K-ASSAY 

KAMIYA BIOMEDICAL COMPANY

KAMIYA BIOMEDICAL COMPANY

Rat High-Sensitive
CRP ELISAFor the quantitative determination of
C-reactive protein in rat biological samples

Cat. No. KT-099
For Research Use Only.

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KAMIYA BIOMEDICAL COMPANYPRODUCT INFORMATION

Rat High-Sensitive CRP ELISA
Cat. No. KT-099
INTENDED USE
The Rat High-Sensitive CRP ELISA is a highly sensitive two-site enzyme-linked immunoassay (ELISA) for the quantitative
determination of C-reactive protein (CRP) in rat biological samples. For research use only.

INTRODUCTION
Acute phase proteins are plasma proteins which increase in concentration following infection, inflammation or trauma.
The first acute phase protein to be recognized was discovered in humans by Tillet and Frances in 1930. This CRP is so
named because it is able to effect precipitation of somatic C-polysaccharide of Streptococcus pneumonia. CRP is analpha globulin protein with a mass of 110,000 to 140,000 daltons, and composed of five identical subunits, which are noncovalently assembled as a cyclic pentamer. It is synthesized in the liver and, in humans, is normally present as a trace
constituent of serum at a level less than 0.3 mg/dL. The level in serum rises quickly following acute tissue damage andcan reach a level 1,000-fold higher within 24 to 48 hours. It also falls very rapidly once the stimulus is removed. It has
been proposed that CRP aids in complement activation, influences phagocytic cell function and augments cell-mediatedcytotoxicity. Investigations over the past few years have shown that quantification of CRP in plasma or serum can provide
valuable information in the detection, prognosis and monitoring of disease not only in humans, but in companion animals
and farm herds as well.

PRINCIPLE
The principle of the double antibody sandwich ELISA is represented in Figure 1. In this assay the CRP present in thesample reacts with the anti-CRP antibodies that have been adsorbed to the surface of polystyrene microtiter wells. Afterthe removal of unbound proteins by washing, anti-CRP antibodies conjugated with horseradish peroxidase (HRP) areadded. This HRP-conjugated antibody forms a complex with the previously bound CRP. Following another washing step,
the enzyme bound to the immunosorbent is assayed by the addition of a chromogenic substrate, 3,3’,5,5’tetramethylbenzidine (TMB). The quantity of bound enzyme is proportional to the concentration of CRP in the sample
tested; thus, the absorbance, at 450 nm, is a measure of the concentration of CRP in the test sample. The quantity of
CRP in the test sample can be interpolated from the calibration curve constructed from the calibrators, and corrected forsample dilution.

Figure 1.

Anti-CRP Antibodies Bound To Solid Phase│
Calibrators and Samples Added│
CRP * Anti-CRP Complexes Formed│
Unbound Sample Proteins Removed│
Anti-CRP-HRP Conjugate Added│
Anti-CRP-HRP * CRP * Anti-CRP
Complexes Formed│
Unbound Anti-CRP-HRP Removed│
TMB Substrate Added│
Determine Bound Enzyme Activity

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KAMIYA BIOMEDICAL COMPANYCOMPONENTS
1. Diluent Concentrate
One bottle containing 50 mL of a 5X concentrated diluent running buffer.
2. Wash Solution Concentrate
One bottle containing 50 mL of a 20X concentrated wash solution.
3. Enzyme-Antibody Conjugate Concentrate
One vial containing 150 µL of a 100X concentrated affinity-purified anti-rat CRP antibody conjugated with HRP instabilizing buffer.
4. TMB Substrate Solution
One bottle containing 12 mL of TMB and hydrogen peroxide in citric acid buffer at pH 3.3.
5. Stop Solution
One bottle containing 12 mL of 0.3 M sulfuric acid.
WARNING: Avoid contact with skin.
6. Microtiter Plate
Twelve removable eight-well micro strips in well holder frame. Wells are coated with affinity-purified anti-rat CRP.
7. Rat CRP Calibrator
One vial containing a lyophilized Rat CRP Calibrator.
8. Positive Control
One vial containing 50 µL of serum with 0.1% sodium azide. See the Control Certificate for the concentration.

