Establishment of an Indirect Competitive Enzyme-Linked Immunosorbent Method for the Detection of Heavy Metal Cadmium in Food Packaging Materials

Heavy metals in food packaging materials have been indicated to release into the environment at slow rates. Heavy metal contamination, especially that of cadmium (Cd), is widely acknowledged as a global environment threat that leads to continuous growing pollution levels in the environment. Traditionally, the detection of the concentration of Cd relies on expensive precision instruments, such as inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma-atomic emission spectrometry (ICP-AES). In this study, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a specific monoclonal antibody was proposed to rapidly detect Cd. The half-inhibitory concentration and detection sensitivity of the anti-cadmium monoclonal antibody of the ic-ELISA were 5.53 ng mL−1 and 0.35 ng mL−1, respectively. The anti-Cd monoclonal antibody possessed high specificity while diagnosising other heavy metal ions, including Al (III), Ca (II), Cu (II), Fe (III), Hg (II), Mg (II), Mn (II), Pb (II), Zn (II), Cr (III) and Ni (II). The average recovery rates of Cd ranged from 89.03–95.81% in the spiked samples of packing materials, with intra- and inter-board variation coefficients of 7.20% and 6.74%, respectively. The ic-ELISA for Cd detection was applied on 72 food packaging samples that consisted of three material categories—ceramic, glass and paper. Comparison of the detection results with ICP-AES verified the accuracy of the ic-ELISA. The correlation coefficient between the ic-ELISA and the ICP-AES methods was 0.9634, demonstrating that the proposed ic-ELISA approach could be a useful and effective tool for the rapid detection of Cd in food packaging materials.


Introduction
Due to its favourable toughness and ductility [1][2][3], cadmium (Cd) is becoming one of the most frequently used elements in the manufacturing of metal, plastic and dyestuff materials, which are commonly used in food packaging, kitchenware, plastics, bottles and boxes [4,5]. Increased public awareness of contamination in food packaging materials has raised global concerns regarding potential heavy metal environmental contamination from packaging [6,7]. Cd pollution has now been identified as one of the most major factors affecting food safety in many countries [8][9][10], especially in nori (restaurant-served and ready-to-eat sushi in the Polish market) [11]. Perhaps more worrisome, massive overpackaging has led to the growing presence of toxic metals such as cadmium, mercury, and lead [12], which now present extreme or even incredible toxicity. Therefore, the quality and safety of food packing materials are closely related to people's health and happiness [13]. and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) were bought from Sigma-Aldrich (St. Louis, MO, USA). 3,3 ,5,5 -Tetramethylbenzidine (TMB) was gained from Dean Biological Co., Ltd. (Zhejiang, China). RPMI 1640 cell culture medium, fetal bovine serum (FBS), HAT supplement (liquid mixture of sodium hypoxanthine, methotrexate, and thymine) and HT supplement (liquid mixture of sodium hypoxanthine and thymidine) were obtained from Life Technologies Co. (New York, NY, USA). 1-(4-isothiocyanatobenzyl) vinyldiamine-N,N,N',N'-tetraacetic acid (ITCBE) was acquired from Dongren Chemical Technology Co., Ltd. (Shanghai, China). The myeloma cell line of Sp2/0 was obtained from the Chinese Academy of Sciences (Shanghai, China). Cell culture plates and 96-well micro-titer plates were obtained from Corning Inc. (New York, NY, USA). We paid Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China) for other conventional chemical reagents.
A phosphate buffer saline (PBS buffer, 10 mM, pH 7.4) was prepared by dissolving 0.2 g NaCl, 1.55 g NaH 2 PO 4 and 0.25 g KH 2 PO 4 in ultra-pure water and diluted to 1 L. Phosphate buffer solution (PBST) was obtained from a PBS buffer (10 mM, pH 7.4) containing 0.05% Tween-20. HEPES (2.649 g) and ITCBE (4.136 g) were dissolved in ultra-pure water and diluted to 1 L, resulting in a HEPES buffer of 10 mM, with pH 9.0. The solution is adjusted to pH 9.0 using an NaOH solution. The food packing material samples were either presented by Professor Jun Wang (Jiangnan University, Wuxi, China) or bought from a supermarket in Hangzhou (China).
72 food packaging materials were collected for Cd detection. These 72 samples could be classified into three categories: ceramic, glass and paper. For each category, 24 samples were prepared. The ceramic group included six ceramic water cups, six ceramic bowls, six ceramic pots and six flat ceramic basins. The glass group included six glass water cups, six glass jars, six small glass wine cups and six high foot glass wine cups. The paper group included six paper trays, six paper cups, six paper bowls and six paper plates. All 72 samples were purchased from the supermarket.

