Development of a Protein Biochip to Identify 6 Monoclonal Antibodies Against Subtypes of Recombinant Human Interferons

Abstract

Recombinant human interferons (rhIFNs) are broadly used as effective therapeutic agents with antiviral, antitumor, and immune-modulating properties. Advances in protein biochip technology have benefited the medical community greatly, making true parallelism, miniaturization, and high throughput possible. In this study, 5 rhIFN proteins (IFN-α1b, IFN-α2a, IFN-α2b, IFN-β, and IFN-γ) were immobilized onto an N-hydroxysuccinimide (NHS)-modified gold-based biochip. The protein biochip was incubated with 6 specific mouse IgG antibodies (AK1, AK2, AK3, AK4, BK1, and CK1) against the human IFNs and then with Cy3-conjugated goat anti-mouse IgG antibody. The results showed that monoclonal antibody AK1 presented a unique binding characteristic to IFN-α1b. AK2 reacted in immunoassays equally with IFN-α2a and IFN-α2b. AK3 detected IFN-α1b, IFN-α2a, and IFN-α2b. AK4 had positive immunological responses directed to both IFN-α1b and IFN-α2b. Monoclonal antibodies BK1 and CK1 recognized epitope of IFN-β and IFN-γ, specifically. The assay specificity of the biochip was further confirmed by enzyme-linked immunosorbent assay (ELISA) and western blotting. Finally, 88 serum samples from patients treated with rhIFN-α2b were simultaneously tested on a single biochip. The result demonstrated that 6.8% (6 of 88 cases) presented positive reactions to anti-IFN-α2b antibodies, indicating that the patients under rhIFN-α2b therapy produced neutralized antibody against the IFN. The biochip format would offer a competitive alternative tool not only for facilitating characterization of IFN subtypes but also potentially for enabling clinical serum detection of corresponding antibodies directed against IFNs.

INTRODUCTION

Type I and type II interferons (IFNs) are defined based on their receptor specificity. Type I interferons are composed of a family of monomeric proteins including IFN-α, IFN-β, IFN-ω, and IFN-τ. The only identified type II interferon is the dimeric IFN-γ.1 –3 Different IFN subtypes activate the same cell surface receptor complex to mediate variable responses.3 –5 IFN-α is produced by monocytes, macrophages, and activated plasmacytoid dendritic cells (DCs)6 and modulates immunologic functions of DCs toward a polarized DC1-type capable of coordinately promoting TH1-type and TC1-type T-cell-mediated immunity.7 Structurally, IFN-α2a and IFN-α2b shared the same sequence of 165 amino acid residues with the exception of an amino acid variance at position 23, where IFN-α2a is arginine, while IFN-α2b is lysine. IFN-β has one subtype. It is composed of 166 amino acids and is mainly produced by fibroblasts and epithelial cells. Mature IFN-γ has 143 amino acids in length and is primarily produced by T cells and NK cells.8 ^, 9 Accumulating evidences demonstrate that minimal variance in amino acid sequences of IFNs affects the biologic activity,10 for example, nature of the ligand–receptor interaction and the subsequent responses.11

Recombinant human IFNs (rhIFNs) have broadly been applied in the treatment of human viral diseases,12 ^, 13 tuberculosis,14 multiple sclerosis,15 leukemia,16 and other tumors.7 ^, 17 However, neutralizing IFN-β antibodies in melanoma patients treated with recombinant and natural IFN-β may occur.18 Also, antibodies to IFN-α2b may develop during rhIFN-α2b therapy.19 Of the antibodies that bind to different epitopes of the IFN molecule, some are neutralizing antibodies (NABs), as measured in antiviral neutralization assays.20 IFN antibody formation might influence IFN therapeutic effect. Thus, developing novel assay techniques will obviously play a important role not only in elucidating pharmacokinetic properties of IFN preparations in human,21 but also in determining IFNs proteins or antibodies in various samples.22

