Based on the previous literature and preliminary experiments to determine the concentration of DMOAP used in the direct method we used, the following experimental procedures were obtained.
Liquid Crystal Debugging Experiment
Experimental Procedure Ⅰ
Pretreatment
Prepare 5% Decon 90 cleaning solution, stir, pour into the slide pool, soak the slides overnight; pour off the overnight cleaning solution, shake it with deionized water to achieve the purpose of washing, repeat 10 times; reload the ionized water was rinsed in the ultrasonic for 15 min and repeated twice; the tweezers were clamped on one end of the slide and washed with deionized water for 3 times, and dried with nitrogen, placed in an oven for 20 min, and taken out in a dry slide pool. Dustproof and spare.
Self-assembly of the upper and lower slides
DMOAP self-assembly of the upper slide: The cleaned slide is immersed in a 0.2% (v/v) DMOAP aqueous solution, allowed to stand at room temperature for half an hour, rinsed with deionized water, dried by N2, and dried at 110℃ for 1 h. Dustproof and spare.
APTES/DMOAP hybrid self-assembly of the lower slide: Immerse the cleaned slide in 3% (v/v) APTES and 1% (v/v) DMOAP in 10 mmol/L acetic acid-sodium acetate solution (pH =5) The solution was incubated at 80℃ for 2 h, rinsed with deionized water, dried with N2, dried at 110℃ for 1 h, then immersed in 1% (v/v) GA solution for 37 h at 37℃. Ion water rinse, N2 blow dry, dustproof and spare.
Fixation of Nanobodies
The Nanobody was dissolved in 0.01 mol/L of PBS buffer (pH=7.4) and configured to have different concentrations of the Nanobody solution. Appropriate amount of Nano-antibody solution was added dropwise to the surface of the lower slide, and reacted at 37℃ for 2 h. After removal, it was washed with 0.01 mol/L PBS buffer (pH=7.4) and deionized water to remove unfixed Nanobody molecules. N2 is blown dry, placed at -20℃ for freezing or dustproof at room temperature.
Production of liquid crystal cell
The treated upper and lower slides were assembled face to face, and the slides were separated by Mylar polyester sheets (intermediate open convex cavity), and the other three sides were fixed with small clips except for the opening direction. A small amount of liquid crystal is injected into the liquid crystal cell from the opening by capillary action, and the liquid crystal is filled with the entire cavity, and then observed by a polarizing microscope. And record the image results.
Optimization of Experimental Conditions
Explore the appropriate antibody concentration
Time:
- 20 July: Antibody concentration 50ng/ml. Antibody concentration 200ng/ml.
- 21 July: Antibody concentration 500ng/ml. Antibody concentration 1000ng/ml.
- 30 July: Antibody concentration 1ng/ml. Antibody concentration 10ng/ml. Antibody concentration 50ng/ml.
Since the antibody itself has a corresponding perturbation effect on the liquid crystal, in order to avoid the influence of the nano-antibody perturbation effect on the experimental results, we carried out an experiment to determine the appropriate concentration of the antibody, and we selected 50 ng/mL, 500 ng/mL, 1000 ng/mL, 2000 ng/mL antibody respectively. The concentration was tested and three parallel experimental groups were set for each concentration. Parallel light and conical light were used for observation. The parallel light observation results (Fig. 1) were all black, indicating that the liquid crystal remained vertical.
Parallel light observations at 50 ng/mL, 200 ng/mL, 500 ng/mL and 1000 ng/mL
Figure 1 Effect of antigen concentration gradient on liquid crystal alignment
Since the experimental results did not result in antibody concentrations that caused the liquid crystals to align in parallel and cause color changes, we continued to increase the concentration for experiments. Experiments were performed at 1 ng/mL and 10 ng/mL to obtain the results of parallel light. Parallel light observations still show all black, and cross-contrast observations have crosshairs. It is indicated that the liquid crystals are still mostly arranged vertically. The critical concentration for changing the liquid crystal from the vertical alignment to the parallel alignment was not found, but it was found that the antibody concentration of 10 ng/mL or less did not cause a change in the color of the liquid crystal.
Parallel light observations at antibody concentrations of 1 ng/mL and 10 ng/mL
Figure 2 Effect of high antibody concentration on liquid crystal alignment
Explore the appropriate antibody concentration
Time:
- 23 Aug: Antibody concentration 125ng/ml. Antibody concentration 2000ng/ml.