Preparation of Cd-ITCBE-BSA and Cd-ITCBE-OVA
Complete antigens were reacted according to Kong et al. [42] and modified with a bi-functional chelating agent ITCBE. The comprehensive synthesis scheme is shown in Figure 1. Cd-1-(4-isothiocyanatobenzyl) vinyldiamine-N,N,N',N'-tetraacetic acid (ITCBE)ovalbumin(OVA) (Cd-ITCBE-OVA) and Cd-1-(4-isothiocyanatobenzyl) vinyldiamine-N,N,N',N'-tetraacetic acid (ITCBE)-bovine serum albumin (BSA) (Cd-ITCBE-BSA) were used as an immunogen and a coating antigen, respectively. Briefly, 2.0 mg ITCBE was dissolved in 0.2 mL DMSO and mixed with 10.0 mg BSA in 10.0 mL HEPES buffer, and then subsequently stirred at room temperature for 24 h. At the end of the reaction, the samples were collected and dialyzed at 4 • C for 24 h with the HEPES buffer in order to purify. After dialysis, the Cd (II) standard solution (1.2 mL, 1 mg/mL) was added drop by drop and the pH was adjusted to 9.0 with NaOH, then stirred at room temperature for 4 h. The conjugates were dialyzed in a HEPES buffer at 4 • C for 24 h, and subsequently in a phosphate buffer (PBS buffer) at 4 • C for 24 h. Afterwards, the collected samples were stored at −20 • C at the dark and were ready to be used. Using OVA instead of BSA, CD-ITCBE-OVA was synthesized in the same way.

Production of Monoclonal Antibody
This study strictly complied with the Regulations of Zhejiang Province on the Management of Experimental Animals (2009) to minimize the suffering of animals. Antibodies were generated in Balb/c mice, as described by Gong et al. [43]. Three Balb/c female mice were purchased from the experimental Animal Centre of Hangzhou Normal University (Hangzhou, China), immunized with Cd-ITCBE-OVA (200 µg in 100 µL sterile PBS) by sub-cutaneous injection and emulsified with an equal volume of Freund's complete adjuvant. In subsequent immunizations (three times, at an interval of every 2 weeks), 100 µg of Cd-ITCBE-OVA conjugate (in 100 µL sterile PBS) with the same volume of Freund's incomplete adjuvant was used. Blood was collected from the posterior orbital vein and stored at 4 • C overnight. Afterwards, the anti-serum was extracted from the blood and centrifuged at 5000 rpm for 8 min. Anti-cadmium monoclonal antibodies were produced by the hybridoma technique. In an RPMI 1640 cell culture medium supplemented with 10% FBS, murine myeloma cells Sp2/0 were maintained at the exponential growth stage. Immune mouse spleen cells were fused with myeloma cells. Hybridomas were selected in a HAT medium (dulbecco's modified eagle medium (DMEM) containing 15% FBS). The cultures of the 96-well plates were maintained in an incubator with 5% CO 2 , and the hybridoma colonies were subsequently expanded in the culture medium. The supernatant of the hybridoma was screened by ic-ELISA, with 10 ng mL −1 cadmium similarly screened for comparison. The established ELISA system is used to select specific cell lines against Cd, and sub-clones are used to continuously cultivate multi-cell line wells to specific single-cell line wells. Then, the stable cells were expanded and cryopreserved in liquid nitrogen. The stable cells were produced for ascites and purified via the octanoic acid-sulfur ammonium method to obtain monoclonal antibodies. The supernatant of hybridoma was screened by ic-ELISA, with 10 ng mL −1 cadmium similarly screened for comparison.

