08 May – 14 May (Week 19)

This week we performed the first attempts at transformation and colony growth.

• On May 8, LB agar plates were prepared, and plasmids were transformed into E. coli using heat shock. Colonies were left to grow while replicating the plasmids.
• On May 12, we prepared 5 mL TB medium supplemented with 5 μL antibiotics(ampicillin) in four tubes. Four single colonies were inoculated. Tubes were incubated overnight.
• On May 13, no growth was observed from the inoculated cultures. Colonies were re-picked and grown again.
• On May 14, after 14h incubation, no signs of bacterial growth were observed. Possible reasons included excessive antibiotic concentration or the culture medium being unsuitable. Switching to LB medium was suggested for future attempts.

19 May – 22 May (Week 20)

This week we restarted transformations and checked plasmid yields.

• On May 19, to avoid troubleshooting variable by variable, we repeated the transformation with E.coli DH5α. Two selective conditions were used: Erythromycin and Ampicillin plates. Cultures were grown ~18 h.
• On May 20, colonies were picked: five tubes from Erythromycin plates and three from Ampicillin plates were inoculated into 5 mL culture medium and incubated at 37 °C.
• On May 21–22, most cultures grew well. For ermGFP plasmid, all five cultures showed growth. For mRFP1 plasmid, only one out of three tubes had stronger growth. Plasmid extraction resulted in low concentrations (30–50 ng/µL). After re-elution, concentrations improved to ~80 ng/µL in some samples, but still insufficient overall.

• Bad news: the plasmid supplier reported they could not construct plasmids with Erythromycin selection.

26 May – 27 May (Week 21)

This week we focused on mRFP1 plasmid growth and plasmid extraction.

• On May 26, six cultures of pLEM415-IdhL-mRFP1 were prepared (3 in TB medium, 3 in LB medium).
• On May 27, two LB cultures showed poor growth. Plasmid extraction was performed on the remaining cultures. Colonies in some tubes displayed visible pink coloration, indicating expression of red fluorescent protein. The plasmid was confirmed to be a high-copy plasmid. NanoDrop results showed good concentration, but the purity ratios were inconsistent. After discussion, two plasmids were selected for submission to the company.

  Table 1. Nanodrop concentration for pLEM415-IdhL-mRFP1 plasmids.

  sample name	ng/µL	A260/A280	A260/A230	A260	A280
  sample 1	570.5	2.08	2.38	11.41	5.47
  sample 2	383.5	2.18	2.23	7.67	3.52
  sample 3	595.5	2.00	2.40	11.91	5.95
  sample 4	637.9	2.07	2.41	12.76	6.17

• A cloning strategy discussion was held:
  • The original plasmid contained the insert: IdhL-FLAG-mRFP1, flanked by restriction sites ApaI (GGGCCC) and ClaI (ATCGAT).
  • Plan: excise the FLAG-mRFP1 fragment and replace it with the designed sequence (IdhL–[our sequence]).

15 July (Week 27)

Deletions and mutations were detected in some constructs, requiring a redesign of the cloning strategy.

06 August (Week 32)

We designed three prototype constructs of anti-IL-23 single-chain variable fragments (scFvs). In addition to these novel prototypes, we also modified the variable regions of two clinically approved IL-23-targeting antibodies into scFv format: Risankizumab, which neutralizes the p19 subunit of IL-23 (hereafter referred to as p19 in this notebook), and Ustekinumab, which targets the p40 subunit (hereafter referred to as p40 in this notebook). All three prototype scFvs, along with the scFv versions of Risankizumab and Ustekinumab, were submitted to a biotechnology company for gene synthesis and recombinant expression.

11 Aug – 19 Aug (Week 33)

This week we focused on plasmid amplification in E.coli DH5α, plasmid extraction, quality check, and subsequent transfection into HEK293T cells.

