STANDARD OPERATING PROCEDURE

CHROMOGENIC METHOD

(End Point Method)

Note:

Method provided in this document is only valid for ACCI, USA Chromogenic kits
(CD060 and C1500). This is controlled document and property of Pharma Canada,
Pakistan. Without permission publishing or copying of this document is not allowed.
1. Purpose

This document describes the procedures to perform endpoint chromogenic

assays with Pyrochrome in a microplate

Background on the test

Limulus Amebocyte lysate is an aqueous extract from the blood cells (amebocytes)
from
the horseshoe crab Limulus polyphemus. In the presence of endotoxin, factors in the
LAL
are activated in a proteolytic cascade that results in the cleavage of a colorless
artificial
peptide substrate present in Pyrochrome LAL. Proteolytic cleavage of the substrate
liberates p-nitroaniline (pNA), which is yellow and absorbs at 405 nm. The test is
performed by adding a volume of Pyrochrome to a volume of sample and incubating the

reaction mixture at 37�C. The greater the endotoxin concentration in the sample,
the
faster pNA will be produced. In the endpoint, the amount of pNA released can be
measured following a fixed incubation period. A standard curve, consisting of
measured
optical density plotted against known standard endotoxin concentration, is used to
determine concentrations in samples. For samples that absorb at 405-410 nm, the
diazo-
coupling method is reacted with nitrite in HCl and then with N-(1 Naphthyl)-
ethylenediamine (NEDA) to form a diazotized magenta derivative that absorbs at a
range
between 540-550 nm.

Definitions

1. EU/mL or IU/mL: Endotoxin Units or International Units per milliliter

(Endotoxin and International are equivalent).
2. . (�Lambda�):
The lowest concentration on the standard curve and determines
the sensitivity of that particular assay.
3. Valid Assay (per USP): An assay that must have a valid standard curve, valid
negative controls and valid positive product controls (PPC), all tested in a least
duplicate.

4. Standard curve: A set of endotoxin concentrations assayed in a test that is used

to determine the endotoxin concentrations of the samples. A standard curve

must consist of at least 3 endotoxin concentrations tested in at least
duplicate. If using a CSE standard curve, do not use a range that is beyond the
potency range stated on the Certificate of Analysis (C of A).
5. Valid Standard Curve: A standard curve that must have a correlation
coefficient, �r�, greater than or equal to 0.980.
6. Negative Controls (NC): LRW or the diluent used to prepare the standard curve
and sample dilutions. Valid negative controls should have significantly less
endotoxin than that of the lowest standard concentration (estimated by
extrapolation of the standard curve).
7. Positive Product Controls (PPC):

Product being tested usually spiked with a concentration at or near the middle
of the standard curve. Each sample and /or sample dilution tested must have a
PPC tested concurrently.Valid PPC concentrations must be within 50-200%
of the nominal value.

8. Double dilution method: A method of making dilutions (standard curve, sample

and PPC), that are prepared in dilution tubes and made two-fold greater than
desired concentrations. The dilutions are then added to the microplate in a 1:2
ratio with LRW, or the sample to dilute the solution to the desired final
concentration.
9. Direct Addition: A method of making dilutions (standard curve, sample and
PPC) that are prepared in dilution tubes or microplate at the desired
concentrations.
10. Sample to lysate ratio: The ratio of sample/diluents volume to lysate volume in

the reaction mixture.

a. A 1:1 ratio: Refers to 1 part sample/diluents to 1 part lysate.

2. Resposibilities

2.1 Microbiologist
2.2 Lab Assistant

3. Abbreviations

EU Endotoxin Units

Std Standard

ET Endotoxin

DF Dilution Factor

4. Requirements

3.1. General

3.1.1. Hot air oven.

3.1.2. Glass pipettes (1,2,5,10 ml)
3.1.3. Micropipette (0.1 ml or 100 ul)
3.1.4. Sterile and pyrogen-free micro-tips
3.1.5. Borosilicate glass tubes
3.1.6. 0.1N HCl
3.1.7. 0.1N NaOH
3.1.8. Photocells (full and half size)
3.1.9. Spectrophotometer
3.1.10. Vortex mixer
3.1.11. Incubator
3.1.12. Tube stands S/S
3.1.13. Beakers
3.1.14. LAL water
3.1.15. Acetic acid 0.8 M
3.1.16. 70% alcohol
3.1.17. Aluminum foil dehydrogenated
3.1.18. Deep freezer (-20oC)
3.1.19. Refrigerator (2-8oC)
3.1.20. Forceps
3.1.21.

