1Human Beta 2-microglobulin ELISA

MATERIAL REQUIRED BUT NOT SUPPLIED

Cat. No.: BA1010
Enzyme Immunoassay for the quantitative determination
Beta 2-microglobulin in human serum, plasma, and urine.

ofUrinary excretion of Beta 2-microglobulin (beta 2M) is a widely used test ofrenal proximal tubular function.1 Proximal tubular dysfunction leads to anincreased urinary concentration. 2The serum level of beta 2-microglobulin is determined by the glomerularfiltration rate and the rate of synthesis. Increased production, with raisedserum levels, is sometimes observed in malignancy--mainly at an advanced
state.2
A strong correlation between lupus nephritis (LN), disease activity, andserum beta 2-microglobulin (b2MG) was observed. Serum b2MG level canbe used as a valuable predictor for LN, clinical disease activity, and damage
score. 3

1.
2.
3.
4.
5.

WARNINGS AND PRECAUTIONS
1.
2.
3.
4.
5.
6.
7.

PRINCIPLE OF BETA 2-MICROGLOBULIN ELISA
The microtiter plate is coated with the antibody specifically binding the Beta
2-microglobulin. The human serum, plasma or urine is incubated in the platewith the capture antibody.
The specimen is washed out and the specifically bound protein is incubatedwith HRP-labelled detection antibody. Unbound reagent is then washedout. Horseradish peroxidase (HRP) bound in the complex reacts with thechromogenic substrate (TMB) creating the blue colour. The reaction isstopped by addition of STOP solution (H2SO4).
The absorbance values are measured at 450 nm (optionally 450/630 nm)
and are proportional to the concentration of the Beta 2-microglobulin thespecimen. The concentration of Beta 2-miroglobulin in unknown samples isdetermined from the calibration curve which is created by plotting theabsorbance values against the standard concentration values.

Glassware and test tubes
Microtiter plate washer
Precision pipettes (various volumes) with tips
Orbital shaker
Microtiter plate reader capable of measuring absorbanceat 450 nm or 450/630 nm with software for data generation

For research use only
For professional laboratory use
The reagents with different lot numbers should not be mixed
To prevent cross sample contamination, use disposable labware
and pipette tipsTo protect laboratory stuff, wear protective gloves andprotective clothing
The substrate solution should remain colourless, keep it
protected from light
The test should be performed at standard laboratory conditions(temperature 25oC ± 2oC).

STORAGE CONDITIONS
1.
2.

The kit must be stored at 2 – 8oC.
The opened components can be stored for one week at 2 – 8oC.

PREPARATION OF REAGENTS

Use new pipette tip for pipetting different reagents and samples
to prevent cross-contamination.
All reagents and samples should be allowed to reach thetemperature 25oC ± 2oC.

Preparation of Standards
Reconstitute lyophilized Human Beta 2-microglobulin Standard in Dilution
Buffer, for the volume information see the Certificate of Analysis. Let it
rehydrate for 15 min prior to use. The concentration of human Beta 2microglobulin in Master Standard is 40 ng/mL.
Prepare set of Standard solution as follows:
Use the Master Standard for serial dilution (as below). Mix each tube
thoroughly before the next transfer. The Dilution Buffer serves as Blank.
Volume of Standard

Kit Contents
Item
Antibody Coated Microtiter Plate
Antibody-HRP Conjugate
Master Standard (lyophilized)
Quality Control A (human serum, lyophilized)
Quality Control B (human serum, lyophilized)
Dilution Buffer
Wash Buffer 15x conc.
Substrate Solution
STOP Solution
v_01

Std1

Standard 40 ng/mL (lyophilized)

Dilution
Buffer1000 µL

Concentration

Std2

250 µL of Std1

250 µL

20 ng/mL

Std3

250 µL of Std2

250 µL

10 ng/mL

Std4

250 µL of Std3

250 µL

5 ng/mL

Std5

250 µL of Std4

250 µL

2.5 ng/mL

Std6250 µL of Std5

250 µL1.25 ng/mLBlank-200 µL

0 ng/mL40 ng/mL

Preparation of Quality Control A and B
Qty.
96 wells
13 mL
1 vial1 vial1 vial2x13 mL
50 mL
13 mL
13 mL

Reconstitute the lyophilized human serum Quality Controls indeionized/distilled water, for the volume information see the Certificate ofAnalysis. Let the QCs rehydrate for 15 min and dilute them 1:300 in Dilution
Buffer, prior to use, see Preparation of samples.

