An ELISA was used for CHOP quantification. Samples containing CHOP were incubated in the wells, followed by incubation with
anti-CHOP antibodies conjugated with horseradish peroxidase (HRP). The HRP enzymatic activity was detected with o-phenylenediamine, and the CHOP was quantiﬁed by reading absorbance at 490 nm in a microtiter plate reader. Based on the principles
of sandwich ELISA, the concentration of peroxidase corresponded to the CHOP concentration. The assay range for the ELISA was
typically 10–320 ng/mL, with intra-assay variability of approximately 10%. CHOP values were reported in units of ng/mL. CHOP
values could be divided by the mAb concentration and the results reported in units of PPM (parts per million; ng of CHOP/mg
Bacteriophage ΦX174 quantification
Host organism E. coli was used for the detection of infectious ΦX174 phage particles. E. coli cells were incubated on agar plates (Soybean-Casein Digest Agar Medium– Trypticase Soy Broth 211043), which served as a base
layer with nutrients. E. coli cells multiplied rapidly and formed a bacterial lawn. Phage particles infect the cells, causing the lysis of E. coli host cells and producing single circular, nonturbid areas called plaques in the bacterial lawn. Each plaque represents the
lysis of a phage-infected bacterial culture and is designated as a plaque-forming unit (pfu), and used to quantitate the number
of infective phage particles in the culture. Plaques must be clearly defined and samples were then diluted several times (1:10)
depending on the phage concentration. During the study, 150 μL of the host cell solution (optical density 2–6) was mixed with
150 μL of sample and top agar (1.3% Tryptikase Soy Agar BD 211043) and the mixture was then distributed to agar plates (4%
Tryptikase Soy Agar BD 211043 in 90 mm petri dishes) and incubated for 18 to 24 hours at 37 °C. Plaque forming units were
counted and the titer of the sample in PFU/mL (plaque forming units per mL) was calculated using Equation 2,
where P is the number of plaques of all countable dilutions, E is the sum of emphasis, D is the lowest evaluated dilution,
and VSample is the sample volume.
The LRV was calculated using Equation 3,
where c0 was the titer of the initial solution and cFT the titer in the flow-through fraction.
The endotoxin level was measured by the kinetic chromogenic method test according to the manufacturer's instructions (Limulus
Amebocyte Lysate Chromogen, Charles River endosafe Endochrome-K R1710K, Lot A4992L 10/2012). The quantification principle
is based on coloration caused by the contact of a sample containing endotoxin with a mixture of lysate and chromogenic substrate.
A β-glucan blocker was added (Lonza N190 Lot 0000132199 01/11). During the 1-hour incubation the extinction coefficient was
measured continuously at 405 nm using a temperature controlled (37 °C) plate reader (Tecan Safire). The reaction rate varies
with endotoxin level and the samples were quantified for endotoxin by comparing the results with the calibration series. The
detection limit of the assay was 0.012 EU/mL. LRV was calculated similarly to phage quantification by measuring the endotoxin
level of the initial solution El0 and the level of endotoxin in the collected flowthrough fractions (ElFT) using Equation 4.