You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


1. Introduction
===============

SARS is a severe acute respiratory infective disease. A safe and effective vaccine would be an important complement to the existing SARS control strategies [@bib1]. Neutralization antibodies against SARS virus in immunized animals are measured by potential serological assays. A well-characterized SARS reference antiserum is of great necessity to provide reliable standardized evaluation and quality control for the assay, and moreover to instruct the development of inactivated vaccine against the SARS virus.

The current report summarizes preparation, characterization and calibration of a SARS reference antiserum, according to the WHO biological reference requirements [@bib2].

2. Materials and methods
========================

2.1. Viral strains and virus manipulation
-----------------------------------------

Viral strains: SARS viral strains Sino1, Sino2, Sino3 and Sino6 were isolated from pharyngeal swabs of clinically confirmed SARS patients at Peking Union Hospital.

SARS virus was propagated on Vero cells in a culture flask cultivated at 37 °C until 75% cells showed cytopathic effect. After three freeze--thaw cycles, virus was harvested and well characterized. The batch of virus was stored at −80 °C as reference virus (manuscript in preparation) for neutralization assay.

After harvest, the SARS virus was inactivated and the total protein was purified by centrifugation, ultrafiltration and gel filtration. The gel filtration elutent was used for Western blot assay.

2.2. Cell line
--------------

Vero cells, a line of African green monkey kidney cells, were obtained from ATCC (American Type Culture Collection). Vero cells were grown at 37 °C in minimum essential medium (MEM), containing 10% (w/v) fetal calf serum and 1% (w/v) glutamine.

2.3. Serum
----------

A panel of positive sera, collected from 20 patients in the convalescent stage of SARS, was heat inactivated at 56 °C for 30 min and stored at −20 °C. In order to verify the inactivation efficiency, Vero cells were inoculated with the inactivated serum and cultivated for three generations. No cytopathic effect was detected. In addition, a panel of serum samples was collected from 10 healthy people for negative control.

2.4. Titration of infective virus
---------------------------------

Vero cells were cultivated in 96-well plates at 37 °C to form confluent monolayer. Cells were inoculated with 200 μl per well of viral suspension that was reconstituted and diluted serially in tenfold steps in MEM containing 2% new-born calf serum and 50 μg/ml penicillin (pH 7.2), with each dilution filling 8-well. The 96-well plates were incubated at 37 °C in 5% CO~2~ incubator. Virus titre was expressed as cell culture infectious dose (CCID), which is the dilution of virus causing half of the cultured cells to produce CPE (cytopathic effect).

2.5. Neutralization assay [@bib3], [@bib4]
------------------------------------------

Heat inactivated sera (two-fold serial dilution from 1:2 to 1:2048, 100 μl for each dilution) were added to equal volume of virus (diluted to 100CCID~50~/μl) and incubated for 1h at 37 °C before being added to confluent monolayers of Vero cells in 96-well plates and incubated at 37 °C in 5% CO~2~ incubator for 1--4 days. During the incubation period the cell cultures were checked for development of CPE.

2.6. Western blot assay
-----------------------

Proteins from SDS-PAGE gels were transferred onto nitrocellulose membrane. For immunostaining of the transferred proteins, the blocked membrane was incubated with convalescent serum. Anti-human antibody conjugated with HRP (horseradish peroxidase, Sigma) was used as the second antibody, and the color developing substrate was DAB.

2.7. ELISA
----------

The convalescent serum of SARS was identified and confirmed by a commercially obtained enzyme-linked immunosorbent assay (ELISA) Kit (Beijing WanTai). The antigenic protein employed in the ELISA kit was part of the recombinant N protein of the SARS CoV (the polypeptide product of the third ORF of the N protein coding region) expressed in an *Escherichia coli* strain. The purified SARS virus protein was immobilized on a 96-well plate. The antiserum against SARS virus was added to the coated wells and incubated. HRP-labelled SARS antigen was then added to react with the bound antiserum. TMB was used for color development.

2.8. Preparation of reference antiserum
---------------------------------------

The serum STS-D-Zhang-05 was mixed thoroughly and filled into ampoules (0.5 ml/ampoule). The serum was freeze-dried and the ampoules were sealed.

2.9. Moisture content
---------------------

The moisture content of freeze-dried reference antiserum was detected by Karl Fischer method described as previously [@bib5]. The moisture content of at least three ampoules was tested separately.

