Insulin, Rev.

1RC

5. Specificity
The antibodies employed in this kit cross react with bovine insulin (20-25%) and porcine insulin but not with
proinsulin of any species or any other insulin complexes.

6. Recovery
Samples have been spiked by adding Insulin solutions with known concentrations in a 1:1 ratio.
Expected value (µIU/ml)
9.85
Recovered (µIU/ml)
8.80
Percentage of Recovery
89.3
Insulin ELISA
41.1 40.4 98.3 Catalog No. IS130D (96 Tests)
53.7 54.2 100.9 INTENDED USE
The Calbiotech Insulin ELISA Kit is intended for the quantitative measurement Insulin in human serum or plasma.
REFERENCES
1. Ashby, J. and Frier, B.: Circulating C-Peptide: Measurement and Clinical Applications. Annals of Clinical Biochemistry. SUMMARY AND EXPLANATION
18:125, 1981 Insulin is the principal hormone responsible for the control of glucose metabolism. It is synthesized in the ß-cells of the
2. Beischer, W.: Proinsulin and C-Peptide in Humans. Hormones in Normal and Abnormal Human Tissues. Volume 3K, islets of Langerhans as the precursor, proinsulin, which is processed to form C-peptide and insulin. Both are secreted in
Fotherby and Pal, S., ed. (Berlin: Walter DeGruyter). pp. 1-43, 1983 equimolar amounts into the portal circulation. The mature insulin molecule comprises two polypeptide chains, the A chain
3. Beyer, J., Krause V., Cordes V.: C-Peptide: Its Biogenesis, Structure, Determination and Clinical Significance. and B chain (21 and 30 amino acids respectively). The two chains are linked together by two inter-chain disulphide
Giornale Italiano di Chimica Clinica 4 Supp. 9:22, 1979 bridges. There is also an intra-chain disulphide bridge in the A chain. Insulin concentrations are severely reduced in
4. Bonger, A. and Garcia-Webb, P.: C-Peptide Measurement: Methods and Clinical Utility. CRC Critical Reviews in insulin-dependent diabetes mellitus (IDDM) and some other conditions such as hypopituitarism. Insulin levels are raised
Clinical Laboratory Sciences. 19:297, 1984. in non-insulin-dependent diabetes mellitus (NIDDM), obesity, insulinoma and some endocrine dysfunctions such as
5. Chevenne D., Ruiz J., Lohmann L., [Link].: Immunoradiometric Assay of Human Intact Proinsulin Applied to Patients Cushing’s syndrome and acromegaly.
with Type 2 Diabetes, Impaired Glucose Tolerance, and Hyperandrogenism. Clinical Chemistry. 40/5:754, 1994
6. Bowsher R. R., Wolny J. D. and Frank B. H.: A Rapid and Sensitive Radioimmunoassay for the Measurement of PRINCIPLE OF THE TEST
Proinsulin in Human Serum. Diabetes. 41:1084, 1992 The Insulin ELISA is a solid phase two-site enzyme immunoassay. It is based on the direct sandwich technique in which
7. Kao P. C., Taylor R. L. and Service F. J.: Proinsulin by Immunochemiluminometric Assay for the Diagnosis of two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule. During
Insulinoma. Jorunal of Clinical Endocrinology and Metabolism. 78:1048, 1994 incubation insulin in the sample reacts with enzyme (HRP)-conjugated anti-insulin antibody and anti-insulin antibody
8. Dhahir F. J., Cook D. B. and Self C. H.: Amplified Enzyme-Linked Immunoassay of Human Proinsulin in Serum bound to micro-titration well. A simple washing step removes unbound enzyme labeled antibody. The bound HRP
(Detection Limit: 0,1 pmol/L). Clinical Chemistry. 38/2:227, 1992 complex is detected by reaction with TMB substrate. The reaction is stopped by adding acid to give a colorimetric
2008-12-18 endpoint that is read using ELISA reader.

MATERIALS PROVIDED 96 Tests

1. Microwell coated with Insulin MAb 12x8x1
2. Insulin Standard 1:1 vial ( ready to use) 2ml
3. Insulin Standards2-6: 5 vials ( ready to use) 1ml
4. Insulin Enzyme Conjugate: 1 vial 1ml
5. Assay Diluent: 1 bottle (ready to use) 14ml
6. TMB Substrate: 1 bottle (ready to use) 12ml
7. Stop Solution: 1 bottle (ready to use) 12ml
8. 20X Wash concentrate: 1 bottle 25ml