• On Aug 11, DH5α cultures harboring the CMV plasmid were inoculated in LB medium and incubated overnight at 37 °C.
• On Aug 12, plasmid extraction was performed from the overnight cultures. NanoDrop measurements were taken to determine plasmid concentration and purity.

  Table 2. Nanodrop concentration for CMV plasmids containing ScFv-IL23 variants (prototype A-C), positive control (PC-p19, and PC-p40).

  Insertion (sample name)	Replicate	ng/µL	A260/A280	A260/A230	A260	A280
  ScFv IL23 prototype A	1-1	507.2	1.88	2.28	10.14	5.41
  ScFv IL23 prototype A	1-2	375.0	1.87	2.23	7.50	5.41
  ScFv IL23 prototype B	2-1	427.2	1.87	2.26	8.54	4.02
  ScFv IL23 prototype B	2-2	536.8	1.88	2.29	10.74	4.58
  ScFv IL23 prototype C	3-1	464.8	1.87	2.26	9.30	5.71
  ScFv IL23 prototype C	3-2	527.1	1.87	2.27	10.54	4.97
  PC-p19	4-1	279.7	1.86	2.20	5.59	3.01
  PC-p19	4-2	295.9	1.86	2.22	5.92	3.18
  PC-p40	5-1	425.4	1.87	2.28	8.51	4.55
  PC-p40	5-2	491.1	1.87	2.26	9.82	5.26

  ps. Each insertion construct was cultured and plasmid DNA extracted in duplicate (labeled -1 and -2). For each pair, the replicate with higher yield/purity was highlighted and selected for downstream use.

• On Aug 14-15, additional plasmid preparations were carried out to obtain sufficient DNA for transfection. Gel electrophoresis confirmed plasmid integrity.
• On Aug 16-17, HEK293T cells were maintained in log-phase growth and seeded at appropriate density for transfection.
• On Aug 19, transfection was performed in HEK293T cells using purified plasmid DNA together with TransIT-X2 transfection reagent, according to the manufacturer’s instructions. The cells were returned to the incubator (37 °C, 5% CO₂) for expression studies.

20 Aug – 23 Aug (Week 34)

This week, we focused on testing IL-23 antibody secretion from transfected HEK293T cells. Samples from both culture supernatants and cell lysates were collected and analyzed by SDS-PAGE followed by Western blotting to evaluate expression and secretion efficiency.

• On Aug 20, culture supernatant and corresponding cell pellets were collected from transfected HEK293T cells.
• On Aug 21, cell lysis was performed to extract intracellular proteins. Both supernatant and lysate fractions were stored at –20 °C for downstream analysis.
• On Aug 22, protein concentration was quantified using BCA assay to normalize sample loading.

Table 3. BCA Assay Protein Quantification Results for NCV and anti-IL23 Samples.

Sample Name	OD562	Conc. (μg/μL)
NCV	0.349	7.992
anti-IL23 scFv 1-1	0.347	7.935
anti-IL23 scFv 2-2	0.338	7.677
anti-IL23 scFv 3-2	0.340	7.735
anti-IL23 scFv p19	0.329	7.420
anti-IL23 scFv p40	0.355	8.164

• On Aug 23, SDS-PAGE was performed to compare protein content in cell lysate.

25 Aug – 29 Aug (Week 35)

This week we carried out Western blot analysis to detect anti-IL23 expression in transfected HEK293T cells.

• On Aug 25, Proteins from SDS-PAGE gels were transferred onto PVDF membranes. Membranes were blocked with blocking buffer (room temperature) and then incubated overnight at 4 °C with primary antibodies (1° Ab):
  • Anti-His tag (mouse) → for detecting His-tagged proteins.
  • Anti-pSTAT3 (Y705, rabbit) → for detecting phosphorylated STAT3 at tyrosine 705.
• On Aug 26, After washing, membranes were incubated with secondary antibodies (2° Ab):
  • Anti-mouse HRP → detecting His-tag primary antibody.
  • Anti-rabbit HRP → detecting pSTAT3 primary antibody.