Conventional methods used to identify subtypes of IFN with mAb technology include enzyme-linked immunosorbent assay (ELISA), western blotting, and neutralized activity.23 ELISA- and western blot-based assays are reliable, relatively cost-effective, and are restricted to the determination of single target specificity.24 Western blot assay appears to be at least as sensitive and specific as the ELISA assay, but is more laborious to perform, needs more serum, and is more expensive. The advantage of western blot is that antibodies can be detected, which may recognize different antigens of a given infectious agent. The disadvantage is the possible identification of nonspecific antigens or cross-reactive epitopes. In addition, conformational epitopes may be lost. In general, western blot is often used to corroborate results from other diagnostic methods. Establishment of simple and specific immunological methods enabling not only to identify subtypes of IFN and to understand pharmaceutical interaction between molecules but also to screen any potential production of anti-IFN antibodies under rhIFN therapy obviously is of great challenge. Currently, advances in protein biochip technology have benefited the medical community greatly, making true parallelism, miniaturization, and high throughput possible, and partially overcoming ELISA limits in serum immunological diagnosis of a variety of diseases and in molecule identification.25 ^, 26 Advantages of protein biochip over conventional methods include: the biochip-based assays enable rapid analysis of a large number of samples in a single experiment; the amount of material needed is very small. Reaction volumes could be 20–40 times lower than the amount that is generally used in conventional 96- or 384-microwell (microtiter) plates; and the signal-to-noise ratio exhibited using microarrays is much better (>10-fold) than that observed for traditional microtiter plate assays.27 Methodological comparison evaluating compatibility of protein biochips, commercial ELISAs, and western blotting in screening cytokines and serum antibodies against pathogens was performed.22 ^, 26 ^, 28 –30 Although an increasing number of expression profiling investigations of IFNs remains being issued at the gene level,31 ^, 32 identification of antibodies against IFN subtypes by protein biochip technique is less available. In this study, based on the gold substrate format of the biochip that we previously reported,22 ^, 26 ^, 35 we developed a protein biochip to validate the potential of discriminating 5 IFN subtypes using 6 corresponding monoclonal antibodies. We evaluated the application of the biochip, ELISA, and western blotting assays in detecting of the IFN proteins.

MATERIALS AND METHODS

Reagents and Biochip

Five standard recombinant human IFN antigens: IFN-α1b, IFN-α2a, IFN-α2b, IFN-β, and IFN-γ were provided by National Institute for the Control of Pharmaceutical and Biological Products of China. Six specific monoclonal antibodies against the individual subtype antigen of rhIFNs and mouse anti-human IgG antibody (2D5) were provided by Anke High Biotechnology Inc. (Hefei, China), of which AK1, AK2, BK1, and CK1 were prepared by the antigens of rhIFN-α1b, rhIFN-α2b, rhIFN-β, and rhIFN-γ, as the protocol described previously.34 AK3 was induced by IFN antigen from natural leukocyte; AK4 was produced by recombinant methionine consensus IFN (r-metIFN-Con1). The purities of the monoclonal antibodies exceed 95%. N-Hydroxysuccinimide (NHS)-modified biochips were supplied by Thermohybaid, Interactiva Division (Ulm, Germany). The biochips were designed on the basis of a standard microscope slide format. Two sets of 96 spots (ie, 192 spots in total) were available for conducting biomolecular interactions. The individual gold spots were separated from each other by a Teflon layer. The hydrophobic Teflon surface prevented cross talk between the adjacent spots and virtually eliminated biochip smearing.35 All other reagents available commercially were of analytical grade and MilliQ grade water was used.

Optimization of Biochip Assays for Immunological Detection of rhIFN-α2b

To optimize the protein coating concentration on biochips, a gradient dilution of recombinant human IFN-α2b was prepared in PBST-BSA buffer: 0.01 M phosphate-buffered saline (PBS, pH 7.4; Sigma, St. Louis, MO), 0.1% Tween 20 (v/v; Sigma), and 0.1% BSA (w/v, bovine serum albumin; Sigma) at the concentrations of 0.098, 0.19, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 μg/mL, and printed on a activated glass slide (1 μL/spot). All surface immobilization and the subsequent steps were performed in humid chambers in order to prevent aqueous evaporation effect. The procedures of antigen immobilization on biochips were carried out at room temperature for 4 h. The biochip was rinsed with sterile water briefly, and then in 0.01 M PBST buffer, 2 min, 3 times. After drying with a stream of nitrogen, the biochip was incubated with 10 μg/mL (1 μL/spot) of the monoclonal antibody against rIFN-α2b in 0.01 M PBST-0.1% BSA buffer (pH 7.4) at room temperature for 1 h. Following rinsing and drying, the antigen–antibody complex on the biochip was detected by Cy3-conjugated goat anti-mouse IgG antibody (Sigma) at a 1:100 dilution (0.01 M PBST-0.1% BSA buffer, pH 7.4) at room temperature for 1 h in the dark. The biochip was washed in PBST buffer and then covered with a cover glass. Image data were obtained by using a fluorescence scanner (GenePix 4100A Scanner; Molecular Devices, Sunnyvale, CA) at wavelength 532 nm for Cy3. The spatial resolution of 16 bits per pixel and a 50-μm pixel size was chosen. The fluorescence intensities obtained from the spots immobilized with 0.01 M PBST-0.1% BSA buffer were used as negative controls. The fluorescence signals are expressed as arbitrary units across a 1-mm² area (AU/mm²). The average fluorescence intensities from 4 spots of individual dilution were quantified with microarray analysis software (GenePix Pro5.0, Axon, USA). Each step of this procedure was optimized for the present study by varying individual parameters.

