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Substitution of antibody withmolecularly imprinted 96-well platein chemiluminescence enzymeimmunoassay for the determination ofchloramphenicol residuesXingjie Dua, Feng Zhanga, Huixiao Zhanga, Yongjia Wena & TuoyaSarena

a College of Fisheries and Life Science, Dalian Ocean University,Dalian 116023, P.R. ChinaPublished online: 30 Jul 2013.

To cite this article: Food and Agricultural Immunology (2013): Substitution of antibodywith molecularly imprinted 96-well plate in chemiluminescence enzyme immunoassay forthe determination of chloramphenicol residues, Food and Agricultural Immunology, DOI:10.1080/09540105.2013.821598

To link to this article: http://dx.doi.org/10.1080/09540105.2013.821598

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Substitution of antibody with molecularly imprinted 96-well plate inchemiluminescence enzyme immunoassay for the determination ofchloramphenicol residues

Xingjie Du, Feng Zhang*, Huixiao Zhang, Yongjia Wen and Tuoya Saren

College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, P.R. China

(Received 16 March 2013; final version received 26 June 2013)

A direct competitive chemiluminescence enzyme immunoassay was developed byusing the molecularly imprinted 96-well plate as an artificial antibody to detectchloramphenicol (CAP). The artificial antibody was synthesised on the wellsurface of MaxiSorp polystyrene 96-well plate and CAP was conjugated with thehorseradish peroxidase. The results showed that the imprinted plate exhibitedantibody-like binding ability. The plate showed fast adsorption rate, 66%adsorption was finished within 20 min. The cross-reactivity for CAP, florfenicoland thiamphenicol were 100%, 1.25% and 2.08%, respectively. And the imprintedplate could be reused for many times without loss of sensitivity. The IC50 and thedetection limit values under optimum conditions were 3092 mg �L�1 and 0.990.01 mg �L�1, respectively. The plate was used to detect CAP in sea cucumber,which showed excellent recoveries ranging from 89% to 98.7%. And the resultcorrelated well with that obtained by the CAP enzyme-linked immunosorbentassay (ELISA) kit.

Keywords: chloramphenicol; chemiluminescence enzyme immunoassay; molecu-larly imprinted 96-well plate; artificial antibody

Introduction

Chloramphenicol (CAP) is considered as a prototypical broad-spectrum antibiotic. It

is effective against a wide variety of Gram-positive and Gram-negative bacteria,

including most anaerobic organisms. As CAP is both cheap and easy to manufacture,

it is widely used to treat serious infections in poultry, livestock and aquatic products.

However, this medication has caused serious side effects, such as fatal blood disorders

(e.g. aplastic anaemia, hypoplastic anaemia) and bone marrow suppression. Because

of its extensive usage, CAP gradually accumulates in edible animal products such as

muscle and kidney, which makes it a potential threat to human health. Therefore, in

many developed countries, CAP use is strictly prohibited and the maximum level for

CAP residues in animal products was allowed to be zero tolerance.

So far, the established methods for the determination of CAP residues in aquatic

products are mostly based on immunoassays and chromatography. Chromatography

(including high performance liquid chromatography (HPLC): Aerts, Keukens &

Werdmuller, 1989; Allen, 1985; Long et al., 1990; Moretti, van de Water, &

Haagsma,1992; GC: Arnold & Somogyi, 1985; Pfenning et al., 2000; or GC-MS:

*Corresponding author. Email: Fengz57@gmail.com

Food and Agricultural Immunology, 2013

http://dx.doi.org/10.1080/09540105.2013.821598

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Gantverg, Shishani, & Hoffman, 2003; Nagata & Oka, 1996) is commonly used due

to its sensitivity, selectivity and low detection limit, but it requires expensive

instrumentation, complicated sample pre-treatment and preparation, as well as

skilled operators, which is not good for extensively prevalence. Chemiluminescence

enzyme immunoassay (CLEIA; Lin, Han, Liu, Xu, & Guan, 2005; Xu, Peng, Hao,

Jin, & Wang, 2006; Zhang, Zhang, Shi, Eremin, & Shen, 2006), which has combined

highly sensitive chemiluminescence determination technique with highly selective

immunoassay, is an analytical technique that is used to analyse all kinds of antigens,

haptens, antibodies or drugs. CLEIA is developed rapidly because of its sensitivity,

selectivity and running many samples simultaneously. However, in this method, the

production of antibodies is particularly difficult and the antibodies show low

stability, poor reproducibility and poor resistibility against harsh environments such

as high temperature, strong acid or base. Therefore, some researchers have attempted

to synthesise artificial antibodies to replace biological antibodies.

