CYGNUS#F410试剂盒说明书-USB迷|专注于互联网分享

2024年4月15日发(作者：赛鸿风)

Immunoenzymetric Assay for the

Measurement of

E. coli Host Cell Proteins

Catalog # F410

This kit is intended for use in determining the presence of E. coli host cell

protein contamination in products manufactured by recombinant expression

in E. coli. The kit is for Research and Manufacturing Use Only and is not

intended for diagnostic use in humans or animals.

Recombinant expression in E. coli is a relatively simple and cost effective

method for production of certain proteins and pDNA. Many of these products

are intended for use as therapeutic agents in humans and animals, and as

such, must be highly purified. The manufacturing and purification process of

these products leaves the potential for contamination by host cell proteins

(HCPs) from E. coli. Such contamination can reduce the efficacy of the

therapeutic agent and result in adverse toxic or immunological reactions and

thus it is desirable to reduce HCP contamination to the lowest levels practical.

Immunological methods using antibodies to HCPs such as Western Blot

and ELISA are conventionally accepted. While Western blot is a useful

method aiding in the identity of HCPs, it suffers from a number of limitations.

Western blot is a complex and technique dependent procedure requiring

subjective interpretation of results. Furthermore, it is essentially a qualitative

method and does not lend itself to obtaining quantitative answers. The

sensitivity of Western blot is severely limited by the volume of sample that

can be tested and by interference from the presence of high concentrations of

the intended product. While Western Blot may be able to detect HCPs in

samples from upstream in the purification process, it often lacks adequate

sensitivity and specificity to detect HCPs in purified downstream and final

product. The microtiter plate immunoenzymetric assay (ELISA) method

employed in this kit overcomes the limitations of Western blots providing on

the order of 100 fold better sensitivity. This simple to use, highly sensitive,

objective, and semi-quantitative ELISA is a powerful method to aid in optimal

purification process development, process control, routine quality control, and

product release testing. This kit is “generic” in the sense that it is intended to

react with essentially all of the HCPs that could contaminate the product

independent of the purification process. The antibodies have been generated

against and affinity purified using a blended lysate of E. coli from a variety of

strains including the commonly used DH5α and BL21 strains. This relatively

mild lysing procedure is intended to obtain HCPs typically encountered in

initial product recovery steps, such as clarification of conditioned media when

the product is secreted, or after osmotic shock or mild detergent and

mechanical disruption, to obtain inclusion bodies and other intracellular

proteins. Western blot was used as a preliminary method and established

that the antibodies reacted to the majority of HCP bands resolved by the

PAGE separation. If you have need of a more sensitive and specific method

to demonstrate reactivity to individual HCPs in your samples Cygnus

Technologies recommends a method we find superior to 2D Western blot.

We term this method 2D HPLC-ELISA. 2D HPLC-ELISA can yield much

better sensitivity and specificity as compared to 2D Western blot. For more

information on this 2D HPLC-ELISA analysis please contact our Technical

Services department.

Special procedures were utilized in the generation of these antibodies to

insure that low molecular weight and less immunogenic contaminants as well

as high molecular weight components would be represented. As such, this kit

can be used as a process development tool to monitor the optimal removal of

host cell contaminants as well as in routine final product release testing.

Because of the high sensitivity and broad reactivity of the antibodies, this

generic kit has been successfully validated for testing of final product HCPs in

many different products regardless of growth and purification process. When

the kit can be satisfactorily validated for your samples, the application of a

more process specific assay may not be necessary, in that such an assay

would only provide information redundant to this generic assay. However, if

your validation studies indicate the antibodies in this kit are not sufficiently

reactive with your process specific HCPs it may be desirable to also develop

a more process specific ELISA. This later generation assay may require the

use of a more specific and defined antisera. Alternatively, if the polyclonal

antibody used in this kit provides sufficient sensitivity and broad antigen

reactivity, it may be possible to substitute the standards used in this kit for

ones made from the contaminants that typically co-purify through your

purification process and thus achieve better accuracy for process specific

HCPs. The suitability of this kit for a given sample type and product must be

determined and validated experimentally by each laboratory. The use of a

process specific assay with more defined antigens and antibodies in theory

may yield better sensitivity however such an assay runs the risk of being too

specific in that it may fail to detect new or atypical contaminants that might

result from some process irregularity or change. For this reason it is

recommended that a broadly reactive “generic” host cell protein assay be

used as part of the final product purity analysis even when a process specific

assay is available. If you deem a more process specific assay is necessary,

Cygnus Technologies is available to apply its proven technologies to develop

such antibodies and assays on custom basis.

