A Model System for Concurrent Detection of Antigen and Antibody Based on Immunological Fluorescent Method

This paper describes a combined antigen/antibody immunoassay implemented in a 96-well plate using fluorescent spectroscopic method. First, goat anti-human IgG was used to capture human IgG (model antigen); goat anti-human IgG (Cy3 or FITC) was used to detect the model antigen; a saturating level of model antigen was then added followed by unlabelled goat anti-human IgG (model antibody); finally, Cy3 labelled rabbit anti-goat IgG was used to detect the model antibody. Two approaches were applied to the concomitant assay to analyze the feasibility.The first approach applied FITC andCy3 when both targets were present at the same time, resulting in 50 ng/mLof the antibody detection limit and 10 ng/mLof antigen detection limit in the quantitativemeasurements of target concentration, taking the consideration of FRET efficiency of 68% between donor and acceptor. The sequential approach tended to lower the signal/noise (S/N) ratio and the detection of the model antigen (lower than 1 ng/mL) had better sensitivity than the model antibody (lower than 50 ng/mL). This combined antigen/antibody method might be useful for combined detection of antigens and antibodies. It will be helpful to screen for both antigen and antibody particularly in the situations of the multiserotype and high-frequency mutant virus infections.


Introduction
Multiplexed detection of antigens in immunoassays has been the most successful strategy using fluorescent probe molecules which are spatially isolated from each other on beads or different locations on a surface [1][2][3].Detection schemes for multiple targets have been extensively studied and are commercially available [4][5][6].The greatest successes have involved spatially isolated formats such as planar arrays, beads, and separate assays which run concurrently [7][8][9][10].Multiple target assays in a single spatial location or on a single bead are not widely used and the detection using these fluorophores at present is still the challenge due to energy transfer effects.Therefore, the needs to separate spectra and more complex experimental protocols are usually required.Fluorescent methods are widely used in the analysis of immunological assays due to the high sensitivity and simple detection formats [8,[10][11][12][13][14][15][16], but multiplexed detection in a spatially homogeneous format has been less successful and faces significant challenges.In particular, when different dyes are used in the system, the energy transfer between the dyes is an inevitable problem leading to variation of fluorescence intensities.Fluorescent dyes based multiplexing detection methods in combined antigen/antibody assays (Ag/Ab) are promising methods permitting simultaneous detection of host antibodies and viral antigens [11][12][13].So, a simple and economic way to carry out the combined Ag/Ab assay in one test is of interest.
In order to investigate this possibility, we developed a simple strategy for concurrent detection of antigen and antibody.The strategy involves two applications of a single sample to an immobilized antibody on the well of a 96well plate.Sample is applied to the immobilized probes and the well is rinsed.Bound antigen is visualized by applying a fluorescent labelled antibody to the antigen.
To subsequently detect the presence of antibodies to the antigen, the plate is then washed with a saturating concentration of antigen.The antigen will bind to the remaining 2 Journal of Spectroscopy antibody sites on the surface of the plate.The plate is rinsed and a second aliquot of sample is applied.If an antibody is presented in the sample it will bind to the antigen and can be detected by adding a specific antibody to the antigen labelled with a second fluorescent dye.In this study, a model system consisting of human IgG as antigen and anti-human IgG as probe was used as virus antigen and antibody, respectively.By using two kinds of fluorescent labelled antibody and two independent fluorescence detection methods, we demonstrate the detection of Ag/Ab, respectively.
Here we used 96-well plate as platform based on the fluorescent microscope.In this combined Ag/Ab assay strategy showed here, we investigated two approaches to the detection of the two targets.When FITC was used for the first target followed by Cy3 for the second one, the system was proved to be a useful system for the investigations of multitarget detection methods.

Equipment and Characteristic.
The microscope (Olympus IX-71, Japan) was linked with the camera (PCO1600, PCO, GMbH, Germany) and imaging spectroscope (PARISS, LightForm Inc., USA).And mercury lamp was used as the excitation light source (Hg light).The immunoassays were carried out using the 96-well plate with the transparent bottom so that the light can pass through (Figure 1(a)).The excitation light passes through the band-pass (BP) excitation filter and the dichroic mirror (DM) reflects it to the 96well plate; then the emission light passes through the DM to the long pass (LP) emission filter before the detection of camera and spectroscope.The fluorescent spectra were collected and the intensities were extracted and analyzed in the operation computer.The equipment's illustration is shown in Figure 1(b).Cy3 labelled IgG was detected with the filter set consisting of a 480-550 nm band-pass (BP) excitation filter, 570 nm dichroic mirror (DM), and 590 nm long pass (LP) emission filter (U-MSWG2; Olympus).Analysis of FITC employed a 470DF35 BP filter, a 515 nm DM, and a 515 LP (IF19-2; Omega).