Molecular imprinting technology (MIT; Fang et al., 2011; Surugiu et al., 2000;

Surugiu, Danielsson, Ye, Mosbach, & Haupt, 2001; Wang et al., 2009; Wang, Tang,

Fang, Pan, & Wang, 2011; Xu, Gao, Zhang, Chen, & Qiao, 2011; Ye & Mosbach,

2001; Ye & Haupt, 2004) has been used to prepare biomimetic mimics that can

imitate the molecular recognition ability of biological antibodies. Molecularly

imprinted polymers (MIPs) are prepared by the polymerisation of a selected

monomer and cross-linker in the presence of target analyte, acting as the template

for assembly of its own recognition sites. After the polymerisation, the templates are

removed from the resulting polymer matrices, and binding sites having the size and

shape complementary to the template are generated. These MIPs are synthesised

with ‘‘tailor-made’’ binding sites for a template and strongly interact with it

(Kareuhanon, Lee, Nimmanpipug, Tayapiwatana, & Pattarawarapan, 2009). Due

to favourable molecular recognition capability and stability, MIPs have been

exploited as artificial antibodies. In this paper, the molecularly imprinted polymer

was directly synthesised on the well surface of a MaxiSorp polystyrene 96-well plate

and used as an artificial antibody to detect CAP residues in sea cucumber with

CLEIA. And the results were validated by the CLEIA with CAP enzyme-linked

immunosorbent assay (ELISA) kit.

Experimental

Materials and reagents

CAP (98%), florfenicol(98%) and thiamphenicol(99.5%) were purchased from

Shanghai Jingchun Reagent Co., Ltd. (Shanghai, China). Other chemicals used for

the polymer synthesis were the solvent tetrahydrofuran ( 99%), the functional

monomer 2-(N,N-Diethylamino) ethylmethacrylate (DEAEM, 99%), the initiator

2,2-Azobisisobutyronitrile (AIBN, 99%, Shanghai Jingchun Reagent Co., Ltd,

Shanghai, China) and the cross-linker ethylene glycol dimethacrylate (EGDMA,

98%, Sigma�Aldrich, USA). Methanol (99.5%) and acetic acid (99%) used for

template extraction were obtained from Shanghai Jingchun Reagent Co., Ltd.

(Shanghai, China). Chemicals used for enzyme conjugate preparation were zinc

powder (90%, Dalian Shenlian Chemical Reagent and Glass Apparatus Co., Ltd,

Dalian, China), horseradish peroxidase (HRP,�300 m mg�1, Shanghai Sangon

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Biotech Co., Ltd, Shanghai, China) and glutaraldehyde (25%, Shanghai Sangon

Biotech Co., Ltd, Shanghai, China). Luminol (98%) and H2O2 (30%) were obtained

from Shanghai Sangon Biotech Co., Ltd (Shanghai, China). Tween-20 was

purchased from Sigma (Sigma-Aldrich, USA). Maxisorp polystyrene 96-well plateswere from Nunc (Roskilde, Denmark). CAP ELISA kit was from Beijing Wanger

Biotech Co., Ltd (Beijing, China).

Solutions

1�PBS: 8.5 g NaCl, 2.2 g Na2HPO4 �12H2O, 0.2 g NaH2PO4 �2H2O, 1 L H2O. PBS/

T: PBS with 0.05% Tween-20. CLIA substrate solution: 0.0886 g Luminol, 100 mL

carbonate buffer solution (70 mL 0.1 mol �L�1 Na2CO3, 30 mL 0.1 mol �L�1

NaHCO3), stored at 2 �88C avoiding light, when used, diluted to 5�10�4 mol �L�1

with 0.1mol �L�1 NaHCO3. 30% H2O2: when used, diluted to 5�10�3mol �L�1 with

Tris-HCl buffer solution (50 mL 0.1 mol �L�1 Tris, 14.7 mL 0.1 mol �L�1 HCL, 35.3

mL H2O). Luminol and H2O2 were mixed at the ratio of 1:1 before use. Wash

solution: 1.5 mL deionised water, 28.5 mL concentrated wash solution. Buffer

solution: 3 mL deionised water, 3 mL concentrated buffer solution.

