Instructions for use

ß2-Microglobulin ELISATM E-4600R

β2-MICROGLOBULIN ELISAINTENDED USE
For the direct quantitative determination of β2-Microglobulin (β2-M) by an enzyme immunoassay in humanserum.
PRINCIPLE OF THE TESTThe principle of the following enzyme immunoassay test follows the typical competitive binding scenario.
Competition occurs between an unlabelled antigen (present in standards, controls and samples) and an
enzyme-labelled antigen (conjugate) for a limited number of antibody binding sites on the microplate. The
washing and decanting procedures remove unbound materials. After the washing step, the enzyme substrate isadded. The enzymatic reaction is terminated by addition of the stopping solution. The absorbance is measured
on a microtiter plate reader. The intensity of the colour formed is inversely proportional to the concentration ofβ2-M in the sample. A set of standards is used to plot a standard curve from which the amount of β2-M insamples and controls can be directly read.
PROCEDURAL CAUTIONS AND WARNINGS
1. Users should have a thorough understanding of this protocol for the successful use of this kit. Reliable
performance will only be attained by strict and careful adherence to the instructions provided.
2. Control materials or serum pools should be included in every run at a high and low level for assessing thereliability of results.
3. When the use of water is specified for dilution or reconstitution, use deionized or distilled water.
4. In order to reduce exposure to potentially harmful substances, gloves should be worn when handling kit
reagents and human specimens.
5. All kit reagents and specimens should be brought to room temperature and mixed gently but thoroughly
before use. Avoid repeated freezing and thawing of reagents and specimens.
6. A standard curve must be established for every run.
7. The kit controls should be included in every run and fall within established confidence limits.
8. Improper procedural techniques, imprecise pipetting, incomplete washing as well as improper reagent
storage may be indicated when assay values for the controls do not reflect established ranges.
9. When reading the microplate, the presence of bubbles in the wells will affect the optical densities (ODs).
Carefully remove any bubbles before performing the reading step.
10. The substrate solution (TMB) is sensitive to light and should remain colourless if properly stored. Instability
or contamination may be indicated by the development of a blue colour, in which case it should not beused.
11. When dispensing the substrate and stopping solution, do not use pipettes in which these liquids will comeinto contact with any metal parts.
12. To prevent contamination of reagents, use a new disposable pipette tip for dispensing each reagent,sample, standard and control.
13. Do not mix various lot numbers of kit components within a test and do not use any component beyond theexpiration date printed on the label.
14. Kit reagents must be regarded as hazardous waste and disposed of according to national regulations.
LIMITATIONS
1. All the reagents within the kit are calibrated for the direct determination of β2- M in human serum. The kit
is not calibrated for the determination of β2-M in saliva, plasma or other specimens of human or animal
origin.
2. Do not use grossly hemolyzed, grossly lipemic, icteric or improperly stored serum.
3. Any samples or control sera containing azide or thimerosal are not compatible with this kit, as they may
lead to false results.
4. Only Standard A may be used to dilute any high serum samples. The use of any other reagent may lead tofalse results.
SAFETY CAUTIONS AND WARNINGS
POTENTIAL BIOHAZARDOUS MATERIAL
Human serum that may be used in the preparation of the standards and controls has been tested and found to
be non- reactive for Hepatitis B surface antigen and has also been tested for the presence of antibodies to HCVand Human Immunodeficiency Virus (HIV) and found to be negative. No test method however, can offer
complete assurance that HIV, HCV and Hepatitis B virus or any infectious agents are absent. The reagentsshould be considered a potential bio-hazard and handled with the same precautions as applied to any blood
specimen.
CHEMICAL HAZARDS
Avoid contact with reagents containing TMB, hydrogen peroxide and sulfuric acid. If contacted with any of thesereagents, wash with plenty of water. TMB is a suspected carcinogen.
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2/8SPECIMEN COLLECTION AND STORAGE
Approximately 0.1 ml of serum is required per duplicate determination. Collect 4 - 5 ml of blood into anappropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4 °C
for up to 24 hours or at -10 °C or lower if the analyses are to be done at a later date. Consider all humanspecimens as possible biohazardous materials and take appropriate precautions when handling.
SPECIMEN PRETREATMENT
This assay is a direct system; no specimen pretreatment is necessary.
REAGENTS AND EQUIPMENT NEEDED BUT NOT PROVIDED
1. Precision pipettes to dispense 20, 50, 100, 150 and 300 μl.
2. Disposable pipette tips.
3. Distilled or deionized water.
4. Plate shaker.
5. Microplate reader with a filter set at 450 nm and an upper OD limit of 3.0 or greater* (see assay procedurestep 10).
REAGENTS PROVIDED
1. AA E-0030

