determination of folic acid in milk, milk powder and energy drink by an indirect immunoassay

Research ArticleReceived: 2 November 2011 Revised: 15 January 2011 Accepted: 17 January 2011 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.5625

Determination of folic acid in milk, milk powderand energy drink by an indirect immunoassayTaichang Zhang,a Huyin Xue,a Bo Zhang,b Yu Zhang,a Pei Song,a Xi Tian,a

Yue Xing,a Peng Wang,a Meng Menga∗ and Rimo Xia∗

Abstract

BACKGROUND: Folic acid (FA) is essential for healthy people (reference daily intake 400 µg day−1) and pregnant women(600 µg day−1). Insufficient intake of FA will increase the risk of neural tube defects in newborns. In this study an indirectenzyme-linked immunosorbent assay was developed for rapid and convenient detection of FA in vitamin-fortified foods.

RESULTS: A carbodiimide-modified active ester method was used to synthesise the immunogen (FA–bovine serum albumin(BSA) conjugate) to raise polyclonal antibodies for FA. The coupling ratio of FA with BSA was determined to be 14 : 1 (molarratio). The detection limit of the immunoassay was 3.0 ng mL−1 in buffer, 3.52 ng mL−1 in energy drink, 11.91 ng mL−1 inmilk and 16.50 ng mL−1 in milk powder. Intra- and inter-assay variability ranged from 6.6 to 15.1%. Analytical recoveries ofFA-spiked samples were 88.3–108.9%.

CONCLUSION: The immunoassay developed in this study can be used as a simple, rapid and accurate method for fastsemi-quantitative and quantitative on-site analysis of FA in food products.c© 2012 Society of Chemical Industry

Keywords: folic acid; folate; polyclonal antibody; immunoassay

INTRODUCTIONFolic acid (FA, (2S)-2-[(4-{[(2-amino-4-hydroxypteridin-6-yl)methyl]amino}phenyl)formamido]pentanedioic acid, vitamin B9)is essential for DNA synthesis, repairment and methylation.1 – 6 Itis also required to produce healthy red blood cells and preventanaemia in humans.7,8 Insufficient intake of FA can lead to mentalconfusion, diarrhoea, irritability, behaviour disorder, headache ornerve damage.9 Besides, insufficient FA intake by pregnant womencan cause neonate neural tube defects.2,10,11 Therefore, to evalu-ate the intake of this nutrient, the United States National Academyof Sciences has set reference daily intakes of FA by healthy adults(400 µg day−1) and pregnant women (600 µg day−1). Althoughfolate is highly enriched in leafy vegetables and animal liver prod-ucts, health organisations worldwide require FA addition in manygrain products, including bread, flour, cereal and rice (Australiafrom 18 September 2009; Canada in 1998; New Zealand from18 September 2009; USA from 1 January 1998). This mandatoryintroduction effectively decreases the risk of neural tube defectsin newborns. Although FA is only lowly toxic, its excess intakewill influence the absorption of vitamin B12, thus inducing B12

deficiency.1 The Ministry of Health of the People’s Republic ofChina has published the regulation ‘Hygienic Standard for the Useof Nutritional Fortification Substances in Foods (GB 14880-94)’.This regulation sets fortification levels of FA in infant formulae(380–700 µg kg−1) and nutritional foods for pregnant women(2000–4000 µg kg−1). Thus it is very important to establish a rapidand efficient method for analysing FA that can be utilised by foodcontrol departments to evaluate the reliability of nutrition labels.

Microbiological assay, high-performance liquid chro-matography (HPLC), flow injection chemiluminescence/spectrophotometry and optical biosensor techniques have beenemployed for determining FA. Microbiological assay is the‘gold standard’ method for FA determination. It uses Lac-tobacillus casei and Streptococcus faecalis to measure totalfolate (5-methyltetrahydrofolate (5-methyl-THF) and FA) andnon-methylated folates (FA, dihydrofolate (DHF) and THF).12 – 15

However, this method cannot distinguish the different forms offolate. Moreover, the experimental procedure is time-consumingand tedious. Although HPLC is accurate, it is complicated interms of sample preparation and operation.16,17 Flow injec-tion chemiluminescence/spectrophotometry cannot be appliedfor FA determination in complex biological samples becauseof its poor separation ability.8,18 To enhance the sensitivityof detection, many research groups have built biosensors forFA analysis (limit of detection of 1–2 ng mL−1 in buffer).10,19,20

