Establishment of a Rapid and Sensitive Chemiluminescence Enzyme

Immunoasay for Aflatoxin M

: Verified by HPLC

Min Li, Renrong Liu

, Lixin Zhu and Yu Jin

School of Life Science, Jiangxi Science & Technology Normal University, Jiangxi, Nanchang, 330013, China

Keywords: Aflatoxin M

, chemiluminescence enzyme immunoassay, high performance liquid chromatography

Abstract: A rapid and sensitive chemiluminescence enzyme immunoassay method (CLEIA) was established to detect

Aflatoxin M

(AFM

) in milk, which was Verified by high performance liquid chromatography (HPLC). It

only takes 30 minutes. Optimized conditions included antibody dilution ratio and enzyme conjugate, ionic

strength, pH value and organic solvent. Results: The 50% inhibitory concentration(IC

) and the detection

limit of the CLEIA were 0.08ng/mL and 0.024ng/mL, the recovery ranged from 86.94% to 114.49% in

dairy products. The correlation was 99.7% between this method and HPLC .

1 INTRODUCTION

AFM

is produced by hydroxylation when mammals

ingesting crops contaminated with Aflatoxin B1. It

has a destructive effect on liver tissue, with Strong

carcinogenicity and mutagenicity. At present, a lack

of effective method for prevention and

detoxification, so the monitoring of AFM

is an

important means to prevent and control.

At present, the analytical method mainly include:

high performance liquid chromatography (Shuib et

al., 2017) time-resolved fluorescence (Gao et al.,

2017), quantum dot immunoassay (Bailey et al.,

2004) and enzyme-linked immunosorbent assay

(Radoi et al., 2008) etc. Instrument method are

expensive, laborious, time-consuming, and sample

pretreatment cumbersome. In recent years, the

limited standard of AFM

is decreasing (eg:

Commission regulation EU No 165, 2010; GB

2761-2017 limits on mycotoxins in food, 2017), so it

is necessary to establish simple, rapid and high

sensitivity detection method to quantify and confirm

the AFM

in dairy products. However,

chemiluminescence enzyme immunoassay is

combination of ELISA and chemiluminescence, the

detection sensitivity is 10 ~ 102 orders of magnitude

than conventional ELISA (Zhao et al., 2009).

2 MATERI ALS AND METHODS

Instruments: Luminoskan Ascent and its software (

Thermo, USA), 96-well white polystyrene plates

(Costar), AFM

immunoaffinity column (Own

laboratory), High performance liquid

chromatography.

Reagents: AFM

standard solutionantigen and

anti-AFM

monoclonal antibody were got from our

own laboratory. IgG-HRP was purchased from

Sigma, Luminol chemiluminescent substrate was

purchased from Helisence (Shanghai, China)

2.1 CLEIA Operation

Chemiluminescent plate were coated with 120μL of

AFM

antigen per well for the night at 4℃ washed 4

times with PBS, added 320μL5% skim milk per

well, which incubated for 2h at 37℃, after washing 4

times, added 50μLstandard solution or sample

solution, 50μL antibody diluotin and 100μL

IgG-HRP per well at 37°C for 30min then washed 4

times, at last added 100μL Luminol

chemiluminescent substrate per well and get the

relative light unit (RLU).

2.2 Sample Preparation

Weighing 1g milk powder in centrifugal tube, adding

standard, adding 5ml acetonitrile (liquid milk take

3ml, add 3ml acetonitrile), vortex 5min and

Li, M., Liu, R., Zhu, L. and Jin, Y.

Establishment of a Rapid and Sensitive Chemiluminescence Enzyme Immunoasay for Aﬂatoxin M1: Veriﬁed by HPLC.

DOI: 10.5220/0008185300510054

In The Second International Conference on Materials Chemistry and Environmental Protection (MEEP 2018), pages 51-54

ISBN: 978-989-758-360-5

centrifugal 15 minute for 4500r/min, remove the

underlying liquid and dry it with a nitrogen blower ,

then dissolve the residue with 5% skimmed milk for

test.

Sample pretreatment for liquid chromatography

is referred to 2016 National Food Safety Standard

determination of Aflatoxin M in Food (GB, 2016) .

3 RESULTS AND DISCUSSION

3.1 Optimization of the AFM

CLEIA

Reaction System

The concentration of the coating antigen was

optimized by the checkerboard titration, the coated

concentration between 0.25μg/mL to 2μg/mL. The

results show that 0.5μg/ml is the best coated

concentration. When antibody concentration was too

low, it led to a small RLUmax. Therefore, antibody

concentrations were selected from 1:2000 to 1:8000.

Finally, the best antibody dilution is 1: 2000.

The RLUmax/IC50 ratio was used as a parameter

to judge the impact of factors. Research the effects

of enzyme dilution , ionic strength, pH value and

methyl alcohol on CLEIA. The results demonstrate

that optimum conditions when enzyme concentration

at 1: 500, ionic strength was 5mM , methyl alcohol

was 0 and pH was 7.0. As show in Figure 1-4. This

indicates that neutral buffer is beneficial to the

combination of AFM

antigen- antibody.