Production of Monoclonal Antibody
This study strictly complied with the Regulations of Zhejiang Province on the Management of Experimental Animals (2009) to minimize the suffering of animals. Antibodies were generated in Balb/c mice, as described by Gong et al. [43]. Three Balb/c female mice were purchased from the experimental Animal Centre of Hangzhou Normal University (Hangzhou, China), immunized with Cd-ITCBE-OVA (200 μg in 100 μL sterile PBS) by sub-cutaneous injection and emulsified with an equal volume of Freund's complete adjuvant. In subsequent immunizations (three times, at an interval of every 2 weeks), 100 μg of Cd-ITCBE-OVA conjugate (in 100 μL sterile PBS) with the same volume of Freund's incomplete adjuvant was used. Blood was collected from the posterior orbital vein and stored at 4 °C overnight. Afterwards, the anti-serum was extracted from the blood and centrifuged at 5000 rpm for 8 min. Anti-cadmium monoclonal antibodies were produced by the hybridoma technique. In an RPMI 1640 cell culture medium supplemented with 10% FBS, murine myeloma cells Sp2/0 were maintained at the exponential growth stage. Immune mouse spleen cells were fused with myeloma cells. Hybridomas were selected in a HAT medium (dulbecco's modified eagle medium (DMEM) containing 15% FBS). The cultures of the 96-well plates were maintained in an incubator A Cd-ITCBE-BSA conjugate was used as the coating coupling. A continuous dilution method was introduced to sub-clone the hybridoma secreting cadmium specific antibody. Colonies of interest were propagated, frozen (kept overnight at −80 • C) in a culture medium containing 10% DMSO and preserved in liquid nitrogen.
The affinity of monoclonal antibodies was determined with ic-ELISA by referring to Kim et al. [44]. The affinity of the monoclonal Abs (K) was as Equation (1): where C Ab-Ag , C Ab and C Ag are the concentrations of the conjugate, antibody and anti-gen, respectively.

Optimal Working Concentration of Anti-Cadmium Monoclonal Antibody and Coating Anti-Gen
96-well plates coated with Cd-ITCBE-BSA were blocked at 4 • C for 24 h [45]. After the reaction, the 96-well plates were dried at 37 • C for 5 h and stored at 4 • C for later use. PBS and the diluted Cd (II) standard material were added to seated 96-well plates at a concentration of 50 µL per well. Afterwards, the anti-cadmium monoclonal antibody and goat anti-mouse-horseradish peroxidase were diluted and added to the closed 96well plates with 50 µL per well. The reaction took place at 25 • C for 30 min, and the plates were washed with PBST three times after the reaction was completed. The TMB chromogenic reagent was then added to the 96-well plates at a concentration of 100 µL per well. The reaction was performed at 25 • C for 10 min, and the stopping solution was added to the plate at 50 µL per well. The OD 450 value was measured at 450 nm with a micro-plate reader [46]. The appropriate OD 450 value was selected, and the optimal working concentration was determined based on the inhibition rate of the Cd (II) standard.