Wash Buffer Concentrate – Requires Preparation X10

Contents:

One vial containing buffer with a non-ionic detergent and a non-mercury preservative.

Volume:50 ml/vial

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.

Preparation:

Dilute 1:10 in distilled or deionized water before use. If the whole plate is to be used dilute
50 ml of the wash buffer concentrate in 450 ml of water.
TMB Substrate - Ready To Use

2. AA E-0055
Contents:

One vial containing tetramethylbenzidine and hydrogen peroxide in a non-DMF or DMSO
containing buffer.

Volume:16 ml/vial

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.
Stopping Solution - Ready To Use

3. AA E-0080
Contents:

One vial containing 1M sulfuric acid.

Volume:6 ml/bottle

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.

Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.

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4. Standards and Controls- Ready To Use.
Listed below are approximate concentrations, please refer to vial labels for exact concentrations:Cat. no.

SymbolStandard A

Concentration
0 mg/l2.0 mlStandard B

0.2 mg/l

0.5 mlStandard C

0.6 mg/l

0.5 mlTM E-4604

Standard D

1.6 mg/l

0.5 mlTM E-4605

Standard E

4 mg/l

0.5 mlTM E-4606

10 mg/lTM E-4651

Standard F
Control 1

0.5 ml
0.5 mlTM E-4652

Control 2

TM E-4601
TM E-4602
TM E-4603

Standard

Refer to vial labels for expectedvalue and acceptable range!

Volume/Vial

0.5 mlContents:

β2-M in a protein-based buffer with a non-mercury preservative. Prepared by spiking buffer
with a defined quantity of β2-M.

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months in unopened vials or as indicated on label. Once opened, the standards should
be used within 14 days or aliquoted and stored frozen. Avoid multiple freezing and thawing
cycles.

5. TM E-4613
Contents:

Assay Buffer - Ready To Use
One vial containing a protein-based buffer with a non-mercury preservative.

Volume:15 ml/vial

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.
Mouse Anti-β2-M Antibody-Coated Break-Apart Well Microplate

6. TM E-4631- Ready To Use
Contents:

One 96 well (12x8) monoclonal antibody-coated microplate in a resealable pouch withdesiccant.

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.

7. TM E-4640

β2-M-Horseradish Peroxidase (HRP) Conjugate Concentrate – Requires
Preparation X50

Contents:

β2-M-HRP conjugate in a protein-based buffer with a non-mercury preservative.

Volume:0.4 ml/vial

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.

Preparation:

Dilute 1:50 in assay buffer before use (eg. 40 μl of HRP in 2 ml of assay buffer). If the wholeplate is to be used dilute 240 μl of HRP in 12 ml of assay buffer. Discard any that is left over.

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ASSAY PROCEDURE
Specimen Pretreatment: NoneAll reagents must reach room temperature before use. Standards, controls and specimen samples should beassayed in duplicate. Once the procedure has been started, all steps should be completed without interruption.
1.

Prepare working solutions of the β2-M-HRP conjugate and wash buffer.

2.

Remove the required number of well strips. Reseal the bag and return any unused strips to therefrigerator.

3.

Pipette 20 μl of each standard, control and specimen sample into correspondingly labelled wells induplicate.

4.

Pipette 100 μl of the conjugate working solution into each well.
(We recommend using a multichannel pipette).

5.

Incubate on a plate shaker (approximately 200 rpm) for 1 hour at room temperature.

6.

Wash the wells 3 times with 300 µl of diluted wash buffer per well and tap the plate firmly against
absorbent paper to ensure that it is dry (The use of a washer is recommended).

7.

Pipette 150 μl of TMB substrate into each well at

8.

timed intervals.