2.10. Stability test
--------------------

The stability of the reference antiserum was tested by an accelerated degradation test using temperature at −20, 37 and 56 °C, for 7, 14, 21 and 28 days of storage, respectively. Potency was determined by neutralization assay. All samples were monitored against the −20 °C stored samples.

3. Results
==========

3.1. Identification of SARS corona virus
----------------------------------------

The viruses were electron microscopically visualized, and appeared to have clearly Corona virus typical characteristics. Cytopathic changes could be seen when the viruses were inoculated on Vero cells, which are susceptible to SARS virus infection. The two virus strains, Sino1 and Sino3, which we used extensively for our research have been sequenced (they have more than 99% similarity with the available SARS CoV virus sequences) and have been accepted by GenBank with the accession number of [AY485277](AY485277) (Sino1) and [AY485278](AY485278) (Sino3). The virus could neutralize convalescent sera. All the above evidence has proved that the virus we used is SARS corona virus.

3.2. Epidemiological identification
-----------------------------------

The antiserum was from a male SARS patient with clear epidemiological history and was confirmed clinically. This male patient was identified as a SARS probable case according to the WHO criteria and was confirmed to be SARS case according to the diagnosis criteria of the Chinese Ministry of Health. He had been infected SARS through contact with a SARS patient at Zhangjiakou No. 2 Hospital affiliated with Zhangjiakou Medical Institute. There were two other patients who had had close contact with him. They also showed SARS symptoms such as fever, cough and dyspnea and pulmonary could be detected by X-ray check. They were also confirmed to be SARS probable cases according to the WHO criteria and were confirmed to be SARS cases according to the diagnosis criteria of the Chinese Ministry of Health. All patients were interviewed to ascertain their contacts with each other.

3.3. Serological assays
-----------------------

Convalescent serum was collected from the patient in 3.2, and was named STS-D-Zhang-05 after inactivation process. Antibody specificity of the serum was identified by ELISA, Western blot assay, and neutralization assay.

The convalescent sera were collected from 20 SARS patients (probable cases according the WHO criteria) in the Inner Mogolia Autonomous Region (NeiMengGu, thus the three letter initial code Nei), the cities of Beijing and Zhang Jiakou (thus the three letter initial codes Jing and Zhang, respectively). The collection was arranged by Chinese Ministry of Health and China CDC. Sera were collected 1--5 months post the onset of the symptoms.

Nineteen sera from the above 20 sera tested gave positive results and all negative ones proved negative. Results are given in [Table 1](#tbl1){ref-type="table"} . The serum STS-D-Zhang-05 had strong positive reaction, confirming SARS antibody specificity. The serum volume of STS-D-Zhang-05 is sufficient for reference preparation and its neutralization potency is close to the potency GMT (1:54) of all the 19 positive sera.Table 1Neutralization test results for 20 convalescent sera from SARS patientsSerum codeNT potencySerum codeNT potencySerum codeNT potencySTS-D-Nei-011:25STS-D-Jing-061:203STS-D-Jing-091:51STS-D-Nei-021:32STS-D-Jing-071:64STS-D-Jing-101:51STS-D-Nei-031:128STS-D-Zhang-011:64STS-D-Jing-171:16STS-D-Nei-041:80STS-D-Zhang-02[a](#tbl1fn1){ref-type="table-fn"}\<1:8STS-D-Jing-251:32STS-D-Nei-051:64STS-D-Zhang-031:128STS-D-Jing-261:16STS-D-Nei-061:51STS-D-Zhang-041:80STS-D-Jing-271:51STS-D-Jing-051:64STS-D-Zhang-051:51[^1][^2]

Western blot assay was further performed to identify the SARS antibody in the serum STS-D-Zhang-05. A band of 48 kDa corresponding to the SARS N protein (Nucleocapsid protein) was detected, suggesting the existence of specific anti-SARS antibody. No bands were detected for the negative control serum. The result is shown in [Fig. 1](#fig1){ref-type="fig"} .Fig. 1Western blot assay. Lane a, pre-stained protein marker (New England BioLabs), the bands from the upmost to the downmost corresponds to 83 kDa, 62 kDa, 48 kDa, 33 kDa, 25 kDa, 17 kDa and 7 kDa, respectively. Lane b, total SARS virus proteins (about two micrograms were applied). The antiserum STS-D-Zhang-05 was used as the primary antibody.