Instrumentation

Full wavelength scanning plot was obtained by UV-VIS spectrophotometer (UV-

1750) produced by Shimadzu, the scanning range was 190�1100 nm, and the

scanning step length was 1 nm.

CAP concentration was analysed via HPLC. The HPLC system produced by

Dalian Elite Analytic Instruments Co., Ltd was equipped with an Elite C18

chromatographic column (4.6�250 mm, 5 mm) and a UV-detector set at 273 nm.

The mobile phase was methanol: pure water �70:30 (v/v) and the flow rate was fixedat 1.0 mL �min�1.

CAP residues in the samples were detected by the BW-300 CLIA plate reader

(Beijing Yadongya Mechanical and Electronic Technology Institute, China)

Synthesis of the molecularly imprinted 96-well plate

The imprinted polymer was directly polymerised on the 96-well plate wells as follows

(Wang et al., 2011): CAP (80 mg, 0.25 mmol) was dissolved in 5 mL tetrahydrofuranas porogen in a 25 mL round-bottom flask, then the functional monomer DEAEM

(200 mL, 1 mmol) was added, and the mixture was sonicated for 20 min. After that,

the cross-linker EGDMA (50 mL, 0.25 mmol) and the initiator AIBN (0.020 g, 0.122

mmol) were added to the mixture and sonicated for 20 min. Then 25 mL of the

mixture was placed in the wells of a 96-well plate. The plate was put in a zip lock bag

and deoxygenated with a stream of nitrogen gas for 15 min. Then the bag was sealed

and irradiated by UV irradiation (l �365nm) for 6 h. After the polymerisation

ended, the 96-well plate was extracted with 100 mL of methanol/acetic acid (3:1, v/v)for 24 h by the ultrasonic cleaner, the eluent was replaced by new methanol/acetic

acid every 4 h. At last, the plate was extracted by 100 mL of the methanol for 6 h to

be free of CAP, which was verified by detection of the methanol eluent using HPLC.

The plate was dried at 508C.

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For comparison, the non-imprinted plate was prepared in the same way without

the template molecule CAP.

Characterisation of the imprinted 96-well plate

To measure the adsorption capacity, the imprinted and non-imprinted wells of the

96-well plate were added with 200 mL of methanol solution containing CAP at

various concentrations (10�120 mg L�1). After mechanically shaking (200 r �min�1

for 60 min at room temperature), the supernatants were measured by HPLC and the

adsorption capacity (Q) was calculated according to the equation being

Q ¼ ðCi � CfÞV ;

where Ci is the initial concentration of the analytes in the solution and Cf is the

final concentration, V is cubage of solution.

Adsorption kinetics of the novel imprinted plate was evaluated by 50 mg �L�1

CAP-methanol solution. The 96-well plates were shaken (200 r �min�1) for differenttime periods (10 �100 min) at room temperature. The final concentration of

supernatants was determined by HPLC.

Synthesis of CAP-enzyme conjugate

CAP-enzyme conjugate was synthesised by the glutaraldehyde method; 100 mg CAPwas dissolved in 10 mL 0.6 mol L�1 HCl. Then 60 mg zinc powder was added in the

mixture. The reaction mixture was incubated for 30 min at 808C. The supernatants

were added slowly with stirring into 2 mL PBS containing 10 mg HRP when it

cooled down to room temperature. And then 0.2 mL 25% glutaraldehyde was added.

The reaction mixture was incubated with stirring for 5 h at room temperature. The

enzyme conjugate solution was then dialysed against PBS at 48C for 3 days and PBS

was changed two times a day. According to the volume of enzyme conjugate, the

same volume of glycerine was added to the solution, and stored at �208C before use.

Optimisation of CAP-enzyme conjugate dilution fold

The concentration of the enzyme conjugate was optimised with dilutions of 1:2000,

1:4000, 1:8000 and 1:16,000; 100 mL of enzyme conjugate in PBS was added into the

imprinted 96 wells and incubated for 1.0 h. After that, the 96 wells were washed withPBS/T solution for four times. During the washing, the chemiluminescent substrate

solution was prepared (luminal and H2O2, 1:1). Then the 96 wells were put in the

CLIA plate reader with 250 mL of substrate solution added to each well and

chemiluminescence values were recorded.