Identifying of IFN Proteins by Biochip

Ten micrograms per milliliter of recombinant human IFN-α1b, IFN-α2a, IFN-α2b, IFN-β, IFN-γ, and human IgG (1 μL/spot in 0.01 M PBST-0.1% BSA, pH 7.4) was individually immobilized on the NHS-modified biochip at room temperature for 4 h. The immunological binding of individual IFN subtype and IgG antigen to the corresponding specific IFN and IgG monoclonal antibodies (10 μg/mL, 0.01 M PBST-0.1% BSA, pH 7.4) was performed, at room temperature for 60 min and then incubated by the Cy3-conjugated goat anti-mouse IgG antibody (Sigma, St. Louis, MO) at a 1:100 dilution (0.01 M PBST-0.1% BSA buffer, pH 7.4) at room temperature for 1 h in the dark. Fluorescence values of IgG and 0.01 M PBST-0.1% BSA were calculated as positive and negative controls. Fluorescence values of 4 times over negative control were evaluated to be positive.

Detecting of IFN Subtypes by ELISA and Western Blotting

Binding antibodies to rhIFN subtypes were measured in a standard ELISA as described previously.22 In brief, polystyrene microtiter plates (Nunc, 96 wells, Denmark) were coated with 100 μL of rhIFN-α1b, rhIFN-α2a, rhIFN-α2b, rhIFN-β, and rhIFN-γ (10 μg/mL, 0.05 M carbonate–bicarbonate buffer, pH 9.6; Sigma) at 4°C overnight. After washing and blocking with 10% bovine serum albumin (w/v; Sigma), the wells were incubated with 100 μL of monoclonal antibody AK1, AK2, AK3, AK4, BK1, and CK1 (10 μg/mL, 0.01 M PBS, pH 7.4, Anke) at 37°C for 90 min. The samples evaluated were performed in duplicate. After washing, HRP-conjugated goat anti-mouse IgG (1:5,000; Sigma) was incubated at 37°C for 60 min. Finally, the wells washed and incubated with enzyme substrate TMB (tetramethylbenzidine; Sigma) and H₂O₂ (30%, v/v; Sigma) at room temperature for 15 min. After stopping the reaction with 2 M H₂SO₄ (50 μL/well), the developing color was quantified on an automatic microtiter plate reader (Bioreader, Model 550, Japan). The results were expressed as optical density units (OD) at 492 nm. Average values from 8 wells containing 0.05 M carbonate–bicarbonate buffer-0.1% BSA were used as negative control. Three times of real values of detectable signals over the negative control were referred as to be positive.

Interactions of subtypes of rhIFN-α1b, rhIFN-α2a, rhIFN-α2b, rhIFN-β, and rhIFN-γ with the corresponding specific monoclonal antibodies (AK1, AK2, AK3, AK4, BK1, and CK1) were detected by western blotting technique. Immunological reaction between human IgG and mouse anti-human IgG antibody was used as a positive control. Ten micrograms of each IFN antigen was loaded onto polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto NC membranes. The membranes were blocked by 5% defatted milk powder and incubated with the IFN antibodies. The specific immunological signals were detected by adding HRP goat anti-mouse IgG (1:5,000; Sigma) and further colorized by DAB and H₂O₂ (30%, v/v; Sigma). The bands were visually evaluated by comparing the intensity to positive control.