The E. coli assay is a two-site immunoenzymetric assay. Samples containing

E. coli HCPs are reacted with a horseradish peroxidase (HRP) enzyme

labeled anti-E. coli antibody simultaneously in microtiter strips coated with an

affinity purified capture anti-E. coli antibody. The immunological reactions

result in the formation of a sandwich complex of solid phase antibody-HCP-

enzyme labeled antibody. The microtiter strips are washed to remove any

unbound reactants. The substrate, tetramethyl benzidine (TMB) is then

reacted. The amount of hydrolyzed substrate is read on a microtiter plate

reader and is directly proportional to the concentration of E. coli HCPs

present.

All reagents should be stored at 2C to 8C for stability until the expiration

date printed.

The substrate reagent should not be used if its stopped absorbance at

450nm is greater than 0.1.

Reconstituted wash solution is stable until the expiration date of the kit.

Component

Anti-E. coli:HRP

Affinity purified antibody conjugated to HRP in a

protein matrix with preservative. 1x12mL

Product #

F411

F412*

F413

F006

F005

F004

Anti-E. coli coated microtiter strips

12x8 well strips in a bag with desiccant

E. coli HCP Standards

Solubilized E. coli HCPs in bovine albumin with preservative.

Standards at 0, 1, 3, 12, 40, and 100ng/mL. 1 mL/vial

Stop Solution

0.5N sulfuric acid. 1x12mL

TMB Substrate

3,3’,5,5’ Tetramethylbenzidine. 1x12mL

Wash Concentrate (20X)

Tris buffered saline with preservative. 1x50mL

*All components can be purchased separately except # F412.

Microtiter plate reader spectrophotometer with dual wavelength capability at

450 & 650nm. (If your plate reader does not provide dual wavelength analysis

you may read at just the 450nm wavelength.)

Pipettors - 25L and 100L

Repeating or multichannel pipettor - 100L

Microtiter plate rotator (150 - 200 rpm)

Sample Diluent (recommended Cat # I028)

Distilled water

1 liter wash bottle for diluted wash solution

absorbance of the undiluted sample may be lower than the highest standard

in the kit, however these samples will fail to show acceptable dilutional

linearity/parallelism as evidenced by an apparent increase in dilution

corrected HCP concentration with increasing dilution. High Dose Hook and

poor dilutional linearity are most likely to be encountered from samples early

in the purification process. If a hook effect is possible, samples should also

be assayed diluted. If the HCP concentration of the undiluted sample is less

than the diluted sample this may be indicative of the hook effect. Such

samples should be diluted at least to the minimum required dilutions (MRDs)

as established by your validation studies using your actual final and in-

process drug samples. The MRD is the first dilution at which all subsequent

dilutions yield the same HCP value within the statistical limits of assay

precision. The HCP value to be reported for such samples is the dilution

corrected value at or greater than the established MRD. The diluent used

should be compatible with accurate recovery. The preferred diluent is our

Cat# I028 available in 100mL, 500mL, or 1 liter bottles. This is the same

material used to prepare the kit standards. As the sample is diluted in I028,

its matrix begins to approach that of the standards, thus reducing any

inaccuracies caused by dilutional artifacts. Other prospective diluents must

be tested for non-specific binding and recovery by using them to dilute the

100ng/mL standard, as described in the “Limitations” section below.

Before relying exclusively on this assay to detect host cell proteins, each

laboratory should validate that the kit antibodies and assay procedure yield

acceptable specificity, accuracy, and precision. A suggested protocol for

this validation can be obtained from our Technical Services Department or

our web site.

