CALBIOTECH Beta-2 Microglobulin ELISA Kit Instructions

CALBIOTECH Beta-2 Microglobulin ELISA Kit

Product Information

Specifications

• Catalog Number: BM010T (96 Tests)
• Intended Use: Quantitative determination of Beta-2 Microglobulin (B2MG) Concentration in Human Serum
• Storage: 2-8°C
• Kit Size: 96 Tests

Product Usage Instructions

Materials Provided:

• Microwells coated Murine monoclonal anti-B2 MG antibody
• B2MG Reference Standards: 0, 0.625, 1.25, 2.5, 5, and 10
• Sample Diluent, 100 ml
• Enzyme Conjugate Reagent, 22 ml
• TMB Reagent (One-Step), 11 ml
• Stop Solution (1N HCl), 11 ml
• Wash concentrate 20X: 1 bottle

Materials Not Provided (Required for Usage):

• Distilled or deionized water
• Precision pipettes
• Disposable pipette tips
• ELISA reader capable of reading absorbance at 450nm
• Absorbance paper or paper towel
• Graph paper

Storage and Stability:

• Store the kit at 2-8°C
• Keep microwells sealed in a dry bag with desiccants
• The reagents are stable until expiration of the kit
• Avoid exposing test reagents to heat, sun, or strong light

Warnings and Precautions:

• Potential for Research Use Only. Not for use in diagnostic procedures.
• For Laboratory use only.
• Avoid internal or external use in humans or animals.
• Avoid eating or drinking within the work area.
• Always wear gloves and a protective lab coat.
• Avoid pipetting by mouth. Handle all specimens and reagents as potentially infectious.

Frequently Asked Questions (FAQ)

• Q: Can I use serum samples demonstrating gross lipemia, gross hemolysis, or turbidity with this test?
  A: No, serum samples demonstrating gross lipemia, gross hemolysis, or turbidity should not be used with this test.

• Q: What is the critical aspect of the wash procedure mentioned in the limitations?
  A: Insufficient washing can result in poor precision and falsely elevated absorbance readings.

• Q: What is the intended use of the Beta 2 Microglobulin ELISA Kit?
  A: The kit is intended for the quantitative determination of Beta-2 Microglobulin (B2MG) Concentration in Human Serum.

INTENDED USE

The Calbiotech, Inc (CBI) Beta 2 Microglobulin ELISA Kit is intended for the quantitative determination of Beta-2 Microglobulin (B2MG) Concentration in Human Serum.

SUMMARY AND EXPLANATION

Human B-2 Microglobulin (B2MG) is an 11.8 kD protein identical to the light chain of the HLA-A, -B, and -C antigen. B2MG is expressed on nucleated cells, and is found at low levels in the serum and urine of normal individuals. B2MG concentrations are increased in inflammatory diseases, some viral diseases, renal dysfunction, and autoimmune diseases. Several publications are available that explain the interpretation of B2MG serum levels in assessing the status of individuals with various clinical conditions.

PRINCIPLE OF THE TEST

The B2MG ELISA test is based on the principle of a solid-phase enzyme-linked immunosorbent assay. The assay system utilizes a unique monoclonal antibody directed against a distinct antigenic determinant on the intact B-2 Microglobulin molecule. Mouse monoclonal anti- B2MG antibody is used for solid phase immobilization (on the microtiter wells). A sheep anti-B2MG antibody is in the antibody-enzyme (horseradish peroxidase) conjugate solution. The diluted test sample is allowed to react first with the immobilized antibody for 30 minutes at 37°C. The sheep anti- B2MG-HRP conjugate is then added and reacted with the immobilized antigen for 30 minutes at 37°C, resulting in the B2MG molecules being sandwiched between the solid phase and enzyme-linked antibodies. The wells are washed with water to remove unbound-labeled antibodies. A solution of TMB Reagent is added and incubated for 20 minutes at room temperature, resulting in the development of a blue color. The color development is stopped with the addition of Stop Solution, changing the color to yellow. The concentration of B2MG is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm.

