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Detection of Penicillinase in Milk by Sandwich Elisa Based Polyclonal and Monoclonal Antibody a

b

Yinli Zhao & Guoxi Li a

College of Bioengineering, Henan University of Technology, Zhengzhou, China

b

Animal Husbandry and Veterinary Engineering, Henan Agricultural University, Zhengzhou, China Accepted author version posted online: 29 May 2015.

Click for updates To cite this article: Yinli Zhao & Guoxi Li (2015): Detection of Penicillinase in Milk by Sandwich Elisa Based Polyclonal and Monoclonal Antibody, Journal of Immunoassay and Immunochemistry, DOI: 10.1080/15321819.2015.1050108 To link to this article: http://dx.doi.org/10.1080/15321819.2015.1050108

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DETECTION OF PENICILLINASE IN MILK BY SANDWICH ELISA BASED POLYCLONAL AND MONOCLONAL ANTIBODY

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Yinli Zhao,1 Guoxi Li2

College of Bioengineering, Henan University of Technology, Zhengzhou , China

2

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Address correspondence to Yinli Zhao, College of Bioengineering, Henan University of Technology, Zhengzhou 450001, China.

ABSTRACT:

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E-mail: [email protected]

A Sandwich ELISA has been developed using polyclonal and monoclonal

antibody for the determination of penicillinase in milk. For this purpose, specific polyclonal and monoclonal antibodies against penicillinase were generated and characterized. Using penicillinase

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standards prepared from 1 to 128 ng/mL, the method indicated that the detection limit of the Sandwich ELISA , as measured in an ELISA plate reader, was as low as 0.86ng/mL of

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penicillinase . For determine the accuracy, raw milk containing 2, 8, 32, and 64 ng/mL of

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penicillinase were tested by Sandwich ELISA. Recoveries were from 93 to 97.5%, and the coefficient of variation [CV (%)] were from 5.55 to 8.38%. For interassay reproducibility, recoveries were from 89.5 to 95.1%, the coefficient of variation [CV (%)] were from 5.26 to

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Animal Husbandry and Veterinary Engineering, Henan Agricultural University, Zhengzhou, China

9.58%. This Sandwich ELISA provides a useful screening method for quantitative detection of penicillinase in milk.

Keywords

Penicillinase, polyclonal antibody, monoclonal antibody, Sandwich ELISA , milk

1

INTRODUCTION Beta-lactams belong to a group of antibiotics that has been widely used in veterinary

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medicine to treat bacterial infections in livestock animals. Even without any signs of disease, these drugs are also administered to animals for preventive purposes. As a result of either illegal use of beta -lactams or non-compliance of producers with existing animal-treatment protocols

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substances may enter the food chain. Previous studies have reported some cases of allergic

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reactions after consumption of foods containing beta-lactams residues. In order to control such veterinary drugs in animals producing food, many countries had established strict regulations, including specific maximum residue limits (MRLs) for each of these substances.[1-4]

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With the rapid growth of the dairy industry and the establishment of strict antimicrobial residue limits in the People's Republic of China's (PRC) milk supply, a "antimicrobial destroyer"

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was introduced into dairy production without regulatory review. According to the literature, About 50% of beta-lactamase activity added to milk remained after pasteurization at 63 ℃ for 30 min.[5]

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For example, to avoid detection of penicillin G, some producers illegally add penicillinase to milk to degrade it into benzylpenicilloic acid (BPA). This degradation product can cause allergic

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(withdrawal times) prior to animal products being available for consumption, residues of these

reactions in humans and, therefore, is a potential hazard to human health. On the other hand, the

abuse of penicillinase also contributed to the abuse of beta lactam antibiotics in milk, and accelerated the spread of drug resistant bacteria. So the penicillinase added in milk represents a

2

public health risk. [6-7] Therefore, in order to ensure milk quality and the health of consumer, the development of detection technology for monitoring the penicillinase is necessary.

[8]

However, enzyme-linked

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Microbiological method is sensitive but time-consuming.