Direct competitive CLEIA procedure

First, 100 mL of standard solution or sample extracts was added into the 96 wells,

except for the blank wells. Then 100 mL of enzyme conjugate in PBS was immediately

added to each well, and the mixture was incubated for 1.0 h. After that, the 96-wells

were washed with PBS/T solution for four times. During the washing, CLIA

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substrate solution was prepared (luminal and H2O2, 1:1). Then the 96 wells were put

in the CLIA plate reader with 250 mL of substrate solution added to each well and

chemiluminescence values were recorded. Finally, the imprinted 96-well plate was

extracted with 100 mL methanol/acetic acid (3:1, v/v) for 3.0 h by an ultrasoniccleaner, followed with 100 mL methanol for 3.0 h, for the next CLEIA procedure.

Standard solution preparation

For the construction of the calibration curve, six kinds of standard CAP-methanol

solution within the range of 0.3�30,000 mg �L�1 were prepared; 30 mg �L�1 CAP-

methanol solution was sequentially diluted to 3000, 300, 30, 3, 0.3 mg �L�1.

Preparation of sample

The sea cucumber sample was purchased from the local supermarket. The coelomic

fluid was extracted by an injector and determined to be free of CAP with HPLC. The

coelomic fluid sample was filtered by a 0.45 mm filter before use. Then it was spiked

with standard CAP-methanol solution (0.05 mg �L�1) at three different volume (0.02 mL,

0.1 mL and 0.2 mL).

The viscera of the sea cucumber were removed and the body wall was cut intopieces and mixed by a household mixer. The body wall did not contain CAP as

determined by HPLC. For spike and recovery studies, 3 g samples were spiked with 1

mg �L�1, 5 mg �L�1 and 10 mg �L�1 CAP- methanol solution, respectively. The samples

were thoroughly mixed, and then allowed to stand at room temperature overnight.

Extraction of sample

The 3.0 g spiked samples were put into three 10 mL centrifugal tubes, respectively.

Then 3.0 mL deionised water was added in the centrifugal tube and shaken to make

the mixture mix up. After the shaking, 6 mL acetic ether was added into the mixture

and shaken for 5 min. Then the centrifugal tubes were put in the centrifuge (4000 r �min�1) for 10 min. After that, 4 mL supernatant was dried by a stream of nitrogen

gas. Then 1.0 mL n-hexane was added to dissolve the dried substance and 1.0 mLdiluted buffer solution was added, strongly shaken for 30 s. And then the tubes were

put in the centrifuge (4000 r �min�1) for 10 min. After that, 50 mL lower phase was

applied for the determination.

Specificity of the imprinted 96-well plate

Cross-reactivity (CR) studies were performed by measuring the competitive curves

and selectivity properties for other structurally related compounds under the

optimised conditions. The florfenicol and thiamphenicol were the competitors with

similar structure and characteristics. Chemical structure of CAP, florfenicol and

thiamphenicol was shown in Figure 1. CR was calculated as the percentage between

the IC50 value for CAP and the IC50 value for the interfering compound with thefollowing equation:

CR% ¼ fIC50 ðCAPÞ=IC50 ðcross� reacting compoundÞg � 100

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Sample analysis and CLEIA with CAP ELISA kit validation

CAP kit was put on the experiment table from the 48C refrigerator at room

temperature for 30 min. The concentrated CAP antibody was diluted according to

the ratio of 1:10. Different samples were numbered and 50 mL sample solution was

added into the same numbered well. Then, 50 mL �well�1 antibody was added. The

wells were put in the light-proof zip lock bag and shaken softly for 2 min. After the

shaking, the wells were placed in the 258C thermostat water bath cauldron and

incubated for 30 min. The liquid in the wells was dropped and the wells were washed

by the cleaning solution (250 mL �well�1) for five times. Then 100 mL �well�1 HRP

antibody was added, shaken softly for 2 min. After that, the wells were placed in the

258C thermostat water bath cauldron and incubated for 30 min. The liquid in the

wells was dropped and the wells were washed for five times. During the washing,

luminal and H2O2 were mixed up according to the ratio of 1:1. The wells were put in

the CLIA plate reader and 250 mL �well�1 substrate solution was added. Then the

chemiluminescence values were recorded. The determination was repeated for six

times. According to the standard curve of CAP, the CAP residue was obtained.