3.2. Test Reagents

1) Chromogenic pyrochrome

2) Respective Endotoxin Standard.

3) Diazo Coupling Reagents (Set of four bottles).

Note: All reagents must be stored in refrigerator at 2-8oC.

5. WASHING & DEPYROGENATION OF MATERIALS

1) After proper cleaning, rinse the test tubes thoroughly with distilled water.

2) Wrap the test tubes properly with Aluminum foil.

3) Depyrogenate the test tubes in an oven at 250oC for 30 minutes or 180 oC
for 2 hours.
6. RECONSTITUTION OF REAGENTS

1. Opening Reagent Vials

Remove the plastic cover. Wipe off with 70% Alcohol around the rubber cap and
top portion of every vial. Remove the aluminum cap with sterile and pyrogen free
forceps.

2. Reconstitution of CSE (Control standard Endotoxin)

Reconstitute CSE with volume specified on the certificate of Analysis. Vortex the
vial for
15 minutes after adding LRW. Must Vortex the CSE prior use for homogeneity.

Verification of Criteria for the Standard Curve

Perform this test to qualify the reagents, laboratory and technician, before
performing any sample testing. Use CSE to prepare at least three endotoxin
concentrations to generate the standard curve. Perform the test using at least
three
replicates of each standard endotoxin concentration. The curve range for endpoint
assays
should not be greater than 1 log. The example given is a curve of two fold
dilution,
ranging from 1.28 to 0.16 EU/mL.

1. Reconstitute the CSE vial following manufacturer�s product insert.

Immediately return to 2-8�C when not in use.

Prepare a fresh set of dilutions from the stock endotoxin solution for each test.
Do
not use previously prepared and stored dilutions unless you have demonstrated the
stability of that range of concentrations. Prepare the endotoxin standards in
dilution tubes.
. The CSE vial contains either:
a. 10 ng/vial.

Reconstitute with LAL reagent water to 50 EU/mL using the volume

obtained from the Certificate of Analysis for the lot combination
(Pyrochrome/CSE) that you are working with.

Initial concentration: 50 EU/mL

. 1:39 (100 �L [50 EU/mL] + 3800 �L LRW)

1.28 EU/mL

. 1:2 (500 �L [1.28 EU/mL] + 500 �L LRW)

0.64 EU/mL

. 1:2 (500 �L [0.64 EU/mL] + 500 �L LRW)

0.32 EU/mL

. 1:2 (500 �L [0.32 EU/mL] + 500 �L LRW)

0.16 EU/mL

Vortex mix
and transfer
0.5 mL

Vortex mix
and transfer
0.5 mL

Vortex mix
and transfer
0.5 mL
Add
0.1
mL

0.5 mL
LRW

STOCK
CSE

50 EU/mL

3.8
mL
LRW

0.5 mL
LRW

0.5
mL
LRW

1.28
EU/mL

0.16
EU/mL

0.32
EU/mL

0.64
EU/mL
b. 0.5 .g/vial

Use the Certificate of Analysis for the lot combination (Pyrochrome /CSE) that
you are working with to:

Obtain the reconstitution volume of LRW, to achieve a CSE concentration of 1000

EU/mL.

OR

Obtain the starting concentration when reconstituting the CSE vial with
5 mL LRW.

Note: The dilution scheme below uses a starting concentration of 1000 EU/mL, the
scheme will have to be modified, if you are starting with a different
concentration.

Initial concentration: 1000 EU/mL

. 1:20 (100 �L [1000 EU/mL] + 1900 �L LRW)

50 EU/mL

. 1:39 (100 �L [50 EU/mL] + 3800 �L LRW)

1.28 EU/mL

. 1:2 (500 �L [1.28 EU/mL] + 500 �L LRW)

0.64 EU/mL

. 1:2 (500 �L [0.64 EU/mL] + 500 �L LRW)

0.32 EU/mL
 . 1:2 (500 �L [0.32 EU/mL] + 500 �L LRW)

0.16 EU/ml

Vortex mix
and transfer 0.1
mL

Vortex mix
and transfer
0.5 mL

Vortex mix
and transfer
0.5 mL

Vortex mix
and transfer
0.5 mL

Add
0.1
mL

STOCK CSE

1000 EU/mL

0.5
mL
LRW

0.5
mL
LRW
0.5
mL
LRW

3.8
mL
LRW

1.9
mL
LRW

1.28
EU/mL

0.64
EU/mL

0.16
EU/mL

0.32
EU/mL

50
EU/mL

NOTE

Prior to dispatching Chromogenic Kit to market every lot is checked by

ACCI and FDA separately as per above dilution method and concentration. So
ACCI recommend above mentioned dilution. By changing dilutions incubation time
period may change.
Reconstitution of Pyrochrome

1. Diazo Reagents

For Diazo kit (CD060) only:

Vial 1a : HCl,

Vial 1 : Sodium Nitrite,

Vial 2 : Ammonium sulfamate,

Vial 3 : NEDA.