MATERIALS REQUIRED BUT NOT PROVIDED
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Precision pipette (2 µL to 200 µL) for making and dispensing dilutions
Test tubes
Microplate washer/aspirator
Distilled or de-ionized H2O
Microplate reader
Assorted glassware for the preparation of reagents and buffer solutions
Timer
Centrifuge for sample collection
Anticoagulant for plasma collection

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

Read the instructions carefully before beginning the assay.
This kit is for research use only.
Great care has been taken to ensure the quality and reliability of this product. However, it is possible that in certain
cases, unusual results may be obtained due to high levels of interfering factors.
Preservatives
 Positive Control contains 0.1% sodium azide.
No additives or preservatives are necessary to maintain the integrity of the specimen. Avoid azide contamination.
Azide and thimerosal at concentrations higher than 0.1% inhibit the enzyme reaction.
Other precautions: Do not interchange kit components from different lots.
 Do not use kit components beyond the expiration date.
 Protect reagents from direct sunlight.
 Do not pipette by mouth.
 Do not eat, drink, smoke or apply cosmetics where reagents are used.
 Avoid all contact with the reagents by using gloves.
 Stop solution contains diluted sulfuric acid. Irritation to eyes and skin is possible. Flush with water after
contact.

REAGENT PREPARATION
1. Diluent Concentrate
The Diluent solution supplied is a 5X concentrate and must be diluted 1:5 with distilled or de-ionized water.
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2. Wash Solution Concentrate
The Wash Solution supplied is a 20X concentrate and must be diluted 1:20 with distilled or de-ionized water. Crystal
formation in the concentrate is not uncommon when storage temperatures are low. Warming of the concentrate to30-35°C before dilution can dissolve crystals.
3. Enzyme-Antibody Conjugate Concentrate
Calculate the required amount of working conjugate solution for each microtiter plate test strip by adding 10 µL
Enzyme-Antibody Conjugate to 990 µL of 1X Diluent for each test strip to be used for testing. Mix uniformly, but gently.
Avoid foaming.
4. TMB Substrate Solution
Ready to use as supplied.
5. Stop Solution
Ready to use as supplied.
6. Microtiter Plate
Ready to use as supplied. Unseal Microtiter Pouch and remove plate from pouch. Remove all strips and wells that will
not be used in the assay and place back in pouch with desiccant and re-seal.
7. Rat CRP Calibrator
Add 1.0 mL of distilled or de-ionized water to the lyophilized Rat CRP Calibrator and mix gently until dissolved. The
calibrator is now at a concentration of 2.70 μg/mL (the reconstituted calibrator should be aliquoted and frozen if
future use is intended). Rat CRP Calibrators need to be prepared immediately prior to use (see the following
chart). Mix well between each step. Avoid foaming.
Calibrator

Concentration(ng/mL)

Calibrator Volume added to
1X Diluent

Volume of
1X Diluent

6
5
4
3
2
1
0

200
100
50
25
12.5
6.25
0

70 μL of Rat CRP Calibrator
300 μL of Calibrator 6
300 μL of Calibrator 5
300 μL of Calibrator 4
300 μL of Calibrator 3
300 μL of Calibrator 2875 μL
300 μL
300 μL
300 μL
300 μL
300 μL
600 μL8. Positive Control
The concentration and recommended dilution are provided on the Control Certificate. Before use, briefly centrifuge thePositive Control to allow all of the liquid to collect in the bottom of the vial.

STORAGE AND STABILITY
1. Complete Kit
The expiration date for the kit is stated on the outer label. The recommended storage temperature is 4°C. Note: Seelong term storage recommendations below for the Rat CRP Calibrator and Positive Control.
2. Diluent
The 5X Diluent Concentrate is stable until the expiration date. The 1X working solution is stable for at least one weekfrom the date of preparation. Both solutions should be stored at 4°C.
3. Wash Solution
The 20X Wash Solution Concentrate is stable until the expiration date. The 1X working solution is stable for at least
one week from the date of preparation. Both solutions should be stored at 4°C.
4. Enzyme-Antibody Conjugate
Undiluted anti-CRP-HRP conjugate should be stored at 4°C in the dark and diluted immediately prior to use. The
undiluted conjugate solution is stable until the expiration date.
5. TMB Substrate Solution

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KAMIYA BIOMEDICAL COMPANYThe TMB Substrate Solution should be stored at 4°C in the dark and is stable until the expiration date. Protect fromlight.
6. Stop Solution
The Stop Solution should be stored at 4°C and is stable until the expiration date.
7. Microtiter Plate
Anti-rat CRP coated wells should be stored at 4°C in the sealed foil pouch with desiccant pack and are stable until the
expiration date.
8. Rat CRP Calibrator
The lyophilized Rat CRP Calibrator should be stored at 4°°C or frozen until reconstituted. The reconstituted
calibrator should be aliquoted and stored frozen (avoid multiple freeze-thaw cycles). The working calibrator
solutions should be prepared immediately prior to use.
9. Positive Control
For storage longer than 7 days keep frozen until the expiration date. Storage for less than 7 days can be at 4°C. Avoid
multiple freeze/thaw cycles.

