1Hemoglobin Assay Kit
Catalog # 6024

For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:

Assay kit to quantify hemoglobin

FORMAT:

96-well ELISA Plate with removeable strips

ASSAY TYPE:

Colorimetric Assay

ASSAY TIME:

15 minutes

STANDARD RANGE:

2 mg/ml to 0.03 mg/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Blood, Serum, and Plasma

RECOMMENDED SAMPLE DILUTIONS:

1:200 (Blood)

CHROMOGEN:

N/A (read at 400 nm)

STORAGE:4°C

VALIDATION DATA:

Intra-Assay (5.7-6.7%)/Inter-Assay (2.7-4.5%)/Spiking Test (103-106%)NOTES:© 2020 Chondrex, Inc. All Rights Reserved, 6024 2.0
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

2Hemoglobin Assay Kit
Catalog # 6024

For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Hemoglobin is a heme protein carried by erythrocytes and transports oxygen from the lungs to the rest of the body. Therefore, low hemoglobin
levels in erythrocytes or low erythrocyte numbers in blood deprives the body of oxygen. This condition is defined as anemia. This oxygen
deficit can cause dizziness or headaches, while severe or long-lasting anemia can damage organs in the body and even result in death.
Anemia is also a complication of various inflammatory diseases such as sepsis (1-3) and autoimmune diseases (4, 5), and has been observedin rodent disease models of inflammatory bowel disease (IBD) (5, 6), colitis (7), sepsis (8), and autoimmune hemolytic anemia (9-12).
As hemoglobin levels correlate to the oxygen carrying-capacity of blood, hemoglobin is a useful marker of anemia to evaluate disease
severity and effectiveness of treatments. A standard hemoglobin assay utilizes the toxic cyanmethemoglobin method. However, Chondrex,
Inc. would like to introduce a safe, non-cyanide hemoglobin assay kit (13, 14). This assay kit correlates well with the cyanmethemoglobin
method and can be used to evaluate anemia in our mouse sepsis model and mouse IBD model, as well as other disease models. Chondrex,
Inc. also provides related products such as FITC-Dextran, mouse anti-bacterial antibody ELISAs, and an HMGB1 Detection ELISA kit. For
more information, please visit www.chondrex.com or contact support@chondrex.com.

KIT COMPONENTS
Item

Quantity

AmountStorage

Bovine Hemoglobin Standard (60241)
Reaction Solution (60242)
ELISA Plate

1 vial
1 bottle
1 plateLyophilized, 2 mg
20 ml

4°C
4°C
4°C

96-well (8-well strips x 12)

ASSAY OUTLINE
Add 50 µl of diluted standards and samples to wells

Add 200 µl of reaction solution to wells

Incubate at room temperaturefor 15 minutes

Read at 400 nm© 2020 Chondrex, Inc. All Rights Reserved, 6024 2.016928 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

PLATE LAYOUT
123456789101112A

22

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B

11

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34

C0.50.5

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D0.25

0.25

S4

S4

S12

S12S20

S20

S28

S28

S36

S36

E

0.125

0.125

S5S5S13

S13

S21

S21

S29

S29

S37

S37

F0.63

0.63

S6S6S14

S14

S22

S22

S30

S30

S38

S38

G0.310.31

S7S7S15

S15

S23

S23

S31

S31

S39

S39

HBB

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40Standards

Samples

NOTES 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.

ASSAY PROCEDURE
1.

Prepare Standard Dilutions: Reconstitute one vial of Standard with 1 ml of distilled water to make a 2 mg/ml standard solution. Then
serially dilute the 2 mg/ml solution with distilled water. For example, mix 250 µl of the 2 mg/ml solution with an equal volume of distilledwater to make a 1 mg/ml solution, and then repeat it five more times to make 0.5, 0.25, 0.125, 0.063, and 0.031 mg/ml solutions.
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 ml2 mg/ml

1 mg/ml

0.5 mg/ml

0.25 mg/ml

0.125 mg/ml

0.063 mg/ml

0.031 mg/ml

Blank

12.

Prepare Sample dilutions: Blood samples should be diluted at 1:200 in distilled water while serum and plasma samples can be assayeddirectly.
NOTE: the sample dilution depends on the animal disease models.

© 2020 Chondrex, Inc. All Rights Reserved, 6024 2.0
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

4

3.

Add Standards and Samples: Add 50 µl of standards, distilled water (blank), and diluted samples to wells in duplicate. Pipet carefully
to avoid bubbles in the wells.

4.

Add Reaction Solution: Add 200 µl of Reaction Solution to each well and tap plate lightly to mix. Pipet carefully to avoid bubbles inthe wells. Incubate for 15 minutes at room temperature.

5.

Read plate: Read the plate at 400 nm (390-405 nm).

CALCULATING RESULTS
1.