MATERIALS NOT PROVIDED

1. Distilled or deionized water
2. Precision pipettes
3. Disposable pipette tips
4. ELISA reader capable of reading absorbance at 450nm
5. Absorbance paper or paper towel
Cat#: IS130D (96 Tests) 6. Graph paper
For Order and Inquiries, please contact
STORAGE AND STABILITY
Calbiotech Inc.,
1. Store the kit at 2 - 8° C.
10461 Austin Dr, Spring Valley, CA, 91978
2. Keep microwells sealed in a dry bag with desiccants.
Tel (619) 660-6162, Fax (619) 660-6970,
3. The reagents are stable until expiration of the kit.
[Link]
4. Do not expose reagent to heat, sun, or strong light.
8°
2°
WARNINGS AND PRECAUTIONS
CEpartner4U, 3951DB; 13. NL. 1. Potential biohazardous materials:
tel: +31 (0)[Link])
 Insulin, Rev.1RC
The calibrator and controls contain human source components, which have been tested and found non-reactive for Example of Standard Data
hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However there is no test method
OD 450 nm Conc. µIU/mL
that can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent. These reagents
should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control/National Institutes Std 1 0.05 0
of Health manual, "Biosafety in Microbiological and Biomedical Laboratories" 1984. Std 2 0.11 6.25
2. This test kit is USA FDA exempt product.
3. Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit reagents are handled. Std 3 0.22 12.5
4. The components in this kit are intended for use as an integral unit. The components of different lots should not be Std 4 0.49 25
mixed.
5. It is recommended that standards, control and serum samples be run in duplicate. Std 5 1.00 50
6. Optimal results will be obtained by strict adherence to this protocol. Accurate and precise pipetting, as well as Std 6 2.11 100
following the exact time and temperature requirements prescribed are essential. Any deviation from this may yield
invalid data. EXPECTED VALUES
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
SPECIMEN COLLECTION HANDLING In a study conducted with apparently normal healthy adults, using the Insulin ELISA the following values are observed:
1. Collect blood specimens and separate the serum immediately. < 25 µlU/ml.
2. Specimens may be stored refrigerated at (2-8° C) for 5 days. If storage time exceeds 5 days, store frozen at (-20°
C) for up to one month. LIMITATIONS OF THE TEST
3. Avoid multiple freeze-thaw cycles. 1. The test results obtained using this kit serve only as an aid to diagnosis and should be interpreted in relation to the
4. Prior to assay, frozen sera should be completely thawed and mixed well. patient’s history, physical findings and other diagnostic procedures.
5. Do not use grossly lipemic specimens. 2. Do not use sodium azide as preservative. Sodium azide inhibits HRP enzyme activities.

REAGENT PRPARATION PERFORMANCE CHARACTERISTICS

1. 20X Enzyme Conjugate: Prepare 1X working dilution at 1:20 with assay diluent as needed, e.g. 0.1 ml of the stock 1. Correlation with a Reference ELISA kit:
conjugate in 1.9 ml of assay diluent is sufficient for 20 wells. The diluted conjugate has to be used the same day. A total of 62 sera were tested by this CBI ELISA and a reference ELISA kit. Results were as follows:
2. 20X Wash Buffer Concentrate: Prepare 1X wash buffer by adding the contents of the bottle to 475 ml of distilled Correlation Slope Intercept
water. Store 1X wash buffer at room temperature. 0.91 0.80 0.24
ASSAY PROCEDURE
Prior to assay, allow reagents to stand at room temperature. 2. Precision
Gently mix all reagents before use. Intra-Assay
1. Place the desired number of coated strips into the holder Mean Standard Coefficient of
Serum No. of Replicates
2. Pipette 25 µl of Insulin standards, control and patient’s sera into appropriate wells. µIU/ml Deviation Variation (%)
3. Add 100µl of working Insulin Enzyme Conjugate to all wells. 1 10 9.26 0.58 6.3
4. Thoroughly mix for 10 sec., it is important to have a complete mixing in this step. 2 10 7.01 0.57 8.1
5. Incubate for 60 minutes at room temperature (18-26° C).
6. Remove liquid from all wells. Wash wells three times with 300 µl of 1X wash buffer. Blot on absorbent paper Inter-Assay
towels. No. of Mean Standard Coefficient of
Serum
7. Add 100 µl of TMB substrate to all wells. Replicates µIU/ml Deviation Variation (%)
8. Incubate for 15 minutes at room temperature. 1 10 6.79 0.58 8.5
9. Add 50µl of stop solution to all wells. Shake the plate gently to mix the solution. 2 10 9.27 0.69 7.4
10. Read absorbance on ELISA Reader at 450 nm within 15 minutes after adding the stopping solution.
3. Linearity
CALCULATION OF RESULTS Two different patient samples were diluted with the "0" calibrator to 1:2, 1:4, 1:8. Insulin values were
The standard curve is constructed as follows: assayed and results were corrected with the dilution factor. The results of these dilution tests are as follows
1. Check Insulin standard value on each standard vial. This value might vary from lot to lot. Make sure you check the Original Value Percentage of Recovery
value on every kit. Serum (µIU/ml) 1/2 1/4 1/8
2. To construct the standard curve, plot the absorbance for the insulin standards (vertical axis) versus the insulin 1 30 95.1 91.5 88.6
standard concentrations in µIU/ml (horizontal axis) on a linear graph paper. Draw the best curve through the 2 71 106 95.5 105
points.
3. Read the absorbance for controls and each unknown sample from the curve. Record the value for each control or
4. Sensitivity
unknown sample.
The sensitivity was determined by calculating the mean plus 2SD of the standard zero point tested 20 times
4. Value above the highest point of the standard are retested after diluting with “0” standard.
in the same run.

Mean Standard Mean + 2SD

Serum No. of Replicates
(µIU/ml) Deviation (Sensitivity)
Zero
20 0.477 0.495 1.467
Standard