Since the preliminary experiment for exploring the appropriate antibody concentration did not find a critical value of the antibody concentration that can change the alignment of the liquid crystal, it is known from the search that the concentration of the antibody below 10 ng/mL does not affect the liquid crystal alignment, so we refer to the literature and related research. It is recommended by field personnel to select a concentration of 500 ng/mL as the initial antibody concentration for detecting the antigen. Since the amount of antigen (β2-MG) in the urine of our current patients with chronic kidney disease is 125 ng/mL in unit conversion, we hope to determine the optimal antibody critical concentration by antigen concentration so that the concentration is lower than 125 ng/mL. The antigen does not allow the liquid crystals to be aligned in parallel, while antigens at concentrations above 125 ng/mL just align the liquid crystals in parallel.
Therefore, our team selected 125 ng/mL, 1000 ng/mL and 2000 ng/mL antigen concentrations for experiments. The number of bright spots in 0.125 ng/mL-2 ng/mL parallel light images increased accordingly, but the overall color was black, and the difference was not large enough (Figure 3). Not enough to determine the standard line of detection. (Note: due to 1000 ng/mL data file corruption, not shown in the figure)
Parallel light observations at antigen concentrations of 125 ng/mL and 2000 ng/mL
Figure 3 Effect of antigen concentration on liquid crystal alignment
DMOAP interference exclusion experiment
Time:
- 25 Aug: Antibody concentration 500ng/ml.
Since the previous experiments used high antibody concentration or high antigen concentration, the liquid crystals could not be arranged in parallel to discolor the observation. We reflect on whether the concentration of DMOAP in the experiment has an interference effect on the experimental results. Since DMOAP plays a role in inducing the vertical alignment of liquid crystals in the experiment, the reason for the analysis may be that DMOAP leads to the failure to obtain ideal experimental results. So we chose 0.2%, 0.4%, 0.6%, 0.8% DMOAP concentration, and the antibody concentration was still 500 ng/mL. The DMOAP concentration was different, and the number of bright spots in the parallel light image was different (Fig. 4). From 0.8% to 0.2% concentration, the main body of the image is black, but the number of bright spots is significantly increased. The number of bright spots is much higher at 0.2%; the number of bright spots on the image is less at 0.8%, which is equivalent to the previous 1% concentration.
Parallel light observations at DMOAP concentrations of 0.2%, 0.4%, 0.6%, and 0.8%
Figure 4 Effect of DMOAP concentration on liquid crystal alignment
Since the experimental results are not ideal, we consider re-selecting the experimental protocol. After consulting the literature and discussing with the professional researchers in the field, we decided to use the competition method. The following is the experimental plan.
Experimental Procedure Ⅱ
Pretreatment
Prepare 5% Decon 90 (5ml Decon 90+95ml water) cleaning solution, stir, pour into the slide pool, soak the slides overnight; pour off the overnight cleaning solution, shake it with deionized water to achieve the purpose of washing, repeat 10 times; reload The ionized water was rinsed in the ultrasonic for 15 min and repeated twice; the tweezers were clamped on one end of the slide and washed with deionized water for 3 times, and dried with nitrogen, placed in an oven for 20 min, and taken out in a dry slide pool. Dustproof and spare.
Self-assembly of the upper and lower slides
DMOAP self-assembly of the upper slide: The cleaned slide is immersed in a 0.2% (v/v) DMOAP aqueous solution, allowed to stand at room temperature for half an hour, rinsed with deionized water, dried by N2, and dried at 110℃ for 1 h. Dustproof and spare.
APTES/DMOAP hybrid self-assembly of the lower slide: Immerse the cleaned slide in 3% (v/v) APTES and 1% (v/v) DMOAP in 10 mmol/L acetic acid-sodium acetate solution (pH =5) The solution was incubated at 80 °C for 2 h, rinsed with deionized water, dried with N2, dried at 110℃ for 1 h, then immersed in 1% (v/v) GA solution for 37 h at 37℃. Ion water rinse, N2 blow dry, dustproof and spare.