  HRP conjugates enabled chemiluminescent detection of target proteins.

• On Aug 27,we performed a second Western blot targeting STAT3 and GAPDH to validate downstream signaling effects and assess protein loading, respectively. Additionally, to prepare for downstream transfection or stimulation assays, we also replaced the culture medium of previously seeded HEK293T cells with Opti-MEM (serum-reduced medium), allowing better compatibility with transfection reagents and minimizing serum interference.

  

• On Aug 29, we collected supernatant and lysate of HEK239T. Samples were concentrated and prepared for Western blotting.

  Table 4. Cell lysate samples (target 110 µg/eppendorf)

  Sample	Conc. (µg/µL)	Lysate (µL)	NETN (µL)	6× dye (µL)
  NCV	11.1133	9.90	56.1	17
  anti-IL-23 1-1	10.96552	10.03	56.0	17
  anti-IL-23 2-2	12.0000	9.17	56.8	17
  anti-IL-23 3-2	11.01478	9.99	56.0	17
  anti-IL-23 4-2	12.54187	8.77	57.2	17
  anti-IL-23 5-2	10.96552	10.03	56.0	17

  ---

  Table 5. Concentrated supernatant samples (target 35 µg/eppendorf)

  Sample	Conc. (µg/µL)	Sup. (µL)	NETN (µL)	6× dye (µL)
  NCV	3.625616	9.65	18.35	7
  anti-IL-23 1-1	3.773399	9.28	18.72	7
  anti-IL-23 2-2	4.26601	8.20	19.80	7
  anti-IL-23 3-2	3.625616	9.65	18.35	7
  anti-IL-23 4-2	4.70936	7.43	20.57	7
  anti-IL-23 5-2	3.330049	10.51	17.49	7

Sep 1 – Sep 7 (Week 36)

This week we focus on the continuation and functional verification for our scFv constructs. We completed Western blot analysis for our samples, and HEK293T cells were subcultured and transfected with additional constructs, followed by supernatant and lysate collection for further protein analysis.

• On Sep 1, we ran the SDS-PAGE for the five samples prepared on Aug 29, which included five anti-IL-23 constructs.
• On Sep 2, ELISA plates were coated with 5ng IL23 to evaluate whether our anti-IL-23 scFv could bind to IL-23 subunits. After coating, ELISA was performed to test antibody-antigen interaction. In parallel, we developed Western blot membranes from the August 29 SDS-PAGE. The blot confirmed whether the transfected cells had successfully secreted scFv antibodies into the supernatant.

  

  

• On Sep 3, we performed ELISA detection for plates coated on September 2. After blocking, we added primary antibodies targeting IL-23 and tag sequences, followed by HRP-conjugated secondary antibodies and TMB substrate for color development. Absorbance was measured to evaluate binding of scFv p19 and p40 to IL-23 subunits. Consistently higher OD450 values were observed in column 2 (p40), indicating stronger or more accessible antibody binding. However, inconsistent blank values (H1 = 0.279, H2 = 0.068) suggest variation in background signal, and data interpretation should be made with caution.

  Table 6. ELISA OD450 values validation of the binding capability of anti-IL-23 scFv p19 and p40 collected on Aug 29.

  Supernatant Protein Amount (μg)	p19	p40
  0	0.279	0.068
  0.3125	0.163	0.377
  0.625	0.241	0.479
  1.25	0.237	0.519
  2.5	0.291	0.620
  5	0.473	0.613
  10	0.454	0.590
  20	0.658	0.591

  

  Table 7. BCA assay of total protein concentration in cell lysates.

  Sample name	OD562	conc. (µg/µL)
  NCV	0.279	11.11
  anti-IL23 1-1	0.276	10.97
  anti-IL23 2-2	0.297	12.00
  anti-IL23 3-2	0.277	11.01
  PC-p19 4-2	0.308	12.54
  PC-p40 5-2	0.276	10.97

  Table 8. BCA assay of total protein concentration in culture supernatant.

