Bovine Albumin
ELISA Kit
Cat. No. E11-113
Lot No. E11-113-230427

Components Supplied•

Bovine Albumin Pre-Coated 96-well Strip Plate, 1 each•

Bovine Albumin Standard, 500 ng/vial, 2 each•

Bovine Albumin Detection Antibody, 12 ml•

20X Dilution Buffer C, 25 ml•

HRP Solution C, 12 ml•

TMB Substrate, 12 ml•

Stop Solution, 12 ml•

20X Wash Buffer, 50 ml•Sealing Tape, 6 sheets

Shelf Life: 6 months from date of sale.
For In vitro laboratory use only. Not for any clinical, therapeutic, or diagnostic
use in humans or animals. Not for animal or human consumption.

Bethyl Laboratories, Inc., www.bethyl.com
Telephone: 800-338-9579 • Fax: 866-597-6105
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Introduction
This enzyme linked immunosorbent assay (ELISA) is for the detection of BovineAlbumin in serum, plasma, milk, colostrum, and cell culture supernatant. This kit
contains sufficient components to quantitate Bovine Albumin in up to 40 samples,tested in duplicate.

Background
Albumin is the most predominant protein in serum of domestic animals andman. It serves several functions including the binding and transporting of fatty
acids, hormones, and metal ions, the maintenance of osmotic pressure and pH,and binding of exogenous toxins and products of lipid oxidation (Bertucci,2002). Bovine and human albumins are carbohydrate-free monomeric proteins
composed of three homologous domains (Peters, 1985 and Sugio, 1999). Overtime, development of large-scale purification methods of bovine albumin havetranslated those functions into diagnostic, cell culture, and microbiologicalapplications, (Mannuzza 2002) allowing for numerous biochemical applications
including ELISAs (Enzyme-Linked Immunosorbent Assay), immunoblots, andimmunohistochemistry. Bovine serum albumin is also used as a nutrient in celland microbial culture.

Principle of the Assay
This kit is based on a sandwich ELISA. Bovine Serum Albumin (BSA) present
in the test sample is captured by anti-Bovine Albumin antibody that has been
pre-adsorbed on the surface of microtiter wells. After sample binding, unboundproteins and molecules are washed off, and a biotinylated detection antibody isadded to the wells to bind the captured albumin. A strepavidin-conjugatedhorseradish peroxidase (SA-HRP) is then added to catalyze a colorimetric
reaction with the chromogenic substrate TMB (3,3’,5,5’-tetramethylbenzidine).
The colorimetric reaction produces a blue product, which turns yellow when thereaction is terminated by addition of dilute sulfuric acid. The absorbance of theyellow product at 450 nm is proportional to the amount of albumin analyte
present in the sample and a four-parameter standard curve can be generated. The
albumin concentrations in the test samples can then be quantified byinterpolating their absorbance from the standard curve generated in parallel withthe samples. After factoring sample dilutions, the albumin concentrations in theoriginal sample can finally be calculated.

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Telephone: 800-338-9579 • Fax: 866-597-6105
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Procedure Overview
1.

2.

Add 100 µl of standard or sample to designated wells.
Note: Run each standard or sample in duplicate.

Cover the plate and incubate at room temperature (20-25°C) for 1 hour.
3.
4.

Add 100 µl of anti-albumin Detection Antibody to each well.

5.

Cover the plate and incubate at room temperature for 1 hour.
6.
7.

8.

Wash the plate FOUR times.

Wash the plate FOUR times.

Add 100 µl of HRP Solution C to each well.

Cover the plate and incubate at room temperature for 30 minutes.
9.

Wash the plate FOUR times.

10. Add 100 µl of TMB Substrate Solution to each well.
11. Incubate the plate in the dark at room temperature for 30 minutes.
12. Stop the reaction by adding 100 µl of Stop Solution to each well.
13. Measure absorbance on a plate reader at 450 nm.

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Additional Materials Required•

Ultrapure water•

Precision pipettors, with disposable plastic tips•

Polypropylene or polyethylene tubes to prepare standard and samples
Do not use polystyrene, polycarbonate or glass tubes.

•A container to prepare 1X Dilution Buffer C•

A container to prepare 1X Wash Buffer•

Multi-channel precision pipettor with disposable plastic tips
Recommended for washing plates•

Disposable reagent reservoirs•

A standard microtiter plate reader for measuring absorbance at 450 nm forend-point ELISA.

