Fig. 11. Improvement of standard curve of mAb-ELISA for D-Mb. MAb 11H against peptide B was used as the capture antibody, and mAb 11E against D-Mb was used as the peroxidase-conjugated antibody (●) or the biotin-conjugated antibody (■).
0.0 0.5 1.0 1.5 2.0 2.5
0 100 200 300
Absorbance at 450 nm
Fig. 12. Specificity of mAb-ELISA. MAb 11H against peptide B was used as the capture antibody, and mAb 11E against D-Mb was used as the biotin-conjugated antibody. Circle (●): beef D -Mb; square (■): pork D-Mb;
and triangle (▲): chicken D-Mb.
0.0 0.5 1.0 1.5 2.0
10 100 1000 10000
Absorbance at 450 nm
Myoglobin concentration (ng/ml) 0
Fig. 13. Standard curve of mAb-ELISA for the beef protein solution.
Beef protein solution was applied to mAb-ELISA. A linear regression line was obtained for the beef protein solution at 0 – 500 ng/ml. The equation was statistically expressed as Y = 0.0015 X + 0.1456, R² = 0.9994, where Y is the absorbance and X is concentration of the beef protein solution.
0.0 0.2 0.4 0.6 0.8 1.0
0 100 200 300 400 500
Absorbance at 450 nm
Protein concentration (ng/ml)
Basic meat
Mixing ratio of beef to basic meat
(%)
Non-heated Heated at 80oC
Heated at 120oC
Beef 100 10.8 3.9 0.6
Pork 10 1.0 0.5 0.1
1 0.11 0.04 0.01
0.1 0.009 0.003 0.000 0.01 0.001 < LOD < LOD
0 < LOD < LOD < LOD
Chicken 10 1.2 0.3 0.1
1 0.08 0.03 0.00
0.1 0.003 0.001 0.000
0.01 < LOD < LOD
0 < LOD < LOD < LOD
LOD: limit of detection *: less than quantification limit.
Beef protein content (μg/ml) Table 1. Determination of beef protein content in model processed foods by pAb-ELISA.
Samples Beef Other
Beef jerky + wheat, soy, apple 126.4
Sausage + pork 43.8
Meat sause + soy 2.8
Meat ball + chicken, pork,
wheat, milk 0.1
Salami - pork, gelatin,
milk, soy < LOD
Pasta sause - milk, soy < LOD
Hamburger steak - chicken, wheat,
soy, apple < LOD Demi glace sause - chicken, wheat, milk,
apple, pork, gelatin < LOD LOD: limit of detection
Beef protein content
(mg/g) Labeling
Table 2. Determination of beef protein contents in several commercial foods by pAb-ELISA.
Model foods
Mean (mg/ml)
S.D.
(mg/ml)
C.V.
(%)
Non-Heated 8.0 8.4 0.3 3.3
Heated at 80oC 3.0 7.5 0.1 1.8
Heated at 120oC 2.2 6.4 0.2 2.8
aThe precision in mAb-ELISA was evaluated by using beef model processed foods, which were prepared with minced beef meat, potato starch, salt and water and
incubated at room temperature (non-heated), 80 oC or 120 oC for 30 min. Mean was calculated from nine replicates of the same sample solution. S.D. = standard
deviation. C.V. = coefficient of variation.
bThe protein content in sample solutions was determined by a 2-D Qunat Protein Assay Kit.
Table 3. The precision of mAb-ELISA using beef-based model processed foodsa. Beef protein content
Protein contentb (mg/ml)
a The precision in mAb-ELISA was evaluated by using beef model processed foods, which were prepared with minced beef meat, potato starch, salt and water and incubated at room temperature (non -heated), 80 oC or 120 oC for 30 min. Mean was calculated from nine replicates of the same sample solution. S.D. = standard deviation. C.V. = coefficie nt of variation.
b The protein content in sample solutions was mesured with a 2 -D Qunat Protein Assay Kit.
Basic meat
Beef 100 8.4 7.5 6.4
Pork 10 0.8 0.5 0.9
1 0.1 0.04 0.1
0.1 0.01 0.01 0.01
0.01 0.001 0.001 0.001
0 < LOD < LOD < LOD
Chicken 10 1.1 0.4 0.5
1 0.1 0.1 0.03
0.1 0.01 0.003 0.01
0.01 0.001 0.000 0.001
0 < LOD < LOD < LOD
LOD: limit of detection .
Table 4. Determination of beef protein content in model processed foods by mAb-ELISA.
Heated at 80oC
Beef protein content (mg/ml) Heated at
120oC Non-heated
Mixture ratio of beef to basic meat
(%)
a PBS (pH 7.2) containing 0.5 % SDS and 2 % 2 -mercaptoethanol (2-ME) or only PBS was used for extraction of proteins in each sample. Protein conte nt of sample solutions was measured with a 2D Quant Protein Assay kit (GE Healthcare), respectively.
b Beef protein content of each sample solution was determined by mAb -ELISA.
c The results evaluated by the commercial ELISA kit were represented that the absorbance of sample solutions was greater than that of beef positive control (+).
Beef extracts
PBS with
SDS & 2-ME PBS
PBS with
SDS & 2-ME PBS
Paste 1 0.8 0.1 0.3 0.001 +
Paste 2 2.0 0.9 0.7 0.004 +
Paste 3 0.8 0.4 0.1 0.001 +
Paste 4 1.4 0.6 0.8 0.015 +
Paste 5 1.6 0.8 2.0 0.010 +
Powder 0.8 0.4 0.1 0.004
-Commercial ELISA kit C Protein content (mg/ml) a Beef protein content (μg/ml) b
Table 5. Determination of beef protein content in beef extracts.
LOD: limit of detection.
a Beef protein content of each sample solution was determined by mAb-ELISA.
b The results evaluated by the commercial ELISA kit were represented that the absorbance of sample solutions was greater than that of beef positive control (+).
Commercial foods Labeling
Beef protein content (mg/g) a
Commercial ELISA kit b
Beef jerky Beef 191.7 +
Hamburger steak 1 Beef 7.5 +
Meatball 1 Beef 2.4 +
Stew de veau Beef extract 0.2
-Demi glace sause Beef extract 0.2
-Tomato sause Beef extract 0.2
-Curry sause Beef extract 0.1
-Roux - < LOD
-Hamburger steak 2 - 0.01
-Meatball 2 - < LOD
-Dry sausage - < LOD
-Ham - < LOD
-Hamburger steak 3 - < LOD
-Chicken meat ball - < LOD
-Table 6. Determination of beef protein content in commercial foods by mAb-ELISA and the commercial ELISA kit.
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