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Microsoft word - 6250.doc

ELISA kits available from ADI:
Human: Adiponectin (Acrp30 and gAcrp30), Albumin, Aldosterone, AFP,
beta-amyloid 1-40/42, Angiogenin, Angiopoietin-2, beta-2M, BMP-7, C- peptide, CRP, Cox-2, Ferritin, PSA, fPSA, GH, IgG, IgM, IgE, IgG1, IgG4, Insulin, NSE, CA125, CA199, CA242, PAP, Resistin, SHBG, LH, FSH, TSH, T3, T4, and Steroid ELISA kits (cortisol, estradiol, testosterone, Mouse Haptoglobin
Monkey: IgM, IgG, IgA, IgE
Rat: Albumin, CRP, IgG, IgM, Alpha-1- Acid glycoprotein
Mouse: Albumin, IgA, IgG, IgG1, IgG2a, IgG2b, IgG3, IgE, IgM, Leptin,
ELISA KIT Cat. No. 6250
Resistin, Acrp30, CRP, Haptoglobin, TNF-alpha Autoimmune Antibody detection kits for ANA, ssDNA, dsDNA, Histone,
Sm, RNP, SSA, SSB, Scl70, Ovalbumin, Cardiolipin, CIC For Quantitative Determination of Haptoglobin
Chicken: IgG, IgM, IgY, Ovalbumin
in Mouse Serum
Turkey: IgG
Bovine: Albumin, IgG, IgM, Lactoferrin, Transferrin

Pig: Albumin, IgG, IgM
Dog: CRP, IgG, IgM

Cat: IgG, IgM
Goat: IgG
Rabbit: CRP, IgG
6203 Woodlake Center Drive • San Antonio• Texas 78244 • USA. Sheep: IgG
Phone (210) 561-9515 • Fax (210) 561-9544 See Details at the web site or Contact ADI Alpha Diagnostic Intl ( 6250/80827A Mouse haptoglobin ELISA KIT Cat. No. 6250
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

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 ( 6250/80827A Alpha Diagnostic Intl ( 6250/80827A WORKSHEET OF TYPICAL ASSAY
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
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
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.
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 ( 6250/80827A Alpha Diagnostic Intl ( 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 ( 6250/80827A Alpha Diagnostic Intl ( 6250/80827A


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