Overview

Our project addresses the critical need for enhanced risk screening and early detection of gynecological diseases. Early diagnosis significantly improves treatment success rates and quality of life after treatments. An array of biomarkers should be studied and included to tackle this challenge, and as a proof-of-concept, we concentrated on two biomarkers, CA125 and IL-6, which are associated with various gynecological diseases. They are intended to be used in biosensors that utilize the antigen-antibody binding mechanism, employing colloidal gold and lateral flow assay (LFA) technology for a visual read-out. We believe this approach will lead to portable, non-invasive, and thus user-friendly gynecological care products.

To learn more about the project background, please refer to our Description [https://2024.igem.wiki/bwya/description ] wiki page.

Design

Biomarker Selection

CA125 and IL-6 were chosen based on their diagnostic relevance and the availability of research supporting their use. CA125 and IL-6 are proteins that, when present in abnormal concentrations, can indicate the presence of disease.

CA125, in particular, is mostly known as an indicator of ovarian cancer, while at the same time, its concentration shows abnormal elevations in many gynecological diseases, such as Endometriosis, Endometrial cancer, Adenomyosis, and etc. The diseases indicated by CA125 covers a wide range of population group.

IL-6 is a common inflammation marker. In fact, gynecological inflammation is significantly more common than cancers in female patients. Such inflammations and infections are also hard to cure and often transform into chronicle conditions, posing a long-term burden on females. Besides, IL-6 is not only associated with inflammations but also indicative of various other conditions, including some cancers.

Detection

We plan to use the lateral flow assay method for a visual read-out upon antigen detection. This is a well-established technique mostly used in point-of-care diagnostics. It is known for its simplicity, requiring only a small sample and providing results within minutes.

Figure. Principle of lateral flow assay test strips. Referenced from Aydin et al, 2020.

The antigen-antibody binding principle is at the heart of our detection method. When the target biomarkers, such as CA125 and IL-6, are present in a sample, they bind to specific antibodies, triggering a visual signal via colloidal gold particles.

Antibody Selection

We selected specific CA125 and IL-6 antibodies from the literature that have demonstrated high affinity and specificity. These antibodies are crucial for the effects of our diagnostic tool.

For CA125 detection, we selected humanized 4H11 (h4H11) antibody against the specific CA125 ectodomain. The ability of the 4H11 to recognize CA125 was proven experimentally by previous works in Fluorescence Activated Cell Sorting (FACs),

CAR-T cell application, and blocking of matrigel invasion [MUC-2024] .

For IL-6 detections, in a newly published work, Biozzi mice were immunized with IL-6 emulsified in sodium chloride and Freund's adjuvant, followed by booster injections, and a final intraperitoneal boost. Splenocytes were harvested and fused with mouse myeloma cells to create hybridomas, which were screened by ELISA. Positive clones were cloned and injected into mice for ascites production. [Celik-2023]

scFv Design

Producing full-length antibodies using biomedical methods usually involve animal agents, which incurred animal welfare concerns and raising production cost. To address this challenge, we opted for single-chain variable fragment (scFv) antibodies. These scFv antibodies are minimal antigen-binding fragments, approximately 25 kDa, and they are composed of the variable domains from both the heavy (VH) and light (VL) chains bridged by a linking sequence. These fragments offer multiple benefits over their full-length monoclonal counterparts. The scFv antibodies can be expressed in simple eukaryotic organisms like yeast and prokaryotic bacteria due to their simplified architecture, and they are not dependent on glycosylation for function. In our experiments, we aim to express the scFv in E. coli chassis.

Fig. This scheme illustrates the principles of scFv design from an antibody, referenced from Monnier et al, 2013.

Build

Expression Optimization

To enhance the expression of our scFv antibodies, we employed various strategies, including codon optimization for E. coli expression. The antibody originates from mammalian protein productions, and codons corresponding to the designed amino acids do not reflect the best expression in E. coli. We used the online tool TI signer (https://tisigner.com/tisigner) to optimize the coding DNA.

