-BSA responded consistently against the antigen. As can be seen from Figure 1a, a sharp rise of titer was observed after the third booster immunisation. After the fourth and fifth booster immunisations, it reached a maximum titer of 1:2¹⁸. Since large numbers of useful eggs were already collected, the detection of antibody titers and IC₅₀ values were stopped at 43 weeks after the first immunisation. With the rise of IgY titer, the IC₅₀ values of the ELISA were decreasing to a relatively low level (Figure 2a). The lowest IC₅₀ value was less than 15 ng/mL, and this low IC₅₀ value remained stable for more than 30 weeks. For rabbit IgG, the maximum IgG titer against SMZ was observed to be 1:2²³ after the third booster immunisation (Figure 1b). It is 32 times higher than IgY. But the changing trends in antibody titer and IC₅₀ values of the ELISA were similar to IgY (Figure 2).

Figure 1. Time-dependent changes in antibody titer of (a) IgY and (b) IgG after immunisation.Each point is the titer of IgY or IgG. The arrows indicate immunisation time points. Antibody titer was defined as the maximum dilution that gave an absorbance of 2.2 times that of the preimmune chickens or rabbits.

Figure 2. Time-dependent changes in IC₅₀ values of (a) IgY and (b) IgG after immunisation.Each point is the IC₅₀ value of IgY or IgG. The arrows indicate immunisation time points.

Therefore, taking both quantity and titer of the two antibodies into consideration, the final productivity of IgY obtained from one chicken was about three times greater than IgG isolated from a rabbit.

3.3. Selectivity determination

In order to determine the specificity of the IgY and IgG, cross-reactivity towards various structure-related sulfonamide analogues was evaluated. Two antibodies (Chicken 1, day 165 and Rabbit 3) with relatively low IC₅₀ values were used in this assay. Table 1 summarised the IC₅₀ values and cross-reactivity of the two antibodies against other sulfonamides. The cross-reactivity testing showed that both IgY and IgG had a high specificity for SMZ. There was no significant cross-reactivity with the other sulfonamides except for sulfamerazine. The IC₅₀ values towards SMZ and sulfamerazine using IgY were 6.76 ng/mL and 53.53 ng/mL, indicating a 12.63% cross-reactivity. In the case of IgG, the IC₅₀ values towards SMZ and sulfamerazine were 4.76 ng/mL and 78.81 ng/mL, meaning 6.04% cross-reactivity. The cross-reactivity study showed that both IgY and IgG had high specificity towards SMZ. But IgG was slightly better than IgY in distinguishing SMZ from sulfamerazine.

Table 1. Cross-reactivity (CR) results of IgY (Chicken 1 day 165) and IgG (Rabbit 3) towards several related sulfonamides.

	IgY		IgG
Sulfonamide	IC50 (ng/mL)	CR (%)	IC50(ng/mL)	CR (%)
Sulfamethazine	6.76	100	4.76	100
Sulfamerazine	53.52	12.63	78.81	6.04
Sulfadimethoxine	361.50	1.87	>1000	<1
Sulfamethoxazole	>1000	<1	>1000	<1
Sulfabenzamide	>1000	<1	>1000	<1
Sulfapyridine	>1000	<1	>1000	<1
Sulfaquinoxaline	>1000	<1	>1000	<1

3.4. Matrix effect

Matrix effect of samples may lead to poor accuracy of immunoassays. An important step for the evaluation of immunoassay is the assessment of matrix effect. To gain basic information on matrix effect of IgY and IgG, standard curves generated in PBS were compared with curves obtained in diluted milk samples. Figure 3 showed that the IC₅₀ values of sulfamerazine using IgY were 6.56, 4.31 and 6.06 ng/mL when dilution factors were 1:2, 1:5 and 1:10, respectively, while the IC₅₀ value of standard curve was 6.76 ng/mL. Though the IC₅₀ values showed minor differences at different dilutions of milk and PBS, the optical density values decreased significantly when the dilution factors were 1:2 and 1:5. Therefore, a 1:10 dilution would be good enough to reduce milk matrix effect on ELISA of IgG. The working range of this method was 1.32–27.81 ng/mL, and the LOD was 0.54 ng/mL. Similar results were obtained using IgG in the milk matrix effect study (Figure 3b). In this case, the IC₅₀ of ELISA using IgG at 1:10 dilution was 4.18 ng/mL, similar to 4.76 ng/mL in PBS. The working range and LOD were 0.68–25.54 ng/mL and 0.24 ng/mL, respectively. The parameters of calibration curves using IgY and IgG in PBS and 10 times diluted milk were shown in Table 2. Generally, it can be presumed that IgY and IgG we obtained in the study had similar tolerance ability for milk matrix.

Figure 3. Calibration curves for sulfamethazine using (a) IgY (Chicken 1 day 165) and (b) IgG (Rabbit 3) prepared in PBS and different dilutions of milk in PBS.

Table 2. Detailed parameters of the calibration curves using IgY (Chicken 1 day 165) and IgG (Rabbit 3) in PBS and milk samples.

	IgY		IgG
Parameter	PBS	Milk samples	PBS	Milk samples
Ab concentration (µg/mL)	1.52	1.52	0.93	0.93
Ag concentration (µg/mL)	0.78	0.78	0.13	0.13
ODmax	1.37	1.34	1.48	1.35
ODmin	0.19	0.15	0.07	0.11
Detection range	1.71–26.75	1.32–27.81	0.46–49.04	0.68–25.54
IC50 (ng/mL)	6.76	6.06	4.76	4.18
LOD (ng/mL)	0.76	0.54	0.11	0.24
Sample dilution	–	10	–	10
R^2	0.99878	0.99876	0.99943	0.99941

3.5. Recovery test

Considering that the calibration curves prepared in 1:10 diluted milk were consistent with those generated in PBS, blank milk samples were fortified with SMZ and then diluted 10 times in PBS. The results of the recoveries of IgY and IgG were summarised in Table 3. The average recoveries (n = 5) for IgY and IgG were in the range of 96.2–131.8% and 101.2–114.4%, respectively. Assay reproducibility was satisfied. For IgY, the coefficient variation (CV) was ranging from 4.8% to 12.0%, while for IgG, it is ranging from 10.0% to 12.1%. These data indicated that both IgY and IgG could be used to determine SMZ in milk samples.

Table 3. Recoveries of sulfamethazine in milk samples using IgY (Chicken 1 day 165) and IgG (Rabbit 3).

Antibody	Fortified (ng/mL)	Measured (ng/mL)	Recovery (%)	CV (%)
IgY	50	65.9	131.8	6.1
	100	110.7	110.7	4.8
	200	192.3	96.2	12.0
IgG	50	57.2	114.4	10.0
	100	113.5	113.5	10.9
	200	202.4	101.2	12.1

4. Conclusion

IgY from chickens against SMZ was successfully produced. The comparison between IgY and rabbit antiserum IgG was also discussed in this study. The properties of IgY are similar to IgG in terms of sensitivity, selectivity and matrix effect. Both antibodies were highly sensitive, providing a LOD 0.54 ng/mL for IgY and 0.24 ng/mL for IgG. The IgY we reported in this study is complementary to the existing mammal antibodies, and it potentially provides a practical and ethical alternative in veterinary drug residue immunoanalysis.

Additional information

Funding

This work was supported by grants from National Natural Science Foundation of China [grant numbers 31372475 and U1301214], Chinese Universities Scientific Fund [grant number 2013QJ048] and Trans-Century Training Programme Foundation for the Talents by the Ministry of Education [grant number NCET-12-0529].