However, these methods usually need well-skilled operatorsand complicated treatment steps, which limit their applica-tion in high-throughput screening tests. A rapid, simple, in-expensive, sensitive and high-throughput method is urgently

∗ Correspondence to: Rimo Xi and Meng Meng, College of Pharmacy and TianjinKey Laboratory of Molecular Drug Research, Nankai University, Tianjin, China.E-mail: [email protected]; [email protected]

a College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research,Nankai University, Tianjin, China

b Tianjin Bioradar Biotechnology Co., Ltd., Tianjin, China

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www.soci.org T Zhang et al.

required, since more and more food products are being fortifiedwith FA.

Microplate titre enzyme immunoassay is an ideal methodfor rapid determination of various residues in biological orfood samples.2 In 1995, Sarma et al.21 prepared polyclonalantibodies of FA to detect FA in plasma. In that study theFA was applied as a clinical marker in therapies to monitorinhibition of DHF reductase. However, the method was nottested on real food products. The aim of our research was todevelop a simple immunoassay for effectively measuring FAfortification in nutritious foodstuffs, especially those for babiesand pregnant women. As far as we know, this is the firststudy to develop a convenient enzyme-linked immunosorbentassay (ELISA) method as a screening test for food products.The laboratory experimental results showed that the methodwas sensitive and specific and could be used in food safetymonitoring.

EXPERIMENTALReagents and immunoreaction buffersBovine serum albumin (BSA), ovalbumin (OVA), 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), folic acid, dihydrofolic acid, tetrahy-drofolic acid, 5-methyltetrahydrofolic acid, methotrexate, pteroicacid, folinic acid and Freund’s complete and incomplete adjuvantswere purchased from Sigma-Aldrich (St Louis, MO, USA). Isobutylchloroformate was obtained from J&K Scientific Ltd (Beijing, China).Goat anti-rabbit immunoglobulin G (IgG)–horseradish peroxidase(HRP) conjugate was purchased from Beijing Wanger Biotechnol-ogy Co., Ltd (Beijing, China). 3,3′,5,5′-Tetramethylbenzidine (TMB)was obtained from Shanghai Sangon Biological Engineering Tech-nology (Shanghai, China).

The following buffers were used in this research: (1) coatingbuffer: 50 mmol L−1 carbonate buffer (15 mmol L−1 Na2CO3 and35 mmol L−1 NaHCO3, pH 9.6); (2) blocking buffer: 1 g/100 mL

OVA in sodium phosphate-buffered saline (PBS, consisting of138 mmol L−1 NaCl, 2.7 mmol L−1 KCl, 1.5 mmol L−1 KH2PO4

and 7 mmol L−1 Na2HPO4) with addition of 0.5 mL L−1 Tween20; (3) washing buffer (PBST): PBS buffer with 0.5 mL L−1

Tween 20; (4) substrate buffer: TMB solution (400 µL of6 mL L−1 TMB/dimethylsulfoxide mixed with 100 µL of 10 mL L−1

H2O2 in citrate/acetate buffer, pH 5.5); (5) stopping solution:2 moL L−1 HCl.

InstrumentationELISA microtitre plates were obtained from JET Bio-filtrationProducts Co. (Guangzhou, China). The absorbance at 450 nmwas measured using a Model 680 microplate reader (Bio-RadLaboratories, Beijing, China). UV data were collected on a U-4100spectrophotometer (Hitachi Co., Kyoto, Japan). Centrifugationwas carried out in a Biofuge Stratos refrigerated centrifuge(Heraeus, Shanghai, China). Hapten–protein conjugate sampleswere lyophilised in an FD-1 freeze-drier (Boyikang TechnologyCorporation, Beijing, China).

Preparation of hapten–protein conjugatesThe immunogen FA–BSA was prepared by a carbodiimide-modified active ester method (Fig. 1). First, 22.7 mg (51.5 µmoL)of FA, 98.7 mg (514.7 µmoL) of EDC and 29.6 mg (257.4 µmoL)of NHS were dissolved in 6 mL of N,N-dimethylformamide (DMF)and incubated for 24 h at room temperature in the dark. To thissolution, 10 mL of PBS (0.1 mol L−1, pH 7.4) containing 100 mg(1.5 µmoL) of BSA was added slowly with stirring, and the mixturewas incubated for 3 h at room temperature. The reaction mixturewas subsequently dialysed (molecular weight cut-off 8–14 kDa)against PBS (0.1 mol L−1, pH 7.4) for 3 days and then againstdistilled water for 3 days. The dialysing medium was changedevery 12 h. The solution in the dialysis bag was lyophilised toobtain FA–BSA conjugate (yield 83.6%).