1:500 1:1000 1:2000 1:3000

0.05

0.06

0.07

0.08

0.09

RLUmax/IC

ration of goat anti-mouse IgG -HRP

20000

25000

30000

35000

RLUmax/IC

Figure 1: Effects of enzyme dilution on CLEIA.

0 5 10 15 20

0.06

0.08

0.10

0.12

RLUmax/IC

PBS(mM)

20000

25000

30000

35000

RLUmax/IC

Figure 2: Effects of ionic strength on CLEIA.

6.0 6.5 7.0 7.5 8.0

0.07

0.08

0.09

0.10

0.11

0.12

RLUmax/IC

PH value

27000

28500

30000

31500

33000

34500

RLUmax/IC

Figure 3: Effects of pH on CLEIA.

0 5 10 15 20

0.25

0.30

0.35

0.40

RLUmax/IC

The concentration of methyl alcohol

6000

8000

10000

12000

14000

RLUmax/IC

Figure 4: Effects of methyl alcohol on CLEIA.

3.2 Establishment of CLEIA Standard

Curve

Based on the optimization results, the standard curve

of AFM

immunoassay was established in Figure 5 .

The linear equation was Y = -55.228 +154.73 (R2 =

0.9916), The IC50 was 0.08ng/ml, the detection limit

was 0.024ng/ml.

1.0 1.5 2.0 2.5 3.0 3.5

100

1.5 2.0 2.5 3.0

100

B/B

(%)

Log(10 x AFM

)

y=-56.021x + 157.06

=0.9904

B/B

(%)

Log(10 x AFM

)

Figure 5: Competitive inhibition curve for AFM

CLEIA.

3.3 Verification by High Performance

Liquid Chromatography

A series of AFM

standard solutions (15, 10, 5, 2, 1,

0.5 and 0.1ng/mL) were prepared with 10%

MEEP 2018 - The Second International Conference on Materials Chemistry and Environmental Protection

acetonitrile aqueous solution, chromatographic

conditions reference to GB of Aflatoxin M1. The

characteristic absorption peak of AFM

was obtained

and retention time was 8.993min in the set

chromatographic conditions. The SNR was 3:1 as the

minimumdetection limit, which was 0.075ng/mL and

the quantitative limit was 0.3ng/mL. As show in

Figure 6.

0 3 6 9 12 15

100

200

300

400

500

600

Peak area (m A u*S)

The concentration of AFM

(ng/ml)

Figure 6: HPLC stand curve for AFM

3.3.1 Recovery Test of Spiked Samples

To verify the accuracy and reliability of CLEIA, the

recovery experiments were carried out in different

dairy products, moreover it made a correlation with

HPLC. As shown in Table 1, the recovery ratio of

CLEIA was 86.94%-114.49, the coefficient of

variation was 0.81%-7.26%; the recovery by HPLC

was 85.25%-98.62%, and the coefficient of variation

was 0.72%-9.64%. Both methods have good

accuracy and Precision.

Table 1: Recoveries of AFM

in diferrent dairy products

(n=4).

Sample

number

CLEIA

HPLC

Spiked

value

(ng/ml)

Recovery

ratio (%)

(%)

Recovery

ratio (%)

(%)

0.1

0.5

0.1

0.5

90.78

109.08

114.49

86.94

5.15

0.81

7.26

3.74

92.76

98.62

96.14

85.25

9.64

0.72

2.95

3.37

3.3.2 Determination of AFM

in Naturally

Sample

Figure 7 shows in adding standard solution of AFM

(0.1ng/ml, 0.5ng/ml, 1ng/ml) in a milk sample ( The

milk sample used does not contain AFM

). Using

CLEIA and HPLC to test. The data show a high

degree of correlation between them (R2 = 0.997).

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

HPLC (ng/ml)

CLEIA (ng/ml)

Value Standa

B Interce 0.0019 0.0117

B Slope 0.9845 0.0189

Adj. R-Square 0.99704

Figure 7: Correlation between the CLEIA and HPLC.

From Table 2, it can conclude that the content of

AFM

in 10 samples detected by CLEIA and HPLC

was lower than the national limit. Besides AFM

was

not detected in ELISA in 10 samples, indicating that

the content of AFM

was lower than the minimum

detection line of this method, which is consistent

with the result of HPLC detection.

Table 2: AFM

detection in naturally sample.

Sample

number

Detected value（ng/mL）

CLEIA

HPLC

0.0089

0.0137

Note: “ ND ”（Not detected）

4 CONCLUSIONS

In the study, CLEIA reaction system has been

comprehensively optimized. Finally, the

immunoassay method of AFM

was established, and

the sensitivity was 0.024ng/ml. The recoveries

ranged from 86.94% to 114.49%, Meanwhile

correlation between the detection results of CLEIA

and high performance liquid chromatography was

99.7%, indicating that established method can be

applied to rapid determination of AFM

in milk.

y = 39.06x - 0.7854

= 0.9999

Establishment of a Rapid and Sensitive Chemiluminescence Enzyme Immunoasay for Aﬂatoxin M1: Veriﬁed by HPLC

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MEEP 2018 - The Second International Conference on Materials Chemistry and Environmental Protection