Supplementary Material for: Prognostic Value of Pre- and Postoperative Anti-p53 Antibody Levels in Colorectal Cancer Patients: A Retrospective Study

<p><b><i>Objective:</i></b> To determine the utility of the post-/preoperative anti-p53 antibody (p53 Ab) ratio as a prognostic factor for colorectal cancer (CRC) recurrence. <b><i>Methods:</i></b> A total of 737 nonmetastatic CRC patients who had undergone R0 resection were retrospectively analyzed. p53 Ab levels were measured within 1 month prior to and at least every 3 months after surgery. Post-/preoperative p53 Ab ratios were calculated, and the optimal ratio cutoff values for predicting recurrence were determined using the Kaplan-Meier method and the log-rank test. <b><i>Results:</i></b> Preoperative p53 Ab elevation was observed in 194 patients (pre-p53 high). Preoperative p53 Ab levels correlated with TNM stage. Re-elevation of p53 Ab levels occurred on recurrence in the pre-p53 high group, but not in the pre-p53 low group (n = 543). In the pre-p53 high group, patients who experienced tumor recurrence exhibited a slow postoperative reduction of p53 Ab levels, and a post-/preoperative p53 Ab ratio >0.4 at postoperative 3 months predicted relapse-free survival. In other words, a p53 Ab level remaining higher than 40% of the preoperative level was an independent and strong risk factor for recurrence in multivariate analyses. <b><i>Conclusion:</i></b> In CRC patients with preoperative p53 Ab elevation, the rate of p53 Ab reduction in the early postsurgical period is a promising prognostic factor for recurrence.</p

Supplementary Material for: Rapid Bioassay for Detection of Thyroid-Stimulating Antibodies Using Cyclic Adenosine Monophosphate-Gated Calcium Channel and Aequorin

<b><i>Background:</i></b> Thyroid-stimulating antibodies (TSAb) are known to be responsible for hyperthyroidism in Graves' disease (GD). The conventional methods to measure TSAb depend on cell-based assays that require cumbersome procedures and a sterilized tissue culture technique. The aim of the present study was to develop a ready-to-use cell-based assay for measuring TSAb activity without requiring sterilized conditions. <b><i>Methods:</i></b> We developed a new assay kit using a frozen Chinese hamster ovary cell line expressing the thyroid-stimulating hormone receptor, cyclic adenosine monophosphate (cAMP)-gated calcium channel and aequorin, tentatively named the aequorin TSAb assay. Activated stimulatory G-protein-coupled adenylate cyclase increases intracellular cAMP, which then binds to the cyclic nucleotide-gated calcium channel. Activation of this channel allows Ca²⁺ to enter the cell, and the influx of Ca²⁺ can be measured with aequorin, which is quantified by a luminometer. Results can be obtained in only 4 h without sterilized conditions. TSAb activities were expressed by international units using the NIBSC 08/204 standard. <b><i>Results:</i></b> Positive results of aequorin TSAb were obtained in 197 of 199 (98.9%) of untreated patients with GD. Only 1 of 42 (2.3%) patients with painless thyroiditis had a weakly positive aequorin TSAb. All 45 patients with subacute thyroiditis and 185 normal subjects showed negative aequorin TSAb. As for chronic thyroiditis, all 52 euthyroid patients showed negative aequorin TSAb, but 8 of 50 (16.0%) hypothyroid patients had a positive reaction. However, these positive reactions were not induced by serum thyroid-stimulating hormone (TSH) and were thought to be induced by the stimulating activity of anti-TSH receptor immunoglobulins. Conventional porcine TSAb and Elecsys thyroid-stimulating hormone receptor antibodies were positive in 69.3 and 95.5% of GD, respectively. <b><i>Conclusion:</i></b> The aequorin TSAb assay was positive in 98.9% of GD and was more sensitive than the conventional assay. This assay can be conducted in only 4 h without sterilized conditions and is practically useful in general clinical laboratories