Precautions•Store all reagents at 2-8ºC. Do not freeze reagents.

•All reagents must be at room temperature (20-25°C) before use.

•Vigorous plate washing with a multi-channel pipettor is recommended.

•Use new disposable pipette tips for each transfer to avoid crosscontamination.

•

Use a new adhesive plate cover for each incubation step.

•

Minimize lag time between wash steps to ensure the plate does not becomecompletely dry during the assay.

•Avoid microbial contamination of reagents and equipment. Automated plate
washers can easily become contaminated and result in erroneous data.

•

Do not contaminate the TMB Solution. Do not expose TMB Substratesolution to glass, foil, or metal. If the solution is blue before use, DO NOTUSE IT.

•

Individual components may contain preservatives. Wear gloves while
performing the assay and follow proper disposal procedures.

Bethyl Laboratories, Inc., www.bethyl.com
Telephone: 800-338-9579 • Fax: 866-597-6105
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Handling and Preparation of Reagents, Standards, and Samples
Preparation of 1X Dilution Buffer C•Prepare 1X Dilution Buffer C by diluting 25 ml of 20X Dilution Buffer C
into 475 ml of ultra pure water. Mix well. Store reconstituted 1X Dilution
Buffer C at 2-8ºC for up to six (6) months. Do not use 1X Dilution Buffer C
if it becomes visibly contaminated during storage.

Preparation of Standard Solutions•Standard solutions should be treated as a biological material and universal
precautions should be followed.

1.

Reconstitute the 500-ng Bovine albumin standard in vial with 1.0 ml of 1X
Dilution Buffer C to achieve a final concentration of 500 ng/ml. Mix well.

2.

Label seven (7) tubes, one for each standard curve point: 167, 55.6, 18.5,
6.17, 2.06, 0.69, and 0 ng/ml. The original vial of re-suspended standard
solution represents the top standard at 500 ng/ml.

3.

Add 300 µl of 1X Dilution Buffer C into each of the seven tubes.

4.

Serially dilute 1:3 by adding 150 µl of the 500 ng/ml standard into the firsttube containing 300 µl of 1X Dilution Buffer C. Mix well. Continue thedilution by adding 150 µl of the previous standard into 300 µl of 1X
Dilution Buffer C in the next tube until the sixth tube (0.69 ng/ml).

5.

The seventh tube containing 300 µl of 1X Dilution Buffer C serves as thezero standard value or blank.

•Using this dilution scheme, only 350 µl of the re-suspended standard
solution should be used. The remaining standard solution can be used foranother assay within the same day. Otherwise, use the second vial oflyophilized standard.

Sample Handling•This ELISA assay can be used for Bovine serum, plasma, milk andcolostrum samples.

•All blood components and biological materials should be handled as
potentially hazardous. Follow universal precautions when handling anddisposing of infectious agents.
Bethyl Laboratories, Inc., www.bethyl.com
Telephone: 800-338-9579 • Fax: 866-597-6105
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100 µl of sample or standard is required per well.

•Samples must be assayed in duplicate each time the assay is performed.

•Store samples to be assayed within 24 hours at 2-8ºC. For long-term
storage, aliquot and freeze samples at -70°C. Avoid repeated freeze-thaw
cycles when storing samples.

•If particulates are present in samples, centrifuge prior to analysis.

•If blood samples are clotted, grossly hemolyzed, lipemic, or the integrity ofthe sample is of concern, make a note on the Plate Template and interpret
results with caution.

Sample Preparation•

The dilution schemes indicated below are only suggestions. Dilutions
should be based on the expected concentration of the unknown sample suchthat the diluted sample falls within the dynamic range of the standard curve.
Prepare one or more dilutions of the sample in 1X Dilution Buffer C using
the same serial dilution technique described below until the desiredconcentration is obtained.

•Serum and plasma – Recommended starting dilution is 1:250,000. With thislarge dilution one must strive for accurate pipetting. A typical dilution
scheme starts by adding 10 µl of plasma into 990 µl of 1X Dilution Buffer
C to give a 1:100 dilution. This is repeated by adding 10 µl of the 1:100
diluted sample to 990 µl of 1X Dilution Buffer C to give 1:10,000 dilution.
Finally, 50 µl of the 1:10,000 diluted sample is added to 1.2 ml of 1X
Dilution Buffer C to result in a 1:250,000 dilution. It is important to mix
thoroughly at each dilution step.