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Some methods for detection of beta-1actamase in milk have been established by now.

immunosorbent assays (ELISA) have been widely used as a screening tool in trace residue

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technical support for large-scale screening purpose.[9-14] Hujer et al. described the production of

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polyclonal anti-SHV-1 and anti-CMY-2 β-lactamase antibodies , antibody recognition determined by immunoblot analysis, polyclonal antibodies as coating antibody, biotinylated polyclonal antibodies as detecting antibodies , developed an enzyme-linked immunosorbent assay for detecting CMY-2 and SHV β-lactamases. This enzyme-linked immunosorbent assay was

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sensitive, but biotinylated detecting antibodies need relatively complex steps.[15-16] Hujer et al. also developed a method of visualizing the SHV β-lactamases in Gram-negative bacilli using

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fluorescein-labeled polyclonal antibodies. This method was suitable for detecting β-lactamases within the cell or bacilli, not in milk.[17] A sandwich ELISA based on an monoclonal antibodies

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(mAb) pair has been described for the detection of Temoneira (TEM) 1 beta-lactamase.[18] In this study, we chose to generate polyclonal and monoclonal antibodies against penicillinase and to

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analysis. As a rapid, special, sensitive and safe biological method, ELISA could provide a

produce a sandwich ELISA for the detection of penicillinase in milk.

3

MATERIALS AND METHODS Chemicals and Materials

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Penicillinases were purchased from the China Institute of Veterinary Drugs Control. BSA,

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OVA, FCA and FIA were purchased from Beijing dingguo changsheng biotech CO.LTD. Goat

anti-rabbit IgG was from Sino-American Biotechnology Co. (Luoyang, China). Ninety-six-well

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Female BALB/c mice (6–8 weeks old) were purchased from the Laboratory Animal Center,

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Zhengzhou University, China, and raised under strictly controlled conditions. Other reagents and solvents were of analytical grade or higher.

Apparatus Microplate Reader was from Bio-Rad (Richmond, CA). Milli-Q water was obtained from Millipore (Bedford, MA). GS-15R refrigerated centrifuge was from BKMAN(US).

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AE260 Electronic analytical balance was from METTLER (Germany).

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Production of Penicillinase pAb

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Immunization of Rabbits. Four male New Zealand white rabbits (2 kg size) were subcutaneously immunized at multiple sites in the back with penicillinase antigen. The initial immunization was subcutaneously injected with 500μg penicillinase in 0.5 mL of NaCl (0.9%)

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culture plates were bought from Greiner (Germany). New Zealand white rabbits (2 kg size) and

and 0.5 mL of Freund’s complete adjuvant. Subsequent booster injections [500μg penicillinase in

0.5 mL of NaCl (0.9%) plus 0.5 mL of Freund’s incomplete adjuvant] were performed 21 days later and then at 21 day intervals. Blood samples were taken from the marginal vein of the ear 14 days after each immunization (from the third immunization onward).

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Production of pAb The antiserum obtained after each booster (from the third immunization onward) was prepared by allowing the blood to clot overnight at 4 ℃, followed by centrifugation to remove particulate material. And serum titers were determined by indirect ELISA. If a

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satisfactory titer was obtained, then stop the immune. Ten days after the last boost, all rabbits were then exsanguinated by heart puncture. The serum was separated from blood cells by storage of the

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obtained was purified by saturated ammonium sulfate (SAS) precipitation method (purified three times using 50, 33, and 33% (v/v) of SAS, respectively), and the precipitate was dissolved in

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normal saline to the original serum volume. And sodium azide was added as a preservative at a final concentration of 0.02% (w/w). The purified serum was then aliquotted and stored at -20℃.[19-21]

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Titer Determination by Indirect ELISA

The titer of the antibody was tested by indirect ELISA, using a procedure described below.

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The microplates were coated with 50 μL/well of penicillinase (5μg /mL ) in 0.05 mol/L

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carbonate-buffered saline (CBS, pH 9.6) at 37℃ for 2 h. After washing three times with 0.01 mol/L PBS (pH 7.2) containing 0.05% Tween 20 (PBST), the plates were blocked with 200 μL/well of 5% normal pig serum in PBST (blocking buffer) at 37℃ for 1 h. Plates were washed

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blood overnight at 4 ℃ and centrifugation with 12000 rpm/min for 20 min. The crude serum

three times again. Samples of rabbits antisera of 50 μL/well with two fold serially diluted in blocking buffer were added and incubated at 37℃ for 15 min. The plates were then washed again as above. A total of 50 μL/ well of 1:1000 diluted goat anti-rabbit IgG-HRP were added in the wells and incubated at 37℃ for 30 min. Plates were washed three times again. Finally, a 50-μL

5

aliquot of HRP substrate, 3,3,5,5,- tetramethylbenzidine (TMB) buffer and chromogen buffer were added and incubated for 10 min at room temperature. Colour development was stopped by 2 mol/L sulfuric acid (50 μL/well) and optical densities (ODs) were measured at 450nm with an

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ELISA plate reader. Absorbances were corrected by blank reading. Preimmune withdrew serum

(the serum before immunization) was used as a negative control, and the penicillinase pAb titer