Serological Screening of IFN-α2b Antibody by the Biochip, ELISA, and Western Blotting

Eighty-eight sera from the patients with solid tumors who received rhIFN-α2b as a maintenance therapy were compared with 4 serum samples from control individuals in a dilution of 1:100 in 0.01 M PBST-0.1% BSA buffer by the biochip. Ten micrograms per milliliter of recombinant hIFN-α2b protein was immobilized on biochip (1 μL/spot) at room temperature for 4 h. After washing and drying, the serological assay was performed at room temperature for 60 min (dilated sera, 1 μL/spot). The binding of rhIFN-α2b-specific serum antibodies were detected by Cy3-conjugated goat anti-human IgG antibody (Sigma) at a 1:100 dilution (0.01 M PBST-0.1% BSA buffer, pH 7.4) at room temperature for 1 h in the dark. Arrays were imaged using a fluorescence scanner (XNA ScanPro 20 microarray scanner, Thermohybaid, UK). Fluorescence values of 3 times over the mean value of the controls were evaluated to be positive. The 6 positive and 2 negative sera of the patients were checked by ELISA and western blotting.

RESULTS

Quantification of Antigens on Biochip

Recombinant human IFN-α2b was immobilized onto a biochip at the concentrations ranging from 0.098 to 100 μg/mL in 0.01 M PBST-0.1% BSA (1 μL/spot). Figure 1 showed that the intensities of fluorescence signals increased as concentrations of immobilized recombinant proteins increased on the biochip. Obviously, the immobilization efficiency of the protein on the biochip was concentration-dependent. The detectable limit of rhIFN-α2b was as little as 1.56 μg/mL per spot. Regression analysis demonstrated that the fluorescence signal intensities (y-axis) appeared a relatively linear to the logarithm of the protein immobilized on the biochip (x-axis, log₁₀ of protein loading). Protein calibration curve showed that coefficient of determination (R ²) was 0.987 within a range from 1.56 to 25 μg/mL. To obtain optimal fluorescence signals in the panel of multiplexed individual antigen assay, the immobilized concentration of each IFN subtype was therefore selected to be 10 μg/mL thereafter. Additionally, there was no significant effect of immobilization temperatures between room temperature and 37°C on fluorescent intensities of the biochip (data not shown). The variation in the chip-to-chip comparison was tested by repeating 3 biochips on 3 days. The variation coefficients ranged between 2.84% and 3.39% in intra-biochip and 3.15% in inter-biochip comparative calculations (Table 1).

Fig. 1.

Optimization of recombinant human interferons (rhIFNs)-α2b coating concentration by biochip. A duplicated dilution of rhIFN-α2b (0.098–100 μg/mL) on a N-Hydroxysuccinimide (NHS)-modified biochip was detected by a Cy3-based immunological assay. The fluorescent intensities of the spots (y-axis, mean ± SD) appeared linear to the logarithms of the protein immobilized on the array (x-axis, log₁₀ of IFN-α2b, μg/mL).

Table 1.

Intra-Biochip and Inter-Biochip Variation

	Day 1	Day 2	Day 3
Average	7,224.9	7,234.2	7,230.3
SD	23.2	24.54	20.58
CV (%)	3.22	3.39	2.84

The chip-to-chip variability was tested by repeating 3 biochips on 3 days. rhIFNα2b coated biochips were incubated with monoclonal anti-rhIFNα2b antibody and Cy3-labeled goat anti-mouse IgG antibody. The average fluorescence signals and standard deviations were calculated from 24 spots on each chip.

Screening of IFN Subtypes by Biochip

The ability of the biochip to differentiate recombinant human IFN subtypes was validated individually on each IFN antibody biochip (8 spots on each, data not shown) and on an integrated (6 IFN antibodies on one) biochip. Human IgG and 0.01 M PBST-0.1% BSA were used as positive and negative controls. Figure 2A showed that monoclonal antibody AK1, BK1, and CK1 specifically recognized IFN-α1b, IFN-β, and IFN-γ. AK2 had positive responses to IFN-α2a and IFN-α2b, but not any others. AK3 reacted equally with IFN-α1b, IFN-α2a, and IFN-α2b. AK4 presented identical immunological reactions to both IFN-α1b and IFN-α2b. The average values of fluorescence intensities from the positive spots in each subtype were 4 times higher than that of the negative control. An acceptable fluorescence background from 0.01 M PBST-0.1% BSA was obtained. Compared with the fluorescence intensities of IFN subtypes by the protein biochip assay, the reaction against IgG molecule appeared weaker, which might result from the affect of the high molecular weight of IgG and/or its spatial structure.