2.3.
Antigen/Antibody Detection.The 96-well plate was rinsed several times with low concentration HCl solution and deH 2 O and PBS before the loading of probes.In brief, the detection was carried out in the following orders showed in Figure 2: (1) Goat anti-human IgG (20 g/mL, in PBS) was loaded at 100 L/well.The plate was then incubated overnight at 27 ∘ C in order to immobilize the IgG.After the immobilization, the wells were blocked with 5% BSA and then washed for several times with PBS-T.
(2) 50 L of human IgG (0-20 g/mL, in PBS) was loaded to the wells.The plate was incubated at 27 ∘ C for 4 hours followed by several rinses with PBS-T.
(3) Excess Cy3 labelled goat anti-human IgG solution was added to the wells (20 g/mL, 100 L/well) and the plate was cultured for 4 hours.The wells were washed with PBS-T, and then the spectra of the wells were taken by the camera and imaging spectrograph.
(4) 100 L human IgG (20 g/mL, in PBS) was added to each well to saturate the unoccupied sites.Then the plate was incubated for 4 hours and rinsed with PBS-T.
(5) 50 L of goat anti-human IgG (0-20 g/mL, in PBS) was loaded to the wells.Then the plate was incubated at 27 ∘ C for 4 hours followed by several rinses with PBS-T.
(6) Excess Cy3 labelled rabbit anti-human IgG (20 g/mL, 100 L/well) was then added to the wells and the plate was incubated at 27 ∘ C for 4 hours.The wells then were rinsed with PBS-T several times and the spectra were measured again.

Methods.
Two methods were used to perform the concomitant detection of antibody and antigen (Figure 2).In both cases, the first step was to expose the sample to a surface prepared with a capture reagent for the antigen; this was followed by the detection of the antigen with a labelled antibody.The preparation was then exposed to a saturating level of antigen and washed and analyte sample was added.The preparation was washed again and detected with a labelled specific antibody.In Method 1, the two detection steps were done with two different fluorophores (FITC and Cy3).In Method 2, Cy3 labelled antibody was used for both detection steps.

Method 1.
When using Method 1, the analysis of the system is straightforward by detecting the individual fluorescent intensity of FITC and Cy3, except for the rare cases where a disease (such as HCV) is escaping the immune system and hence both antigen and antibodies are present.In this case energy transfer between the two fluorophores results in lower fluorescence from FITC-IgG and sensitized emission of Cy3.In this system, FITC behaves as a donor and Cy3 acts as an acceptor.The effects of energy transfer are presented as energy transfer efficiency () and normalized sensitized emission (SE). is given by where   is the intensity of the donor in the absence of the acceptor and   is the intensity of the donor in the presence of the acceptor [14][15][16].SE is computed as Label 1 = Cy3 labeled rabbit anti-goat IgG Label 2 = Cy3 labeled goat anti-human IgG where   is the intensity of the acceptor in the presence of the donor and   is the intensity of the acceptor in the absence of the donor.has the same efficiency in both reporters, the increased intensity (Δ) between step 1 (I 1 ) and step 2 (I 2 ) is Δ indicates the existence of antibody in the sample and the intensity represents the concentration of that antibody (Figure 2).The factor of two arises from the specific design of our model system; however, other designs are possible where this would not be a requirement.Therefore, in this method, only one Cy3 fluorescent signal is employed and the intensities of these two detection steps are collected and calculated to obtain the analyte concentration.Figure 3: Fluorescence resonance energy transfer when two dyes Cy3-IgG and FITC-IgG were used in this combined Ag/Ab immunoassay system.(a) Cy3 fluorescent intensity differences (Δ) when the concentration of FITC-IgG was changed from 0, 1.0, 2.0, 5.0, 7.5, and 10.0 g/mL.Cy3-IgG was fixed at 2.0 g/mL.480-550 nm band-pass excitation filter, 570 nm DM, and 590 nm long pass emission filter sets were used.Exposure time was 600 ms.Other conditions were the same.(b) FITC fluorescent intensity differences (Δ) when the concentration of Cy3-IgG was changed from 0, 1.0, 2.0, 5.0, 7.5, and 10.0 g/mL.470DF35 excitation filter, 515 nm DM, and 515 LP emission filter sets were used.FITC-IgG was fixed at 2.0 g/mL.Exposure time was 600 ms.Other conditions were all the same.