  Sample name	OD562	Conc. (µg/µL)
  NCV	0.127	3.63
  anti-IL23 1-1	0.130	3.77
  anti-IL23 2-2	0.140	4.27
  anti-IL23 3-2	0.127	3.63
  PC-p19 4-2	0.149	4.71
  PC-p40 5-2	0.121	3.33

• On Sep 4, HEK293T cells were subcultured to expand stocks and prepare conditioned medium for subsequent treatment of Caco‑2 cells. In parallel, ELISA plates were coated with 5ng IL23 to valid the binding capability of anti‑IL‑23 scFv 1‑1, p19 and p40 collected from culture supernatant, then incubated at 4 °C overnight for the next day’s detection.
• On Sep 5, we performed ELISA detection for plates coated on September 4. After blocking, we added primary antibodies targeting IL-23 and tag sequences, followed by HRP-conjugated secondary antibodies and TMB substrate for color development. Absorbance was measured to evaluate binding of scFv 1-1, p19 and p40 to IL-23 subunits.

  The signals do not decrease monotonically down the columns as expected for a serial dilution. This indicates a technical issue rather than true binding behavior. Because the concentration trend is inconsistent, these OD values are not reliable for quantitative comparison. We will repeat the assay with the corrected workflow and document blank‑subtracted, monotonic standard curves before interpreting sample binding.

  In parallel, we transfected HEK293T cells with new constructs, which will later be used to treat Caco-2 cells in downstream functional assays.

  Table 9. ELISA OD450 values for validating the binding capability of scFvs collected from culture supernatants on Aug 29.

  Supernatant Protein Amount (μg)	anti-IL23 scFv p19 sup	anti-IL23 scFv p40 sup	anti-IL23 scFv 1-1 sup
  20	0.835	0.708	0.725
  10	0.464	0.681	0.545
  5	0.487	0.742	0.715
  2.5	0.463	0.853	0.314
  1.25	0.704	0.676	0.109
  0.625	0.285	1.230	0.386
  0.3125	0.260	1.102	0.118
  0	0.113	0.778	0.105

  

• On Sep 6, we replaced the culture medium of HEK293T cells transfected on September 5. The transfection was intended to produce scFv antibodies for subsequent Caco-2 treatment and analysis.The old medium was carefully aspirated, and replaced with serum-reduced medium (Opti-MEM) to support continued protein expression.Cells were incubated at 37 °C, 5% CO₂ for a total of 72 hours, allowing sufficient time for antibody secretion into the supernatant, which will be harvested for downstream Western blot and ELISA assays.

Sep 8 – Sep 14 (Week 37)

This week focused on downstream functional assays for anti-IL-23 scFv constructs. We harvested supernatants and lysates from transfected HEK293T cells and continued protein detection via Western blot. In parallel, E. coli DH5α was cultured for plasmid amplification. On September 12, we treated Caco-2 cells with IL-23 and scFv samples to evaluate downstream signaling. We then collected cells for Western blot analysis of pSTAT3 and JAK expression. ELISA coating and detection were also carried out to monitor binding activity.

• On Sep 8, supernatants were harvested from HEK293T cells transfected with five different anti-IL-23 scFv constructs (#1–p40), as well as a negative control vector (NCV). All samples were rapidly frozen in liquid nitrogen and stored for future use. A BCA assay was performed to quantify total protein concentrations.Samples were diluted and mixed with loading buffer according to calculated volumes to normalize total protein input to 15 µg per lane for SDS-PAGE. A new gel was cast for downstream Western blot analysis.

