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Package Insert
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WHM Biologics
Ranibizumab Kit
(Lucentis)
Enzyme immunoassay (EIA) for the simultaneous
quantitative or qualitative detection of Lucentis.
M Inc.
WHM
WBiologics
Redwood City CA, 94063
[email protected]
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at 4-6 weeks intervals.
* The long-term local and systemic effect of Ranibizumab is
unknown because of its long half-life and its suppression
of VEGF (vascular endothelium growth factor). Therefore
the need for a sensitive and accurate assay is imperative to
understand and prevent potential damaging side effects.
3 Principle
The Ranibizumab (Lucentis) Assay is a solid-based enzyme
immunoassay with a sequential addition and incubation
steps in its protocol. The test is a colorimetric microtiter
plate enzyme immunoassay for the direct determination
of Ranibizumab in serum or plasma. The assay protocol
includes:
1. Incubation/shaking of the sample with the microtiter well
coated plate
2. Discarding contents of the wells, followed with addition
of enzyme conjugate, shaken and followed by a wash
sequence
3. Formation of blue color with TMB substrate
4. Addition of 3N HCl to stop the enzyme/substrate reaction
giving a yellow color. The color intensity is measured as
optical density (absorbance) on a microtiter plate reader at
450nm.
* The absorbance value is directly proportional to the
concentration of Ranibizumab in the sample.
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4 Materials Provided
1. Lucentis Assay Kit Components (96 tests)
2. Lucentis Microwell Strips (1 plate of 96 coated wells)
3. Lucentis Enzyme Conjugate Reagent (8ml)
4. Rinse Solution Concentrate (50ml)
5. Substrate/Chromogen Reagent (11ml)
1 Intended Use
6. Stop Solution/ 3N HCL (10ml)
The Ranibizumab (Lucentis) EIA test kit is an in-vitro
7. Stock standard/Negative standard (10 ml)
enzyme immunoassay for the quantitative determination
8. Standard Calibrator Concentrated (100ng/ml) 1ml
of Lucentis in serum or plasma.
2 Introduction
* Ranibizumab (trade name Lucentis, Genentech/Roche) is
an angiogenesis inhibitor, a drug that slows the growth of
new blood vessels. It is used for the treatment of diseases
of the eye in macular degeneration, diabetic retinopathy
and retinopathy of prematurity.
* The intraocular dose is 1.25 mg to 2.5 mg in 100 ul (0.1ml)
injections. The lower dose is usually preferred in premature
newborns to prevent retinal blindness.
Lucentis microwell strips and enzyme
conjugate are provided as matched set. Do
not interchange components of kits with
different lot numbers.
* The higher dose(s) is for adults with macular degeneration
Other Materials and Instrumentation Required
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Squirt bottle for dispensing 500ml of rinse solution
Graduated cylinder for measuring 450ml
Distilled water
Single channel pipette and tips for dispensing 60ul
Multi-channel pipettes, tips and reservoirs or repeating
pipettes and tips for dispensing 50ul, 75ul, and 100ul
Absorbing toweling
Clock or timer
Microtiter plate reader that is capable of reading optical
density (OD) of 450nm and printing absorbance values
Tubes for diluting samples
Microtiter Plate Shaker
Preparation of Assay Components
* The Rinse Solution Concentrate must be diluted before
use; pour the vial contents into a large squirt bottle and
add 450 ml of di-water to make the rinse solution.
* The other Lucentis Assay reagents are provided ready
to use.
* Calibrator Dilution: 100ng/ml 50/ml, 25ng/ml, 12.5ng/
ml, 6.25ng/ml, and 3.125ng/ml
Storage of Assay Components
* Store the reagents refrigerated at 2-8º C (36-46º F) Close
the reagents vials when not in use.
* After dilution, store the rinse solution at room
temperature.
* Store the microtiter strips/wells at room temperature or
refrigerated in the airtight bag with provided desiccant.