Standard Curve and Sensitivity Analysis
The optimal working concentration of the anti-cadmium monoclonal antibody and goat anti-mouse-horseradish peroxidase were determined based on Liu's reference [47]. The specific concentrations of Cd (II) standard substance (0, 0.33, 1.0, 3.0, 9.0, 27.0 and 81.0 ng mL −1 ) and goat anti-mouse-horseradish peroxidase were added to the blocked 96-well plates, respectively, and the reactions were carried out at 25 • C for 30 min. After the reaction, the 96-well plate was washed with PBST three times and the TMB chromogenic reagent was added to the plate at a concentration of 100 µL per well and subsequently performed at 25 • C for 10 min. The stop solution was added to the 96-well plate at 50 µL per well, and the OD value was measured at 450 nm with a micro-plate reader. The experiment was repeated 12 times. The logarithm of Cd (II) standard concentration and binding ratio were set as the abscissa and ordinate, respectively. The semi-logarithmic standard curve and regression standard curve were established, and the half-inhibition concentration was IC 50 . IC 50 is a very important datum in the ic-ELISA standard curve, which is an S-shaped curve. In ic-ELISA, the OD value of the control well without an inhibition substance is B0, and that of the well with an inhibition substance is B. B/B0% is known as the binding rate, and the corresponding inhibitory substance concentration is called IC 50 when the binding rate is 50%. The half-inhibitory concentration (IC 50 ) and detection sensitivity (IC 10 ) values of 50% and 10% inhibition rates were calculated by the formula, and the sensitivity of the standard curve was analysed.

Minimum Detection Limit and Minimum Limit of Quantification
A suitable variety of food packing materials samples were selected from the market and tested by ICP-AES. From the results, 10 food packing materials samples without Cd (II) were randomly selected and identified as negative samples. Negative samples were examined 10 times using the ELISA method established in this study. The mean value of the negative ELISA samples assay plus three standard deviations (SD) was taken as the limit of detection (LOD). The mean value of negative ELISA sample test plus six times the standard deviation (SD) was represented as the minimum limit of quantitation (LOQ).

Precision and Accuracy of Ic-ELISA Analysis
The precision of the ic-ELISA could be reflected by the fluctuation of the OD 450 value of each well in the plate and the OD 450 value of the same concentration between the plates [42]. That is to say, the coefficient of variation (CV) was calculated by measuring the OD 450 value, and each group of values came from 12 parallel experiments.
The accuracy analysis of ic-ELISA established in this study could be verified by adding the recovery rate method. Cd (II) standards were added to these negative food packing material samples at gradient concentrations of 0, 100, 200 and 400 ng mL −1 , respectively. Then, the negative food packing material samples were detected via ICP-AES. The recovery rate was calculated for analysis [49]. Ten parallel experiments were respectively repeated to calculate the average value added and recovery rate after extraction. The ratio of actual sample concentration to theoretical concentration was used as the recovery rate.

Comparison of Ic-ELISA with ICP-AES
To verify the applicability of ic-ELISA, ICP-AES and ic-ELISA were used to analyse the spiked and actual samples of food packing material, respectively. 400 µL of 10% HCl and 400 µL of 0.1 M HNO 3 were added to a 200 mg food packing material debris sample. The mixture was shaken well and placing it in a 100 • C constant temperature metal bath for 10 min. After heating, the supernatant was centrifuged at 13,000 rpm for 10 min. Soon afterwards, the same volume of the HEPES buffer was added to the neutralization solution for standby use.
The Cd (II) standard was added to the 10 negative samples in order to achieve the final specific concentrations (50, 100, 200 and 400 ng mL −1 ). These above added negative samples were analysed by ic-ELISA and ICP-AES, respectively.
ICP-AES and ic-ELISA were used to detect 12 kinds of food packing material samples. According to the test results, the test results of ic-ELISA and ICP-AES were used as the ordinate and abscissa, respectively. The regression curve was established with respect to the test results of the two methods. The correlation between the two methods was reflected by the correlation coefficient (R 2 ) in the regression equation.