The standards used in this assay are comprised of E. coli HCPs

solubilized by mechanical disruption and detergent. 1D Western blot

analysis of the antibodies used in this kit demonstrates that they recognize

the majority of distinct PAGE separated bands seen using a sensitive

protein staining method like silver stain or colloidal gold. Because the

majority of HCPs will show sufficient antigenic conservation among all

strains of E. coli this kit should be adequately reactive to HCPs from your

strain. The antibodies used in this kit were generated against HCPs

obtained predominately from DH5α and BL21 strains but they have been

shown to react with the majority of HCP from all strains of E. coli tested.

However, there can be no guarantee that this assay will detect all proteins

or protein fragments from your process. If you desire a much more

sensitive and specific method than western blot to detect the reactivity of

the antibodies in this kit to your individual HCPs Cygnus is pleased to offer

a service for fractionation of HCPs using 2-D HPLC methods followed by

detection in ELISA.

Certain sample matrices may interfere in this assay. The standards used

in this kit attempt to simulate typical sample protein and matrices. However

the potential exists that the product protein or other components in the

sample matrix may result in either positive or negative interference in this

assay. High or low pH, detergents, urea, high salt concentrations, and

organic solvents are some of the known interference factors. It is advised

to test all sample matrices for interference by diluting the 100 ng/mL

standard, 1 part to 4 parts of the matrix containing no or very low HCP

contaminants. This diluted standard when assayed as an unknown should

give a value of 15 to 30 ng/mL. Consult Cygnus Technologies Technical

Service Department for advice on how to quantitate the assay in

problematic matrices.

Avoid the assay of samples containing sodium azide (NaN

) which will

destroy the HRP activity of the conjugate and could result in the under-

estimation of HCP levels.

For Research or Manufacturing use only.

Stop reagent is 0.5N H

. Avoid contact with eyes, skin, and clothing.

At the concentrations used in this kit, none of the other reagents are

believed to be harmful.

This kit should only be used by qualified technicians.

Bring all reagents to room temperature.

Dilute wash concentrate to 1 liter in distilled water, label with kit lot and

expiration date, and store at 4C.

1. Complete washing of the plates to remove excess unreacted reagents is

essential to good assay reproducibility and sensitivity. We advise against the

use of automated or other manual operated vacuum aspiration devices for

washing plates as these may result in lower specific absorbances, higher

non-specific absorbance, and more variable precision. The manual wash

procedure described below generally provides lower backgrounds, higher

specific absorbance, and better precision. If duplicate CVs are poor, or if the

absorbance of the 0 standard minus a substrate blank is greater than 0.300,

evaluate plate washing procedure for proper performance.

2. High Dose Hook Effect or poor dilutional linearity may be observed in

samples with very high concentrations of HCP. High Dose Hook Effect is due

to insufficient excess of antibody for very high concentrations of HCPs

present in the samples upstream in the purification process. Samples greater

than 200 g/mL may give absorbances less than the 100 ng/mL standard. It

is also possible for samples to have certain HCPs in concentrations

exceeding the amount of antibody for that particular HCP. In such cases the

800-F410, Rev. 1, 8/21/12 E. coli HCP ELISA Product Insert

The assay is very robust such that assay variables like incubation times,

sample size, and other sequential incubation schemes can be altered to

manipulate assay performance for more sensitivity, increased upper

analytical range, or reduced sample matrix interference. Before modifying

the protocol from what is recommended, users are advised to contact our

technical services for input on the best way to achieve your desired goals.

The protocol specifies use of an approved microtiter plate shaker or rotator

for the immunological steps. These can be purchased from most

laboratory supply companies. Alternatively, you can purchase an

approved, pre-calibrated shaker directly from Cygnus Technologies. If you

do not have such a device, it is possible to incubate the plate without

shaking however it will be necessary to extend the immunological

incubation step in the plate by about one hour in order to achieve

comparable results to shaking protocol. Do not shake during the 30-

minute substrate incubation step, as this may result in higher

backgrounds and worse precision.

Bring all reagents to room temperature. Set-up plate spectrophotometer to

read dual wavelength at 450nm for the test wavelength and ~650nm for

the reference.

Thorough washing is essential to proper performance of this assay.

Automated plate washing systems or other vacuum aspiration devices are

not recommended. The manual method described in the assay protocol is

preferred for best precision, sensitivity and accuracy. A more detailed

discussion of this procedure can be obtained from our Technical Services

Department or on our web site.

