1Rat Albumin Detection ELISA Kit
Catalog # 3020
For Research Use Only - Not Rat or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA Kit to quantify rat albumin

FORMAT:

96-well ELISA Plate with removeable strips

ASSAY TYPE:

Sandwich ELISAASSAY TIME:

3.5 hours

STANDARD RANGE:

100 - 1.6 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Urine and Serum

RECOMMENDED SAMPLE DILUTIONS:

1:500 (at least)

CHROMOGEN:

OPD (read at 490 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

Intra-Assay (2.1-6.5%)/Inter-Assay (4.1-8.7%)/Spiking Test (91-94%)

NOTES:© 2022 Chondrex, Inc. All Rights Reserved, 3020 2.2
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

2Rat Albumin Detection ELISA Kit
Catalog # 3020
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Albumin is the most abundant protein in plasma, accounting for more than half of the measured serum protein. It consists of a single
polypeptide chain of 585 amino acids with a molecular weight of 66.5 kDa. Because of its high molecular weight and concentration inplasma, albumin is critical in maintaining plasma osmotic pressure.
In human, plasma albumin levels are significantly decreased in patients with severe liver disease (2) or poor nutritional status (5) asalbumin synthesis takes place exclusively in the liver (1). Alternatively, low plasma albumin levels occur due to increased capillaryleakage caused by critical illnesses such as kidney disease (3), inflammation (4), sepsis, and major surgical stress (6). In addition,
excretion of albumin into urine (albuminuria) is very low in healthy individuals, but it is significantly increased by renal damage inglomerulonephritis (7) and diabetic nephropathy (8,9).
In fact, rat disease models of sepsis (10), glomerulonephritis (11), and diabetic nephropathy (12) demonstrate comparable albuminuria
with human patients, suggesting that rat urinary albumin levels are useful for evaluating disease severity and treatment efficacy.
Chondrex, Inc. provides a Rat Albumin Detection ELISA Kit (Cat # 3020) and a Rat Urinary Protein Assay Kit (Cat # 9040) dependingon the purpose of the study.

KIT COMPONENTS
Item

Quantity

AmountStorage

Rat Albumin Standard (30201)

1 vial100 ng, lyophilized-20°C

Capture Antibody (30202)

1 vial

50 µl, 1 mg/ml-20°C

Detection Antibody (30203)

1 vialLyophilized-20°C

Solution A - Coating Buffer (9052)

1 bottle

10 ml

-20°C

Solution C - Sample/Standard/Secondary Antibody Dilution Buffer (30106)

1 bottle

50 ml

-20°C

OPD (90021)

2 vials

Lyophilized-20°C

Chromogen Dilution Buffer (90022)

1 bottle

20 ml-20°C

Stop Solution - 2N Sulfuric Acid (9016)

1 bottle

10 ml

-20°C

Wash Buffer, 20X (9005)

1 bottle

50 ml

-20°C

96-Well ELISA Plate

1 each8-well strips x 12-20°C© 2022 Chondrex, Inc. All Rights Reserved, 3020 2.2
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

3

ASSAY OUTLINE
Add 50 ml of diluted Capture Antibody Solution

Incubate at 4 C overnight.
Wash plates.
Add 50 ml of diluted Standards and Samples
Incubate at room temperature for 2 hours.
Wash plates.

Add 50 ml of diluted Detection Antibody Solution
Incubate at room temperature for 1 hour.
Wash plates.

Add 50 ml of diluted OPD Solution
Incubate at room temperature for 10 minutes.

Add 50 ml of diluted Stop Solution

Read plates at 490 nm/630 nm

PLATE MAPPING
123456789101112A

100

100

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B

5050

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34C25

25

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D12.512.5

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E

6.3

6.3

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

F

3.13.1

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

G

1.6

1.6

S7

S7

S15

S15

S23

S23

S31

S31

S39

S39

H

B

B

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

Standards

Samples© 2022 Chondrex, Inc. All Rights Reserved, 3020 2.2
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

4NOTES BEFORE USING ASSAY
NOTE 1: It is recommended that the standard and samples be run in duplicate.
NOTE 2: Warm up all buffers to room temperature before use.
NOTE 3: Crystals may form in Wash Buffer, 20X when stored at cold temperatures. If crystals have formed, warm the wash buffer by placing
the bottle in warm water until crystals are completely dissolved.
NOTE 4: Measure exact volume of buffers using a serological pipet, as extra buffer is provided.
NOTE 5: Cover the plate with plastic wrap or a plate sealer after each step to prevent evaporation from the outside wells of the plate.
NOTE 6: For partial reagent use, please see the assay protocol’s corresponding step for the appropriate dilution ratio. For example, if theprotocol dilutes 50 µl of a stock solution in 10 ml of buffer for 12 strips, then for 6 strips, dilute 25 µl of the stock solution in 5 ml of buffer.
Partially used stock reagents may be kept in their original vials and stored at -20⁰C for use in a future assay.
NOTE 7: This kit contains animal components from non-infectious animals and should be treated as potential biohazards in use and fordisposal.