We also composite the coding sequence with a strong T7 promoter. It is derived from the T7 bacteriophage and is recognized by the T7 RNA polymerase, which transcribes the gene at a much faster rate than the host cell's polymerase. Aided with a lac operator (LacO), the expression is muted without external induction of iPTG, which allows the E. coli to grow to the best condition of the exponential phase. Upon induced expression, T7 promoter can facilitate 5 times heterogenous expression than generic proteins in E. coli. We chose the BL21 strain of E. coli for its robust expression capabilities. The pET28a expression vector was selected for its efficiency in producing recombinant proteins under the control of the T7 promoter.

Test

Fermentation Conditions

We selected fermentation conditions based on literature empirical conditions, including temperature (20 C), media (common LB media) and induction timing (OD600 0.6-0.8), and induction strength (0.3 mM). These mild conditions allow more time so that the protein can properly fold into the desired structures.

SDS-PAGE Analysis

SDS-PAGE was used to confirm the successful expression of the scFv antibodies. The gel electrophoresis results provided visual evidence of protein expression levels and purity. The anti-IL-6 scFv is anticipated to be 27.09 kDa, and the anti-CA125 scFv is anticipated to be 28.38 kDa.

Protein Purification

We purified the protein using Tris-HCl buffer, guanidine hydrochloride and imidazole denaturing solution, as well as a column packed with nickel resin that resists denaturation. The fermented bacteria were resuspended, sonicated, and the supernatant and precipitation were separated, with the precipitation being redissolved. Samples of both the supernatant (sup. ) and the precipitation (prec.) were taken for analysis.

Learn

The SDS-Page results from our TEST showed the successful expression of the designed scFv. One of the main challenges we faced was the production of insoluble proteins. This is often the case when the protein originates from mammalian origins. The lack of complex cell functions in E. coli undermines the synthesis and folding of these proteins. There exist various strategies to improve solubility, including changes in expression conditions and the choice of new expression chassis.

To further enhance protein solubility and expression, we considered using the Shuffle T7 strain, which is designed to improve the solubility of recombinant proteins. BL21 and Shuffle T7 are both E. coli strains used for recombinant protein expression, but they have distinct features. BL21 is suitable for general protein expression, especially those without disulfide bonds. In contrast, Shuffle T7 is genetically engineered for efficient disulfide bond formation in the cytoplasm, making it ideal for proteins requiring proper folding. It harbors mutations in trxB and gor, essential for a redox environment conducive to disulfide bond formation, and expresses DsbC, which aids in correct folding. Additionally, Shuffle T7 has a chromosomal copy of T7 RNAP for high-level expression and lacks lon and ompT proteases, reducing proteolytic degradation. In summary, for proteins that need correct disulfide formation, Shuffle T7 is preferred, while BL21 is fit for easily soluble proteins.

Solubilisation of Inclusion Bodies

The previous experimental results showed that after the production of anti-IL-6 and anti-CA125 scFv, a lot of proteins existed in the form of inclusion bodies. In order to solubilize the protein within the inclusion bodies, we attempted purification with non-denaturing agents.

Experiment procedure:
1、Collect cell pellets after bacteria culture fermentation, resuspend the pellets with 20 mM Tris-Cl, and lysis with ultrasonication;
2、Separate supernatant and precipitations, and dissolve the precipitations with non-denaturing agents.
3、Pass the above re-dissolved solution through Ni column and wash with 5 column volume.
4. Elute with 2 to 3 column volumes of elution buffer.
5. Collect the protein eluate and concentrate it.

Non-denaturing solvent/wash buffer:
1% (w/v) N-lauroyl sarcosine (NLS) (Celik et al., 2023) ， 300 mM NaCl ，50 mM Tris-Cl ，pH 8.0

Elution Buffer：
0.1% (w/v) NLS ，300 mM NaCl, 50 mM Tris-Cl ，300 mM Imidazole ，pH 8.0

The SDS-Page run after purification and elution shows a clear band for the anti-CA125 scFv and anti-IL-6 scFv, proving they can be properly solubilized.

We estimated the production quantity of anti-IL-6 scFv to be 10mg/L (0.5 mg protein from 50 mL bacteria culture) and anti-CA125 scFv to be 4 mg/L (0.2 mg protein from 50 mL culture).