Incubate the plate on a plate shaker at room temperature for 15-20 minutes.
(or until Standard A attains dark blue colour for desired OD).

9.

Pipette 50 μl of stopping solution into each well at the same timed intervals as in step 7.

10.

Read the plate on a microplate reader at 450 nm within 20 minutes after addition of the stopping solution.
If the OD exceeds the upper limit of detection or if a 450nm filter is unavailable, a 405 or 415nm filter may
be substituted.
The optical densities will be lower, however, this will not affect the results ofspecimen/control samples.

CALCULATIONS
1. Calculate the mean optical density of each standard duplicate.
2. Draw a standard curve on semi-log paper with the mean optical densities on the Y-axis and the standard
concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5-parameter curveis recommended.
3. Calculate the mean optical density of each unknown duplicate.
4. Read the values of the unknowns directly off the standard curve.
5. If a sample reads more than 10 mg/l then dilute it with Standard A at a dilution of no more than 1:20. The
result obtained should be multiplied by the dilution factor.
TYPICAL TABULATED DATA
Sample data only. Do not use to calculate results.
StandardABCDEFUnknown

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OD 1
2.542
2.037
1.006
0.447
0.206
0.116
0.469

OD 2
2.558
2.043
1.021
0.441
0.212
0.112
0.456

Mean OD
2.550
2.040
1.014
0.444
0.209
0.114
0.453

Value (mg/l)
00.2
0.6
1.6
4
10
1.57

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TYPICAL STANDARD CURVE
Sample curve only. Do not use to calculate results.

2.6

OD 450nm2.1
1.6
1.1
0.6
0.1

0.2

0.6

1.6

410B2-M (mg/L)

PERFORMANCE CHARACTERISTICSSENSITIVITY
The lower detection limit is calculated from the standard curve by determining the resulting concentration ofthe mean OD of Standard A (based on 10 replicate analyses) minus 2 SD. Therefore, the sensitivity of the β2MICROGLOBULIN ELISA kit is 0.1 mg/l.
SPECIFICITY (CROSS-REACTIVITY)
The following compounds were tested for cross-reactivity with the β2-MICROGLOBULIN ELISA kit with β2-M
cross-reacting at 100%.
Compoundβ2-Microglobulin
Human IgG

% Cross-Reactivity
100< 0.00001

INTRA-ASSAY PRECISION
Three samples were assayed ten times each on the same Standard curve. The results (in mg/l) are tabulated
below:
Sample
123

Mean
0.78
3.43
15.63

SD
0.08
0.03
0.01

CV %
5.5
6.4
2.9

INTER-ASSAY PRECISION
Two samples were assayed ten times over a period of four weeks. The results (in mg/l) are tabulated below:
Sample
12

Mean
0.92
3.64

SD
0.09
0.14

CV %
9.5
3.8

RECOVERY
Spiked samples were prepared by adding defined amounts of β2-MICROGLOBULIN ELISA to two serum samples(1:1). The results (in mg/l) are tabulated below:
Sample1 Unspiked+4+12+32
2 Unspiked+4+12+32

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Obs. Result
1.70
3.21
7.94
17.05
2.51
3.25
8.14
17.69

Exp. Result
2.85
6.85
16.85
3.26
7.26
17.26

Recovery %
112.6
115.9
101.2
99.7
112.1
102.5

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LINEARITY
Two serum samples were diluted with Standard A. The results (in mg/l) are tabulated below:
Sample
1
1:5
1:10
1:20
2
1:5
1:101:20

Obs. Result
15.96
3.14
1.57
0.77
17.71
3.63
1.94
1.00

Exp. Result
3.19
1.60
0.80
3.54
1.77
0.89

Recovery %
98.4
98.1
96.3
102.5
109.6
112.4

EXPECTED NORMAL VALUESEach laboratory should collect data and establish their own range of expected normal values.
GroupMales (Age 24 - 70)
Females (Age 19 - 45)
Postmenopausal Females
Young Males and Females(Age 3 - 17)