Immobilization of antigen
The antigen was dissolved in 0.01 mol/L of PBS buffer (pH = 7.4) and configured to have different concentrations of the antigen solution. An appropriate amount of the antigen solution was added dropwise to the surface of the lower slide and reacted at 37℃ for 2 h. After taking out, they were washed with 0.01 mol/L PBS buffer (pH=7.4) and deionized water, and the unfixed antigen molecules were removed, dried with N2 and stored at -20℃.
Production of liquid crystal cell
The treated upper and lower slides were assembled face to face, and the slides were separated by Mylar polyester sheets (intermediate open convex cavity), and the other three sides were fixed with small clips except for the opening direction. A small amount of liquid crystal is injected into the liquid crystal cell from the opening by capillary action, and the liquid crystal is filled with the entire cavity, and then observed by a polarizing microscope. And record the image results.
Nanobody binds to immobilized antigen
According to the competitive immunoassay method, an appropriate amount of a certain concentration of the nano-antibody solution was sequentially added dropwise to the slide to which the antigen was immobilized, and reacted at 37℃ for 1 h. Rinse with 0.01 mol/L PBS buffer (pH=7.4) and deionized water to remove non-specific adsorbed substances, and dried with N2. A liquid crystal cell was prepared according to the method (4), and a change in color and brightness of the liquid crystal film was observed using a polarizing microscope and image analysis was performed.
Experiment of Suitable Antigen Concentration
Time:
- 30 Aug: Antigen concentration 500ng/ml; Antigen concentration 1000ng/ml; Antigen concentration 2000ng/ml; Antigen concentration 4000ng/ml.
- 2 Sept: Antigen concentration 100ng/ml; Antigen concentration 200ng/ml; Antigen concentration 300ng/ml; Antigen concentration 400ng/ml.
We investigated the effect of antigens of different concentrations on the vertical alignment of liquid crystals without antibodies. The methods of antigen-antibody immobilization on glass were collected from the literature of silkworm (crosslinked glutaraldehyde).The antigen was dissolved in 0.01 mol/L of PBS buffer (pH = 7.4) and configured to have 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L, 1mg/L, 2mg/L and 4mg/L of the antigen solution to have experiments with 1% DMOAP. For details of subsequent treatment, please refer to the third step of competitive immunoassay method. Antigen concentration gradient is small in the first five groups while antigen concentration gradient of the last three groups is big. Three parallel experiments were carried out in each group, and we chose the best picture to integrate them together. We observed bright spots separately under quadruple and tenfold objective. The specific results of the experiment are recorded as follows:
a. Images of 0.1mg/L, 0.2mg/mL, 0.3mg/L, 0.4mg/L, 0.5mg/L, 1mg/L, 2mg/L, 4mg/L of antigens in proper order under a quadruple objective
b. Images of 0.1mg/L, 0.2mg/mL, 0.3mg/L, 0.4mg/L, 0.5mg/L, 1mg/L, 2mg/L, 4mg/L of antigens in proper order under a tenfold objective
From the image, we can see that there is little difference among 0.1mg/L, 0.2mg/L and 0.3mg/L of antigens. It showed that the antigens in this concentration range have almost no effect on the vertical alignment of liquid crystal. The number of bright spots increased, but not more differences among 0.5mg/L-4mg/L. It suggested that the molecular weight of antigen may be too small to affect the vertical alignment of liquid crystal. Therefore, the effect of antigens of 0.5mg/L concentration on the vertical alignment of liquid crystals is good.
Experiment of Suitable Nanobody Concentration
Time:
- 8 Sept: Antigen concentration 500ng/ml, antibody concentration 500 ng/ml; Antigen concentration 500ng/ml, antibody concentration 1000 ng/ml; Antigen concentration 500ng/ml, antibody concentration 1500 ng/ml; Antigen concentration 500ng/ml, antibody concentration 2000 ng/ml.
- 9 Sept: Antigen concentration 500ng/ml, antibody concentration 250 ng/ml; Antigen concentration 500ng/ml, antibody concentration 500 ng/ml.
- 10 Sept: Antigen concentration 500ng/ml, antibody concentration 500 ng/ml.