Method 1: Detection of Antigen with FITC and Antibody with Cy3
. The behaviour of the concomitant assay was investigated by varying the amounts of antigen and antibody which were detected by two different labels (FITC and Cy3, resp.).
In this format, quantitative measurements of the individual antigen and antibody signals were complicated by concentration dependent on intensity.The effect of energy transfer on the observed intensities was also investigated by holding the amount of Cy3 constant while varying the amount of FITC and then holding the amount of FITC constant while varying the amount of Cy3 (Figure 3).In the first instance (Figure 3(a)), the concentration of FITC-IgG was increased from 0 to 15.0 g/mL while maintaining Cy3-IgG at 2 g/mL.Under these conditions, the Cy3 signal increases by more than a factor of 2 as the result of energy transfer.A similar but opposite effect occurred when the concentration of Cy3-IgG was increased from 0 to 10 g/mL while the FITC-IgG was held constant; the fluorescent intensity of FITC-IgG decreased by 68% with a standard deviation less than 5% (Figure 3(b)).The background noise in these tests was lower than 1%.These results demonstrated that as the surface coverage of the two labelled antigens increases, the proximity between fluorophores increases which leads to efficient FRET.Therefore, in this system, when the two dyes are applied in the detection, the fluorescent intensity has to be calculated before the concentration-intensity plots.1.These five possibilities may cover most of the clinical diagnostic scenario based on detection of antigen and/or antibody in the sample.For example, in case 1, the results indicate the existence of an active antigen in the sample; in case 2, the results indicate the coexisting of antigen and antibody, which is rare in practice (as it is not possible that both antigen and antibody exist in the same sample); and, for cases 3 and 4, they indicate the existing of either antigen or antibody, while, in case 5, they mean there is no detectable antigen and antibody in the sample.

Limits and Solutions.
Due to the energy transfer, it is impossible to apply standard calibration procedures or use the direct detection method in this system.But it is still possible to obtain the analyte concentration by calibration using ( 1) and ( 2) discussed in Section 2.4.1.In order to show the practical solutions for this method, more tests had been carried out.
As antigen was added in a decreased concentration, two types of results were expected: presence or absence of FITC signal, depending on the concentration of the antigen.The absence of FITC signal indicates the absence of any detectable concentration of the antigen and vice versa.Series of antigen (human IgG) concentrations were tested to show the detection limit of this assay (Figure 5(a)).The results clearly showed that, with the increasing of antigen concentration, the fluorescence intensity increases in the range of 0 ng/mL-3.0g/mL (linear relation); but when the antigen concentration was increased beyond 1.0 g/mL, the intensity did not increase corresponding to the concentration.This may be because the antigen in the sample saturates the probes immobilized on the surface of the well when the concentration was higher than 1.0 g/mL (Figure 5(b)).In this case, it means that the sample contains high concentration of active antigen when the fluorescence intensity was in the scope of nonlinear range (antigen concentration was higher than 1.0 g/mL).
From the fluorescent signal, one can see that the lower limit obtained by this method was down to 50 ng/mL (Figure 5(a)).The intensity increases with the increasing of human IgG concentration (antigen in this case) and there was a linear correlation ( 2 = 0.998;  = 3) between the intensity and antigen concentration in the range of 50 ng/mL∼1 g/mL.
Following the antigen detection, antibody detection was carried out by changing the filter for Cy3.The signal of Cy3 indicates the existence of antibody in the sample and intensity represents the concentration of antibody (Figure 6).This was similar to the described antigen detection procedure, where there were two possibilities for the antibody detection: absence or presences of the Cy3 signal after the loading of Figure 5: The FITC-labelled anti-human IgG immunoassay and its work curve in this system.(a) Spectra of human IgG in series concentration in the reactions.From curves 1 to 7 the human IgG concentration was 0 ng/mL, 30 ng/mL, 50 ng/mL, 100 ng/mL, 0.2 g/mL, 0.4 g/mL, 0.5 g/mL, 1.0 g/mL, 1.5 g/mL, 2.0 g/mL, and 3.0 g/mL.(b) The correlation of fluorescent intensity and concentration of human IgG.The data were repeated for 3 times.It should also be noted that energy transfer will only be a problem when both Ab and Ag are present in a sample and at least one of them is present at high concentration.In the absence of these two conditions, the doubly labelled approach will be successful.In the clinical practices, the positive species have either high concentration of antigens with low concentration of antibody or high concentration of antibody with low concentration of antigen.Either case will be in the applicable conditions for this strategy as discussed above.