UV-Scanning Identification of Cd-ITCBE-BSA and Cd-ITCBE-OVA
The prepared Cd-ITCBE-BSA and Cd-ITCBE-OVA were identified using UV scanning [50]. The characteristic peak of Cd-ITCBE was found at 245.0 nm (1.43 mg/mL) and that of BSA was found at 280.0 nm (1.0 mg/mL). However, the coated anti-gen Cd-ITCBE-BSA (4.2 mg/mL) had a characteristic peak at 275 nm. Obviously, the characteristic peak of Cd-ITCBE-BSA was significantly different from those of Cd-ITCBE and BSA. These properties indicate that Cd-ITCBE was successfully coupled to the BSA carrier protein. The result is shown in Figure 2a. Similarly, Cd-ITCBE-OVA (2.1 mg mL −1 ) was successfully synthesized. The result is shown in Figure 2b.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Identification of Cd-ITCBE-BSA and Cd-ITCBE-OVA
The prepared Cd-ITCBE-BSA and Cd-ITCBE-OVA were identified by SDS-PAGE electrophoresis. The results showed that the Cd-ITCBE-BSA band was found after the appearance of the BSA band, reflecting a hysteresis. It showed that the relative molecular weight of Cd-ITCBE-BSA was greater than that of BSA. Based on this phenomenon, it could be inferred that Cd (II) was successfully conjugated to BSA by the bi-functional chelator ITCBE. The identification results were consistent with those obtained via UV scanning. The result is shown in Figure 3a. The Cd-ITCBE-OVA band had a significant hysteresis relative to OVA, indicating that its relative molecular weight was much greater than that of OVA. In accordance with this result, it could be concluded that Cd (II) was triumphantly coupled to OVA via the bi-functional chelator ITCBE. The result is shown in Figure 3b.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Identification of Cd-ITCBE-BSA and Cd-ITCBE-OVA
The prepared Cd-ITCBE-BSA and Cd-ITCBE-OVA were identified by SDS-PAGE electrophoresis. The results showed that the Cd-ITCBE-BSA band was found after the appearance of the BSA band, reflecting a hysteresis. It showed that the relative molecular weight of Cd-ITCBE-BSA was greater than that of BSA. Based on this phenomenon, i could be inferred that Cd (II) was successfully conjugated to BSA by the bi-functiona chelator ITCBE. The identification results were consistent with those obtained via UV scanning. The result is shown in Figure 3a. The Cd-ITCBE-OVA band had a significan hysteresis relative to OVA, indicating that its relative molecular weight was much greater than that of OVA. In accordance with this result, it could be concluded that Cd (II) was triumphantly coupled to OVA via the bi-functional chelator ITCBE. The result is shown in Figure 3b.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Identification of Cd-ITCBE-BSA and Cd-ITCBE-OVA
The prepared Cd-ITCBE-BSA and Cd-ITCBE-OVA were identified by SDS-PAGE electrophoresis. The results showed that the Cd-ITCBE-BSA band was found after the appearance of the BSA band, reflecting a hysteresis. It showed that the relative molecula weight of Cd-ITCBE-BSA was greater than that of BSA. Based on this phenomenon, i could be inferred that Cd (II) was successfully conjugated to BSA by the bi-functiona chelator ITCBE. The identification results were consistent with those obtained via UV scanning. The result is shown in Figure 3a. The Cd-ITCBE-OVA band had a significan hysteresis relative to OVA, indicating that its relative molecular weight was much greate than that of OVA. In accordance with this result, it could be concluded that Cd (II) wa triumphantly coupled to OVA via the bi-functional chelator ITCBE. The result is shown in Figure 3b.

Identification of Anti-Cadmium Monoclonal Antibody
Eight stable clones emerged after the fusion, and the most sensitive clones from the plate, as well as the clone 3A2, were chosen for ascite generation and further ELISA optimization. The affinity of anti-cadmium monoclonal antibody was 3.6 × 10 −5 by ELISA.

Determination of Optimal Working Concentration of Monoclonal Antibody and Coated Anti-Gen for Cd (II)
The inhibition rate was calculated from the measured OD 450 value, the blank value and the standard displayed value of 10 ng mL −1 Cd (II). The result is shown in Table 1. The results highlighted that the reaction values and inhibitory rates of Cd-ITCBE-BSA with different dilution concentrations were different from those of the anti-cadmium monoclonal antibody. Then, the maximum inhibition rate of the 10 ng mL −1 Cd (II) standard was selected within the range of OD 450 values. Therefore, the dilution multiples of the coating anti-gens Cd-ITCBE-BSA and the anti-cadmium monoclonal antibody were 1:50,000 and 1:20,000, respectively. It was chosen as the optimal working concentration.