After the experiment of suitable antigen concentration, we found that the effect of antigens of 0.5mg/L concentration on the vertical alignment of liquid crystals is good. Therefore, we take the concentration of antibody solution as 0.5mg/L as the premise to investigate suitable nanobody concentration. An appropriate amount of 0.5mg/L, 1mg/L, 1.5mg/L and 0.5mg/L concentration of the nano-antibody solution was sequentially added dropwise to the slide to which the antigen was immobilized. For details of subsequent treatment, please refer to the fifth step of competitive immunoassay method .Three parallel experiments were carried out in each group, and we chose the best picture to integrate them together under both quadruple and tenfold objective. As the results are good, we intend to explore the lowest limit of antibody concentration by immersion（Add the liquid to a clean Petri dish and immerse the slide in）. The specific results of the experiment are recorded as follows:
a. Images of 0.5mg/L, 1mg/L, 1.5mg/L and 2mg/L of Nanobodys in proper order under a quadruple objective
b. Images of 0.5mg/L, 1mg/L, 1.5mg/L and 2mg/L of nano-bodys in proper order under a quadruple objective
c. Images of 0.25mg/L and 0.5mg/L in proper order by immersion
Verify Antigen Concentration Experiment
Time:
- 12 Sept: Antigen concentration 1000ng/ml, antibody concentration 1000 ng/ml; Antigen concentration 500ng/ml, antibody concentration 1000 ng/ml; Antigen concentration 62.5mg/L, antibody concentration 5000 ng/ml; Antigen concentration 62.5 mg/L, antibody concentration 1000 ng/ml;Antigen concentration 31.25 mg/L, antibody concentration 500 ng/ml; Antigen concentration 31.25 mg/L, antibody concentration 1000 ng/ml.
Due to the instability of the experimental results we have done before, we need to detect the antigen concentration and antibody concentration, and use several extreme antigen concentrations to determine whether there is a problem with our antigen concentration. And considering the damage of the slide and the slide pool, in the self-assembly of the slide, the slide was dried at 110℃ for 1 h and changed to dry at 80℃ for 1 h. At the same time, the antigen fixation process still adopts the method of soaking.
We changed the fixed antigen concentration to 1 mg/L and the corresponding antibody to 1000 ng/mL (shown in Figure (5)a). Four parallel experiments were performed. We also made a fixed antigen to 62.5mg / L, 31.25mg / L, and explored the results of the two antigen concentrations, the combined antibody was 500ng / ml and 1000 ng / ml (Figure (5) b, c, d, e), did four experiments, four experiments in each group. Finally, we carried out the experimental results when the fixed antigen was 500 ng/mL and the antibody was 1000 ng/mL (shown in Figure (5)f). Two parallel experiments were performed to determine whether there was a deviation in the results of our previous experiments.
a. Antigen concentration 1 mg/L, antibody concentration 1000 ng/mL result image
b. Antigen concentration 62.5 mg/L, antibody concentration 500 ng/mL result image
c. Antigen concentration 62.5 mg/L, antibody concentration 1000 ng/mL result image
d. Antigen concentration 31.25 mg/L, antibody concentration 500 ng/mL result image
e. Antigen concentration 31.25 mg/L, antibody concentration 1000 ng/mL result image
f. Antigen concentration 500ng/mL, antibody concentration 1000 ng/mL result image
Figure 5 Verification of extreme antigen concentration
The result can be that the combination of the two can disturb the alignment of the liquid crystal. At the same time, the antigen is fixed.
Explore the Appropriate Concentration of Nanobodies Again
Time:
- 15 Sept: Antigen concentration 500ng/ml, antibody concentration 1000 ng/ml; Antigen concentration 500ng/ml, antibody concentration 1500 ng/ml.
Following the last experiment, we thought that 500 ng/mL antigen fixation is currently reasonable. So we did the effect of antibody concentration of 1000 ng/mL and 1500 ng/mL at an antigen concentration of 500 ng/mL (Figure (6) a, b), where we will self-assemble the upper and lower slides. And considering the damage of the slide and the slide pool, in the self-assembly of the slide, the slide was dried at 110℃ for 1 h and changed to dry at 80℃ for 1 h. At the same time, the antigen fixation process still adopts the method of soaking. Three sets of parallel experiments were performed separately.
