Detection Method

ELISA

Assay Time

Approximately 3 hours (primary incubation) + 15 minutes substrate incubation

Storage Temperature

2-8 °C (kit); Anti-E.coli HCP-E Microtiter Strips 2-8 °C (stable for 60 days); Reconstituted Calibration Standard 2-8 °C (stable for 30 days)

Product Description

This product is a one-step, solid-phase, double-antibody sandwich Enzyme-Linked Immunosorbent Assay (ELISA) intended to detect residual host cell proteins (HCPs) from E.coli protein expression strains (originated from BL21) in bioprocess samples. Polyclonal capture antibody (sheep anti-E.coli HCP affinity antibody) is pre-coated on microtiter strips. Calibration standards and samples are simultaneously incubated with HRP-conjugated anti-E.coli HCP detection antibody (one-step format). HRP catalyzes TMB oxidation to produce a colorimetric signal measured at 450 nm (with long-wavelength correction 620–650 nm). Concentrations are calculated by a 4-parameter logistic (4PL) fit to the calibration curve. The kit is supplied for research use only and is not intended for clinical diagnostic use.

Technical Specifications

Parameter	Details
Format / Throughput	96 tests per kit; 8-well × 12 strips
Detection principle	One-step double-antibody sandwich ELISA with HRP/TMB colorimetric detection (read at 450 nm, reference 620–650 nm)
Linearity & Range	1.5–243 ng/mL, R2 > 0.990
Lower Limit of Quantitation (LLOQ)	1.5 ng/mL
Calibration curve model	4-parameter logistic (4PL); example parameters A=2.41626, B=1.09106, C=89.16833, D=0.01118, R2=0.99995 (example data)
Specificity / Cross-reactivity	No cross-reactivity observed with CHO, Vero, HEK293T, and Hansenula polymorpha HCPs (as tested)
Assay incubation conditions	Add 100 µL HRP detection reagent + 100 µL sample/standard per well; incubate on microplate thermoshaker at 600 rpm for 3 hours at room temperature; wash; add 100 µL TMB and incubate 15 minutes at room temperature in dark; stop and read
Reading	Absorbance measured at 450 nm with correction wavelength set at 620–650 nm; subtract NCS (blank) and use 4PL fit
Recommended sample pH	pH 6.5–8.5 (measurements may be compromised outside this range)
Minimum recommended pipetting volume for dilutions	Minimum volume 5 µL recommended for accurate dilutions
Reading window after stop	Complete plate reading within 30 minutes after termination

Features

One-step ELISA format: Simultaneous addition of calibration standard/sample and HRP-conjugated detection antibody reduces hands-on time and simplifies workflow.
High sensitivity: LLOQ of 1.5 ng/mL with a linear range up to 243 ng/mL.
Strain-targeted specificity: Polyclonal antibodies generated against E.coli protein expression strains (BL21 origin) to improve detection of residual HCPs from these expression hosts.
Pre-coated strips: Microtiter strips are pre-coated with capture antibody and supplied in sealed bag with desiccant for convenience and reproducibility.
Validated lack of cross-reactivity: No cross-reactivity observed with CHO, Vero, HEK293T, or Hansenula polymorpha HCPs (as tested).
Complete kit: Includes calibration standard, HRP-conjugated detection antibody, diluent, wash buffer concentrate, substrate, stop solution and sealing films.

Applications

Quantitation of residual host cell proteins (HCPs) in products expressed in E.coli BL21 protein expression strains
In-process samples, harvested bulk, drug substance and drug product testing during downstream processing
Assay development and validation for bioprocess monitoring of E.coli-expressed biologics (e.g., IL, rhIFN, rhGM-CSF, rhTNF, EGF/FGF/PDGF)
Spike/recovery and method validation studies for residual HCP measurement (research use)

Kit Contents

E.coli HCP‑E Calibration Standard (PNB003), lyophilized (2 bottles)Store lyophilized at 2-8 °C; reconstitute with 500 µL Reconstitution Solution; reconstituted standard stable 2-8 °C for up to 30 days (single use recommended)