Method 2: Detecting Ag and Ab
Using a Single Fluorophore and Two Measurements.The behaviour of the model antigen (human IgG) was investigated by varying the concentration in a series from 0 to 20 g/mL, the intensity in a nearly linear fashion at low concentration followed by saturation (Figure 7(a)), but we can see from Figure 7(b) that the intensity did not increase in ratio to the target concentration after 1.0 g/mL, which means the amount of antigen in the sample can saturate the probe which was preimmobilized on the wells.And also the results showed that the as-used 96well plate has very good preference in the low background noise (less than 0.5%).For 96-well plate, the lower detection limit is determined by the signal/noise.Antigen could still be distinguished from background noise down to 30 ng/mL (Figure 7(c)).In theory, the detection limit is lower than 1 ng/mL according to equation (4): where  is Student's -test ( = 3, 99% confidence level) and  is standard deviation;  is linear slope in Figure 7(c).
After the antigen detection, excess antigen was loaded to the well to saturate the unoccupied probes.In this model system, Cy3 labeled rabbit anti-goat IgG was used as the antibody detection reporter.Antibody was loaded into the wells at concentrations from 50 ng/mL to 20 g/mL.Results (Figure 8(a)) clearly show that the fluorescent intensity increases according to the antibody concentration.From the results (Figure 8(b)) we can see Δ increased when the antibody concentration was in the range of 50 ng/mL to 1.0 g/mL in this model system.

Application of Method 2.
In this system, the first detection is aimed at the antigen, which has better sensitivity for low titer of antigen in real samples from clinic diagnostic; and the second is aimed at antibody which usually has higher titer so that it is easier to be detected in detection 2. From the results we show above, it appears that there is a possibility for the combined Ag/Ab immunoassay in one system when only one Cy3 label marked antibody is applied to the test system.

Conclusion
In a word, an approach to detect the antigen and antibody concomitantly is demonstrated in this paper.By comparing the relative fluorescent intensity, Method 1 proposed in this paper has better sensitivity in the detection of antigen than Journal of Spectroscopy in the detection of the antibody, and it is applicable to the clinic species in which usually the antigen concentration is low.But the idea of using one fluorophore both for antigen and for antibody reporter will simplify the detection system.And the one test for both antigen and antibody is also a more economic format for the combination of Ag/Ab immunoassay than most of reported methods which have separate steps to test antigen and antibody.And also five cases were concluded by one fluorophore and two detection strategies in a cheap and simple method.The sensitivity can be comparable to the commercial plate-based methods, and it will be helpful to screen for both antigen and antibody particularly in the situations of the multiserotype and highfrequency mutant virus infections.

Figure 1 :
Figure 1: Schematic illumination of antigen and antibody concurrent immunoassay based on the 96-well plate (a) and the fluorescent microscopy based experimental setup (b).

Figure 2 :
Figure 2: The procedures of this model diagnostic strategy in the immunoassay.

2. 4 . 2 .
Method 2. For Method 2, there are two possibilities at each detection step: presence or absence of the Cy3 fluorescence with the intensity varying with the amount of detected analyte.At the first detection step the Cy3 intensity indicates the concentration of antigen directly.The fluorescence intensity of the second detection step has two parts: (i) antigen detected with Cy3 labelled goat anti-human IgG in the first antigen detection and (ii) antibody detection reporter (Cy3 labeled rabbit anti-goat).Assuming that Cy3

1 =Figure 4 :
Figure 4: The scheme of this model diagnostic strategy and its operation procedures.

Figure 6 :
Figure6: The Cy3 labelled antibody immunoassay and its work curve in this system.(a) The concentration of human IgG that was employed to the wells was 0 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL, 0.5 g/mL, and 1 g/mL.(b) Work curve of fluorescent intensity and human IgG.The tests were repeated for 3 times.

Figure 7 :
Figure 7: The antigen detection fluorescent intensities (a) and the relation between intensities and higher concentrations (b) and lower concentration (c).For (a) and (b) the exposure time was 600 ms and for (c) it was 8000 ms.

Table 1 :
Five possibilities in this analytical strategy.
Method 1   3.2.1.Applications.In order to analyze the detection possibilities in this model, the detailed strategy was showed in Figure4.From the above results of the proposed concurrent detection of Ag/Ab assay, it appears that there are five possibilities, which are shown in Table