Results and discussion

Characterisation of the novel imprinted 96-well plate

The isothermal adsorption of imprinted and non-imprinted 96-well plate was

performed by a series of CAP-methanol solution (10 �130 mg �L�1), which is

shown in Figure 2. The figure shows that the non-imprinted plate adsorbed less CAP

than the imprinted plate. The adsorption capacity of both of them was increasing

O2N

OH

OH

NH O

Cl Cl

Chloramphenicol

S

NH

Cl

Cl

F

O

O

O

OH

Florfenicol

Thiamphenicol

S

OH

NH

Cl

Cl

OH

O

O

O

H3C

Figure 1. Chemical structure of CAP, florfenicol and thiamphenicol.

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with the initial concentrations of CAP increasing. And at 120 mg �L�1, the

adsorption capacity of the imprinted plate was 1.51 mg �well�1 while the non-

imprinted plate was only 0.85 mg �well�1.

Adsorption kinetics of the imprinted 96-well plate was examined at 50 mg L�1

concentration (Figure 3). It indicated that the imprinted plate showed fast

adsorption rate, 66% adsorption was finished within 20 min, and the adsorption

equilibrium was reached within 50 min.

Condition optimisation

In order to improve the sensitivity and precision of CLEIA method, the preparing

solvent and thickness of the imprinted plate were optimised by a series of

experiments. Different ratios of methanol (0%, 5% and 10%) contained in PBSsolution were tested by comparing the standard curves. Results indicated that the

method showed a higher sensitivity when PBS solution contained 5% methanol. And

0 10 20 30 40 50 60 70 80 90 100 110 120 1300.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

CAP NCAP

Ads

orpt

ion

capa

blili

ty (

g µw

ell–1

)

Intitial CAP concentration (mg L–1)

Figure 2. Adsorption isotherms of the imprinted and non-imprinted 96-well plate.

0 10 20 30 40 50 60 70 80 90 1000.0

0.2

0.4

0.6

0.8

1.0

Ads

orpt

ion

capa

blili

ty (

gw

ell–1

)

Time (min.)

µ

Figure 3. Kinetic adsorption plot of the imprinted 96-well plate.

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the sensitivity decreased with the concentration of methanol decreasing. Therefore,

5% methanol in PBS solution was chosen to be the preparing solvent for the routine

analysis of CAP in the sea cucumber samples.

According to previous reports for the immunoassay, the concentration of

immobilised antibodies on the 96-well plate may have a direct effect on the

sensitivity of the direct competitive ELISA (Li, Wang, Lee, Allan, & Kennedy,

2004). In order to study the effect of the concentration of immobilised antibodies on

the assay performance, three kinds of volume (10 mL �well�1, 25 mL �well�1 and 50

mL �well�1) were added into the 96-well plate with different polymerisation time (6 h,

12 h, 18 h and 24 h). Results showed that the method had a higher sensitivity with

25 mL �well�1 and polymerisation time for 6 h.

Optimisation of CAP-enzyme conjugate concentration

The optimum reagent concentration was defined as that which gave the maximum

chemiluminescence intensity with the minimum reagent expense. The RLUmax/IC50

ratio was shown to be a useful parameter with which to estimate the effect of a

certain factor on the CLEIA performance, the highest ratio indicating the highest

sensitivity (Mercader & Montoya, 1999). It is shown in Figure 4 that the IC50

decreased with the CAP �HRP concentration decreasing and the RLUmax also

decreased with the CAP�HRP concentration decreasing. According to the highest

RLUmax/IC50 ratio, 1:4000 of CAP�HRP concentration was selected.

1:2000 1:4000 1:8000 1:160000

5

10

15

20

25

30

35

IC50

Enzyme conjugate dilution fold

0

2

4

6

8

10

12

RL

Um

ax/IC50

Figure 4. Optimisation of CAP-enzyme conjugate concentration.

Table 1. Specificity of the imprinted 96-well plate (mean9SD, n �3).