All standards, controls, and samples should be assayed at least in

duplicate.

Maintain a repetitive timing sequence from well to well for all assay steps

to insure that all incubation times are the same for each well.

Make a work list for each assay to identify the location of each standard,

control, and sample.

It is recommended that your laboratory assay appropriate quality control

samples in each run to insure that all reagents and procedures are correct.

You are strongly urged to make controls in your typical sample

matrix using HCPs derived from your cell line. These controls can be

aliquoted into single use vials and stored frozen for long-term

stability.

If the substrate has a distinct blue color prior to the assay it may have

been contaminated. If the absorbance of 100L of substrate plus 100L of

stop against a water blank is greater than 0.1 it may be necessary to

obtain new substrate or the sensitivity of the assay may be compromised.

Strips should be read within 30 minutes after adding stop solution since

color will fade over time.

Assay Protocol

1. Pipette 25µL of standards, controls and samples into wells indicated

on work list.

2. Pipette 100µL of anti- E. coli:HRP (#F411) into each well.

3. Cover & incubate on rotator at ~ 180rpm for 90 minutes at room

temperature, 24°C ± 4°C.

4. Dump contents of wells into waste. Blot and gently but firmly tap over

absorbent paper to remove most of the residual liquid. Overly

aggressive banging of the plate or use of vacuum aspiration devices in

an attempt to remove all residual liquid is not necessary and may cause

variable dissociation of antibody bound material resulting in lower ODs

and worse precision. Fill wells generously to overflowing with diluted

wash solution using a squirt bottle or by pipetting in ~350µL. Dump and

tap again. Repeat for a total of 4 washes. Wipe off any liquid from the

bottom outside of the microtiter wells as any residue can interfere in the

reading step. Do not allow wash solution to remain in wells for longer

than a few seconds. Do not allow wells to dry before adding substrate.

5. Pipette 100µL of TMB substrate (#F005).

6. Incubate at room temperature for 30 minutes. DO NOT SHAKE.

7. Pipette 100µL of Stop Solution (#F006).

8. Read absorbance at 450/650nm.

Quality Control

Precision on duplicate samples should yield average % coefficients of

variation of less than 10% for samples in the range of 3-100ng/mL. CVs for

samples < 3 ng/mL may be greater than 10%.

For optimal performance the absorbance of the substrate when blanked

against water should be < 0.1.

It is recommended that each laboratory assay appropriate quality control

samples in each run to insure that all reagents and procedures are correct.

Example Data

Well #

Contents

Zero Std

1ng/mL

3ng/mL

12ng/mL

40ng/mL

100ng/mL

sample A

sample B

The standards may be used to construct a standard curve with values

reported in ng/mL “total immuno-reactive HCP equivalents” (See Limitations

section above). This data reduction may be performed through computer

methods using curve fitting routines such as point-to-point, cubic spline, or 4

parameter logistic fit. Do not use linear regression analysis to interpolate

values for samples as this may lead to significant inaccuracies! Data

may also be manually reduced by plotting the absorbance values of the

standard on the y-axis versus concentration on the x-axis and drawing a

smooth point-to-point line. Absorbances of samples are then interpolated

from this standard curve.

800-F410, Rev. 1, 8/21/12

Abs. at

450nm

0.066

0.068

0.099

0.098

0.155

0.153

0.396

0.393

1.190

1.179

2.481

2.505

0.733

0.740

0.152

0.151

Mean

Abs.

0.067

0.099

0.154

0.395

1.185

2.493

0.737

0.152

ng/mL

HCP equivs.

24.1

2.9

E. coli HCP ELISA Product Insert

Cygnus Technologies has validated this assay by conventional criteria as

indicated below. This validation is generic in nature and is intended to

supplement but not replace certain user and product specific qualification and

validation that should be performed by each laboratory. At a minimum each

laboratory is urged to perform a spike and recovery study in their sample

types. In addition, any of your samples types containing process derived

HCPs within or above the analytical range of this assay should be evaluated

for dilutional linearity to insure that the assay is accurate and has sufficient

antibody excess for your particular HCPs. Each laboratory and technician

should also demonstrate competency in the assay by performing a precision

study similar to that described below. A more detailed discussion of

recommended user validation protocols can be obtained by contacting our

Technical Services Department or at our web site.

