Novel Immunolatex Possessing A Mixed-PEG/Antibody. Coimmobilized Surface: High-performance Latex

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1 Novel Immunolatex Possessing A Mixed-PEG/Antibody Coimmobilized Surface: High-performance Latex Immunodiagnostics of Ferritin Xiaofei Yuan 1,2,3, Keitaro Yoshimoto 1,2,3, and Yukio Nagasaki 1,2,3,4,5* 1 Graduate School of Pure and Applied Sciences, University of Tsukuba, Ten-noudai 1 1 1, Tsukuba, Ibaraki , Japan; 2 Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), University of Tsukuba, Ten-noudai 1 1 1, Tsukuba, Ibaraki , Japan; 3 Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Ten-noudai 1 1 1, Tsukuba, Ibaraki , Japan; 4 Master School of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 5 Satellite Laboratory of International Center for Materials Nanoarchitechtonics (MANA) in National Institute for Materials Science.(NIMS), Ten-noudai 1 1 1, Tsukuba, Ibaraki , Japan. * To whom correspondence should be addressed. Tel: Fax: nagasaki@nagalabo.jp. S1. Reaction time of N6-PEG-5k with the LA complex. To simplify the preparation procedure for the LAP complex, it is desirable to shorten reaction time of the N6-PEG polymer as much as possible, particularly for large-scale industrial production. With this goal in mind, a comparison of the immunoreactivity, size and ζ-potential of the LAP complex (ECR = 0, 1:2, 1:1 and 2:1), which was separately prepared by allowing N6-PEG-5k (0.1 % w/v in the solution, the proper concentration for all these four 1

2 candidates) to react with the LA complex for 30 min and 60 min, was carried out immediately upon preparation and after several hours at 4 C. Insignificant differences in response to 100 ng/ml ferritin, size and ζ-potential were observed, even after 6 h at 4 C, suggesting that similar forms of LAP complex were obtained although the reaction times of N6-PEG-5k with the various forms of LA complex (i.e. the LA complex with versatile ECR values) were different. On the other hand, all of these forms, which were prepared using different ECR values, seemed stably dispersed since no decrease in immune response or increase in particle size was detectable during the experiment (data not shown). Consequently, a 30-min reaction time for N6-PEG-5k (0.1 % w/v in the solution) was used to prepare the LAP complex, unless otherwise stated. S2. ECR value. It is crucial to use the optimal amount of EDC to activate carboxylated latex particles in order to simplify the procedure for preparing various latex complexes, especially since no purification was performed in this study. If the amount of EDC is not sufficient to activate almost all of the carboxyl groups, the activation procedure will be inefficient. Conversely, excess free EDC may attack the subsequently added antibody. Four candidates, ECR = 0, 1:2, 1:1 and 2:1, were used to separately activate the latex particles, followed by successive reactions with anti-ferritin for 1 h and N6-PEG-5k (0.1 % w/v in solution) for 30 min to obtain four forms of LAP complex. 2

3 First, the amount of LAP complex to be used in the immune response measurements was determined. As representative data, the results for the LAP complex at ECR = 1:1 are shown in Figure S-1a. With increasing ferritin concentration, all the responses corresponding to different volumes of LAP complex proportionally increased at the beginning and then separately tended to plateau. The larger the amount of latex complex, the higher the response, as can be reasonably expected, but using 50 and 60 µl of LAP complex resulted in an almost constant response when the ferritin concentration was lower than 120 ng/ml. This phenomenon suggests that using 50 µl of LAP complex may result in the correct identification of ferritin concentrations below 120 ng/ml. This is why 50 µl of various forms of the complex should be used in response measurements, as described elsewhere. Figure S-1b shows the immune response yields of these four forms of LAP complex (ECR = 0, 1:2, 1:1 and 2:1) as a function of the ferritin concentration. Figure S-1c is an amplification of a part of Figure S-1b. Obviously, the LAP complex at ECR = 0 was not as active as the other forms, as indicated by its low response yield, particularly in the range of ng/ml of ferritin. The LAP complex at ECR = 2:1 reached a plateau, which was lower than those of the other two forms of LAP complex, at ECR = 1:1 and 1:2, in the range of ng/ml of ferritin, possibly due to the excess EDC present 3

4 in this system. The LAP complex at ECR = 1:1 seemed to behave in a manner similar to that at ECR = 1:2, except that it was slightly more reactive when the ferritin concentration was lower than 60 ng/ml (Fig. S-1c). Considering that the anti-ferritin load will be increased in the future, which needs more activated carboxyl groups for antibody immobilization, ECR = 1:1 was selected as the optimal ratio used to activate the latex particles. The quantification of anti-ferritin (see S3) was carried out for the LAP complex at ECR = 0 and 1:1 to explain the low yield of the LAP complex at ECR = 0. Compared to the LAP complex at ECR = 1:1 (Table S-1), there is more free anti-ferritin (2.8 µg/ml) and less coupled anti-ferritin (8.6 µg/ml) in the LAP complex at ECR = 0, indicating that the activated latex particles capably promoted anti-ferritin location. The chain density of the N6-PEG-5k layer, which closely relates to the response of the LAP complex, as described in the Introduction section, was also identified by using the n-lp (ECR = 0) and LP (ECR = 1:1) complexes (see S3) with the assumption that the LP and LAP (ECR = 1:1) and the n-lp and LAP (ECR = 0) complexes possess a similar chain density of the PEG layer. As shown in Table S-2, the chain density of the PEG layer was determined to be and 0.22 chains/nm 2 for the LAP complex at ECR = 0 and 1:1, respectively, evidently suggesting that the activated latex particles also favor a compact 4