Reconstitute Vial 1 with entire contents of vial 1a.

Reconstitute Vial 2 with 4 ml of Distilled water/ LRW.

Reconstitute vial 3 with 4 ml of Distilled water / LRW

PRE-TEST PREPARATIONS

A) pH of the sample

The pH of the reaction mixture (Sample + pyrochrome reagent ) should be

between 6.0 to 8.0. The sample should be adjusted by pyrogen free 0.1N NaOH or
0.1 HCL

Procedure:

. Gently tap the vial of Pyrochrome to cause loose LAL to fall to the bottom.
. Break the vacuum by lifting the gray stopper.
. Do not contaminate the mouth of the vial.
. Remove and discard the stopper; do not inject through or reuse the stopper.
. A small amount of LAL on the stopper will not affect the test.
. Reconstitute a vial of Pyrochrome with 3.2 mL of Pyrochrome Reconstitution
Buffer or Glucashield�buffer.
. Reconstitute the Pyrochrome 5 minutes prior to use.
. Swirl vial to insure homogeneity but avoid vigorous mixing that may cause
excessive
foaming and a loss of activity.
. Cover the vial with Parafilm� M and store 2-8�C when not in use.
. Pyrochrome must be used within 8 hours of reconstitution.
. Reconstituted Pyrochrome may not be frozen.
. Add 50 �L of each standard concentration and negative control to their assigned
wells
in the 96-well microplate or test tubes (10 X 75).
. Cover the reaction tube to reduce risk of contamination.
. Using a repeater pipette (e.g. Eppendorf. ) with a 2.5 mL Combitip., add 50 .L
reconstituted Pyrochrome to all test tubes starting with the negative controls,
then the
standard curve from lowest to highest endotoxin concentration.
. Be sure not to splash and add the LAL reagent to all test tubes within 2 minutes.

INCUBATION

. Place covered test tube in heated Pyroblock�/incubator/water bath and incubate

for
appropriate amount of time.
. Note that the incubation time will depend on the range of the standard curve.
. Use �Recommended Incubation Times for Endpoint Assays with Pyrochrome��
sheet supplied with the kit for the specific lot of Pyrochrome reagent and CSE
used,
for guidance in choosing the incubation time. Preliminary tests may be necessary to

determine the correct incubation period.

REACTION STOPAGE

1.2. Reaction Stoppage by Acetic acid.

. Stop the reaction by adding 50 % acetic acid.
. Prepare a 50% acetic acid solution by mixing equal volumes of LRW and
glacial acetic acid, or use a stock solution of 50% acetic acid.
. Using a repeater pipette (e.g. Eppendorf�), add 25 �L of 50% acetic acid
to all test tubes used in the assay immediately following the end of the
incubation time (�30 seconds).
. Cape COD don�t recommend to use sample/ pyrochrome quantity 0.1 ml
each.

Reaction Stopage by Diazo Coupling (For End Point)

. Reconstitute the Diazo coupling reagents while the test is incubating, but
not more than 10 minutes before addition to the plate
. Transfer 50 �L of vial #1 (sodium nitrite reconstituted with 0.48N
hydrochloric acid) to all wells used in the assay following the end of the
incubation time (�30 seconds), with a repeating pipette. Manually shake
the plate gently.
. Transfer 50 �L of vial #2 (reconstituted ammonium sulfamate) to all wells
used in the assay, using a new tip on the repeating pipette. Manually
shake the plate gently.
. Transfer 50 �L of vial #3 (reconstituted NEDA) to all wells used in the
assay, using a new tip on the repeating pipette.

. Manually shake the covered test tubes gently to stop the reaction.
. Insert the reaction tubes/ microplate in the spectrophotometer/reader.