  Table 10. BCA assay and sample loading volumes for concentrated HEK293T supernatants (target 15 µg antibody per lane in WB)

  Sample	OD562	Conc. (µg/µL)	Volume of sample (µL)	PBS (µg)	6× dye (µL)
  NCV	0.118	1.78	8.43	3.57	3
  anti-IL23 scFv 1	0.126	2.06	7.27	4.73	3
  anti-IL23 scFv 2	0.114	1.64	9.16	2.84	3
  anti-IL23 scFv 3	0.125	2.03	7.39	4.61	3
  anti-IL23 scFv p19	0.132	2.28	6.59	5.41	3
  anti-IL23 scFv p40	0.123	1.96	7.66	4.34	3

  Table 11. BCA assay for cell lysates.

  Sample	OD562	Conc. (µg/µL)
  NCV	0.467	15.107
  anti-IL23 scFv 1	0.440	14.055
  anti-IL23 scFv 2	0.420	13.276
  anti-IL23 scFv 3	0.432	13.744
  anti-IL23 scFv 4	0.499	16.354
  anti-IL23 scFv 5	0.426	13.510

• On Sep 9, we proceeded with the western blot. After protein transfer to PVDF membranes, we incubated the membrane overnight at 4 °C with the primary antibody (mouse anti-His tag).
• On Sep 10, we performed several parallel tasks:
  1. 1. Western blot development: After overnight incubation with primary antibody, the membrane was washed and incubated with HRP-conjugated secondary antibody. A chemiluminescent substrate was added, and the membrane was imaged to observe scFv expression and secretion patterns.
  2. 2. Caco-2 cell maintenance: We found the Caco-2 cell will grow better after changing the medium. So we changed the medium for the Caco-2 culture. Cell density was examined under the microscope to assess readiness for treatment.
  3. 3. E. coli DH5α culture: We inoculated E. coli DH5α in selective LB medium to propagate plasmids for future transfection or cloning. Overnight shaking culture was started at 37 °C.

  

• On Sep 11, we performed plasmid extraction (miniprep) from overnight E. coli DH5α cultures grown on September 10. Plasmids were purified using a commercial miniprep kit according to standard protocol. After extraction, we measured DNA concentration and purity using Nanodrop spectrophotometry.

  Table 12. Nanodrop result for plasmid 1 to 5, 3 tubes per plasmids.

  Sample	Concentration (ng/µL)	A260/A280	A260/A230	A260	A280
  1-1	347	1.87	2.22	6.94	3.72
  1-2	303.6	1.86	2.26	6.07	3.26
  1-3	265.9	1.86	2.18	5.32	2.87
  2-1	253.6	1.86	2.20	5.07	2.73
  2-2	289.7	1.86	2.20	5.79	3.12
  2-3	240.3	1.86	2.24	4.81	2.58
  3-1	348.5	1.86	2.23	6.97	3.74
  3-2	347.6	1.87	2.26	6.95	3.73
  3-3	366.3	1.86	2.24	7.33	3.93
  4-1	306.3	1.86	2.17	6.13	3.30
  4-2	224.2	1.85	2.14	4.48	2.43
  4-3	282.2	1.86	2.20	5.64	3.04
  5-1	275.1	1.86	2.20	5.50	2.97
  5-2	320.8	1.86	2.24	6.42	3.45
  5-3	339.8	1.86	2.26	6.80	3.65

• On Sep 12, we stimulated HEK293T cells with recombinant IL-23 and simultaneously treated them with anti-IL-23 scFv antibodies collected on August 29 for 72 hours. These scFv constructs included multiple prototypes previously verified through Western blot and ELISA. The goal of this experiment was to evaluate the functional activity of the antibodies in neutralizing IL-23–mediated signaling. By mimicking an inflammatory environment with IL-23 stimulation, we aimed to observe downstream changes.

Sep 15 - Sep 21 (week 38)

This week focused on functional validation of anti-IL-23 scFv antibodies through IL-23 stimulation, Western blot analysis of downstream signaling (e.g., STAT3, NF-κB, JAK2), and ELISA-based detection.

