Lewin Bio Submits IPO Prospectus Highlighting Ultrasensitive iPCR-Based IL-33 Biomarker Detection Platform Validated by Genentech
Genentech
Pharmaceutical R&D Manufacturer
Company website:www.lewinbio.com
In drug development and clinical trials, capturing rare, low-abundance biomarkers in extremely small sample sizes has always been a bottleneck for detection technologies. Recently, the Genentech team published a new study in the Journal of Pharmaceutical and Biomedical Analysis: they optimized and validated aImmuno-PCR (iPCR) Ultra-Sensitive Detection Method, which can be used to detect the activity (reduced form) of IL-33 in human serum, plasma, and aqueous humor.
Clinical Significance of IL-33
IL-33Is an important cytokine in inflammation, infection, and ophthalmic diseases.(such as AMD, Age-related Macular Degeneration)and play a key role. It induces Th2 cells to produce type 2 cytokines by binding to the cell surface receptors ST2 and IL-1, triggering pro-inflammatory responses. In clinical studies, active IL-33(Reduced form)The concentration is extremely low (often below pg/mL), and soluble ST2 may be present in the sample.（sST2）Interference Detection. Especially in ophthalmic research, the sample volume of aqueous humor is extremely small (usually requiring high dilution for testing),Traditional ELISA has limited sensitivity and is difficult to detect in rare samples such as aqueous humor.。
Although the ELISA method previously developed by the team can tolerate sST2 interference, the lower limit of detection（LLOQ）At merely 8.2 pg/mL, IL-33 in the aqueous humor could not be detected — this is precisely the core motivation for developing the iPCR method.
Technological Breakthrough:
Immuno-PCR（iPCR）Breakthrough Detection Limits
The core advantage of iPCR lies in "powerful synergy": combining the specificity of antibodies with the signal amplification capability of PCR, replacing the "enzymatic optical signal" in traditional ELISA with a "DNA amplification signal," achieving a qualitative leap in sensitivity.
This study focuses on the initial iPCR method(For basic research)Nine key optimizations were carried out to meet the stringent requirements of clinical biomarker testing, with the core improvements and effects as follows:
Optimization Direction | Specific Adjustments | Core Effects |
Detection Range | Standard Curve from 156-40,000 fg/mL extended to 61-250,000fg/mL | Dynamic range increased 6.25 times, covering a wider concentration range of clinical samples. |
SampleStability | Add 100 μg/mL anti-IL-33 monoclonal antibody to the diluent. | Prevent IL-33 oxidation and maintain its active state |
Anti-pollution capability | Replace with centrifugal Blue®Plate Washer, Number of washes from 13 times Increased to 33 times | Reduce Cross-Contamination, Improve Repeated Testing Accuracy |
Background Control | Reduce the concentration of biotin conjugates (200→50 ng/mL) and purify oligonucleotide-antibody conjugates using SEC. | Reduce background Ct value, Reduce False Negatives |
Incubation Time | Streptavidin plate incubation time from 20min Extended to 60 minutes | Improve Detection Reproducibility |
Clinical-Grade Validation: Reliability of the iPCR Method
The research team validated the reliability of the method from five key dimensions based on clinical biomarker validation guidelines such as "Crystal City V/VI" (data based on seven independent experiments):
Accuracy and Precision
· Inter-batch Coefficient of Variation for Reference Standards（%CV）10%-22%, Quality Control Samples(High / Medium / Low Concentration + Endogenous Control)The %CV is 12%-19%;
· Total Error（%RE+%CV）All within acceptable limits, no significant deviations.
Lower Limit of Quantification (LLOQ) and Upper Limit of Quantification (ULOQ)
· After dilution correction, the LLOQ was 977 fg/mL.（≈1pg/mL）, ULOQ is 1,000,000 fg/mL（1ng/mL）；
· Can accurately detect reduced IL-33 at the "zeptogram/milliliter" level, with a sensitivity more than 8 times higher than the first-generation ELISA.
Sample Suitability
· Serum/Plasma Samples: IL-33 Detection Rate Reaches 97%-100%(Covering healthy individuals and patients)；
· Aqueous humor samples: IL-33 was detected in only 1/20 human aqueous humor samples and 0/5 New Zealand rabbit aqueous humor samples.(Note: The concentration of IL-33 in aqueous humor is extremely low and may be easily oxidized.)。
Parallelism and Repetition
· Parallelism: 10 serum samples from disease patients(1 case of AMD, 4 cases of asthma, 5 cases of rheumatoid arthritis)At dilutions of 1:4 and 1:8, 90% of the samples showed %CV ≤ 30%, demonstrating consistent detection response between endogenous IL-33 and the recombinant standard.
· Repeatability: 8 cases of chronic obstructive pulmonary disease（COPD）Patient plasma samples were tested by two analysts within two days, with all samples showing %RE ≤ 30% and excellent inter-batch stability.
Clinical Application: What Can It Bring to Drug Development?
Directly Support Eye Disease Research
The team used this method to detect a Phase 1 clinical study.(NCT04615325, Evaluation of the Safety/Pharmacokinetics of Anti-IL-33 Fab in the Treatment of AMD)Although IL-33 was not detected in the aqueous humor samples, it provided a tool for subsequent "low-dose drug efficacy monitoring" — after all, the ability to "precisely detect 'very low concentrations'" is itself a clinical need.
Wide Applicability
The design concept of this method can be extended to the detection of other low-concentration cytokines, and is particularly suitable for:
·Matrix with extremely small sample size (e.g., cerebrospinal fluid, aqueous humor);
·Low-abundance inflammatory factors(such as IL-4, IL-13, etc.)；
· Clinical Studies Requiring Ultra-Sensitive Detection(Such as early disease diagnosis, drug dosage optimization)。
Original link: https://doi.org/10.1016/j.jpba.2025.117095
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