Reading on Spectrophotometer (For end point assay)

*
Take the reading of sample at 405 nm to 410 nm for samples which reaction is
stopped by acetic acid.
*
While for samples which reaction is stopped by Diazo coupling take reading
at 540 nm to 550 nm.

CALCULATION

When interpreting the data of the samples, they have to be compared relative to
the standard curve. the curve is essentially defined by measured OD value (not the
theoretical values of the endotoxin concentration).
Linear Equation Formula is; X = y � c/m

X = concentration of endotoxin in sample

Y = OD of sample

c & m (value will be obtained after standard curve formation by

interpolating data (Absorbance of positive replicates and concentration of
replicates) in linear equation.

Please note that the equation for the linear regression line could change with each

standard curve performed

Interpreting the results

. In order for the test to be valid the endotoxin concentration of negative

controls
(estimated by extrapolation of the standard curve) should be significantly less
than that of the lowest standard concentration.
. The correlation coefficient for the standard curve should have an absolute value
greater than 0.980.
. Endotoxin concentrations can only be reported for the range from the lowest to
the highest standard endotoxin concentration. Results for samples that lie outside
of this range should be reported as either less than the lowest standard
concentration (not detectable) or greater than the highest standard concentration.
 FREQUENTLY ASKED QUESTIONS

1. Cape COD is claiming 50 test for 50 test vial but actually test are 52 to 54 as
lypholized material (lysate) get soluble in it, likewise claim is regarding
chromogenic as 60 test kit but actually test goes to 64 to 70. Then why claim
is 50 or 60 ?
Ans: . The vials of each reagent come with a little bit of an overfill. This is to
account
for pipetting errors and traces of the solution left on the walls of the glass
vials. So the
number of tests (=number of reactions) per vial is a guarantee that the end user
should
get out of a single vial if it is used appropriately.

2. Is it necessary to form standard curve during each chromogenic testing kit

for end point method or we can form standard curve for one time for each
lot? Means is it each lot base or kit base .

Ans: Per USP chapter <854> a full chromogenic standard curve should be performed
with every single test. This is to take into account the variability of the test as
the final
OD measured (in the case of endpoint methods) may potentially be quite different
between different tests. As extrapolation of the sample test results is not
allowed, one can
only avoid this by performing a full standard curve with each test (one per test/
microplate).

3. Is ACCI every lot/batch of Gel clot is tested by FDA in their own lab before
dispatch to market or not? Or just its FDA approved product.

Ans: Every single batch manufacturerd is submitted to FDA for testing before we are

granted to release it to the market.

4. When it can give false positive results and for which products?

Ans: False positive happens only in the presence of serine proteases (such as
trypsin
which acts as the clotting enzyme) and an overwhelming presence of certain size
1,3-beta
glucan (which then activates the alternative pathway � Factor G).
 (1,3)-�-D-glucan contamination may occur from product contact with the following:

Common Sources/Products that may contain glucans are;

Naturally derived raw materials, Cellulose filters/materials, Collagen products,

Products
derived from fungi, bacteria or algae, Cotton Medical devices (surgical
sponges/packaging, wound dressing etc.), Yeastolates, Acetates, Citrates, Sugars
(Glucose preparations), Biologicals (albumin, plasma protein, immunoglobin,
preparations, coagulation factors, cell culture fluid), rDNA yeast protein,
Oligonucleotide
drugs, Saline Preparations, Water for injection (WFI), Paper products.

5. Can we verify our chromogenic results by parallel testing via Gel clot
method?

Ans: Yes, you can verify your end point chromogenic results by the gel clot test,.
The expectation is that the results will be within 2-fold of each other. This is
the
2-fold error of the test as defined by the USP chapter <85>: the recovery of
positive product controls is expected to be within 50-200% (for the photometric
methods). The gel clot labeled sensitivity is required to be confirmed within
2lambda to 1/2lambda)

6. We get reading of a sample on spectrometer one time it gives us optical

density then we take the reading of same sample again. It gives different
reading even with positive replicates. Although stock solution is same. Why?

Ans: I would expect difference in the OD values due to the use the cuvette. Once
the reaction is stopped and developed, the enzymatic reaction no longer proceeds
so there is no more increase in OD due to endotoxin. Any variability in the OD
should not be significant and may be more related to the instrumentation and
handling the cuvette. We don�t have much experience with this kit on the cuvette
format as all of the testing we perform here is on a 96 well microplate and a plate

reader. The repeated OD values may be taken there without removing the plate
from the reader. I would consider the first read as the �real� result.