Fig. 2.

Identification of interferons (IFNs) by the protein biochip and western blotting. Recombinant human IFN-α1b, IFN-α2a, IFN-α2b, IFN-β, IFN-γ, and human IgG were individually immobilized on a N-Hydroxysuccinimide (NHS)-modified biochip (10 μg/mL, 1 μL/spot). Immunological complex of individual IFN subtype and IgG antigen to the corresponding specific IFN and IgG monoclonal antibodies on the biochip was reacted with Cy3-conjugated goat anti-mouse IgG antibody and detected by using a fluorescence scanner at wavelength 532 nm. Fluorescence intensities of human IgG and dilution buffer were used as positive and negative controls. The fluorescence signals are expressed as arbitrary units across a 1-mm² area (AU/mm²) (A). Interactions of subtypes of rhIFN-α1b, rhIFN-α2a, rhIFN-α2b, rhIFN-β, and rhIFN-γ with the corresponding monoclonal antibodies were detected by western blotting (B).

Comparison of Biochip, ELISA, and Western Blotting in Detecting IFNs

To verify the accuracy and the detection limits of the biochip, the experiment was evaluated by IFN protein-specific ELISA and western blotting. Table 2 showed immunological reactions of each individual antibody to relevant IFNs by the protein biochip was completely proportional to that of ELISA. The correlation coefficient (R ²) between fluorescence values by biochip method and OD values by ELISA assay at the positive spots was 0.984, indicating that the protein biochip (Fig. 2A), ELISA, and western blotting (Fig. 2B) were quite compatible.

Table 2.

Methodological Comparison of Biochip and ELISA

	Biochip					ELISA
	α1b	α2a	α2b	β	γ	α1b	α2a	α2b	β	γ
AK1	6,858	1,524	1,423	1,342	1,465	0.805	0.174	0.142	0.161	0.081
AK2	1,654	7,189	7,228	1,325	1,245	0.112	0.894	0.895	0.109	0.094
AK3	6,878	6,947	6,975	1,584	1,241	0.810	0.841	0.863	0.157	0.141
AK4	7,014	945	6,945	1,024	1,134	0.880	0.123	0.828	0.124	0.118
BK1	998	973	854	6847	764	0.114	0.120	0.161	0.803	0.089
CK1	875	836	765	624	7052	0.142	0.152	0.137	0.144	0.884

Comparison of biochip and ELISA techniques in identifying of interferons (IFNs) subtypes. Biochip presented in complete accordance with those of ELISA. The correlation coefficient (R ²) between fluorescence values by biochip method and OD values by ELISA assay at the positive spots was 0.984.

Serological Detection of IFN-α2b Antibody in the Patients Treated With Interferon α2b by Biochip, ELISA, and Western Blotting

The feasibility of the protein biochip format to screen potential neutralized anti-IFN α2b antibodies in the patients under the treatment of rhIFN-α2b was evaluated accordingly. The result showed that 6 of 88 patients tested presented positive reactions (6.8%, Fig. 3A). The seroimmunological reactions in the positive patients were investigated by western blotting (Fig. 3B) and ELISA (Fig. 3C). The results were in consistence with that of the biochip.

Fig. 3.

Screening of patients’ sera. Sera (1:100 dilution, 1 μL/spot) from 88 patients under the treatment of recombinant human interferons (rhIFNs)-α2b were spotted on a rhIFN-α2b-coated biochip (10 μg/mL; 1 μL/spot). Four control sera (the 4 spots of the upper right corner of the biochip) and PBST-BSA buffer (the 4 spots of the lower right corner) were used as negative control and background. After incubation with Cy3-conjugated goat anti-human IgG antibody (Sigma, St. Louis, MO) at a 1:100 dilution (0.01 M PBST-0.1% BSA buffer, pH 7.4), the fluorescence intensity was shown. The scanned fluorescence intensity was calculated and given as AU/mm. Each point represented the result of an individual patient serum. Three times higher than mean fluorescence value of control sera were referred to as positive. The incidence of positive IFN-α2b antibodies in the patients was 6.8% (6 of 88 cases) (A). The 6 positive sera and 2 negative sera of the patients were investigated by western blotting (B) and ELISA (C). Note: numbers in ELISA table represented the same sample numbers as shown in western blotting.