The coating antigen FA–OVA was prepared via a mixed acidanhydride reaction (Fig. 1). The carboxylic acid group in the FAmolecule was activated by isobutyl chloroformate and reactedwith amino groups on OVA. First, 49 mg of FA was dissolved in3 mL of dry DMF, then 29.1 µL (122.2 µmoL) of tri-n-butylaminewas added and the mixture was stirred for 30 min at 4 ◦C.Subsequently, 20.2 µL (155.6 µmoL) of isobutyl chloroformatewas added and the mixture was allowed to react for 1 h at roomtemperature. The activated FA was then slowly added to 10 mL ofPBS containing 100 mg (2.2 µmoL) of OVA and stirred overnight atroom temperature. The FA–OVA conjugate obtained was purifiedby dialysis and lyophilised as described above (yield 67.2%).

Production of polyclonal antibodiesTwo male New Zealand white rabbits (1.5–2 kg) were subcuta-neously immunised at multiple sites on their back with FA–BSAconjugate. For initial immunisation, 0.5 mg of conjugate dissolvedin 0.5 mL of physiological saline was emulsified with the same vol-ume of Freund’s complete adjuvant and then subcutaneously

C + N

O

O

HOEDC/DMF

room temperature,24h

N

O

O

OC

OBSA-NH2

C

OHN BSAOH

O

FA FA FA

Folic acid NHS FA-NHS FA-BSA conjugate(Yield=83.6%)

room temperature,3 h

FA+OVA-NH2Tri-n-butylamine

Cl O

O

O O

O O

Folic acid Isobutyl chloroformate FA-OVA conjugate(Yield=67.2%)

FA C

OHN OVA



C OH

O

FA

Figure 1. Synthesis of immunogen (FA–BSA conjugate) and coating antigen (FA–OVA conjugate).

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Determination of folic acid by indirect immunoassay www.soci.org

12

3

200 250 300 350 4000.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5A

bsor

banc

e (A

)

Wavelength (nm)

1. FA-BSA (280, 345nm)2. FA (280, 349nm)3. BSA (279nm)

Figure 2. UV spectra of folic acid (FA), bovine serum albumin (BSA) andFA–BSA conjugate.

1 10 100 1000

0.2

0.4

0.6

0.8

1.0

B/B

0

Concentration of folic acid (ng mL-1)

Figure 3. Relationship between inhibition ability (B/B0) and analysedconcentration of folic acid (n = 5).

10 1000.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

B/B

0

Folic acid (ng mL-1)

y=1.1167-0.4424xR2=0.9933

Figure 4. Linear calibration curve of folic acid determination by developedicELISA method (n = 5).

injected at ten sites. For subsequent booster immunisations,0.25 mg of conjugate in 0.5 mL of physiological saline and 0.5 mLof Freund’s incomplete adjuvant was injected 20 days later and at15 day intervals thereafter. Seven days after the fifth immunisa-tion the animals were bled to collect antiserum, which was thencentrifuged and stored at −20 ◦C.

Table 1. Coefficients of variation for detection of folic acid standardsolutions

Measured level (ng mL−1)FA solution Detection Inter-assay(ng mL−1) number Day 1 Day 2 Day 3 variation (%)