•Colostrum - Recommended starting dilution is 1:10,000.

•Milk - Recommended starting dilution is 1:5,000.

•Cell Culture Supernatant – Should be determined by investigator.

Preparation of 1X Wash Buffer•Prepare 1X Wash Buffer by diluting 20X Wash Buffer in ultrapure water.
For example, if preparing 1 L of 1X Wash Buffer, dilute 50 ml of 20X
Wash Buffer into 950 ml of ultrapure water. Mix well. Store reconstituted
1X Wash Buffer at 2-8ºC for up to six (6) months. Do not use 1X Wash
Buffer if it becomes visibly contaminated during storage.
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Telephone: 800-338-9579 • Fax: 866-597-6105
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Assay Procedure
Sample Incubation•Determine the number of strips required. Leave these strips in the plateframe. Place unused strips in the foil pouch with desiccant and seal tightly.
Store unused strips at 2-8°C. After completing assay, keep the plate frame
for additional assays.

•Use a Plate Template to record the locations of the standards and unknown
samples within the wells.

1.

Add 100 µl of appropriately diluted standards or samples to each well. Run
each standard, sample, or blank in duplicate.
Note: Serum, plasma, milk, colostrum, and cell culture supernatantsamples must be diluted prior to testing (see Sample Preparation sectionabove).

2.

Carefully cover the wells with a new adhesive plate cover and incubate forone (1) hour at room temperature, 20-25°C.

3.

Carefully remove the adhesive plate cover and wash FOUR times with 1X
Wash Buffer, as described in the Plate Washing section below.

Plate Washing•Using a multi-channel precision pipettor with disposable plastic tips

1.

Pour the appropriate volume of the 1X Wash Buffer into a BSA-free washtrough or solution basin. Set the multi-channel pipettor to dispense between
200 and 300 mcl of solution into each well.

2.

Slowly aspirate the 1X Wash Buffer from the wash trough or solution basin
into the tips of the multi-channel pipettor. Carefully dispense the 1X Wash
Buffer into the appropriate wells of the plate. Repeat until all wells havebeen filled. Then aspirate the wash solution from each well using the multichannel pipettor and discard the wash solution and tips.

3.

Repeat wash step number 2 three more times. To prevent the possibility ofcross contamination, it is recommended that new disposable plastic tips beused with each step of the wash procedure.

•NOTE: A plate washer or squirt bottle may be used only if it is dedicated toBSA-free assays. Use of a plate washer or squirt bottle that has used BSA
from other applications may cause cross contamination in this ELISA.

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Telephone: 800-338-9579 • Fax: 866-597-6105
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Incubation with Detection Antibody•Only remove the required amount of Detection Antibody reagent for thenumber of strips being used.

1.

Add 100 µl of Detection Antibody to each well containing standard, sample
or blank. Mix well by gently tapping the plate several times.

2.

Carefully attach a new adhesive plate cover and incubate the plate for one
hour at room temperature, 20-25°C.

3.

Carefully remove the adhesive plate cover, discard plate contents and wash
FOUR times with 1X Wash Buffer (see Plate Washing section above).

Incubation with HRP Solution C•Remove only the required amount of HRP Solution C for the number ofstrips being used.

1.

Add 100 µl of HRP Solution C to each well containing standard, sample orblank.

2.

Carefully attach a new adhesive plate cover and incubate plate for 30
minutes at room temperature, 20-25°C.

3.

Carefully remove the adhesive plate cover, discard plate contents and wash
FOUR times with 1X Wash Buffer (see Plate Washing section). Blot off
any residual liquid at the bottom of the wells that might interfere later withthe absorbance readings.

Incubation with TMB Substrate and Stopping the Reaction•Remove only the required amount of TMB Substrate Solution and StopSolution for the number of strips being used.

•Do NOT use glass pipette to measure the TMB Substrate Solution. Do NOT
cover the plate with aluminum foil or metalized mylar. Do NOT return
leftover TMB Substrate to bottle. Do NOT contaminate the unused TMB
Substrate Solution. If the solution is blue before use, DO NOT USE IT!