2

Preparation of Wash Buffer 1x3.

Prepare a working solution of Wash Buffer by adding 50 mL of Wash Buffer
15x conc. to 700 mL of deionized/ distilled water (dH2O). Mix well. Store at
4oC for two weeks or at -20oC for long term storage.

Accuracy

Dilution linearity
SampleDilution

Measuredconcentration(ng/mL)
2195
1048
495
235
1295
586
324
138

Expected
concentration(ng/mL)
1097
549
274
647
324
162

Yield(%)

Measuredconcentration(ng/mL)
1059
4186
2937
1934

Expected
concentration(ng/mL)
4059
2559
1809

Yield (%)

1

Preparation of samples

2x4x8xHuman serum, plasma or ur1ne may be used with this assay. For long-term
storage the samples should be frozen at minimum -70oC. Lipemic or
haemolytic samples may cause false results.
Recommended dilution of serum and plasma is 1:300. It is recommended
to use the two-step dilution.
Dilution A (15x) for both singlets and duplicates: 5 µL of samples + 70 µL ofDilution Buffer.
Dilution B (20x): 10 µL of Dilution A + 190 µL of Dilution Buffer, for singlets;
15 µL of Dilution A + 285 µL of Dilution Buffer, for duplicates.
Recommended dilution of urine is 1:10, i.e., 20 of samples + 180 µL of
Dilution Buffer, for singlets, 30 of samples + 270 µL of Dilution Buffer, forduplicates.
Do not store the diluted samples.

22x4x8x96
90
86
91
100
85

Spiking Recovery
SampleSpike(ng/ml)

1

3000
1500
750

103
115
107

ASSAY PROCEDURE

3.

4.
5.
6.
7.
8.

Prepare the reagents as described in the previous chapter.
Pipette 100 µL of set of Standards, Quality Controls, diluted
Samples and Dilution Buffer = Blank into each well. Incubate for2 hours at 25oC ±2oC, shaking at 300 rpm.
Wash the wells 3-times with 1x Wash Buffer (350 µL/well). When
finished, tap the plate against the paper towel to remove theliquid completely.
Pipette 100 µL of HRP-labelled Antibody Conjugate into eachwell. Incubate for 1 hour at 25oC ±2oC, shaking at 300 rpm.
Wash the wells as described in point 3.
Pipette 100 µL Substrate solution, incubate for 10 min,at 25oC ±2oC. Avoid exposure to the light during this step.
Pipette 100 µL of STOP solution.
Read the signal at 450 or 450/630 nm within 10 min.

PERFORMANCE CHARACTERISTICS
Samples used in the tests were diluted 1:300 as recommended and assayed.
The results are multiplied by the dilution factor.
1.

Sensitivity

The limit of detection, defined as a concentration of humanBeta 2-microglobulin giving absorbance higher than absorbance ofblank + 3 standard deviations, is better than 0.42 ng/mL of sample.
2.

Typical standard curve
The standard curve needs to be measured in every test. Most of themicroplate reader can automatically calculate the analyte concentration
using 4-parameter algorithm or alternative functions to fit the standard
points properly. The concentrations need to be multiplied by the dilution
factor, either automatically by reader or manually.

3.5

Human Beta 2 microglobulin Standard Curve

3Absorbance (450/630 nm)

1.
2.

2.5
21.5
10.5
01.25

Precision

Mean (ng/mL)
1049
2178

SD49131

CV (%)
56v_01

Mean (ng/mL)
1191
1469

1020

40

RESOURCES
1 Davey PG, Gosling P. beta 2-Microglobulin instability in pathological urine. Clin Chem.

1982 Jun;28(6):1330-3. PMID: 6176371.
2 Karlsson FA, Wibell L, Evrin PE. beta 2-Microglobulin in clinical medicine. Scand J Clin

Inter-assay (Run – to – run)
Sample125Concentration (ng/mL)

Intra-assay
Sample122.5

Lab Invest Suppl. 1980;154:27-37. PMID: 6163193.

SD11588CV (%)
1063 GamalDM, Badr FM, Taha SIAEF, Moustafa NM, Teama MAEM. Serum beta-2
microglobulin as a predictor of nephritis, disease activity, and damage score insystemic lupus erythematosus: a cross-sectional study. Rheumatol Int. 2022 Oct 7. doi:
10.1007/s00296-022-05221-1. Epub ahead of print. PMID: 36205758.