ASSAY PROCEDURE
1.

Coat Plate with Capture Antibody: Dilute one vial of Capture Antibody with 5 ml of Coating Buffer (Solution A). Alternatively, dilute
according to the table below. Add 50 µl of capture antibody solution to each well and incubate at 4°C overnight. Any leftover Capture
Antibody Stock Solution may be stored at -20°C for future assays.
Strip #2
468
10122.

Capture Antibody (µl)
81725334250

Solution A (ml)
0.8
1.7
2.5
3.3
4.2
5.0

Prepare Standard Dilutions: The recommended standard range is 1.6-100 ng/ml. Dissolve one vial of mouse albumin standard in 1
ml of Sample/Standard/Secondary Antibody Dilution Buffer (Solution C) for the 100 ng/ml standard. Then serially dilute it with Solution
C. For example, mix 250 µl of the standard (100 ng/ml) with an equal volume of Solution C to make a 50 ng/ml solution, and then repeat
it five more times for 25, 12.5, 6.25, 3.125, and 1.6 ng/ml solutions. The remaining 100 ng/ml standard stock may be stored at -20°C
for use in a second assay. Chondrex, Inc. recommends making fresh serial dilutions for each assay.
1000 ml
250 ml

250 ml

250 ml

250 ml

250 ml

250 ml23456781000 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml100 ng/ml

50 ng/ml

25 ng/ml

12.5 ng/ml

6.3 ng/ml

3.1 ng/ml

1.6 ng/ml

Blank

1

© 2022 Chondrex, Inc. All Rights Reserved, 3020 2.216928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

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support@chondrex.com

53.

Prepare Sample Dilutions: Centrifuge the samples at 10,000 rpm for 5 minutes to remove insoluble materials in urine or serum
samples. Dilute samples 1:500-1:100,000 with Solution C depending on the estimated albumin content in the samples. It is
recommended to use 2-3 different dilutions if the sample albumin level is unknown.
NOTE: Due to the high dilution of samples, 1% BSA/PBS pH 7.4 can be used for sample dilution. However, the final diluted samples
for ELISA should include at least 50% of Solution C. For example, if samples are diluted at 1: 20,000; take 10 µl of sample and add to990 µl of 1% BSA/PBS pH 7.4 (1:100). Then repeat this dilution for a 1:10,000 dilution. Mix 100 µl of the diluted sample (1:10,000) withan equal volume of Solution C for a 1:20,000 dilution.

4.

Dilute Wash Buffer: Dilute 50 ml of 20X wash buffer in 950 ml of distilled water (1X wash buffer). Wash the plate with 1X wash buffer
at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blotting on a paper
towel to remove excess liquid. Do not allow the plate to dry out.

5.

Add Standards and Samples: Add 50 µl of Solution C (blank), standards, and samples to designated wells in duplicate and incubate
at room temperature for 2 hours.

6.

Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty theplate by inverting it and blotting on a paper towel to remove excess liquid. Do not allow the plate to dry out.

7.

Add Detection Antibody Solution: Dissolve one vial of Detection antibody in 5 ml of Sample/Standard/Secondary Antibody Dilution
Buffer (Solution C). Alternatively, dissolve one vial of Detection antibody in 50 µl of Solution C and dilute accordingly. Add 50 µl ofDetection antibody solution to each well and incubate at room temperature for 1 hour. Any remaining detection antibody stock solution
can be stored at -20°C for use in a second assay
Strip #24681012Detection Antibody (µl)
81725334250

Solution C (ml)
0.8
1.7
2.5
3.3
4.2
5.0

8.

Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty theplate by inverting it and blotting on a paper towel to remove excess liquid. Do not allow the plate to dry out.

9.

Add OPD Solution: Dissolve one vial of OPD in 10 ml of Chromogen Dilution Buffer just prior to use. Add 50 µl of OPD solution to allwells immediately after washing the plate and incubate for 10 minutes at room temperature.

10. Stop: Stop the reaction with 50 µl of 2N Sulfuric Acid (Stop Solution) to each well.
11. Read Plate: Read the OD values at 490 nm. If the OD values of samples are greater than the OD values of the highest standard, reassay the samples at a higher dilution. A 630 nm filter can be used as a reference.

© 2022 Chondrex, Inc. All Rights Reserved, 3020 2.2
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

6

CALCULATING RESULTS
1.