Then we also made a set of antigen concentration of 500 ng / ml, antibody concentration of 1500 ng / ml (Figure (6) c). However, the method of soaking the immobilized antigen was not adopted, but the previous dropping method was employed.
a. Antigen concentration 500ng/mL, antibody concentration 1000 ng/mL result imag.
b. Antigen concentration 500ng/mL, antibody concentration 1500 ng/mL result image
c. Antigen concentration 500ng/mL, antibody concentration 1500 ng/mL result image（Antigen drop fixation）
Figure 6 Verifying the nanobody concentration experiment again
The experimental results showed that the antibody concentration was responsive at 1000 ng/mL and 1500 ng/mL, and the results were obvious. Among them, the method of dropping antigen is more stable.
The Influence of GA and APTES
Time:
- 19 Sept: The influence with or without APTES and GA.
Since the previous results were more pronounced, we then explored the impact of GA and APTES on the results. We skipped the steps of immobilizing the antigen and dropping the antibody, directly performing the GA addition step after self-assembly of the slide, and exploring the effect of GA (shown in Figure (7)a). The impact of APTES is to observe the observations made after self-assembly (Figure (7)b).
a. APTES inquiry results
b. GA inquiry results
Figure 7 The impact of APTES and GA
It can be seen from the results that APTES and GA are negligible for liquid crystal disturbance.
Hydrophilic Experiment of Slide
In order to investigate how hydrophilic the slides were before and after antigen addition, we conducted a measurement of the contact angle experiment. Among them, we will self-assemble the upper and lower slides, and the upper and lower slides will be dried at 110℃ for 1 h, changed to be dried at 80℃ for 1 h. The method of immobilizing the antigen is to adopt a soaking method.
No antigen was added, and the contact angle of 50 ng/mL antigen with 500 ng/mL antigen was shown (Fig. 8 (8) a, b, c).
a. Unfixed antigen |
b. Antigen concentration 50ng/mL |
c. Antigen concentration 500ng/mL |
Figure 8 Results of the hydrophilicity test of the lower slide
It can be concluded that as the concentration of the immobilized antigen increases, the slide changes from hydrophobic to hydrophilic.
Study on Antigen Concentration and Fixed Time
Time:
- 26 Sept: Antigen concentrations of 500 ng/ml and 750 ng/ml were fixed for 12 h.
- 27 Sept: Antigen concentrations of 500 ng/ml and 750 ng/ml were fixed for 8.5 h.
After the discussion of the previous results, we discussed with the teacher, and then explored whether there is a problem with the antigen fixed time. We performed experimental results at antigen concentrations of 50 ng/mL and 500 ng/mL and fixed for 8.5 h and 12 h (Figures (9) a, b, c, d). Among them, we will self-assemble the upper and lower slides, and the upper and lower slides will be dried at 110℃ for 1 h, changed to be dried at 80℃ for 1h. The antigen fixation method is fixed by dropping.
a. Antigen 50ng/mL fixed 8.5h result image
b. Antigen 500ng/mL fixed 8.5h result image
c. Antigen 50ng/mL fixed 12h result image
d. Antigen 500ng/mL fixed 12h result image
Figure 9 Results of antigen fixation time
The experimental results show that the fixed time has a great relationship with the degree of liquid crystal disturbance. The longer the time, the more serious the disturbance.
Experiment of Molecular Biology
Sept. 23
Transformation our two plasmids of BL21 cells. And Pipette 100 μL of the transformation onto a plate with antibiotic as appropiate (kanamycin 50 µg/ mL and 100 µg/mL for ampicillin).
Sept. 24
Picking up a single colony inoculation into 100 mL LB liquid medium (with antibiotic). 37℃, 220 rpm for 16 hours.
Sept. 25
(1) Measured OD600, the value was less than 0.5, pipetted 1 mL cultures to 100 mL new LB liquid medium (with antibiotic). 37℃, 220 rpm for 4 hours.
(2) Measured OD600, but the value was less than 0.5 too.
Sept. 26 ~ Sept. 28
We continued to train the cells, but the OD600 has been low, so we discussed the reasons for the failure and made a series of improvements to the steps.
Sept. 29
We repeat the steps of Sept. 24.
Sept. 30
Picking up a single colony inoculation into 5 mL LB liquid medium (with antibiotic). 37℃, 220 rpm for 16 hours.
Oct. 1
Picking up a single colony inoculation into 5 mL LB liquid medium (with antibiotic). 37℃, 220 rpm for 16 hours.
Oct. 2
When we reconfigured new medium, we found that the two pH meters we used were higher than the actual value of 2, so we switched to the pH test paper.