Standard Curve Linear Range and Detection Sensitivity
The semi-logarithmic standard curve of Cd (II) ic-ELISA was established according to the experimental chromogenic numerical results. The results showed that the standard curve of the Cd (II) semi-logarithm was a typical S curve in the range of 0.33~81 ng mL −1 . The result is shown in Figure 4a. The standard curve of linear regression was established. The result is shown in Figure 4b. The linear regression equation was y = −33.158x + 74.936 (R 2 = 0.9824), indicating that the logarithm of the standard concentration of Cd (II) had a good correlation with the binding rate.

Specificity Analysis of Ic-ELISA
The cross reaction of anti-cadmium monoclonal antibody with other heavy metal ions would increase the interference of false positives. The calculation formula of the CR value was as Equation (2): CR (%) = (IC 50 (Cd (II))/IC 50 (compounds)) × 100%. ( The result is shown in Table 2. The results showed that IC 50 = 5.53 ± 0.76 ng mL −1 and IC 10 = 0.35 ± 0.24 ng mL −1 based on the standard curve. The CR of the anti-cadmium monoclonal antibody compared to those of the other 12 heavy metal ions was less than 0.1%. The results showed that the anti-cadmium monoclonal antibody had little cross-reactivity

Specificity Analysis of Ic-ELISA
The cross reaction of anti-cadmium monoclonal antibody with other heavy metal ions would increase the interference of false positives. The calculation formula of the CR value was as follows: CR (%) = (IC50 (Cd (II))/IC50 (compounds)) × 100%.
The result is shown in Table 2. The results showed that IC50 = 5.53 ± 0.76 ng mL −1 and IC10 = 0.35 ± 0.24 ng mL −1 based on the standard curve. The CR of the anti-cadmium monoclonal antibody compared to those of the other 12 heavy metal ions was less than 0.1%. The results showed that the anti-cadmium monoclonal antibody had little crossreactivity with the other 12 heavy metal ions. It is clear that the ic-ELISA method established in this study has good specificity.

Analysis of Limits of Detection and Limits of Quantitation
Ten negative food packing material samples were detected by ICP-AES. As a control, the ic-ELISA established in this paper was used to determine Cd (II) in food packing material samples and repeated 10 times. The mean and standard deviation of 10 negative food packing material samples were calculated and analysed. The LOD and LOQ of Cd (II) residue in food packing material samples were established by ic-ELISA method. The results show that the LOD and LOQ of Cd (II) residue were 30.53 ng mL −1 and 35.24 ng mL −1 , respectively. The result is shown in Table 3. Therefore, the minimum limit of quantitation of the ic-ELISA method established could meet the detection of residual Cd (II) in food packing material samples.

Precision and Accuracy of Ic-ELISA Analysis
The precision of ic-ELISA could be analysed by the CV values. The CV value is defined as the difference between the test material of the same stage and the test material of different stages. When the coefficient of variation was less than 10%, the experiment was stable. The CV values for each gradient concentration were calculated from 12 sets of parallel experiments, and the total mean values were calculated. The results made clear that the intra-and inter-board variation coefficients were 7.20% and 6.74%, respectively. The result is shown in Table 4. The CV values of both were less than 10%. This indicated that the ic-ELISA established in our paper had good precision. The accuracy of ic-ELISA could be analysed by adding the recovery rates of Cd (II) standard at different concentrations in negative food packing material samples. After the addition of 0, 100, 200 and 400 ng mL −1 Cd (II) standards to the negative food packing material samples, the average recoveries were 92.34% ± 4.26%, 89.03% ± 10.80% and 95.81% ± 11.40%, respectively. The result is listed in Table 5. The CV values of the 10 check duplications were 4.61, 11.68 and 9.74%, respectively. On the basis of the result of our ic-ELISA method, the recovery rate and the repeat CV value were 89.03~95.81%, and 4.61%~11.68%, respectively. The apparent high accuracy can meet the requirement for the rapid detection of Cd (II) residue in food packing material samples.