5 PEG modification. The low immunoreactivity of the LAP complex at ECR = 0 may therefore be imputed to both its lower anti-ferritin load and the low chain density of the PEG layer. These results agree well with previous reports, and furthermore stress the necessity to activate latex particles when constructing a high-performance LAP complex Response (Abs550nm/s) a ECR = 1:1 LAP 60 µl LAP 50 µl LAP 40 µl LAP 30 µl Ferritin conc. (ng/ml) Response (Abs 550nm/s) b Ferritin conc. (ng/ml) ECR = 0 ECR = 1:2 ECR = 1:1 ECR = 2:1 5

6 10 4 Response (Abs 550nm/s) ECR = 0 ECR = 1:2 ECR = 1:1 ECR = 2:1 c Ferritin conc. (ng/ml) Figure S-1. (a) Immune response of the LAP complex (ECR = 1:1; reaction time of N6-PEG-5k with latex particles: 30 min) measured by using different volumes of the LAP complex. (b) Immune response of four forms of LAP complex (ECR = 0, 1:2, 1:1 and 2:1) as a function of the ferritin concentration in the solution. (c) Amplification of a part of Figure S-1b. Table S-1. Quantification of free and coupled anti-ferritin in the LAP complex using the Micro BCA method. Adsorption time of anti-ferritin (min) Anti-ferritin in supernatant (µg/ml) Anti-ferritin on latex particles (µg/ml) Located anti-ferritin/ total anti-ferritin a (%) a Total anti-ferritin = 10.3 µg/ml 6

7 Table S-2. Quantification of free and coupled N6-PEG-5k in the LAP complex using the Micro BCA method. LAP complex N6-PEG-5k conc. a (% w/v) In supernatant Located on latex particles Density of PEG layer b (chain/nm 2 ) ECR = ECR = 1: a Total N6-PEG-5k conc. = 0.1 % w/v. b The surface area of the latex particle was calculated based on the assumption that it is a sphere with a diameter of 250 nm; M n of N6-PEG-5k = 6000 g/mol. S3. Quantification of anti-ferritin and N6-PEG-5k using the Micro BCA method. The anti-ferritin in an LAP complex suspension was quantified by using the copper reduction/bicinchoninic acid reaction (the Micro BCA method). After one round of centrifugation (15000 rpm, 25 C, 20 min) of 450 µl of LAP complex suspension to precipitate the particles, 300 µl of the supernatant were taken out and reacted with the same volume of BCA analytical reagent (Pierce Biotechnology, Inc., US) at 60 C for 1 h. Then, 300 µl of phosphate buffer solution (buffer A; 10 mm, ph = 7.4) were added to re-suspend the precipitated particles by ultra-sonication, and 300 µl of this re-suspended LAP complex were reacted with the BCA analytical reagent in the same way. After the reaction, the absorbance at 562 nm of the test sample from the supernatant was measured on a PL-2500 spectrophotometer (Shimadzu, Japan). For the sample from the re-suspended LAP complex, the elimination of the particles from the solution is required before the absorbance measurement because of the interference from the light 7

8 scattering signal of the particles. This elimination was carried out using a centrifugal device (MWCO = 300K or 100K, rpm, 5 min, Pall Life Sciences, MI, USA). Because N6-PEG-5k was found to slightly react with the BCA reagent, similarly to anti-ferritin, its dedication, which was obtained by using the LP complex as a control, was subtracted from that of anti-ferritin before the calculation of the anti-ferritin concentration using the anti-ferritin calibration curve. The procedure for quantifying N6-PEG-5k was similar to that for quantifying anti-ferritin, except that the LAP complex (ECR = 0 and 1:1) was exchanged for the LP and n-lp complexes, respectively. (The calibration curve of N6-PEG-5k is shown in Figure S-2.) In this case, the activated latex particles (ECR = 1:1) and the non-activated ones (ECR = 0) were separately used as a control, in order to subtract the dedications of the EDC (in the supernatant) and particles (in the precipitate) from that of N6-PEG-5k, although these interferences were insignificant. 8

9 0.3 y = x R² = Abs. 562nm N6-PEG-5k conc. (% w/v) Figure S-2. Calibration curve of N6-PEG-5k in buffer A (10 mm, ph = 7.4 phosphate buffer solution). The N6-PEG-5k concentration shown on the horizontal axis was the utilized concentration. 9

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