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Alpha Diagnostic Intl (www.4adi.com) 6250/80827A
Mouse haptoglobin ELISA KIT Cat. No. 6250 DILUTION OF SAMPLES
Samples containing more than 125 ng/ml HAPTOGLOBIN should be
further diluted and re-tested. The results obtained should be multiplied
Kit Components, 96 tests
by the appropriate dilution factor. It is possible to use normal saline or
Anti-Mouse haptoglobin coated strip plate
PBS for sample dilution if larger volumes of samples are taken for dilution
Mouse haptoglobin Reference Standard (2 ug/ml),
CALCULATION OF RESULTS
Calculate the mean absorbance for each duplicate. Draw the standard
Anti-mouse haptoglobin-HRP Conjugate, 11 ml
curve on semi-log graph paper by plotting net absorbance values of
standards against appropriate HAPTOGLOBIN concentrations. Read off
the HAPTOGLOBIN concentrations of the control and patient samples.
Multiply the values by the dilution factor of the samples. If samples were
diluted 1:20K then the values must be multiplied by 20,000 and results
PERFORMANCE CHARACTERISTICS Detection Limit: The minimum HAPTOGLOBIN concentration
detectable using this assay is below 1 ng/ml. The detection limit is
defined as the value deviating by 2 SD from the zero standard.
NTRODUCTION
The liver produces haptoglobin and secretes it into the blood. When red
Expected Values: Mouse HAPTOGLOBIN levels in serum may vary
blood cel s are destroyed, the hemoglobin is released. Haptoglobin binds
from 0.1-2 mg/ml. Each laboratory should establish testing ranges for the
to the released hemoglobin. Macrophages wil then bring the haptoglobin-
hemoglobin complex to the liver, where the haptoglobin and hemoglobin
are separated and the iron is recycled. This process destroys the
Specificity: The antibodies used in this kit are specific for mouse
haptoglobin. When red blood cel s are actively being destroyed, the rate
haptoglobin and have shown no cross-reactivity with other serum
of haptoglobin destruction by the liver wil outpace the rate at which new
haptoglobin is created, and the levels of haptoglobin in the blood wil
Species Crossreactivity: Cross-reactivity was tested with animal sera
at dilutions of 1:100. Rat, Dog, G. pig, Horse, sheep and goat haptoglobin
Haptoglobin is an acute phase reactant protein. Its level increases during
sera did not show good reactivity. Rabbit, bovine, goat, sheep, human,
acute conditions such as infection, injury, tissue destruction, some
monkey sera were significantly positive. Since we only tested the sera and
cancers, burns, surgery, or trauma. Its level decreases during such
not the purified haptoglobin, it is not possible ascertain the extent of
conditions as chronic liver disease, hematoma, hemolytic anemia.
crossreactivity. But the above information should provide some measure of anti-
mouse haptoglobin reactivity with the other species.
ADI’s mouse Haptoglobin ELISA provides is a rapid, specific and
sensitive assay for measuring mouse Haptoglobin in serum or other
Alpha Diagnostic Intl (www.4adi.com) 6250/80827A
Alpha Diagnostic Intl (www.4adi.com) 6250/80827A
WORKSHEET OF TYPICAL ASSAY PRINCIPLE OF THE TEST
Mouse HAPTOGLOBIN ELISA kit is based on binding of Mouse
Calculated
HAPTOGLOBIN from samples to two antibodies, one immobilized on the
Stds/samples
microtiter wel plates, and other conjugated to the enzyme horseradish
peroxidase. After a washing step, chromogenic substrate is added and colors
Negative Diluent Control
developed. The enzymatic reaction (color) is directly proportional to the amount
of HAPTOGLOBIN present in the sample. Adding stopping solution terminates
Standard A 1.95 ng/ml
the reaction. Absorbance is then measured on a microtiter wel ELISA reader at
450 nm. and the concentration of HAPTOGLOBIN in samples and control is
Standard B 3.9 ng/ml Standard C 7.8 ng/ml MATERIALS AND EQUIPMENT REQUIRED Standard D 15.6 ng/ml
Adjustable micropipet (5-1000 ul) and multichannel pipet with disposable plastic
tips. Reagent troughs, plate washer (recommended) and ELISA plates Reader.
Standard E 31.2 ng/ml Standard F 62.5 ng/ml PRECAUTIONS AND SAFETY INSTRUCTIONS
The Mouse HAPTOGLOBIN ELISA Kit is for research use only.
Standard G 125 ng/ml
Stop Solution contains 1% sulfuric acid. Fol ow good laboratory practices, and
avoid ingestion or contact of any reagent with skin, eyes or mucous
= 7.8 ug/ml
membranes. Al reagents may be disposed of down a drain with copious
NOTE: These data are for demonstration purpose only. A complete
standard curve must be run in every assay to determine sample values.
MSDS for TMB, sulfuric acid, if not already on file, can be requested or obtained
Each laboratory should determine their own normal reference values.
SPECIMEN COLLECTION AND HANDLING
Col ect blood by venipuncture, al ow clotting, and separating the serum by
centrifugation at room temperature. Do not heat inactivate the serum. If sera
can not be immediately assayed , store frozen for up to six months. Avoid
repeated freezing and thawing of samples. It is also possible to use plasma for
REAGENT PREPARATION
1. Dilute the Sample Diluent 1:10 with water (10 ml diluent in 90ml water).
Dilute only the required reagent. Store diluted solution at 2-8o C for 3-4
2. Dilute Wash Buffer (10x stock). Dilute the entire 60 ml with distil ed or
deionized water to 540 ml water (total volume 600 ml). Store at room
temperature for the entire use of the kit.