N46
25147

Mean (mg/l)
1.58
1.46
1.61
1.13

Range (mg/l)
1.15 - 3.85
0.73 - 3.56
1.28 - 2.34
0.89 - 1.36

REFERENCES
1. Ling TGI, Mattiasson B. A General Study of the Binding and Separation in Partition Affi nity Ligand Assay.
Immunoassay of Beta 2-Microglobulin. J of Immunol Methods. 1983; 59(3):327–37.
2. Evrin PE, et al. A Turbidimetric Immunochemical Method for Determination of Serum β2-Microglobulin
Using a Centrifugal Analyzer. Clinica Chemica Acta. 1986; 155(2):151–7.
3. Curry R, et al. Rapid Semi-Quantitative Isolation of Beta-2- Microglobulin From Urine. J of ImmunolMethods. 1981; 47(3):365–73.
4. Francioli P, et al. Beta 2-Microglobulin and Immunodefi ciency in a Homosexual Man. New Engl J Med.
1982; 307(22):1402–3.
5. Martinez-Brú C, et al. Beta 2-Microglobulin and Immunoglobulins Are More Useful Markers of Disease
Progression in HIV Than Neopterin and Adenosine Deaminase. Ann Clin Biochem. 1999; 36:601.
6. Tachibana K, et al. A Two-Site Sandwich Radioimmunoassay of Beta 2-Microglobulin With Monoclonal
Antibodies. J of Immunol Methods. 1984; 75(1):43–51.
7. Uotila M, et al. Two-Site Sandwich Enzyme Immunoassay With Monoclonal Antibodies to Human AlphaFetoprotein. J of Immunol Methods. 1981; 42(1):11–5.
8. Brodsky FM, et al. Characterization of a Monoclonal Anti-Beta 2- Microglobulin Antibody and its Use in theGenetic and Biochemical Analysis of Major Histocompatibility Antigens. Eur J Immunol. 1979; 9(7):536–45.
9. Nilsson K, et al. Involvement of Lymphoid and Non-lymphoid Cells in the Production of β2-Microglobulin–aHomologue of the Constant Domains of IgG. Nature. 1973; 244:44.
10. Leclair K, et al. A Rapid Method for Isolation of Antigenically Active Human Cell Surface Antigens
Associated with Beta 2- Microglobulin Using a Monoclonal Antibody. J of Immunol Methods. 1981;
41(2):137–44.
11. O’ Reilly D, et al. A Simple, Precise and Sensitive Nephelometric Assay for Beta 2-Microglobulin in BodyFluids. J of Immunol Methods. 1983; 57(1–3):265–73.
12. Revillard JP, et al. Lyon Medical. 1979; 241:681.
13. Wibell L, et al. Serum 2 -Microglobulin in Renal Disease. Nephron. 1973; 10(5):320–31.
14. Burnett RW. Accurate Estimation of Standard Deviations for Quantitative Methods Used in ClinicalChemistry. Clin Chem. 1975; 21(13):1935–8.
15. Sönnerborg AB, et al. Elevated Neopterin and Beta 2-Microglobulin Levels in Blood and Cerebrospinal Fluid
Occur Early in HIV-1 Infection. AIDS. 1989; 3(5):277–83.
16. Domingo P, et al. Prognostic Value of Serum Beta 2-Microglobulin in HIV Infection. Lancet. 1992; 340:371.
17. Lacey JN, et al. Serum Beta 2-Microglobulin and Human Immunodefi ciency Virus Infection. AIDS. 1987;
1(2):123.
18. Bhalla RB, et al. Abnormally High Concentrations of Beta 2 Microglobulin in Acquired Immunodefi ciency
Syndrome (AIDS) Patients. Clin Chem. 1983; 29(8):1560.
19. Lambin P, et al. Neopterin and Beta 2-Microglobulin in Serum of HIV-Seropositive Subjects During a TwoYear Follow-Up. Clin Chem. 1988; 34(6):1367–8.
20. Fahey JL, et al. The Prognostic Value of Cellular and Serologic Markers in Infection with Human
Immunodefi ciency Virus Type 1. New Engl J Med. 1990; 322(3):166–72.
21. Check JH, et al. Falsely Elevated Steroidal Assay Levels Related to Heterophile Antibodies Against VariousAnimal Species. Gynecol Obstet Invest. 1995; 40(2):139–40.

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