• On Sep 15, HEK293T cells previously treated with IL-23 and scFv antibodies were harvested for Western blot analysis. Protein lysates were prepared and gels were cast for SDS-PAGE.
• On Sep 16, To investigate the downstream signaling response to IL-23 stimulation, the Western blot membrane from the previous protein extraction was sectioned into three parts and probed for inflammatory pathway targets.

  Table 13. The Western blot membrane was cut into three sections to probe multiple targets in parallel. Primary antibodies included anti-pSTAT3, NF-κB p65, JAK2, p-NF-κB, STAT3, CDK6, and GAPDH. Each blot was incubated with 1° antibodies (1:1000) and appropriate HRP-conjugated 2° antibodies (1:10,000) for detection.

  Sample	1°Ab (1:1000)	2°Ab (1:10000)
  1-1	p-STAT3	HRP-Rabbit
  1-2	NF-κB (p65)	HRP-Rabbit
  1-3	GAPDH	HRP-Rabbit
  2-1	p-JAK2	HRP-Rabbit
  2-2	p-NF-κB (p-p65)	HRP-Rabbit
  2-3	CDK6	HRP-Rabbit

• On Sep 17, The Western blot was developed and imaged to assess activation of inflammatory signaling pathways in response to IL-23. In parallel, the culture medium was replaced, and internal control antibody staining was performed. On the same day, IL-23 ELISA plates were coated for the next assay.
• On Sep 18, When reviewing the setup, we found that PBS buffer had been mistakenly used instead of the proper coating buffer for the ELISA plate. To correct this issue, new IL-23 ELISA plates were re-coated with the correct buffer. Internal controls for Western blot analysis—including JAK2, STAT3, and GAPDH—were also performed. In addition, new Caco-2 cells were subcultured for upcoming experiments, and SDS-PAGE gels were prepared for a Western blot to evaluate the inflammatory response in cells treated with IL-23 for six hours.

  Table 14. Validation of internal controls (JAK2, STAT3, GAPDH) for Western Blot normalization.

  Sample	1°Ab (1:1000)	2°Ab (1:10000)
  1-1	JAK2	HRP-Rabbit
  2-1	STAT3	HRP-Rabbit
  2-3	GAPDH	HRP-Rabbit

  

• On Sep 19, we treated Caco-2 cells with 20 μg and 40 μg of IL-23 for 6 hours, respectively, to determine the optimal inflammatory response time. Additionally, we performed ELISA on the supernatant collected on Sep 8 to achieve an ideal OD450 reading. However, the ELISA results did not align with our expectations. The anti-IL-23 scFv #1 showed an inconsistent OD pattern, while no significant signal was detected for the anti-IL-23 scFv p19 or p40. This outcome was unexpected, as previous assays have confirmed that both p19 and p40 possess IL-23 binding capability. Upon review, we identified a technical issue in this ELISA run: the HRP-conjugated secondary antibody for rabbit IgG was mistakenly added instead of the HRP–anti-mouse antibody. The error was promptly corrected by washing the plate and re-adding the proper HRP–anti-mouse antibody. Given this procedural error, the current results are not considered reliable, and the assay will be repeated to validate the findings.

  Table 15. ELISA quantitative assessment OD450 values of scFv binding activity, collected from culture supernatant on Sep 8.

  Supernatant Protein Amount (μg)	1	P19	P40
  0	0.081	0.091	0.096
  0.3125	-	0.083	0.111
  0.625	0.099	0.096	0.146
  1.25	0.116	0.091	0.117
  2.5	0.144	0.092	0.14
  5	0.593	0.097	0.153
  10	0.091	-	-

  

• On Sep 21, we coated IL-23 ELISA plates to test the cell lysate collected on Sep 8.

Sep 22 - Sep 26 (week 39)

This week, we focused on determining the optimal inflammatory response time of Caco-2 cells and aimed to obtain ideal ELISA OD450 readings.