3. Standard preparation-it is provided as lyophilized stock. See detailed
A typical assay Curve (do not use this for calculating sample values)
Alpha Diagnostic Intl (www.4adi.com) 6250/80827A
Alpha Diagnostic Intl (www.4adi.com) 6250/80827A
STORAGE AND STABILITY
Label or mark the microtiter wel strips to be used on the plate.
The microtiter wel plate and al other reagents, if unopened, are stable at 2-8oC
3. Pipet 100 ul standards and diluted samples into appropriate wel s.
until the expiration date printed on the label. After opening the kit components,
Mix gently, and incubate at room temperature (20-25oC) for 60
the shelf life is approximately 2 months.
minutes on an orbital shaker (100-150 rpm). If an automated shaker
is not available, the plate can be mixed manual y every few minutes.
TEST PROCEDURE (ALLOW ALL REAGENTS TO REACH ROOM
4. Remove or aspirate the plate contents and wash the wells 4-5 times with 300 ul of 1x wash buffer using an automated washer. If
1. Reconstitute the lyophilized Reference Standard with the amount of
washing manual y then dump the plate contents and tap over paper
distil ed water indicated on the vial label. The stock concentration wil
towels , add wash buffer, shake the contents of 5-10 seconds and
be 2 ug/ml. Store unused Reference Standard at -20°C.
repeat the steps. Tap the plate over fresh paper towels between
2. Prepare liquid standards using the fol owing dilution scheme. Label 8
microcentrifuge tubes as 125, 62.5, 31.2, 15.6, 7.8, 3.9, 1.95, and 0
5. Pipette 100 ul of Ab-enzyme conjugate into each wel . Mix gently,
and incubate for 30 minutes at room temperature as in step 3.
6. Wash the wells 4-5 times as in step 4. Tap the plate over fresh
31.25 ul of 2
paper towels to remove traces of liquid from the last washing step.
Std G (125 ng/ml) ug/ml stock
6. Add 100 ul of TMB Substrate into each wel . Mix gently. Cover the Std F (62.5 ng/ml) 250 ul of Std G
plate and incubate for 20 minutes at room temperature. Blue color
develops. This step can be reduced or increased by + 5 minutes to keep
Std E (31.2 ng/ml)
the color within reading range. If your ELISA reader cannot read above
Std D (15.6 ng/ml)
A450 of 2.00 then reduce the incubation time.
Std C (7.8 ng/ml) 250 ul of Std D
7. Stop the reaction by adding 100 ul of stop solution to al wel s. Mix Std B (3.9 ng/ml) 250 ul of Std C Std A (1.95 ng/ml)
8. Measure the absorbance at 450 nm using an ELISA reader. Color is
stable for at least 30 minutes after stopping.
Notes: When preparing the serial dilutions of the standards, gently mix NOTES: Read instructions careful y before the assay. Do not al ow
the standards for 5-10 seconds and then take aliquots to make further
reagents to dry on the wel s. Careful aspiration of the washing solution is
dilutions. Fol owing the above dilution scheme, you wil have 250 ul of al
essential for good assay precision. Since timing of the incubation steps
standards (B-F) and 500 ul of Std. A. You would need 200 ul of each
is important to the performance of the assay, pipet the samples without
interruption and it should not exceed 5 minutes to avoid assay drift. If
more than one plate is being used in one run, it is recommended to
Diluting the mouse serum samples 1:20,000-1:40,000 (use 1x Sample
include a standard curve on each plate. The unused strips should be
Diluent) wil bring most samples into the testing range. For those testing
stored in a sealed bag at 2-8oC. Addition of the HRP substrate solution
out of the range dilute accordingly. We recommend the fol owing dilution
starts a kinetic reaction, which is terminated by dispensing the stopping
solution. Therefore, keep the incubation time for each wel s the same by
1. Take 10 ul of samples and 990 ul of 1x diluent and mix for 1:100
adding the reagents in identical sequence. Plate readers measure
absorbance vertical y. Do not touch the bottom of the wel s.
2. Take 10 ul of 1:100 dilution and 990 ul of 1x diluent for 1:10,000
3. take 200 ul of 1:10,000 dilution and 200 ul of diluent for 1:20,000
Alpha Diagnostic Intl (www.4adi.com) 6250/80827A
Alpha Diagnostic Intl (www.4adi.com) 6250/80827A
Express Scripts/Medco Prescription Plan Information For Drug Coverage Review, Prior Authorization Process and Personalized Medicine Information The endowed health plan offers faculty and staff members and their families a very comprehensive prescription drug program at relatively low costs to the consumer. Prescription drugs have become an important part of health plan coverage. The prescrib
Quesito 1 1) In merito al lotto n. 817 IMIGLUCERASI si specifica che il prezzo a base d’asta da Voi indicato è di qualche millesimo inferiore a quello indicato nelle liste di rimborsabilità AIFA. Risposta: Si rettifica il lotto 817 nel seguente modo: prezzo base d’asta unitaria: € 664,38 Importo complessivo annuo: € 143.506,08 Importo cauzione provvisoria: € 5.740,24 2) Ste
ELISA kits available from ADI: 
WORKSHEET OF TYPICAL ASSAY