Binding Interaction Verification

To verify whether the designed scFv antibodies can bind to their corresponding antigens, we used a simpler method, native PAGE, instead of more complex techniques such as ELISA or co-immunoprecipitation. We incubated the purified scFv antibodies with the purchased IL-6 and CA125 antigen proteins at room temperature for 2 hours and then performed native-PAGE under non-denaturing conditions.

In the native PAGE results, it is evident that the position of the CA125 protein bands shifted after incubation of the CA125 antigen with our anti-CA125 scFv. The CA125 antigen protein has an original size of approximately 80 kDa. Due to native PAGE being a non-denaturing electrophoresis, the protein is not linearized and appears at a position slightly higher than its expected size.

The anti-CA125 scFv, on the other hand, is a much smaller protein, with a size of about 28 kDa. We believe that due to the binding between the two, the CA125 antigen protein was pulled down by the smaller scFv, causing the shift in band position. Therefore, we infer that our designed scFv can effectively bind to the target antigen.

On the other hand, the results with IL-6 are nonconclusive. The IL-6 antigen protein we purchased was reported to be around 20 kDa. The antigen protein showed a consistent band on the PAGE results. However, after inoculation with the scFv antibodies, the resulting bands showed no discernible shifts. It could be that the scFv antibody does not have a high enough affinity for the antigen, or due to inappropriate electrophoresis conditions such as pH values. The conditions can affect protein migration and interactions. It invites us to further study and characterize the scFv antibodies.

References

[MUC-2024] Lee, K., Perry, K., Xu, M. et al. Structural basis for antibody recognition of the proximal MUC16 ectodomain. J Ovarian Res 17, 41 (2024).

[Celik-2023] Şahinbaş, D., & Çelik, E. (2023). Enhanced production and single-step purification of biologically active recombinant anti-IL6 scFv from Escherichia coli inclusion bodies. Process Biochemistry, 133, 151-157.

[scFv-1] Monnier PP, Vigouroux RJ, Tassew NG. In Vivo Applications of Single Chain Fv (Variable Domain) (scFv) Fragments. Antibodies. 2013; 2(2):193-208.

[Liu-2017] Liu D., Zhang L., Indima N., Peng K., Li Q., Hua T., Tang G. CT and MRI findings of type I and type II epithelial ovarian cancer. Eur. J. Radiol. 2017;90:225–233.

[Karimi] Karimi-Zarchi M, Dehshiri-Zadeh N, Sekhavat L, Nosouhi F. Correlation of CA-125 serum level and clinico-pathological characteristic of patients with endometriosis. Int J Reprod Biomed. 2016 Nov;14(11):713-718.

[Zou-2006] Zou, S., & Yu, H. (2006). Clinical application of serum CA-125 level detection in gynecological diseases. Medical Clinical Research, 23(2), 151-152

[Zhang-2012] Zhang, D., Hu, Y., Zhang, D., Xu, K., & Lu, J. (2012). Variations in serum CA125, CA15-3, and CA19-9 levels during the menstrual cycle in women of reproductive age. Journal of Radioimmunology, (6), 690-690.

[Munezz-2020] Munezza AK and Mohammad M. Recent Advances in Electrochemical and Optical Biosensors Designed for Detection of Interleukin 6. Sensors 2020, 20, 646.

[Bellone-2005] Bellone S, Watts K, Cane' S, Palmieri M, Cannon MJ, Burnett A, Roman JJ, Pecorelli S, Santin AD. High serum levels of interleukin-6 in endometrial carcinoma are associated with uterine serous papillary histology, a highly aggressive and chemotherapy-resistant variant of endometrial cancer. Gynecol Oncol. 2005 Jul;98(1):92-8.

[Aydin-2020] Aydın, Beyza & Kul, Yaren. (2020). DESIGN OF ANTIGEN DETECTING IMMUNOCHROMATOGRAPHIC RAPID TEST KIT AGAINST SPIKE GLYCOPROTEIN OF SARS-CoV-2. 10.13140/RG.2.2.16968.96001.]