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of the background.[22-23]

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Production of monoclonal antibody Against Penicillinase

Immunization of Mice. six BALB/c female mice were immunized with Penicillinase. The initial immunization was subcutaneously injected with 50μg penicillinase in 0.1 mL of NaCl (0.9%) and 0.1 mL of Freund’s complete adjuvant. Subsequent booster injections [50μg

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penicillinase in 0.1 mL of NaCl (0.9%) plus 0.1 mL of Freund’s incomplete adjuvant] were performed 21 days later and then at 21 day intervals. The titer of the Antisera was tested by indirect

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ELISA too. The mouse showing the highest Titer received a fifth injection intraperitoneally (i.p.).

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Three days later, the spleen of the injected mouse was removed for hybridoma production. Cell fusion and hybridoma screening. Cell fusion procedures were carried out according to the procedure described by Zhao, Y.[24] Briefly, the spleen of the immunized mouse was removed

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was defined as the reciprocal of the dilution that resulted in an absorbance value that was twice that

and the splenocytes isolated and fused with NS0 cells using PEG1500. The fused cells were then distributed into 96-well culture plates, in which mouse peritoneal macrophages were prepared on the day before the fusion and were grown with the selective HAT medium. Selected clones were

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subcloned by limiting dilution. Ascite fluids were produced in paraffin-primed BALB/c mice and the titer of ascitic fluid was tested by indirect ELISA .

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A Sandwich ELISA based polyclonal and monoclonal antibody

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Ninety-six-well microplates were coated with anti- penicillinase pAb diluted in coating

buffer (100 μL/well) and subsequently incubated at 4 °C overnight. Following incubation, the

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blocked with blocking buffer (200 μL/well) at 37 °C for 1 h. Following another washing step, 100

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μL of a serially diluted penicillinase standard solution or sample extract solution was added to each well, and the microplate was incubated at 37 °C for 30min. Subsequently, 100 μL of antipenicillinase mAb was added to each well, and the plate was incubated for 30min at 37 °C. After washing the plate three times, 100 μL of anti-mouse IgG-HRP(1:1000)was added to each well, and

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the plate was incubated for 30min at 37 °C. After washing the plate three times, 100 μL of TMB substrate solution was added to each well at room temperature for 15 min . The reaction was

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stopped by adding 2 M sulfuric acid (50 μL/well). Absorbance was measured at 450 nm in a

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microplate reader.

The Standard Curve and Sensitivity of the Sandwich ELISA

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wells were washed three times with washing buffer; the free binding sites in the wells were

To prepare the standard curve of penicillinase in PBS (pH7.2) buffer, penicillinase solutions

were diluted with the PBS to 1, 2, 4, 8, 16, 32, 64,128 ng/mL. The standard curve was fitted based

on the average of four separate assays in quadruplicate. The limit of detection (LOD) was defined

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as the lowest analyte concentration that was determined by the mean background levels plus three times standard deviation (B 0 +3s) .[25-26]

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Recovery of Penicillinase in Raw Milk Samples

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Raw milk, purchased from the dairy farm , was penicillinase free based on the cylinder plate method. For recovery test, penicillinase-spiked solutions were prepared by dissolving penicillinase

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centrifuged at 4,000 × g at RT for 10 min. After centrifugation, milk fat was removed from the

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supernatant, and the milk was immediately analyzed by Sandwich ELISA. The recoveries of penicillinase from the spiked raw milk were calculated based on the standard curve constructed by above .[24, 27]

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RESULTS AND DISCUSSION

Production of Anti- penicillinase pAbs

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Four rabbits injected with penicillinase both produced the final antisera exhibiting high titer

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values after the fourth injection. The titer was defined as the reciprocal of the dilution that resulted in an absorbance value that was twice that of the background, the titer was about of 1:256 000. Therefore, their seras were mixed together for preparation of the pAbs .

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in raw milk to give final concentrations of 2, 8, 32, and 64ng/mL. The milk samples were

Production of Anti- penicillinase mAbs After cell fusion, the anti- penicillinase mAbs in the supernatant from the cell culture plates

were detected by indirect ELISA. the titer of 4A1D5 Cell line was about of 1:32 000. These cells

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were intraperitoneally injected into mice, and the ascitic fluid produced was removed for preparation of the mAbs. the titer of ascitic fluid was about of 1: 512 000.