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

2.

Subtract the “blank” (B) values from the averaged OD values of standards and test samples in Step 1

3.

Plot the OD values of standards against the concentration of hemoglobin (mg/ml). Using a log/log plot will linearize the data. Figure 1
shows a representative experiment where the standard range is from 0.03 - 2 mg/ml.

4.

The concentration (mg/ml) of hemoglobin in test samples can be calculated using regression analysis. Multiply the calculated
hemoglobin levels by the sample dilution factor to obtain the hemoglobin concentration (mg/ml) in the original sample specimen. For
additional assistance, please download a sample calculation worksheet from www.chondrex.com.
NOTE: Conversions: 1 mg/ml hemoglobin equals 100 mg/dl hemoglobin, 15.6 µM, 0.1%, or 1000 ppm.

Figure 1 - A Typical Standard Curve for the Hemoglobin Assay Kit
1.000

OD 400 nm

R² = 0.9997

0.100

0.010
0.01

0.10

1.00

10.00

Hemoglobin Concentration (mg/ml)

VALIDATION DATA
Reproducibility Data for the Hemoglobin Assay Kit
Test At
0.06 mg/ml
0.3 mg/ml
1 mg/ml
Intra-Assay CV (%)
5.7
6.7
6.3
Inter-Assay CV (%)
2.7
4.2
4.5
Spiking Test*
106%
103%
105%
*Standard was added with known amounts of Hemoglobin and then diluted with distilled water for assaying hemoglobin.

© 2020 Chondrex, Inc. All Rights Reserved, 6024 2.016928 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

5TROUBLESHOOTING
For frequently asked questions about assays and ELISAs, please see Chondrex, Inc.’s Assay FAQ for more information.

REFERENCES
1.

D. Kempe, A. Akel, P. Lang, T. Hermle, R. Biswas, et al., Suicidal Erythrocyte Death in Sepsis. J Mol Med (Berl) 85, 273-81 (2007).

2.

J. A. Russell, Management of sepsis. New England Journal of Medicine 355, 1699-1713 (2006).

3.

M. Piagnerelli, K. Z. Boudjeltia, B. Gulbis, M. Vanhaeverbeek, J. L. Vincent, Anemia in sepsis: The importance of red blood cell
membrane changes. Transfusion Alternatives in Transfusion Medicine 9, 143-149 (2007).

4.

G. Weiss, L. Goodnough, Anemia of Chronic Disease. N Engl J Med 352, 1011-23 (2005).

5.

F. de la Morena, J. P. Gisbert, [Anemia and inflammatory bowel disease]. Rev Esp Enferm Dig 100, 285-293 (2008).

6.

M. Cantorna, C. Munsick, C. Bemiss, B. Mahon, 1,25-Dihydroxycholecalciferol Prevents and Ameliorates Symptoms of Experimental
Murine Inflammatory Bowel Disease. J Nutr 130, 2648-52 (2000).

7.

B. R. Sartor, Insights into the Pathogenesis of Inflammatory Bowel Diseases Provided by New Rodent Models of Spontaneous Colitis.
Inflammatory Bowel Diseases 1, 64-75 (1995).

8.

S. Rivera, T. Ganz, Animal Models of Anemia of Inflammation. Semin Hematol 46, 351-7 (2009).

9.

M. B. Jordan, N. van Rooijen, S. Izui, J. Kappler, P. Marrack, Liposomal clodronate as a novel agent for treating autoimmune hemolytic
anemia in a mouse model. Blood 101, 594-601 (2003).

10. L. Fossati-Jimack, L. Reininger, Y. Chicheportiche, R. Clynes, J. Ravetch, et al., High Pathogenic Potential of Low-Affinity
Autoantibodies in Experimental Autoimmune Hemolytic Anemia. J Exp Med 190, 1689-96 (1999).
11. D. Meyer, C. Schiller, J. Westermann, S. Izui, W. Hazenbos, et al., FcgammaRIII (CD16)-deficient Mice Show IgG Isotype-Dependent
Protection to Experimental Autoimmune Hemolytic Anemia. Blood 92, 3997-4002 (1998).
12. D. Schirmer, S. Song, J. Baliff, S. Harbers, R. Clynes, et al., Mouse Models of IgG- And IgM-mediated Hemolysis. Blood 109, 3099-107(2007).
13. O. van Assendelft, W. Zijlstra, Observations on the Alkaline haematin/detergent Complex Proposed for Measuring Haemoglobin
Concentration. J Clin Chem Clin Biochem 27, 191-5 (1989).
14. V. Shah, B. Shah, G. Puranik, Evaluation of Non Cyanide Methods for Hemoglobin Estimation. Indian J Pathol Microbiol 54, 764-8(2011).

© 2020 Chondrex, Inc. All Rights Reserved, 6024 2.0
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