Neutralization assay demonstrated that the serum is capable of neutralizing four SARS-CoV strains ([Table 2](#tbl2){ref-type="table"} ).Table 2Neutralization potency of the serum STS-D-Zhang-05 for four SARS CoV strainsSARS CoV viral strainNeutralization potencySino11:64Sino21:32Sino31:16Sino61:64

3.4. Preparation of SARS reference antiserum
--------------------------------------------

After the above identification, STS-D-Zhang-05 was designated to be SARS reference antiserum candidate. The serum, which had a volume of about 100 ml, was filled into ampoules, freeze-dried, sealed, and tested for residual moisture following the WHO requirements [@bib2]. In total 163 ampoules were obtained. This batch of reference antiserum, assigned a lot number of 20031009, was stored at −20 °C and was in the charge of a special keeper.

3.5. Calibration of SARS reference antiserum
--------------------------------------------

Neutralization assay was used to calibrate the reference antiserum. Repetitive titrations were performed by different persons at three different times. Statistic analysis identified the range and GMT (geometric mean titre) of the antibody potency. Results are given in [Table 3](#tbl3){ref-type="table"}, [Table 4](#tbl4){ref-type="table"} .Table 3Results from titration by different persons at different timesTestNumber of experimentsGMTA111:52.3B41:50.6C61:54.8Table 4Results from titration by the same person at the same time (intra-test) or at different times (inter-test)Test number[a](#tbl4fn1){ref-type="table-fn"}ResultIntra-testInter-test11: 801: 5121: 441: 4031: 641: 644--1: 645--1: 516--1: 517--1: 328--1: 51Geometric mean1: 60.81: 49.4Coefficient of variance0.2880.214[^3]

3.6. Potency definition of SARS reference antiserum
---------------------------------------------------

The potency of SARS reference antiserum is defined to be 52.7 U. The geometric mean titre is 1:52.7, which is derived from 21 independent experiments. The 95% confidence interval was found to lie between 1:47.6 and 1:62.2. The Inter-laboratory GCV is 29%.

3.7. Moisture content
---------------------

The moisture of the freeze-dried SARS reference antiserum was determined to be 0.5%.

3.8. Stability test on SARS reference antiserum
-----------------------------------------------

Results on the stability of SARS reference antiserum at 37 and 56 °C are given as [Table 5](#tbl5){ref-type="table"} . More experiments on stability are still in progress.Table 5Results on the stability of SARS reference antiserum[a](#tbl5fn1){ref-type="table-fn"}Experimental duration (day)0714212835Neutralization Antibody Potency at different temperatures −20 °C1:651:451:451:451:451:45 37 °C1:651:511:32[a](#tbl5fn1){ref-type="table-fn"}1:511:511:51 56 °C1:651:231:231:161:161:13[^4]

Results in [Table 5](#tbl5){ref-type="table"} show that neutralization antibody potency has not decreased substantially after SARS reference antiserum was kept at 37 and 56 °C for 0--35 days. Therefore, the storage condition for SARS reference antiserum is designated to be −20 °C in the dark, and the time period before expiration is primarily designated to be 1 year.

4. Discussion
=============

The world has to be prepared to face a possible resurgence of local SARS epidemic. Although progress has been made worldwide to develop a safe and effective human vaccine for SARS prevention, there still lacks international references for and quality control and expressing test results. A recent WHO SARS laboratory workshop has agreed on the urgent need for quality control, standardization of test protocols and reagents, the need for verification of initial cases/clusters in non-epidemic periods, and the establishment of international reference and verification laboratories [@bib6]. Antibody capable of neutralizing the virus has been discovered in the convalescent serum from SARS patients, suggesting its role in the immunity to SARS infection. In order to fulfill the needs for standardization, we have been working on a potential reference antiserum in an effort to search for a vaccine to combat SARS.

A SARS reference antiserum, which will serve as a reference in different serological methods in immunized animals, has been successfully prepared, tested and calibrated.

Qualitative tests including ELISA and Western blot assay and neutralization assay have made it clear that the reference antiserum has satisfied the basic criteria for references. According to the titration results, the neutralization antibody unit of the reference antiserum is 52.7 U. Primary results on stability showed that neutralization antibody potency of the reference antiserum has not decreased substantially up to 3 weeks. The stability test is still in progress.

This newly developed reference antiserum is so far only for in-house use. For acceptance to become an international reference candidate, more has to be done to comply with the WHO regulations on references, such as collaborative titration of the serum and methodology verification.

[^1]: The potency GMT of all the 19 sera is 1:54.

[^2]: This is the negative serum.

[^3]: Test number indicates independent measurements.

[^4]: The results in this table are from single test with no repetition, and there might be deviation in results, as the neutralizing activity on day 14 at 37 °C is lower than that on days 21, 28 and 35.