Compounds IC50 (mg �L�1) CR (%)

CAP 3092 100

Florfenicol 4000920 1.25

Thiamphenicol 2400914 2.08

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Cross-reactivity

The specificity of CLEIA was evaluated by performing competitive assays with other

structurally related compounds. CR was calculated by the percentage between the

IC50 value for CAP and the IC50 value for the interfering compound, and the result is

shown in Table 1. The table shows that the imprinted plate had a higher selectivity

for CAP than other related compounds, and CR for florfenicol and thiamphenicol

was 1.25 and 2.08, respectively. This may result from the imprinting effect, the

difference of the molecular interaction and the different structure. The �OH of the

template CAP reacted with the oxygen atom of the functional monomer DEAEM.

After CAP was removed from the resulting polymer matrices, binding sites having

the size and shape complementary to the template CAP were generated. It is shown

in Table 1 that the imprinted plate had a higher selectivity for thiamphenicol than

florfenicol because the chemical structure of thiamphenicol was more closely related

to CAP than that of florfenicol.

0

10

20

30

40

50

60

70

80

0.1 10000010000100010010

Inhi

biti

on (

%)

1

(a)

0.01 0.1 1 1010500

11000

11500

12000

12500

13000

13500

14000

Rel

ativ

e lig

ht in

tens

ity

(b)

CAP concentration ( /L)gµ

CAP concentration ( /L)gµ

Figure 5. Standard curves of CAP. (a) CLEIA with the imprinted 96-well plate as the

artificial antibody. (b) CLEIA with the CAP ELISA kit.

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Standard curve of CAP

The standard competition curve was established with the logarithm of concentration

of CAP standard solution as x-coordinate and the inhibition as y-coordinate, which

is shown in Figure 5(a). According to the standard curve of CAP, the IC50 value was

3092 mg �L�1. The limit of detection (LOD) which was calculated as a concentration

that gives a 10% inhibition (IC10) of colour development for the CLEIA with the

imprinted 96-well plate as the artificial antibody was 0.990.01 mg �L�1.The standard curve of CAP obtained by the CAP ELISA kit is shown in Figure

5(b). It is shown that CAP standard solution showed linear correlation ranging from

0.05 to 4.05 mg �L�1. The LOD was 0.05 mg �L�1.

Sample analysis and CLEIA with CAP ELISA kit validation

The suitability and applicability of the CLEIA with the artificial antibody were

evaluated by the measurement of real samples. The samples were determined with

three replicates for each concentration. It is shown in Table 2 that the recovery

ranged from 89% to 98.7%, indicating that the CLEIA with the artificial antibody

had relative applicability.

The accuracy of the CLEIA method was validated by comparative analysis of the

spiked samples with CAP ELISA kit (Table 2), and no significant differences wereobserved between the two methods.

Conclusions

In this paper, a molecularly imprinted polymer which efficiently and selectively

bound CAP specifically was prepared on the surface of 96-well plate by the bulk

polymerisation. The molecular template was labelled with the enzyme HRP, and

according to the direct competitive ELISA, a chemiluminescence enzyme immu-

noassay method was established for the determination of CAP residues in the sea

cucumber based on the molecularly imprinted polymer of CAP as an artificial

antibody. This method exhibited excellent performance in real sample analysis with

the IC50 and the detection limit values being 3092 mg �L�1 and 0.990.01 mg �L�1,respectively. The artificial antibody prepared in this study showed high selectivity

and strong specificity like the biological antibody. Besides, the biological antibody

had the advantages of low cost, ease of preparation, high stability and strong

resistibility over harsh environments such as high temperature, strong acid or base.

Table 2. Results of CAP residues by the CLEIA with the artificial antibody and CLEIA with

CAP ELISA kit (mean9SD, n �3).

Sample Spiked level (mg �L�1) CAP kit (mg �L�1) CLEIA (mg �L�1)

Coelomic fluid 1 0.9890.01 0.9090.02

5 4.8990.01 4.7990.02

10 9.8790.01 9.3490.02

Body wall 1 0.9590.02 0.8990.03

5 4.8190.05 4.7390.04

10 9.8490.04 9.2890.07

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Substitution of the biological antibody in the kit with the MIP showed a great

application prospect. Although the sensitivity of the method was low, the sensitivity

and accuracy of the MIP-based immunoassays would be improved with the

development of MIT.

References

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