10 1 11.23 10.19 9.07 10.63

2 10.37 9.23 10.10 6.02

3 8.07 10.10 10.19 12.68

4 10.19 8.23 8.67 11.39

5 9.32 10.01 10.56 6.24

6 10.10 11.85 9.83 10.35

7 11.85 10.66 11.01 5.47

8 10.01 11.67 10.56 7.87

9 8.85 9.01 9.07 1.27

10 9.66 10.37 9.01 7.03

Intra-assay variation (%) 10.97 11.09 8.21 Average 7.89

50 1 52.28 50.34 53.47 3.04

2 48.85 47.04 42.64 6.92

3 43.29 44.87 41.38 4.05

4 50.72 40.76 58.45 17.75

5 41.38 56.37 51.88 15.42

6 59.23 51.11 49.41 9.86

7 48.07 45.64 46.33 2.68

8 47.75 48.85 40.15 10.39

9 41.69 46.88 42.64 6.32

10 52.67 40.45 47.20 13.09


100 1 93.65 101.73 81.86 10.81

2 89.85 89.02 85.76 2.45

3 97.71 103.73 102.42 3.13

4 96.72 84.96 88.19 6.75

5 102.37 92.40 98.63 5.15

6 96.81 87.37 81.10 8.94

7 80.34 90.69 95.02 8.51

8 114.48 98.74 104.84 7.49

9 99.55 112.30 110.33 6.39

10 106.26 93.26 82.62 12.59


Immunoassay proceduresThe immunoassay was performed in a 96-well polystyrenemicrotitre plate using an indirect ELISA (iELISA) procedure. Todetermine the antibody titre, the coating antigen FA–OVA(1 µg mL−1, 100 µL per well) in carbonate buffer (pH 9.6) wassteadily attached to the solid polystyrene surface by physicalabsorption and incubated overnight at 4 ◦C. Then the plate waswashed three times with 350 µL of washing buffer to removeunbound coating antigen. The uncoated active sites of polystyrenesurface in each well were blocked with 250 µL of blocking buffer.Then 100 µL of diluted antiserum in PBS was added and incubatedfor 0.5 h. After washing the plate a further three times, 100 µL ofHRP-conjugated goat anti-rabbit IgG (1 : 2000 in PBS) was addedand incubated for 0.5 h. Then 100 µL of TMB substrate solutionwas added to each well. After incubation for 15 min the enzymaticreaction between HRP and TMB was immediately stopped byadding 50 µL of stopping solution, and the absorbance at450 nm was measured with a microplate reader. Unless specificallyindicated, all incubations were performed at 37 ◦C.

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Table 2. Cross-reactivity of folic acid antibody to related compounds

Compound Structure IC50 (ng mL−1) CR (%)

Folic acid

N

NHN

N

H2N

NH

O

NHO

OHHO

O O 15.5 100

Pteroic acid

N

NN

HN

H2N

NH

O

OHO 10.7 145.3

Dihydrofolic acid

NH

NN

HN

H2N

NH

O

NHO

OHHO

O O 44.0 35.4

Tetrahydrofolic acid

NH

HNN

HN

H2N

NH

O

NHO

OHHO

O O 84.1 18.5

Methotrexate

N

NN

N

H2N

N

NH2

NHO

OHHO

O O 4423 0.35

5-Methyltetrahydrofolic acid

N

HNN

N

H2N

NH

OH

NHOOHHO

O O 4948 0.31

Folinic acid

N

HNN

N

H2N

NH

OH

NHO

OHHO

O O

OH

28076 0.06



Table 3. Limits of detection of folic acid analysis based on assay of20 blank food samples

Measured value (ng mL−1)

Sample number Milk Milk powder Energy drink

1 9.85 9.88 1.47

2 9.55 9.22 2.24

3 9.37 10.62 2.69

4 9.02 9.60 2.07

5 9.37 11.67 2.65

6 7.91 12.59 2.49

7 8.11 9.70 1.19

8 9.85 10.51 0.71

9 7.96 12.19 1.02

10 8.86 13.05 1.59

11 10.76 12.82 1.57

12 10.49 14.09 1.13

13 10.30 9.49 1.72

14 10.56 10.21 0.87

15 8.64 14.71 1.80

16 10.23 9.77 1.86

17 9.08 12.23 2.34

18 9.20 13.29 2.01

19 9.25 9.53 1.98

20 8.80 9.36 0.97

Average 9.36 11.23 1.72

SD 0.85 1.76 0.60

LOD 11.91 16.50 3.52

The concentration of FA was determined by an indirectcompetitive ELISA (icELISA) procedure. The difference betweenicELISA and iELISA is that in the icELISA method, after blocking,50 µL of analyte solution (FA standard solution or food sample)was mixed with 50 µL of antiserum of appropriate dilution andadded to the well. Then HRP-conjugated goat anti-rabbit IgG andsubstrate solution were added as described above. Competitivecurves were obtained by plotting the normalised signal B/B0

against the logarithm of analyte concentration, where B0 is theabsorbance value for the solution without analyte and B is theabsorbance value for the solution with analyte.