Average the duplicate OD values for the blank, standards, and test samples.

2.

Subtract the “blank” (B) values from the averaged OD values in step 1.

3.

Plot the OD values of standards against the concentration of rat albumin (ng/ml). Using a log/log plot will linearize the data. Figure
1 shows a representative experiment where the standard range is 1.6 - 100 ng/ml.

4.

The ng/ml of rat albumin in test samples can be calculated using regression analysis.

Figure 1 - A Typical Standard Curve for the Rat Albumin Detection ELISA Kit
10.000y = 0.0207x0.9393
R² = 0.9963

OD 490 nm

1.000

0.100

0.010
1.0

10.0

100.0Rat Albumin Concentration (ng/ml)

ASSAY VALIDATION
Table 1 - Reproducibility Data for the Rat Albumin Detection ELISA Kit
Test
2.7 ng/ml
12 ng/ml
48 ng/ml
Intra-Assay CV (%)
5.6
6.5
2.1
Inter-Assay CV (%)
4.1
8.7
7.1
Spike Test* (%)
94%
94%91%* Known amounts of rat albumin were added to a normal rat urine pool and then diluted with Sample/Standard/Secondary Antibody
Dilution Buffer (Solution C).

TROUBLESHOOTING
For frequently asked questions about assays and ELISAs, please see Chondrex, Inc.’s ELISA FAQ for more information.

© 2022 Chondrex, Inc. All Rights Reserved, 3020 2.2
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

7

REFERENCES
1.

K. Oettl, V. Stadlbauer, F. Petter, J. Greilberger, C. Putz-Bankuti, et al., Oxidative Damage of Albumin in Advanced Liver Disease.
Biochim Biophys Acta 1782, 469-73 (Jul-).

2.

B. Don, G. Kaysen, Serum Albumin: Relationship to Inflammation and Nutrition. Semin Dial 17, 432-7 (2004).

3.

L. Miller, C. Bly, M. Watson, W. Bale, The Dominant Role of the Liver in Plasma Protein Synthesis; A Direct Study of the Isolated
Perfused Rat Liver With the Aid of lysine-epsilon-C14. J Exp Med 94, 431-53 (1951).

4.

J. Eustace, B. Astor, P. Muntner, T. Ikizler, J. Coresh, Prevalence of Acidosis and Inflammation and Their Association With Low Serum
Albumin in Chronic Kidney Disease. Kidney Int 65, 1031-40 (2004).

5.

H. Moshage, J. Janssen, J. Franssen, J. Hafkenscheid, S. Yap, Study of the Molecular Mechanism of Decreased Liver Synthesis ofAlbumin in Inflammation. J Clin Invest 79, 1635-41 (1987).

6.

A. Fleck, G. Raines, F. Hawker, J. Trotter, P. Wallace, et al., Increased Vascular Permeability: A Major Cause of Hypoalbuminaemia in
Disease and Injury. Lancet 1, 781-4 (1985).

7.

Y. Li, J. Wang, X. Zhu, Q. Feng, X. Li, X. Feng, et al., Urinary Protein Markers Predict the Severity of Renal Histological Lesions inChildren With Mesangial Proliferative Glomerulonephritis. BMC Nephrol 13, 29 (2012).

8.

B. Roshan, R. Stanton, A Story of Microalbuminuria and Diabetic Nephropathy. J Nephropathol 2, 234-40 (2013).

9.

D. Newman, M. Mattock, A. Dawnay, S. Kerry, A. McGuire, et al., Systematic Review on Urine Albumin Testing for Early Detection ofDiabetic Complications. Health Technol Assess 9, iii-vi, xiii-163 (2005).

10. C. Adembri, E. Sgambati, L. Vitali, V. Selmi, M. Margheri, et al., Sepsis Induces Albuminuria and Alterations in the Glomerular Filtration
Barrier: A Morphofunctional Study in the Rat. Crit Care 15, R277 (2011).
11. S. Zhang, H. Xin, Y. Li, D. Zhang, J. Shi, et al., Skimmin, a Coumarin From Hydrangea Paniculata, Slows Down the Progression ofMembranous Glomerulonephritis by Anti-Inflammatory Effects and Inhibiting Immune Complex Deposition. Evid Based Complement
Alternat Med 2013, 819296 (2013).
12. R. Ma, L. Liu, X. Liu, Y. Wang, W. Jiang, L. Xu, et al., Triptolide Markedly Attenuates Albuminuria and Podocyte Injury in an Animal
Model of Diabetic Nephropathy. Exp Ther Med 6, 649-656 (2013).

© 2022 Chondrex, Inc. All Rights Reserved, 3020 2.2
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com