Methods are needed that are able to discriminate between ruminant and non-ruminant PAPs for facilitating a safe re-introduction of non-ruminant PAPs in aqua feed. Therefore, two immunoassays, MELISA-TEK® and Reveal, have been tested in an interlaboratory study for their capability to detect ruminant PAPs processed according to European conditions. 15 non-ruminant animal protein samples were spiked at 0.5%, 1.0% and 2.0% with ruminant material, sterilised at either 133 oC or 137 oC, and combined with the six basic materials as blanks. Fourteen participants passed the entrance test and investigated the 21 samples. For both MELISA-TEK® and Reveal specificity and sensitivity were at 97% or higher. Concordance and accordance were also higher than 95%, except for the concordance of the blank samples with Reveal (94.2%). The results indicate that both assays are validated at 0.5% and higher for the detection of ruminant PAPs (sterilised at 133 oC) with non-ruminant PAPs as matrix. Given the 2% upper limit of ruminant PAPs in non-ruminant PAPs for avoiding an increase in BSE incidents, these methods are fit for monitoring non-ruminant PAPs intended for aqua feed.

1. Immunoassay validation for monitoring ruminant
proteins in aquafeed
L.W.D. van Raamsdonk, M. Bremer, RIKILT Wageningen
R. Margry, R. van Kaathoven, CCL, Veghel

2. Animal proteins monitoring
•
Microscopy: screening; visible particles
•
PCR: identification; DNA
•
Near Infrared spectroscopy / microscopy
•
Immunoassays: screening or confirmation; proteins
•
Other
L.W.D. van Raamsdonk et al., Beijing, Feedsafety conference, 12-9-2012

3. Animal proteins in aquafeed
Aquafeed
Non-ruminant PAP
25%
2%
Fishmeal
10% gut content
Feed for weaning
ruminants
5%
L.W.D. van Raamsdonk et al., Beijing, Feedsafety conference, 12-9-2012

4. Animal proteins in aquafeed
EFSA critical limit: 2% ruminant-PAP in
non-ruminant PAP (EFSA, 2011)
Concentration of PAP in ruminant feed:
0.25 * 0.1 * 0.05 = 0.00125%
LOD microscopy: 0.0025 %
(Veys et al., 2009)
EFSA critical limit: 0.1% AP in
feed (EFSA, 2011)
Current scenario: 0.000025% of
ruminant PAP in ruminant feed
L.W.D. van Raamsdonk et al., Beijing, Feedsafety conference, 12-9-2012

5. Animal proteins in aquafeed
Non-ruminant PAP
aquafeed
monitoring
2%
< 2%:
Certification for aquafeed
25%
fishmeal
10%
Ruminant feed
5%
< 0.1%:
Approved as feed
L.W.D. van Raamsdonk et al., Beijing, Feedsafety conference, 12-9-2012

6. Immunoassays
(EFSA, 2011)
L.W.D. van Raamsdonk et al., Beijing, Feedsafety conference, 12-9-2012

7. Immunoassay validation; in house validation MELISA-TEK
Matrix: porcine proteins
RR 1 (133°C)
18.0
RR 3 (141°C)
RR 4 (145°C)
Mean OD sample - Mean OD blank (450nm)
RR 2 (137°C)
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
0.0
0.5
1.0
Spike rate (%)
1.5
2.0
L.W.D. van Raamsdonk et al., Beijing, Feedsafety conference, 12-9-2012

8. Immunoassay validation; in house validation MELISA-TEK
•
Specificity and robustness
TN
FP
Porcine PAP
27
0
Poultry PAP
21
1
Other material *
9
0
Milk powder
2
0
Threshold: mean OD ratio + 3 * SD
*: blood plasma, porcine DCP/TCP, feather meal, fish meal, gelatine
L.W.D. van Raamsdonk et al., Beijing, Feedsafety conference, 12-9-2012

9. Immunoassay validation; interlaboratory study
•
MELISA-TEK and Reveal
•
16 participants
•
Three parts: training samples, entrance test, final study
•
After entrance test: 14 participants
•
Period: January to May 2012
•
AOAC guidelines, as far as applicable
L.W.D. van Raamsdonk et al., Beijing, Feedsafety conference, 12-9-2012

10. Immunoassay validation; results Melisa-Tek
•
Accuracy (n=84)
133 ºC
Blank
137 ºC
98.8%
0.5%
100%
1.0%
100%
100%
2.0%
100%
100%
L.W.D. van Raamsdonk et al., Beijing, Feedsafety conference, 12-9-2012

11. Immunoassay validation; results Reveal
•
Accuracy (n=84)
133 ºC
Blank
137 ºC
97.0%
0.5%
100%
1.0%
98.8%
98.8%
2.0%
100%
100%
L.W.D. van Raamsdonk et al., Beijing, Feedsafety conference, 12-9-2012

12. Immunoassay; future strategy
•
Current situation: performance limit (0.5% < 2.0%) approved
•
Future target: performance limit: 0.1%
•
Option: concentration experiments for target (= troponine)
L.W.D. van Raamsdonk et al., Beijing, Feedsafety conference, 12-9-2012

13. Immunoassay; conclusions
•
Immunoassays (Melisa-Tek and Reveal) are validated at
0.5% and higher with PAP as matrix
•
A strategy with immunoassays will avoid the presence of
positive signals for legally applied ingredients
•
The design of the study can be used as guideline for future
studies with qualitative results
•
Application of immunoassays after muscle concentration
could be feasible to reach the 0.1% performance limit
L.W.D. van Raamsdonk et al., Beijing, Feedsafety conference, 12-9-2012

14. THANK YOU