Assay specificityThe specificity of the assay was investigated by cross-reactivity(CR) experiments. Six compounds structurally related to FA wereselected for testing CR. Standard solutions of each compoundwere prepared and subjected to icELISA. CR was expressed as %IC50 value based on 100% response of FA and calculated as follows:

CR (%) = (IC50 of FA/IC50 of cross-reacting compound) × 100

Determination of FA in food samplesMilk, milk powder and energy drink were purchased fromlocal supermarkets for evaluation. The milk was defatted bycentrifugation at 10 000 × g for 10 min at room temperature.The defatted milk was diluted fivefold with PBS. The milk powder(10 g) was dissolved in distilled water (90 mL) and centrifugedat 10 000 × g for 10 min at room temperature. The fat layer wasdiscarded and the upper liquid was diluted ten times with PBS.The energy drink (1 mL) was diluted ten times with PBS.

Twenty blank samples of each matrix were subjected to icELISA.Coefficients of variation were determined by the analysis of milkand milk powder samples fortified with FA at 20, 50 and 100 ng g−1

and energy drink samples fortified with FA at 10, 50 and 100 ng g−1.The analytical recovery (%) for each group was calculated asfollows:

recovery (%) = (concentration measured/

concentration fortified) × 100

RESULTS AND DISCUSSIONHapten conjugationFA is a small molecule with a relative molecular mass of 441.4. Inthis study we chose the carboxylic acid group in the FA moleculeas the active site for coupling with carrier proteins to form theimmunogen and coating antigen. As illustrated in Fig. 1, the freecarboxylic acid group on FA was linked to amino groups on BSAby a carbodiimide-modified active ester method,22 – 24 while thecoating antigen FA–OVA was prepared via a mixed acid anhydridereaction. These reactions resulted in an amide bond betweenthe FA molecule and the carrier protein. Sarma et al.21 modifiedBSA with ε-aminocaproic acid to prepare FA immunogen. TheFA/ε-aminocaproic acid-modified BSA conjugate showed higherspecificity than FA–BSA conjugate. The modified BSA had to bedialysed, lyophilised, purified on a Sephadex G-50 column andlyophilised to obtain the purified product. Before use for couplingwith FA, the modified BSA must be tested again to compare thenumber of amino groups with those in free BSA.21 In comparison,the synthesis protocol employed in our study is much simpler.

To confirm the conjugation reaction, UV spectra of FA, BSA, OVA,FA–BSA and FA–OVA were measured. As can be seen in Fig. 2,BSA showed an absorbance maximum at 279 nm. After removingfree FA by dialysis, absorbance maxima of FA–BSA appeared at280 and 345 nm. Compared with the UV spectrum of FA solution(λmax1 = 280 nm, λmax2 = 349 nm), the successful conjugation ofFA with BSA was confirmed. The UV spectrum of FA–OVA (notshown) was similar to that of FA–BSA. Assuming that the molarabsorbance of the hapten in free and conjugated forms is thesame, the molar ratio of hapten to protein was estimated using theUV–visible spectrophotometric method.25 The hapten/proteinmolar ratio was directly calculated from the molar absorbancecoefficient (ε):

hapten/protein molar ratio = (εconjugate − εprotein)/εhapten

The characteristic wavelength of the hapten (λ = 349 nm) wasselected to determine absorbance values of the hapten, carrierprotein and conjugate at some known concentrations, then valuesof molar absorbance coefficient (ε) were calculated. Finally, themolar ratio of FA to BSA was determined to be 14 : 1, while that ofFA to OVA was approximately 8 : 1. Thus a higher coupling ratioof FA with BSA was calculated, and this result is consistent withpreviously published papers.25 – 27

Antibody titre and sensitivityThe titre of the antibody was defined as the reciprocal of thedilution that resulted in an absorbance value twice that of thebackground. The antibody titre in this study was measured by theiELISA method. From a checkerboard experiment the optimalconcentration of coating antigen FA–OVA was found to be



Table 4. Recoveries and coefficients of variation of icELISA method to detect folic acid in food products

Sample Spiked FA (ng mL−1) Detected folic acid (ng mL−1) Mean recovery (%) CV (%)