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The Standard Curve and Sensitivity of the Sandwich ELISA ELISA

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Checkerboard titrations were performed to determine the optimized diluted concentration of pAbs and mAbs. The optimal concentrations of the coated pAb were determined to be 4μg/mL ,

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The sensitivity of the Sandwich ELISA was determined by measuring the responses to

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penicillinase standard samples. The logarithm of penicillinase concentration and the average value of absorbance produced a sigmoidal dose-response curve, the equation was Y= 0.833x + 0.157 ( R2= 0.992 ) (Table 1, Figure 1 ). The detection limit was defined as the lowest concentration of penicillinase, it was determined by the mean background levels plus three times standard deviation

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( B0+3s ). Based on the standard curve, this assay had a detection limit of 0.86ng/mL and a linear

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range of 0.86-128 ng/mL.

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Recovery and Intra- and Interassay Precision of the Sandwich ELISA For recovery test, penicillinase-spiked solutions were prepared by

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and the diluted concentration of mAb is 1:32 000 (v/v) dilution.

dissolving penicillinase in the raw milk to give final different concentrations of 2, 8, 32, and 64 ng/mL. To determine the accuracy, raw milk containing 2, 8, 32, and 64 ng/mL of penicillinase were tested by Sandwich ELISA. Raw milk samples values calculated from the standard curve. Recoveries were from 93.00 to 97.50%, and the coefficient of variation [CV (%)] were in the range of 5.55-8.38%. For interassay reproducibility, four different batches of the test microtiter plate

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were used for quadruplicate measurements of the samples. Recoveries were from 89.5 to 95.10%, the coefficient of variation [CV (%)] were in the range of 5.26-9.58%. The results are shown in Table 2.

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These results indicate that our developed sandwich ELISA is accurate and reliable for detecting penicillinase in milk.

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In this study, we established a Sandwich ELISA based on polyclonal antibody and mAbs for the

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detection of penicillinase in milk. The limit of detection and linear dynamic range of the method were 0.86ng/mL and 0.86-128 ng/mL, respectively. Wang,W. et,al. established a sandwich ELISA for β-lactamase detection based on an mAb pair. The LOD and linear dynamic range of the method were 4.17 ng/mL and 5.5–100 ng/mL, respectively.[18] So this Sandwich ELISA is more sensitive

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than Wang,W. reported. Considering that it was sensitive, accurate, low production costs, short

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production cycle, etc., this Sandwich ELISA can be used as a screening method to detect

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penicillinase in milk. FUNDNG

This work is financially supported by Henan University of Technology PhD Research Fund ( No.

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CONCLUSIONS

150408)and the Education Department of Henan Province ( No. 14A230004) .

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FIGURE 1. Standard curve for the detection of penicillinasea aSandwich ELISA standard curve for penicillinase in PBS buffer solution using the polyclonal rabbit antibody (4μg/mL) and mAbs(1:32000 dilution). Each point represents the average of 4 replicates. The X-axis is

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expressed as the logarithm of penicillinase concentration, Y-axis is expressed as absorbance

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at 450nm.

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TABLE 1 Penicillinase concentration and the average value of absorbance at 450nm

0

2

0.301

4

0.602

t

0.215

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1

8

ed

16

64

ce

128

pt

32

0.361 0.635

0.903

0.843

1.204

1.208

1.505

1.456

1.806

1.712

2.107

1.856

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concentration(ng/mL)

average value of absorbance at 450nm

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logarithm of penicillinase concentration

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penicillinase

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TABLE 2

Recovery and Intra- and Interassay Precision of the Sandwich ELISA for

Penicillinase Spiked in raw milk (n=4)a

Intra -assay

Inter-assay

Penicillinase mean ± SD

CV

mean ± SD

recovery

CV

(ng/mL)

(%)

(%)

(ng/mL)

(%)

(%)

2

1.86±0.12

93.00±6.00

6.45

1.79±0.15

89.50±7.50

8.38

8

7.67±0.60

95.88±7.50

7.28

7.42±0.39

92.75±4.88

5.26

32

30.25±1.68 94.53±5.25

5.55

29.15±2.02 91.09±6.31

6.93

64

62.40±5.23 97.50±8.17

8.38

60.85±5.83 95.10±9.11

9.58

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Raw milk were spiked with penicillinase at 2, 8, 32, and 64 (ng/mL). Intra-assay variation was

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a

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recovery

(ng/mL)

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determined by four replicates on a single day. Interassay variation was determined by four

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replicates on four different batches in 4 weeks.

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spiked

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Detection of Penicillinase in Milk by Sandwich ELISA Based Polyclonal and Monoclonal Antibody.

A sandwich ELISA has been developed using polyclonal and monoclonal antibody for the determination of penicillinase in milk. For this purpose, specifi...
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