INDICATIONS OF INSTABILITY
If the test is performing correctly, the results observed with the calibrator solutions should be within 20% of the expectedvalues.

SPECIMEN COLLECTION AND HANDLING
Blood should be collected by venipuncture. The serum should be separated from the cells after clot formation by
centrifugation. For plasma samples, blood should be collected into a container with an anticoagulant and thencentrifuged. Care should be taken to minimize hemolysis, excessive hemolysis can impact your results. Assay
immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles.
For any sample that might contain pathogens, care must be taken to prevent contact with open wounds. No additives orpreservatives are necessary to maintain the integrity of the specimen. Avoid azide contamination.

ASSAY PROTOCOL
Dilution of Samples
Due to the high-sensitive nature of the assay each sample should be diluted before use for a normal assay. A 1:12,000
dilution is appropriate for most serum/plasma samples. For absolute quantification of samples that yield results outside
the range of the calibration curve, a lesser or greater dilution might be required. If unsure of sample level, a serial
dilution with one or two representative samples before running the entire plate is highly recommended.
To prepare a 1:12,000 dilution of sample, transfer 5 µL of sample to 495 µL of 1X Diluent. This gives you a 1:100 dilution.
Mix thoroughly. Then dilute this by transferring 5 µL to 595 µL of 1X Diluent. You now have a 1:12,000 dilution of yoursample. Make sure to mix thoroughly at each stage.

Procedure
1. Bring all reagents to RT before use.
2. The Calibrators and the test sample(s) should be loaded into the ELISA wells as quickly as possible to avoid a shift in
OD readings. Using a multichannel pipette would reduce this occurence.
3. Pipette 100 μL of
Calibrator 0 (0.0 ng/mL) in duplicate
Calibrator 1 (6.25 ng/mL) in duplicate
Calibrator 2 (12.5 ng/mL) in duplicate
Calibrator 3 (25 ng/mL) in duplicate
Calibrator 4 (50 ng/mL) in duplicate
Calibrator 5 (100 ng/mL) in duplicate
Calibrator 6 (200 ng/mL) in duplicate
4. Pipette 100 μL of diluted Positive Control (in duplicate) into pre-designated wells.
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5. Pipette 100 μL of diluted sample (in duplicate) into pre-designated wells.
6. Incubate the Microtiter Plate at 22°C (RT) for ten (10 ± 2) minutes. Keep plate covered and level during incubation.
7. Following incubation, aspirate the contents of the wells.
8. Completely fill each well with appropriately diluted Wash Solution and aspirate. Repeat three times, for a total of four
washes. If washing manually: completely fill wells with diluted Wash Solution, invert the plate and pour/shake out thecontents in a waste container. Follow this by sharply striking the wells on absorbent paper to remove residual Wash
Solution. Repeat three times for a total of four washes.
9. Pipette 100 μL of appropriately diluted Enzyme-Antibody Conjugate to each well. Incubate at RT for ten (10 ± 2)
minutes. Keep plate covered in the dark and level during incubation.
10. Wash and blot the wells as described in Steps 7 and 8.
11. Pipette 100 μL of TMB Substrate Solution into each well.
12. Incubate in the dark at RT for precisely five (5) minutes.
13. After five minutes, add 100 μL of Stop Solution to each well.
14. Determine the absorbance at 450 nm of the contents of each well within 30 minutes. Calibrate the plate reader tomanufacturer's specifications.
The absorbance of the final reaction mixture can be measured up to 2 hours after the addition of the Stop Solution.
However, good laboratory practice dictates that the measurement be made as soon as possible.

RESULTS
1. Subtract the average background value from the test values for each sample.
2. Using the results observed for the calibrators construct a calibration curve. The appropriate curve fit is that of a fourparameter logistics curve. A second order polynomial (quadratic) or other curve fits may also be used.
3. Interpolate test sample values from calibration curve. Correct for sample dilution factor to arrive at CRP concentrationin original sample.

QUALITY CONTROLIn accord with good laboratory practice, the assays for specific CRP require meticulous quality control. Each laboratory
should use routine quality control procedures to establish inter- and intra-assay precision and performance characteristics.

LIMITATION OF THE PROCEDURE
1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete
understanding of the information contained in the package insert instructions and with adherence to good laboratory
practice.
2. Factors that might affect the performance of the assay include proper instrument function, cleanliness of glassware,
quality of distilled or de-ionized water, and accuracy of reagent and sample pipetting, washing technique, incubation
time or temperature.

FOR RESEARCH USE ONLY

KAMIYA BIOMEDICAL COMPANY
12779 Gateway Drive, Seattle, WA 98168
Tel: (206) 575-8068
Fax: (206) 575-8094
Email: LifeScience@k-assay.com
www.k-assay.com

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