Milk 20 19.8 21.7 18.1 17.7 22.5 99.8 10.8

23.1 20.8 21.9 22.8 17.3 105.9 11.1

21.3 19.3 20.9 16.8 19.6 97.8 9.0

22.0 21.1 17.4 19.1 22.2 101.9 10.1

17.2 20.3 22.6 22.3 18.3 100.7 11.8

20.8 17.8 19.3 22.5 21.2 101.7 8.9

50 48.9 42.8 52.9 40.8 45.8 92.5 10.4

46.3 51.9 58.8 45.1 42.5 97.8 13.3

54.9 47.4 41.5 43.5 44.2 92.6 11.3

49.6 42.0 45.6 53.7 51.4 96.9 9.6

53.7 41.1 53.0 48.5 43.3 95.8 11.8

42.3 40.9 45.3 53.1 54.3 94.4 13.1

100 95.4 111.4 99.8 88.9 100.6 99.2 8.3

97.7 86.1 108.8 100.9 88.0 96.3 9.7

94.8 98.6 101.6 113.7 89.9 99.7 9.0

83.5 95.7 100.9 97.8 86.1 92.8 8.2

96.7 114.6 91.8 97.7 88.9 97.9 10.2

98.8 89.8 96.7 107.8 88.9 96.4 8.0

Milk powder 20 18.4 20.5 17.5 21.8 23.4 101.5 11.8

16.2 18.7 23.5 20.1 16.0 94.6 16.3

19.3 16.8 21.6 22.9 20.2 100.9 11.6

21.1 17.3 22.3 19.0 20.0 99.7 9.6

22.5 21.3 19.1 16.9 21.9 101.7 11.4

20.8 20.9 16.6 22.2 21.5 101.9 10.8

50 41.3 58.8 52.4 53.9 47.5 101.6 13.1

54.5 48.7 44.6 52.8 47.3 99.2 8.1

43.2 51.5 45.0 55.6 43.6 95.6 11.6

45.7 50.9 45.4 40.9 57.9 96.3 13.5

40.4 48.3 43.6 42.3 46.1 88.3 7.0

51.3 49.3 57.2 45.9 59.7 105.3 10.8

100 81.4 96.2 91.0 82.7 98.2 89.9 8.5

85.2 89.2 91.9 103.3 96.2 93.2 7.4

105.9 94.8 98.7 89.6 91.5 96.1 6.8

95.7 88.7 83.1 94.8 82.7 89.0 7.0

91.9 111.4 81.0 94.8 95.7 95.0 11.5

87.4 80.2 100.7 97.4 88.3 90.8 9.1

Vitamin juice 10 8.4 8.7 8.8 9.0 11.9 93.6 15.1

8.6 10.7 10.8 9.6 10.6 100.7 9.3

9.4 9.9 11.5 10.4 8.6 99.6 10.9

9.4 10.3 10.0 11.3 9.2 100.3 8.2

9.7 10.7 11.3 11.0 11.8 108.9 7.4

9.8 10.9 8.9 10.2 11.7 102.9 10.6

50 53.4 49.0 41.8 54.3 50.7 99.7 10.0

52.8 49.3 55.0 57.0 44.1 103.3 9.8

44.9 56.1 48.2 58.8 43.7 100.7 13.4

46.5 55.4 53.9 51.5 45.3 101.0 8.8

52.4 42.6 45.7 47.2 55.6 97.4 10.8

57.2 49.8 58.1 41.2 51.3 103.1 13.2

100 104.4 89.4 90.8 105.6 81.5 94.3 10.9

84.1 117.6 102.4 115.8 109.7 105.9 12.8

96.2 88.1 104.0 87.7 91.2 93.4 7.3

100.8 108.9 97.4 113.2 98.9 103.8 6.6

83.8 95.9 111.9 90.1 85.1 93.3 12.2

94.0 82.8 99.3 102.0 114.0 98.4 11.6



1 ng mL−1, and a suitable dilution of goat anti-rabbit IgG–HRPwas 1 : 2000. Serum taken before immunisation was used as anegative control. Under the optimal conditions the final antibodyhad a titre of >1 024 000.