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
6. Graph paper

STORAGE AND STABILITY

1. Store the kit at 2-8 C.
2. Keep microwells sealed in a dry bag with desiccants.
3. The reagents are stable until the expiration of the kit.
4. Do not expose test reagents to heat, sun or strong light.

WARNINGS AND PRECAUTIONS

1. Potential for Research Use Only. Not for use in diagnostic procedures.
2. For Laboratory use.
3. Not for Internal or External Use in Humans or Animals.
4. There should be no eating or drinking within the work area.
5. Always wear gloves and a protective lab coat.
6. No pipetting should be done by mouth. Handle all specimens and reagents as potentially infectious and biohazardous.
7. Do not add sodium azide to samples as a preservative.
8. Do not use external controls containing sodium azide.
9. Use disposable pipette tips to avoid contaminating chromogenic substrate reagent. Discard reagent if it turns blue.
10. Do not pour the chromogenic substrate back into container after use.
11. Do not freeze reagents.
12. Do not mix reagents from different kit lot numbers.
13. Keep reagents out of direct sunlight.
14. Handle stop reagent with care, since it is corrosive.
15. Bring all reagents to room temperature.
16. Viscous forensic samples should always be diluted in phosphate-buffered saline or distilled water prior to pipetting.
17. Ensure the bag containing the micro-plate strips and desiccant is sealed well, if only a few strips are used.

SPECIMEN COLLECTION AND HANDLING

1. Blood should be drawn using standard venipuncture techniques and the serum should be separated from the red blood cells as soon as practical. Avoid grossly hemolytic, lipidic or turbid samples.
2. Typicaly, specimens should be capped and may be stored for up to 48 hour at 2-8 C prior to assaying. Specimens held for a longer time can be frozen at -20 C for up to 6 months prior to assay. Thawed samples should be inverted several times to mix prior to testing.
3. Collect urine samples and store at 2-8 C for up to 5 days or at -20 C for longer periods. Urine samples are diluted 1:10 by adding 50 l  urine to 450l sample diluent. Use same assay procedure as for serum test.

REAGENT PREPARATION

Prepare 1X Wash buffer by adding the contents of the bottle (25 ml, 20X) to 475 ml of distilled or deionized water. Store at room temperature (20-25  C).

PREPERATION FOR ASSAY

1. All reagents should be brought to room temperature (20-25 C) before use. All reagents should be mixed by gently inverting or swirling prior to use. Do not induce foaming.
2. Reconstitute each lyophilized standard with 1.0 ml-distilled water. Allow the reconstituted material to stand for at least 20 minutes and mix gently. Reconstituted standards will be stable for up to 30 days when stored sealed at 2-8 C.

ASSAY PROCEDURE FOR SERUM AND PLASMA

1. Samples of patient serum, plasma and control serum need to be diluted before use for best results. Prepare a series of small tubes (such as 1.5 ml microcentrifuge tubes) and mix 10 l serum with 1.0 ml Sample Diluent (101 fold dilution). Do not dilute the standards, they have already been pre-diluted
   101 fold.

2. Secure the desired number of coated wells in the holder.

3. Dispense 20 ul of standards, diluted specimens, and diluted controls into appropriate wells.

4. Dispense 200 ul of Sample Diluent into each well.

5. Thoroughly mix for 30 seconds. It is very important to mix them completely.

6. Incubate at 37°C for 30 minutes.

7. Remove the incubation mixture by flicking plate contents into a waste container.

8. Remove liquid from all wells. Wash wells three times with 300 uL of 1X wash buffer. Blot on absorbance paper or paper towel.