Comparison of Ic-ELISA and ICP-AES Detection in Spiked Sample
The ICP-AES and ic-ELISA established in this paper were used to compare the difference between the two methods. The Cd (II) standards were added as experimental samples to negative food packing material samples of 50, 100, 200 and 400 ng mL −1 , respectively. The ICP-AES and ic-ELISA tests were repeated six times in parallel. In Figure 5, the detection results of the ordinate diagram of ic-ELISA and the abscissa diagram of ICP-AES were arranged. The regression curve analysis was provided, and the regression equation was Y = 0.9026X. The correlation coefficient of the two methods was R 2 = 0.9668. The t-test between the ic-ELISA and ICP-AES detections in the spiked samples was also performed, and the results show that there was no significant difference between these two groups.
difference between the two methods. The Cd (II) standards were added as experimental samples to negative food packing material samples of 50, 100, 200 and 400 ng mL −1 , respectively. The ICP-AES and ic-ELISA tests were repeated six times in parallel. In Figure  5, the detection results of the ordinate diagram of ic-ELISA and the abscissa diagram of ICP-AES were arranged. The regression curve analysis was provided, and the regression equation was Y = 0.9026X. The correlation coefficient of the two methods was R 2 = 0.9668.The t-test between the ic-ELISA and ICP-AES detections in the spiked samples was also performed, and the results show that there was no significant difference between these two groups.

Statistical Analysis of the 72 Actual Samples
As mentioned before, the 72 actual samples consisted of three categories of materials-ceramic, glass and paper. The mean values of Cd contained in the ceramic, glass and paper groups were 826.0 ± 133.0 ng g −1 , 276.2 ± 164.3 ng g −1 , and 45.5 ± 33.8 ng g −1 , respectively. Box-plot analysis of the 72 actual samples is presented in Figure 6, where the x-axis represents the three categories (ceramic, glass and paper) and the y-axis represents the Cd concentration detected in these samples.

Statistical Analysis of the 72 Actual Samples
As mentioned before, the 72 actual samples consisted of three categories of materialsceramic, glass and paper. The mean values of Cd contained in the ceramic, glass and paper groups were 826.0 ± 133.0 ng g −1 , 276.2 ± 164.3 ng g −1 , and 45.5 ± 33.8 ng g −1 , respectively. Box-plot analysis of the 72 actual samples is presented in Figure 6, where the x-axis represents the three categories (ceramic, glass and paper) and the y-axis represents the Cd concentration detected in these samples.
The ICP-AES and ic-ELISA established in this paper were used to compare the difference between the two methods. The Cd (II) standards were added as experimental samples to negative food packing material samples of 50, 100, 200 and 400 ng mL −1 , respectively. The ICP-AES and ic-ELISA tests were repeated six times in parallel. In Figure  5, the detection results of the ordinate diagram of ic-ELISA and the abscissa diagram of ICP-AES were arranged. The regression curve analysis was provided, and the regression equation was Y = 0.9026X. The correlation coefficient of the two methods was R 2 = 0.9668.The t-test between the ic-ELISA and ICP-AES detections in the spiked samples was also performed, and the results show that there was no significant difference between these two groups.