• On Sep 22, we performed a western blot to assess the inflammatory response of Caco-2 cells treated with IL-23 for 6 hours. Additionally, we measured the OD450 values via ELISA using cell lysate collected on Sep 8. The ELISA performed with the Sep 8 lysate demonstrated a consistent dose-dependent curve, particularly for p19 and p40, suggesting effective dilution and binding. Based on this result, we will re-evaluate the Aug 29 lysate under the same conditions to confirm antibody performance and reproducibility.

  Table 16. Second ELISA quantitative assessment OD450 values of scFv binding activity, collected from culture supernatant on Sep 8.

  Supernatant Protein Amount (μg)	#1	p19	p40	NCV	#2	#3
  0	0.086	0.088	0.087	-	-	-
  1.25	0.083	0.209	0.787	-	-	-
  2.5	0.084	0.275	0.746	-	-	-
  5	0.085	0.341	0.819	-	-	-
  10	0.092	0.556	0.985	0.084	0.081	0.086
  20	0.089	0.693	1.188	0.096	0.084	0.086

  

• On Sep 23, we reviewed the Western blot results to assess the inflammatory response of Caco-2 cells treated with IL-23 for 6 hours. Additionally, we subcultured Caco-2 cells for upcoming experiments and coated a new ELISA plate to analyze the lysate collected on Aug 29.

  Table 17. The Western blot membrane was cut into three sections to probe multiple targets in parallel. Primary antibodies included anti-pSTAT3, NF-κB p65, JAK2, p-NF-κB, STAT3, CDK6, and GAPDH. Each blot was incubated with 1° antibodies (1:1000) and appropriate HRP-conjugated 2° antibodies (1:10,000) for detection.

  Sample	1°Ab (1:1000)	2°Ab (1:10000)
  1-1	p-STAT3	HRP-Rabbit
  1-2	NF-κB (p65)	HRP-Rabbit
  1-3	GAPDH	HRP-Rabbit
  2-1	p-JAK2	HRP-Rabbit
  2-2	p-NF-κB (p-p65)	HRP-Rabbit
  2-3	GAPDH	HRP-Rabbit

  

• On Sep 24, we treated Caco-2 cells with IL-23 for 15 minutes, 30 minutes, 1 hour, and 3 hours, respectively, to determine the optimal time point for inflammatory response. Additionally, we performed ELISA and recorded the OD450 values using the cell lysate collected on Aug 29. And we prepared a SDS-PAGE gel for western blot tomorrow.

  Table 18. OD450 value from ELISA using lysate collected on Aug 29.

  Supernatant Protein Amount (μg)	NCV	#1	#2	#3	p19	p40
  0	0.097	0.084	0.142	0.093	0.092	0.105
  6.25	0.118	0.102	0.100	0.103	0.286	0.476
  12.5	0.150	0.122	0.100	0.121	0.357	0.601
  25	0.127	0.122	0.110	0.146	0.483	0.882
  50	0.154	0.152	0.144	0.156	0.819	1.115

  

• On Sep 25, we ran SDS-PAGE for Western blot analysis.

  Table 19. The Western blot membrane was cut into three sections to probe multiple targets in parallel. Primary antibodies included anti-pSTAT3, NF-κB , p-JAK2, p-NF-κB, β actin, and GAPDH. Each blot was incubated with 1° antibodies (1:1000) and appropriate HRP-conjugated 2° antibodies (1:10,000) for detection.

  Sample	1°Ab (1:1000)	2°Ab (1:10000)
  1-1	p-STAT3	HRP-Rabbit
  1-2	NFKB	HRP-Rabbit
  1-3	GAPDH	HRP-Rabbit
  2-1	p-JAK2	HRP-Rabbit
  2-2	p-NFKB	HRP-Rabbit
  2-3	β actin	HRP-Rabbit

• On Sep 26, we visualized and analyzed the Western blot membranes to assess the expression of inflammatory markers.