Than we repeat the steps of Sept. 24 again.
Oct. 3
(1) Today, We did the colony PCR and agarose gel. The methods are at the Protocols. But our retules was not good. We thought that was because we were’t boiled the cells.
(2) We picked up a single colony inoculation from the plate into 5 mL LB liquid medium (with antibiotic). 37℃, 220 rpm for 16 hours.
Oct. 4
(1) Pipetted 150 mL cultures to 15 mL LB liquid medium (with antibiotic). 37℃, 220 rpm for 4-5 hours. Measured OD600. When OD600=0.5, we did the Bacteria Preservation.
(2) At 18:00, add CuSO4 and Ara to 15mL medium (50μM/L for CuSO4 and 0.2% for Ara, 18℃, 220 rpm for 1 hour，and than add IPTG to medium for 0.2mM, 18℃, 220 rpm 16 hours.
Oct. 5
(1) Centrifuged the cultures and resuspended the cells in PBS. Ultrasonic broken the cells, Centrifuged again and take the supernatant in a refrigerator at 4 °C
(2) Repeated the steps of Sept. 24.
Oct. 6
We found that the E.coli BL21 was failed so we want to extracted plasmids from E.coli DH-5α.
(3) Pipetted 50 μL cultures to 5 mL new LB liquid medium (with antibiotic). 37℃, 220 rpm for 16 hours.
Oct. 7
Today, we did the plasmids extraction and agarose gel. But the result was not well. So we wanted to do this step again.
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Figure 10 1~7 are our sample, 8 is Maker. |
Oct. 8
Activation of DH-5α single colony on a 10.2-day conversion plate.
Oct. 9
Extracted plasmids again，and we got a good result.
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Figure 11 1-7 are our sample. 9-10 are Makers. |
| Sample | ng/μL | A260/A280 | A260/A230 | A260 | A280 |
| 1 | 49.4 | 1.67 | 2.43 | 0.99 | 0.59 |
| 2 | 75.5 | 1.82 | 2.55 | 1.51 | 0.83 |
| 3 | 66.6 | 1.83 | 4.63 | 1.33 | 0.73 |
| 4 | 65.9 | 1.82 | 4.57 | 1.32 | 0.72 |
| 5 | 70.1 | 1.76 | 2.55 | 1.40 | 0.80 |
| 6 | 102.8 | 1.86 | 2.59 | 2.06 | 1.11 |
| 7 | 68.9 | 1.75 | 2.37 | 1.38 | 0.79 |
Figure 12 Concentration of each sample
Oct. 10
Transformation our two plasmids of BL21 cells. And Pipette 100 μL of the transformation onto a plate with antibiotic as appropiate (kanamycin 50 µg/ mL and 100 µg/mL for ampicillin).
Oct. 11
Picking up a single colony inoculation into 100 mL LB liquid medium (with antibiotic). 37℃, 220 rpm for 16 hours. Than we did the Bacteria Preservation and expanded cultures.
Oct. 12
pipetted 500 μL cultures to 50 mL new LB liquid medium (with antibiotic). 37℃, 220 rpm for 4-5hours.
(1) Measured OD600.
(2) We repeated the steps of Oct. 4 (2).
Oct. 13
(1) Repeated the steps of 10.5(1). Take the supernatant and make it pH=4， Centrifuged again. Take 100μL supernatant to a tube and add 5μL fluorescent dye to it. Wrpa the tube with tin foil 37℃ for 6 hours. Resuspending the sample with PBS for the above precipitation.
(2) Add 50 mM C18 acylhdrazine to 5 mL DMSO. Add 100μL the mixture to 1 mL supernatant. for two tubes. One at 37℃ for 3 hours. Another at 20℃ for 3 hours.
Oct. 14
We did the SDS-PAGE. The order as follow:
- Fluorescent dye modification
- 2.C18 acylhdrazine modification for 37 ℃
- C18 acylhdrazine modification for 20 ℃
- Acidified supernatant
- Maker
- Maker
- Broken supernatant
- Post-crushing precipitation
- Induced bacterial solution
- Inductive bacteria solution
The result was as follow.
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Figure 12 The result of SDS-PAGE |
Oct. 15
Determination of protein concentration of prepared nanometer antibody.