Anti‑E.coli HCP‑E Microtiter Strips (PNA003) 8‑well × 12 stripsSealed with desiccant at 2-8 °C; store unused strips in bag with desiccant; stable for up to 60 days after opening

Reconstitution Solution (PNC002) 1 × 1.5 mL2-8 °C

Diluent (PNE004) 2 × 25 mL2-8 °C; use for dilution of calibration standards, HRP (100×) and samples

Wash Buffer Concentrate (10×) (PNF001) 2 × 25 mL2-8 °C; dilute 1:10 with ultra-pure water before use; may crystallize at low temps—redissolve at 37 °C if necessary

Anti‑E.coli HCP‑E:HRP (100×) (PNN001) 1 × 120 µL2-8 °C; centrifuge before use; dilute 100× in Diluent to prepare 1× prior to assay; prepare fresh

TMB Substrate (PND002) 1 × 12 mL2-8 °C; equilibrate to room temperature 20 minutes before use; keep protected from light; discard if colored

Stop Solution (PNI002) 1 × 6 mL (1 M HCl)2-8 °C; corrosive—handle with eye/skin protection

Sealing Film (PNK001) 3 piecesRoom temperature; keep clean

* Total: 96 tests/Kit | Method: One-step double-antibody sandwich ELISA (colorimetric HRP/TMB)

Attention

• For research use only (RUO); not intended for clinical diagnostic use.

• Use sterile disposable tips, tubes and reservoirs; recommended to wipe bench and equipment with 75% ethanol before and after use.

• Do not mix reagents from different lot numbers.

• Use fresh ultra‑pure water; water temperature should not exceed 37 °C when preparing wash buffer.

• Reconstituted Calibration Standard and prepared 1× Anti‑E.coli HCP‑E:HRP are recommended for single use due to instability; prepare freshly before each experiment.

• TMB Substrate should be colorless; discard and contact supplier if colored.

• Stop Solution is 1 M HCl — avoid contact with eyes, skin and clothing; wear appropriate PPE.

• Avoid samples containing sodium azide (NaN3) as it will deactivate HRP and lead to underestimation of HCP levels.

• Recommended sample pH 6.5–8.5; outside this range results may be compromised.

• Minimum recommended pipetting volume for dilutions: 5 µL to ensure accuracy.

• Seal microplate immediately after sample loading to prevent evaporation; avoid drying wells before substrate incubation.

• Complete plate reading within 30 minutes after stopping the reaction.

Quality Management & Certifications

Quality System

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QMS (ISO, GMP)

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Quality Advantages

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Quality Control Process

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📂 Technical Resources & Downloads

PDF Product Manual ↓ PDF MSDS / SDS ↓

Frequently Asked Questions (FAQ)

Q1: Is this kit suitable for detection of HCPs from any E.coli strain?

This kit is specifically designed and validated for E.coli protein expression strains originated from BL21. Use with other E.coli strains requires method validation; the kit is not suitable for E.coli cloning strains or samples processed by alkaline lysis.

Q2: How should I prepare and store the calibration standard?

Reconstitute each lyophilized Calibration Standard bottle with 500 µL Reconstitution Solution, gently mix and let stand for ~5 minutes. Use to prepare serial dilutions as described. Reconstituted standard may be stored at 2–8 °C for up to 30 days but single use is recommended due to potential instability.

Q3: What is the assay sensitivity and dynamic range?

The assay LLOQ is 1.5 ng/mL with a validated linear range of 1.5–243 ng/mL (R2 > 0.99 in internal testing).

Q4: What sample types are compatible and how should they be prepared?

Compatible sample types include in-process samples, harvested bulk, drug substance and drug product. Samples should be clear and transparent; remove particulates by centrifugation or filtration, avoid repeated freeze-thaw cycles, and adjust pH to within 6.5–8.5. Dilute samples into the calibration range and verify suitability using dilution series and spike recovery.

Research Use Only

✔

Research Use Only (RUO) – This product is intended for laboratory research purposes only and not for clinical or regulatory diagnostic use.

✔

Not intended for use in USDA or FDA regulated diagnostic testing or official compliance testing.

E.coli (Protein Expression Strains) HCP ELISA Kit (One-step ELISA) SHENTEK®