Statistical Analysis of the 72 Actual Samples
As mentioned before, the 72 actual samples consisted of three categories of materials-ceramic, glass and paper. The mean values of Cd contained in the ceramic, glass and paper groups were 826.0 ± 133.0 ng g −1 , 276.2 ± 164.3 ng g −1 , and 45.5 ± 33.8 ng g −1 , respectively. Box-plot analysis of the 72 actual samples is presented in Figure 6, where the x-axis represents the three categories (ceramic, glass and paper) and the y-axis represents the Cd concentration detected in these samples. As observed in Figure 6, the ceramic group has the highest Cd value, while the paper group has the lowest Cd value, which is interesting. Analysis of variance (ANOVA) was also applied, and the result demonstrated that there is a significant difference among these three groups of food packaging materials at the 0.01 level.

Comparison of Ic-ELISA and ICP-AES Detection in Actual Sample
Detection results of the food packing samples indicated that the ic-ELISA method established could be an effective tool in the detection of actual samples. The feasibility of the ic-ELISA method was reflected by testing actual samples. Six identical samples were taken from each actual sample and repeated three times using ic-ELISA and ICP-AES. Figure 7 exhibits the test results of the ordinate graph of ELISA and the abscissa graph of ICP-AES. The regression equation was Y = 1.0122 X and the correlation coefficient of the two methods was R 2 = 0.9634, indicating that the detection values of ic-ELISA and ICP-AES were very similar. That is to say, the detection value of ic-ELISA is close to that of the actual sample value. The t-test between ic-ELISA and ICP-AES detection in the actual samples was also performed, and the results show that there was no significant difference between these two groups.
As observed in Figure 6, the ceramic group has the highest Cd value, while the paper group has the lowest Cd value, which is interesting. Analysis of variance (ANOVA) was also applied, and the result demonstrated that there is a significant difference among these three groups of food packaging materials at the 0.01 level.

Comparison of Ic-ELISA and ICP-AES Detection in Actual Sample
Detection results of the food packing samples indicated that the ic-ELISA method established could be an effective tool in the detection of actual samples. The feasibility of the ic-ELISA method was reflected by testing actual samples. Six identical samples were taken from each actual sample and repeated three times using ic-ELISA and ICP-AES. Figure 7 exhibits the test results of the ordinate graph of ELISA and the abscissa graph of ICP-AES. The regression equation was Y = 1.0122 X and the correlation coefficient of the two methods was R 2 = 0.9634, indicating that the detection values of ic-ELISA and ICP-AES were very similar. That is to say, the detection value of ic-ELISA is close to that of the actual sample value. The t-test between ic-ELISA and ICP-AES detection in the actual samples was also performed, and the results show that there was no significant difference between these two groups.

Conclusions
A rapid detection method for the determination of Cd in food packing material at ng mL −1 levels was developed in ic-ELISA. This new approach can be completed in 40 min with simple operation. Based on the standard curve established, the UV wavelength of the enzyme marker can be quantitatively analysed. IC50 and specificity are similarly easy to calculate and analyse. The cross-reactivity with other heavy metals was low, and iC-ELISA showed better sensitivity and specificity. As shown by comparison of the results of ic-ELISA and ICP-AES, ic-ELISA can significantly improve the reliability of immunoassaying, and the precision and accuracy of Cd detection. This method provides a rapid, accurate and economical alternative tool for the detection of Cd content in food packing materials.

Conclusions
A rapid detection method for the determination of Cd in food packing material at ng mL −1 levels was developed in ic-ELISA. This new approach can be completed in 40 min with simple operation. Based on the standard curve established, the UV wavelength of the enzyme marker can be quantitatively analysed. IC50 and specificity are similarly easy to calculate and analyse. The cross-reactivity with other heavy metals was low, and iC-ELISA showed better sensitivity and specificity. As shown by comparison of the results of ic-ELISA and ICP-AES, ic-ELISA can significantly improve the reliability of immunoassaying, and the precision and accuracy of Cd detection. This method provides a rapid, accurate and economical alternative tool for the detection of Cd content in food packing materials.

Institutional Review Board Statement:
This research did not require ethical approval, and we chose to exclude this statement.

Informed Consent Statement: Not applicable.
Data Availability Statement: We can provide and share experimental data in order to enable other authors to achieve best practices in archiving research data.