  

Sep 29 - Oct 5 (week 40)

In this week, we focused on evaluating the inflammatory response of Caco-2 cells under IL-23 stimulation and assessing the functional effects of anti-IL-23 scFv candidates.

• On Sep 29, Caco-2 cells were subcultured in preparation for IL-23 stimulation and downstream analysis. In addition, ELISA plates were coated with the IL-23 capture antibody in preparation for analyzing the supernatant collected on Aug 29.
• On Sep 30, Cells were switched to serum-free DMEM and stimulated with IL-23 for 30 minutes. Anti-IL-23 scFvs were added for functional testing at the same time. After treatment, cell lysates were collected for Western blot. In parellel, an ELISA was performed using the supernatant we collected on Aug 29. SDS-PAGE gels were prepared for the following day's protein separation.

  Table 20. OD450 value from ELISA using supernatant collected on Aug 29.

  Supernatant Protein Amount (μg)	NCV	#1	#2	#3	#19	#40
  0	0.092	0.075	-	-	0.077	0.078
  0.625	-	-	-	-	-	0.536
  1.25	0.095	0.161	-	-	0.238	0.492
  2.5	0.085	0.11	-	-	0.304	0.763
  5	0.129	0.077	0.079	0.075	0.279	0.844
  10	0.09	0.088	0.083	0.077	0.366	1.1
  20	0.08	0.126	0.099	0.076	0.59	-

  

• On Oct 1, SDS-PAGE was performed to separate proteins from the treated lysates. Proteins were transferred to membranes, which were incubated with primary antibodies targeting key inflammatory markers.

  Table 21. The Western blot membrane was cut into three sections to probe multiple targets in parallel. Primary antibodies included anti-pSTAT3, NF-κB , p-JAK2, p-NF-κB, and GAPDH. Each blot was incubated with 1° antibodies (1:1000) and appropriate HRP-conjugated 2° antibodies (1:10,000) for detection.

  Sample	1° Antibody (1°Ab)	2° Antibody (2°Ab)
  1-1	p-STAT3	HRP-Rabbit (1:5000)
  1-2	NFKB	HRP-Rabbit
  1-3	GAPDH	HRP-Rabbit
  2-1	p-JAK2	HRP-Rabbit
  2-2	p-NFKB	HRP-Rabbit (1:5000)
  2-3	GAPDH	HRP-Rabbit

• On Oct 2, Membranes were incubated with HRP-conjugated secondary antibodies and visualized by chemiluminescence. Western blot results were analyzed to assess inflammation-related signaling changes under IL-23 and scFv treatment.

  

• On Oct 4, ELISA was performed to assess the binding performance of anti-IL-23 scFv #1-3 that were produced and purified by a biotechnology company using HEK293 cells.(Note: our lab typically uses HEK293T for scFv expression.) And we used p19 which was collected from culture supernatant on Sep 2 in contrast with these three prototypes. This test was conducted to verify the functional activity of the externally sourced and purified scFvs.

  Table 22. OD450 value from ELISA using scFv produced by biotechnology company.

  	p19	#1	#2	#3
  0	0.066	0.069	0.073	0.073
  0.15625	0.105	0.077	0.106	-
  0.3125	0.107	0.082	0.074	-
  0.625	0.122	0.073	0.077	-
  1.25	0.159	0.078	0.072	-
  2.5	0.201	0.093	0.077	-
  5	0.274	0.098	0.085	-
  10	0.484	0.106	0.111	-
  0.051563	-	-	-	0.078
  0.051563	-	-	-	0.078
  0.103125	-	-	-	0.075
  0.20625	-	-	-	0.069
  0.4125	-	-	-	0.071
  0.825	-	-	-	0.075
  1.65	-	-	-	0.078
  3.3	-	-	-	0.088