Characterization and Prevention of the Adsorption of Surfactant Protein D to Polypropylene

<div><p>Surfactant Protein D (SP-D) is a multifunctional protein present in the lung and in respiratory secretions. In the process of developing new experimental approaches to examine SP-D function, we observed that SP-D adsorbs to polypropylene tubes to a great extent, thereby depleting SP-D from the solution. Although it is well known that proteins adsorb nonspecifically to plastic, this effect is usually diminished by treatments to make the plastic “low-retention” or “low-binding”. However, these treatments actually increased the binding of SP-D to the plastic. In addition, this adsorption affected the results of several assays, including proteolytic cleavage assays. In order to block SP-D from adsorbing to polypropylene and the effects caused by this adsorption, we coated the tubes with bovine serum albumin (BSA), as is commonly performed for ELISAs. This coating greatly diminished the amount of SP-D sticking to the plastic, providing an inexpensive and effective method for preventing adsorption and the artifacts resulting from this adsorption.</p></div

Depletion of SP-D in Solution During Serial Transfers.

<p>5 µl aliquots were taken from a serially transferred solution of SP-D after each incubation, analyzed by Western blot, and quantified using ImageJ software.</p

Detection of SP-D in serum and plasma collected using various anticoagulants.

<p>a) SP-D concentrations were measured in samples collected into four different vacutainers during a single blood draw. b) Measured values of samples were normalized to the SP-D concentration in serum for each patient in order to compare the effect of the anticoagulant on the measured SP-D concentration. An asterisk (*) denotes values are significantly different by Wilcoxon signed rank test (for EDTA, p = 0.0156, and for Citrate, p = 0.0078).</p

Depletion of SP-D during Extended Incubations.

<p>5 µl aliquots were taken from SP-D solutions after incubating at the indicated time/temperature, analyzed by Western blot, and quantified using ImageJ software (A). Values were normalized to the average of the “4 hours @ RT” group. The SP-D content of tubes was analyzed and compared to the total amount of SP-D in the supernatant and tube (B). An asterisk (*) denotes p≤0.05 when comparing columns.</p

Influence of calcium concentration on detection of recombinant SP-D and native SP-D in serum and plasma.

<p>a) Recombinant SP-D (rSP-D), serum, and EDTA plasma were assayed in twofold dilutions of [Ca²⁺] or [EDTA] (concentrations are displayed in mM). For the inclusion of calcium with recombinant SP-D, Hank's Balanced Salt Solution (HBSS) was used as a buffer instead of PBS. Recombinant SP-D was assayed at 5 ng/mL while serum and plasma were diluted twofold. Each line represents an independent experiment. b) ELISAs were performed with detection reagents diluted in HBSS without calcium chloride (-CaCl²) or with 5 mM calcium chloride (+CaCl²). Wash buffer also either lacked or included calcium chloride. Recombinant SP-D was assayed at 10 ng/mL and Heparin plasma from four subjects was pooled, and sample values were extrapolated from a single standard curve under the conditions lacking calcium. n = 3 samples per group.</p

Comparison of SP-D values in samples stored in various conditions.

<p>Serum and heparin plasma samples collected during a single draw were either analyzed fresh or aliquoted and stored at 4°C, −20°C, or −80°C.</p

Adsorption of SP-D to Polypropylene Pipet Tips.

<p>5 µl aliquots were taken from a serially aspirated/dispensed solution of SP-D after each round of pipetting, analyzed by Western blot (representative blot shown in A), and quantified using ImageJ software (B). The control consisted of 6 aliquots taken without pipetting. The means were found to be significantly different by One-way ANOVA (p = 0.044).</p

Published values for [SP-D] in the blood.

<p>a) A substantial amount of variation in the average [SP-D] in the serum/plasma of healthy control population exists between studies using the same or different ELISA configurations. Three configurations (Yamasa <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Greene1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Greene2" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Nagae1" target="_blank">[17]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Kuwano1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Lin1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Ilumets1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Mutti1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Krane1" target="_blank">[38]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Takahashi2" target="_blank">[43]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Yanaba1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Janssen1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Koopmans1" target="_blank">[50]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Kawasaki1" target="_blank">[59]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-AlSalmi1" target="_blank">[61]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Haddam1" target="_blank">[77]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Ihn1" target="_blank">[84]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Stapleton1" target="_blank">[89]</a>, BioVendor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Glas1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Lomas1" target="_blank">[31]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-ElDeek1" target="_blank">[36]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Hant1" target="_blank">[46]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Vizcaya1" target="_blank">[52]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Bejvl1" target="_blank">[56]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Wutzler1" target="_blank">[64]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Engstrom1" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Wang1" target="_blank">[72]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Shiels1" target="_blank">[75]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Aul1" target="_blank">[76]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Gil1" target="_blank">[79]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-FernandezLuna1" target="_blank">[80]</a>, and Holmskov et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Hohwy1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Olesen1" target="_blank">[39]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Hoegh1" target="_blank">[53]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Wu1" target="_blank">[55]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-LethLarsen1" target="_blank">[58]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Heidinger1" target="_blank">[62]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Sorensen1" target="_blank">[66]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Zhao1" target="_blank">[69]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Hoffmann1" target="_blank">[70]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Gravholt1" target="_blank">[78]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Nybo1" target="_blank">[85]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Hoegh3" target="_blank">[86]</a>) were compared. b) The range of healthy control [SP-D] greatly varied from study to study. Values shown are either median and interquartile range (IQR), median and 95% confidence interval (95%), or mean and standard deviation (SD). c) The calculated fold increase from the average healthy [SP-D] and average [SP-D] during IPF <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Greene1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Honda1" target="_blank">[16]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Kuwano1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Lin1" target="_blank">[21]</a>, CF <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Sims1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Krane1" target="_blank">[38]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Olesen1" target="_blank">[39]</a>, cigarette smoking <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Mutti1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Lomas1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Wang1" target="_blank">[72]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Shiels1" target="_blank">[75]</a>, or sclerosis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Elhaj1" target="_blank">[42]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Asano1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111466#pone.0111466-Hant1" target="_blank">[46]</a> was different between publications. An asterisk (*) denotes p≤0.001 by one-way ANOVA with Tukey's multiple comparison test.</p

Use of various diluents for the recombinant SP-D standard.

<p>A 1 µg/mL recombinant SP-D stock was diluted 1∶99 in various matrices and then serially diluted 2 fold in the same matrix. Mean and standard deviation for three independent experiments are shown. An asterisk (*) denotes values are significantly different (p≤0.01) from both the 1% BSA and 10% FBS values by one-way ANOVA using Tukey's multiple comparison test.</p

Adsorption of SP-D to Variously Treated Polypropylene Tubes.

<p>The amount of adsorbed SP-D was measured using ImageJ software to analyze Western blots of samples taken from uncoated (A) or BSA-coated (B) regular, low retention, and low binding tubes. The amount was calculated using untreated SP-D as a standard. An asterisk (*) denotes p≤0.05 when comparing columns.</p