Description

Our original Pichia pastoris HCP ELISA kit (F140) 2G has a broad reactivity to peptide epitopes and a small population of antibodies that recognize Pichia glycosylation epitopes. In cases where the recombinant protein product is also glycosylated, there is the potential for some cross-reactivity which may result in overestimation of true HCP or indeterminate HCP concentrations due to non-linearity of sample dilution.

The antibody used in this kit is the same polyclonal antibody used in the original kit, except that it has undergone an additional affinity absorption step to remove antibodies against glycosylation epitopes and is more specific than the Pichia kit. first-generation pastoris (F140). Use Sample Diluent, Item # I028, with these kits; available separately.

Size: 96 T

Sample: Pichia pastoris host cells

Species Reactivity: Pichia pastoris

Intended Use

This kit is designed to determine the presence of Pichia pastoris protein impurities in products manufactured by recombinant expression in Pichia pastoris host cells.

Kit Contents

• Anti-P. pastoris: HRP. Affinity-purified goat antibody conjugated to HRP in a preserved protein matrix. 1x12mL
• Anti-P. pastors coated microtiter strips. 12×8 well strips in a desiccant bag.
• P. pastoral rules. HCP from P. pastoris solubilized in a bovine serum albumin matrix with a preservative. Standards at 0, 1, 4, 20, 75 and 250 ng/mL. 1ml/vial
• Stop solution. 0.5M sulfuric acid. 1x12mL
• TMB Substrate. 3,3′,5,5′ tetramethylbenzidine. 1x12mL
• Concentrated wash (20X). Tris-buffered saline with preservative. 1x50mL
• All components can be purchased separately except Anti-P. pastoris coated microtiter strips.

Storage

1. All reagents should be stored at 2°C to 8°C to be stable until the expiration date printed on the kit.
2. Reconstituted Wash Solution is stable through the kit expiration date.
3. After prolonged storage, you may notice a salt precipitate and/or a yellowish colour in the wash concentrate. These changes will not affect the performance of the assay. To dissolve the precipitate, mix the Wash Concentrate well and dilute as directed in the “Reagent Preparation” section.

Performance Characteristics

At a minimum, each laboratory is encouraged to perform a growth and recovery study on their sample types. Additionally, any of your sample types that contain process-derived HCPs within or above the analytical range of this essay should be evaluated for dilutional linearity to ensure the assay is accurate and has sufficient excess antibodies for your particular HCPs. Each laboratory and technician must also demonstrate proficiency in the assay by performing a precision study similar to the one described below.

Precision

Within (n=16 replicates) and inter-assay (n=5 assays) precision was determined in 3 groups with low (~4 ng/mL) and high (~75 ng/mL) concentrations. %CV is the standard deviation divided by the mean and multiplied by 100.

• Intra-assay (%CV): low-3.0; High-4.2
• Inter-assay (%CV): Low-1.6; High-2.8

Sensitivity

• The lower limit of detection (LOD) is defined as the concentration corresponding to a signal two standard deviations above the mean of the zero standards. The LOD is ~0.1 ng/mL.
• The lower limit of quantification (LOQ) is defined as the lowest concentration at which the concentration coefficients of variation (CV) are less than 20%. The LOQ is ~0.5 ng/mL.