acceptable recovery defined as between 80-120%. The standards used in

this kit contain 8mg/mL of bovine serum albumin intended to simulate non-

specific protein affects of most sample proteins or pDNA products. However

very high concentrations of some products (often in the 2-5 mg/mL range)

may interfere in the accurate measurement of HCP’s. In general, extremes in

pH (<5.0 and >8.5), high salt concentration, high polysaccharide

concentrations, and most detergents can cause under-recovery. Each user

should validate that their sample matrices yield accurate recovery. Such an

experiment can be performed, by diluting the 100ng/mL standard provided

with this kit, into the sample matrix in question as described in the

“Limitations” section.

Hook Capacity

Increasing concentrations of HCPs > 100 ng/mL were assayed as unknowns.

The hook capacity, defined as that concentration which will give an

absorbance reading less than the 100 ng/mL standard was >200 g/mL.

Sensitivity

The lower limit of detection (LOD) is defined as that concentration

corresponding to a signal two standard deviations above the mean of the

zero standard. LOD is ~0.2 ng/mL.

The lower limit of quantitation (LOQ) is defined as the lowest concentration,

where concentration coefficients of variation (CVs) are <20%. The LOQ is <1

ng/mL.

Cygnus Technologies also offers kits for the extraction and detection of E.

coli Host Cell DNA. The following kits are available:

Precision

Both intra (n=20 replicates) and inter-assay (n=5 assays) precision were

determined on 3 pools with low (~3ng/mL), medium (~12ng/mL), and high

concentrations (~40ng/mL). The % CV is the standard deviation divided by

the mean and multiplied by 100.

Pool Intra assay CV Inter assay CV

Low 3.3% 7.7%

Medium 1.9% 5.5%

High 2.7% 6.5%

Residual Host Cell DNA extraction:

Cat # D100W DNA Extraction Kit in 96 deep well plate

Cat # D100T DNA Extraction Kit in microfuge tubes

Extraction and PCR amplification of E. coli Host Cell DNA for use with

user supplied master mix:

Cat # D415W DNA Extraction Kit in 96 deep well plate

Cat # D415T DNA Extraction Kit in microfuge tubes

Residual E. coli Host Cell DNA extraction and detection using

PicoGreen dye:

Cat # D410W DNA Extraction Kit in 96 deep well plate

Cat # D410T DNA Extraction Kit in microfuge tubes

To place an order or to obtain additional product information contact Cygnus

Technologies:

Cygnus Technologies, Inc.

4701 Southport Supply Rd. SE, Suite 7

Southport, NC 28461 USA

Tel: 910-454-9442

Fax: 910-454-9443

Email: techsupport@

______________________________________________________________

Specificity/Cross-Reactivity

1D Western blot and ELISA analysis against several strains of E. coli (DH5α,

BL21, JM109, TOP10F, K12, & MC1061) indicate that most of the proteins

are conserved among all strains. Thus, this assay should be useful for

detecting HCP’s from other E. coli cell lines. Each end user must validate that

this kit is adequately reactive and specific for their samples. 1D Western blot

is highly orthogonal to ELISA and to non-specific protein staining methods

such as silver stain or colloidal gold. As such, the lack of identity between

silver stain and western blot does not necessarily mean there is no antibody

to that protein or that the ELISA will not detect that protein. If you desire a

much more sensitive and specific method than Western blot to detect the

reactivity of the antibodies in this kit to your individual HCPs Cygnus is

pleased to offer a service and/or consultation on fractionation of HCPs using

2 Dimensional HPLC methods followed by detection in the ELISA. This

method has been shown to be much at least 100 fold more sensitive than

Western blots in detecting antibody reactivity to individual HCPs. The same

antibody as is used for both capture and HRP label can be purchased

separately.

Cross reactivity has not been extensively investigated with this kit. You

should evaluate components in your samples for positive interferences such

as cross reactivity and non-specific binding. Negative interference studies are

described below.

Recovery/ Interference Studies

Various buffer matrices were evaluated by adding known amounts of E. coli

HCP preparation used to make the standards in this kit. Because this assay

is designed to minimize matrix interference most of these buffers yielded