The sensitivity to free FA was determined using the icELISAmethod. Aliquots (50 µL) of FA standard solution in PBS (0, 0.4,1, 6.25, 12.5, 25, 50, 100, 300 and 1000 ng mL−1) were analysed.Under the optimised conditions a competitive inhibition curve wasconstructed (Fig. 3). The concentration of FA solution resulting in50% inhibition of the maximum signal is expressed as IC50. Asshown in Fig. 3, the IC50 value for this method was 15 ng mL−1.The limit of detection (LOD, IC10) was measured as 3.0 ng mL−1. Alinear calibration curve was obtained with FA in the concentrationrange 1–100 ng mL−1 (Fig. 4). This LOD value was comparableto that obtained from electrochemical magneto sensors (LOD =2.7 ng mL−1) in buffer.3

For precision assessment, FA standard solutions (10, 50 and100 ng mL−1) were detected ten times on one day and threedifferent days for the calculation of intra- and inter-assay variationsrespectively. As shown in Table 1, the intra-assay coefficient ofvariation (CV) ranged from 8.95 to 12.56% in three days, andthe inter-assay CV was less than 9.0%. There was no significantdifference in intra- or inter-assay variation for each level.

Antibody specificitySix similar compounds (pteroic acid, dihydrofolic acid, tetrahydro-folic acid, methotrexate, 5-methyltetrahydrofolic acid and folinicacid) were tested with the icELISA method to characterise the speci-ficity of the antibody. As seen in Table 2, the antibody showedhigh cross-reactivity with pteroic acid (CR = 145.3%), which meansthat the pentanedioic acid side chain is a characteristic deter-minant structure. For dihydrofolic acid and tetrahydrofolic acid,however, the hydrogen addition in the pteridin ring significantlydecreased their binding affinity with the antibody. Therefore thepeteridin ring is probably one of the characteristic structures.The high cross-reactivity of pteroic acid, dihydrofolic acid andtetrahydrofolic acid with FA antibody was similar to that reportedpreviously.21 Introduction of a methyl (5-methyltetrahydrofolicacid) or aldehyde group (folinic acid) at the nitrogen atom in thepteridin, or a methyl group (methotrexate) in the side chain ofpteridin all inhibited its binding to FA antibody and resulted inlow affinity. Based on these results, we can conclude that themain antigen determinant for FA antibody is the structural moiety[(2-amino-4-hydroxypteridin-6-yl)methyl]amino.

ELISA performance in food productsMilk, milk powder and energy drink (vitamin juice) purchasedfrom local supermarkets were used as the matrices. Sampleswere treated as described above and analysed by the icELISAmethod. The FA level was then determined according to thelinear calibration curve (Fig. 4). The results were compared withthose obtained in buffer. The LOD value is defined as the averageconcentration determined in 20 blank samples plus three timesthe standard deviation (SD) (Table 3). The LOD for energy drink(3.52 ng mL−1) was as low as that in buffer (3.0 ng mL−1), whileit was a little higher for milk (11.91 ng mL−1) and milk powder(16.50 ng mL−1).

Based on the LOD results, the spiked level of FA was set at20–100 ng mL−1 in milk and milk powder and 10–100 ng mL−1

in vitamin juice. Analytical recoveries of FA-spiked food sampleswere 88.3–108.9%, while CV values ranged from 6.6 to 15.1%(Table 4).

CONCLUSIONAn ELISA has been developed for rapid detection of FA in foodsamples. The polyclonal antibody for FA showed a high titre(>1 024 000), and the indirect competitive immunoassay reacheda low LOD of 3.0 ng mL−1 in buffer and an IC50 of 15 ng mL−1. CVvalues were 8.95–12.56% intra-assay and below 9.0% inter-assay.The method showed high specificity, with no obvious cross-reactivity for other related compounds except pteroic acid. TheLOD values were 3.52 ng mL−1 for energy drink, 11.91 ng mL−1

for milk and 16.50 ng mL−1 for milk powder. In summary, theimmunoassay developed in this study can be used as a simple,rapid and accurate assay for fast semi-quantitative and quantitativeon-site analysis of FA in food products.

ACKNOWLEDGEMENTSWe would like to thank the National Natural Science Founda-tion (81173017, 31101277), the National High-Tech Research andDevelopment Program of China (863 Program, 2010AA10Z402,2007AA06A407), the Tianjin Science and Technology Pro-gram (09ZCKFSH07500), the Scientists–Company CooperationProject of the Ministry of Science and Technology of China(SQ2009GJA0002591) and the Fundamental Research Funds forthe Central Universities (65011121) for their financial support.

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determination of folic acid in milk, milk powder and energy drink by an indirect immunoassay

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