* Do not freeze the reagents or the microwell strips. Avoid
prolonged exposure to temperatures above 32º C.
* When stored as directed, the reagents and microwell
strips are stable until the expiration date on the kit.
Improper storage of reagents and microwell strips can
affect assay performance.
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Note: Calibrator 100ng/ml should be diluted using the
negative standard calibrator provided to the following
concentrations:
a) 100ng/ml b) 50ng/ml c) 25ng/ml d) 12.5ng/ml
e) 6.25ng/ml f) 3.125ng/ml
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5 Preparation and Storage of
Assay Components
6 Precautions
* Do not use the kit after the expiration date.
* Stop Solution contains 3N hydrochloric acid, which causes
burns and is harmful, if inhaled, swallowed, or absorbed. In
9 Assay Procedure
1. Bring all specimens and assay components to room
A representative standard curve is exhibited in the
following:
temperature (20-25º C , 68-77º F) and swirl each vial.
Ensure that the rinse solution has been prepared by diluting
the Rinse Solution Concentrate.
* Handle all materials and patient samples as if potentially
2. Place Lucentis Microwells securely in the holder for each
infectious.
sample, control and standard to be run.
* Always wear gloves when handling reagents and samples.
Note: If the plate is not used completely, place the non-used
strips/wells back in the bag with the desiccant and seal
7 Specimen Collection, Preparation,
Note:
For reliable and consistent results, perform the following
and Storage
steps
at a steady pace without interruptions.
Either serum or plasma can be used with the Lucentis
3.
Dispense
60ul of sample (plasma), control, standards and
Assay Kit. Samples may exceed the calibration range of
calibrators
into each well.
the assay kit. Pre-dilution of the samples with wash buffer
4.
Place
the
plate
on a shaker for 45 minutes.
may be required. Serum or plasma samples may be stored
5.
Flick
out
the
liquid
contents of the plate into an appropriate
for a few days at 2-8º C. Samples can be frozen at -20º C
waste
container.
Add
75ul of Lucentis Enzyme conjugate. •
for extended time periods. Multiple freeze/thaw cycles of•
6.
Place
plate
on
a
shaker
for 15 minutes.
Note: the standard curve is for the purpose of illustration
samples should be avoided.
7. Flick out the liquid contents of the plate into an appropriate
only and should not be used to calculate unknowns.
8 Calibration and Quality Control
waste container. Wash all the wells approximately 7-10
11 Limitations
Calibrate and run controls with each batch of samples.
times under cold running tap water. Flick out the tap water
* TBD
Run calibrators and controls in duplicate until satisfactory
after each wash.
precision is established. Analyzed control values and trends
12
Cross-reactivity/ Interference
8. Fill each well to rim with rinse solution. Flick out the rinse
using appropriate statistical methods.
*
No
Crossreactivity with: Rab-IgG, Human-IgG,
solution, then pound the plate dry on an absorbent towel.
Mouse-IgG,
Rat-IgG and Pig-IgG.
9. Add 100ul of Substrate/Chromogen Reagent to each well.
Tap the plate gently to mix. Incubate 30 minutes at room
temperature.
10. Add 50ul of Stop Solution. Tap the plate gently to mix.
11. Immediately read the plate on a microtiter plate reader at
10 Calculation of Results
1. Calculate the average Absorbance at 450nm value for each
sample and standard.
• 2. Construct a standard curve by plotting the average•
Due to the biological properties of Lucentis and
Avastin this assy with detect both compounds.
DO NOT quantitate Lucentis samples against
Avastin Kit and Vice Versa
absorbance for each calibrator against its concentration
(ng/ml) on graph paper. Plot absorbance on the vertical (y)
axis and concentration on the horizontal (x) axis.
3. Using the average absorbance value for each sample,
determine the corresponding Lucentis concentration from
the standard curve.
Note: Multiply the sample dilution factor with the
corresponding sample concentration from the graph to
obtain the actual sample concentration
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