9. Strike the wells sharply onto absorbent paper or paper towels to remove all residual liquid droplets.

10. Dispense 200 ul of Enzyme Conjugate Reagent into each well. Gently mix for 10 seconds.

11. Incubate at 37°C for 30 minutes.

12. Remove the contents and wash the plate as described in step 7, 8, and 9.

13. Dispense 100 ul TMB Reagent into each well.

14. Gently mix for 10 seconds.

15. Incubate at room temperature in the dark for 20 minutes.

16. Stop the reaction by adding 100 ul of Stop Solution to each well.

17. Gently mix for 10 seconds. It is important to make sure that all the blue color changes to yellow color completely.

18. Read absorbance at 450m with a microtiter well reader within 15 minutes.

CALCULATION OF RESULTS FOR SERUM AND PLASMA

1. Calculate the mean absorbance value (A450) for each set of reference standards, controls and patient samples.
2. Construct a standard curve by plotting the mean absorbance obtained from each reference standard against its concentration in ug/ml on graph paper, with absorbance values on the vertical or Y axis, and concentrations on the horizontal or X axis.
3. Use the mean absorbance values for each specimen to determine the corresponding concentration of B2MG in ug/ml from the standard curve.

ASSAY PROCEDURE FOR URINE TEST

1. Urine Samples need 10 fold Dilution with the Sample Diluent (i.e. 50 ul urine + 450 ul Sample Diluenti).
2. Follow the same Assay Procedure for the Serum/Plasma Test from step 2 to step 18.

CALCULATION OF RESULTS FOR URINE TEST

1. Calculate the mean absorbance value (A450) for each reference standard, control and patient sample.
2. Construct a standard curve by plotting the mean absorbance obtained from each reference standard against its concentration in ug/ml on graph paper, with absorbance values on the vertical or Y axis, and concentrations on the horizontal or X axis.
3. Use the mean absorbance values for each specimen to determine the corresponding concentration of ß2MG in ug/ml. Divide the calculated values by 10.1 (Since the B-2 Microglobulin standards have been prediluted 101 fold, the results obtained from urine samples should be further divided by 10.1).
4. For instance, if the calculated value for a urine sample from the standard curve is 2.40 ug/ml; then the real value will be 2.40 ug/ml ÷ 10.1 = 0.238 ug/ml.3.

LIMITATIONS OF THE PROCEDURE

1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the package insert instructions and with adherence to good laboratory practice.
2. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
3. Serum samples demonstrating gross lipemia, gross hemolysis, or turbidity should not be used with this test.

Example of a Standard Curve
Results of a typical standard run with absorbency readings at 450 nm are shown in the Y axis against B2MG concentrations shown in the X axis. This standard curve is for illustration only, and should not be used to calculate unknowns. Each user should obtain his or her own data and standard curve.

REFERENCES

• Berggard | and Beam AG. 1968. Isolation and properties of a low molecular weight B-2 globulin occurring in human biological fluids. J Biol Chem 243: 4095-4103.
• Grey HM, Kubo RT, Colon SM, Poulik MD, Cresswell P, Springer T, Turner M and Stronminger JL. 1973. The small subunit of HL-A antigens is B2-microglobulin. J Exp Med 138: 1608-1612. Nakamuro K, Tanigaki N and Pressman D., 1973. Multiple common properties of human B2-microglobulin and the common portion
• fragment derived from HL-A antigen molecules. Proc Natl Acad Sci 70: 2863-2865.
• Evrin PE and Wibell L., 1972. The serum levels and urinary excretion of B2-microglobulin in apparently healthy subjects. Scand J Clin Lab Invest 29:69-74.
• Crisp AJ, Coughlan RJ, Mackintosh D, Clark B, and Panayi GS. 1983. B2-microglobulin plasma levels reflect disease activity in rheumatoid arthritis. J Rheumatol 10: 954-956.

Cat#: BM010T (96 Tests)
For Order and Inquiries, please contact
Calbiotech Inc., 1935 Cordell Ct., El Cajon, CA 92020
Tel 619-660-6162, Fax 619-660-6970,
www.calbiotech.com

References

• Calbiotech - Contract Development and Manufacturing for Life Science
• Calbiotech - Contract Development and Manufacturing for Life Science

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