DISCUSSION

A number of methodologies capable of screening trace amount of IFN molecules are developed22 ^, 28 ^, 30; however, less information of precisely detecting subtypes of IFNs by protein biochip technology is available. In this study, we successfully identified 6 kinds of IFN mAbs directed to IFN-α1b, IFN-α2a, IFN-α2b, IFN-β, and IFN-γ, by a novel protein biochip. The detection limit of rhIFN-α2b on the biochip was estimated to be 1.56 μg/mL on each spot by the Cy3-based assay, revealing a higher sensitivity and more symmetrical signal images. A linear correlation between the fluorescence signal intensities and the logarithm of the IFN-α2b concentration was obtained. Coefficient of determination from standard calibration curves of rhIFN-α2b was measured at R ² = 0.987, suggesting that Cy3-based protein biochip assay was capable of detection of this specific protein. Furthermore, the variation test in the chip-to-chip comparison showed that variation coefficient among the biochips was 3.15%, indicating that this biochip format was highly reproducible.

The antigenic diversity between highly homologous IFN subtypes has a wider functional significance of IFNs.3 Utilizing mAb technology enables to target a single epitope and to recognize relatively minor changes in antigen structure.23 ^, 36 In this study, a panel of mAbs against major human IFN subtypes was characterized. The regions that the IFN mAbs recognized were precisely defined by the 5 IFN proteins (IFN-α1b, IFN-α2a, IFN-α2b, IFN-β, and IFN-γ). Monoclonal antibody AK1 was directed to recombinant IFN-α1b, not overlapping with epitopes of other mAbs. AK2 reacted only with IFN-α2a and IFN-α2b. AK4 not only definitely distinguished IFN-α, IFN-β, and IFN-γ as a whole, but also identically discriminated one substitute of amino acid at position 23 between IFN-α2a and IFN-α2b. Moreover, BK1 and CK1 specifically recognized individual IFN-β and IFN-γ, respectively. Therefore, by virtue of the 6 monoclonal antibodies, the 5 subtypes of IFN, IFN-α1b, IFN-α2b, IFN-α2b, IFN-β, and IFN-γ, could be successfully differentiated. Furthermore, the assay specificity of the biochips was evaluated by ELISA and western blotting. The results also showed that the immunological reactions of IFNs by the protein biochip were completely proportional to those by ELISA and western blotting. The correlation coefficient (R ²) between fluorescence values at the positive spots by biochip method and OD values of the identical immunological reaction by ELISA assay reached 0.984. Obviously, the protein biochip, ELISA, and western blotting were quite compatible in identification of INF subtypes. Different from larger reaction volume (100–200 μL) of diluted sera and reaction reagents and assay protocols by ELISA and western blot, the biochip format required as little as 1 μL of diluted sera on each well to detect specific antibodies. It would be one of the reasons why various signal intensities produced by the 3 methods occurred in Figure 3; therefore, a standardized amount of loaded samples and harmonized protocols should be carefully taken into account further when the sensitivity of the methodologies needs to be compared. In this study, simultaneous on-biochip assay of 96 or 192 samples (96 × 2 sets one biochip) would be suited to most routine clinical diagnostics.

Potential formation of antibodies against natural IFN37 and recombinant IFN22 is a common event in the patients under IFN treatment and often affects IFN therapeutic efficacy. In this study, 6 of 88 cases (6.8%) under the treatment of rhIFN-α2b showed positive anti-IFN-α2b antibody reaction, indicating that administration of recombinant human IFN-α2b do induced the production of neutralized antibody by the patients. Therefore, dynamic detecting of serum antibody directed to a given IFN used by biochip technique would be of importance in therapeutic decision making during the treatment course. A clinical trial in screening relevant IFN antibodies in the patients under IFN therapy needs to be investigated further.

In conclusion, we developed a novel protein biochip to identify 6 monoclonal antibodies against human IFNs and thereby differentially recognized epitopes available on the IFNs. The protein biochip, ELISA, and western blotting were comparable in IFN assays. The protein biochip provided a useful platform for analyzing IFN structures and characterization and would become a feasible tool in detecting serum corresponding potential antibodies in the patients under treatment of IFNs.

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

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