1.

Add 100 µl of TMB Substrate Solution into each well and allow theenzymatic reaction to develop a blue color at room temperature (20-25ºC)in the dark for 30 minutes. Do NOT cover plate with a plate sealer.

2.

Stop the reaction by adding 100 µl of Stop Solution to each well. Tap plategently to mix. The solution in the wells should immediately change in color
from blue to yellow.
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Absorbance Measurement
Note: Wipe the underside of the wells with a lint-free tissue.
1.

For end-point ELISA, measure the absorbance on an ELISA plate reader set
at 450 nm within 30 minutes after the addition of the Stop Solution.

Calculation of Results•Duplicate absorbance values should be within 10% of each other. Careshould be taken when interpreting data with differences in absorbancevalues greater than 10%.

1.

Prepare a standard curve to determine the amount of albumin in an
unknown sample. Plot the average absorbance obtained for each standard
concentration on the vertical (Y) axis versus the corresponding albumin
concentration on the horizontal (X) axis using curve-fitting software.

2.

Calculate the albumin concentration in unknown samples using the preparedstandard curve. Determine the amount of albumin in each unknown sample
by noting the albumin concentration (X axis) that correlates with theabsorbance value (Y axis) obtained for the unknown sample.

3.

If the sample was diluted, multiply the observed albumin concentration by
the dilution factor to determine the concentration of albumin in the original,undiluted sample.
Note: Most plate readers come with appropriate templates and curve-fitting
software. The standard curve of this assay can be fitted into a 4-parameter
curve fitting equation that can be programmed to calculate and display atable (or tables) consisting of the raw absorbance readings, net absorbancereadings, the analyte concentration in the assay solution, dilution factors,
and analyte concentration in the original unknown sample.

Bethyl Laboratories, Inc., www.bethyl.com
Telephone: 800-338-9579 • Fax: 866-597-6105
9Performance Characteristics
Typical Standard Curve•This typical standard curve was generated using the Bovine Albumin
ELISA Kit Protocol. This standard curve is for demonstration only. A
standard curve must be generated for each assay.

Assay Range: 0.69 – 500 ng/ml•Suggested standard curve points are 500 ng/ml, 167 ng/ml, 55.6 ng/ml, 18.5ng/ml, 6.17 ng/ml, 2.06 ng/ml, 0.69 ng/ml, and 0 ng/ml

Specificity•By immunoelectrophoresis and ELISA the antibodies in this kit react
specifically with Bovine Albumin, not with other Bovine immunoglobulins
or other Bovine serum proteins. Cross-reactivity with other species has notbeen tested.

Bethyl Laboratories, Inc., www.bethyl.com
Telephone: 800-338-9579 • Fax: 866-597-6105
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Warranty

Products are warranted by Bethyl Laboratories, Inc. to meet stated productspecifications and to conform to label descriptions when used, handled andstored according to instructions. Unless otherwise stated, this warranty is limited
to six months from date of sale. Bethyl Laboratories sole liability for the productis limited to replacement of the product or refund of the purchase price. Bethyl
Laboratories products are supplied for research applications. They are notintended for medicinal, diagnostic or therapeutic use. The products may not be
resold, modified for resale or used to manufacture commercial products without
prior written approval from Bethyl Laboratories, Inc.

Background References
Bertucci, C, and E. Domenici. 2002. Reversible and Covalent Binding of Drugs
to Human Serum Albumin: Methodological Approaches and Physiological
Relevance. Curr. Med. Chem. 9:1463-1481.
Peters, T. 1985. Serum Albumin. Adv. Prot. Chem. 37:161-245.
Sugio, S. 1999. Crystal Structure of Human Serum Albumin at 2.5 A
Resolution. Prot. Eng. 12:439-446.
Mannuzza, Frank J, and Joseph G. Montalto. 2010. Is Bovine Albumin Too
Complex to Be Just a Commodity? BioProcess International, Vol. 8, No. 2, pp.
40–43.

Rev 221202

Bethyl Laboratories, Inc., www.bethyl.com
Telephone: 800-338-9579 • Fax: 866-597-6105
11Plate Templates
123456789101112123456789101112A
B
C
DEF
GHA
B
CDEF
GH

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Telephone: 800-338-9579 • Fax: 866-597-6105
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