TURBIDIMETRIC IMMUNOASSAY
FOR DETERMINATION OF C-REACTIVE PROTEIN

SUMMARY

C-reactive protein (CRP) is an acute phase protein synthesized in the liver. Its rate of synthesis increases within hours of acute injury or the onset of inflammation and may reach as high as 20 times the normal levels. A rapid fall of CRP indicates recovery. The degree of elevation of CRP level directly reflects the mass or activity of inflamed tissue. And its ability to fall to normal levels on resolution of the condition renders quantified CRP values to be a good indicator to allow rapid selection of appropriate anti-inflammatory therapy in several rheumatic diseases, which are, clinically difficult to assess.

Apart from indicating inflammatory disorders, CRP levels helps in differential diagnosis, in the management of neonatal septicemia and meningitis where standard microbiological investigations are difficult. CRP levels rise invariably after major surgery, but fall to normal within 7-10 days. Absence of this fall is indicative of septic or inflammatory postoperative complications. Serum CRP concentration provides useful information in patients with myocardial infarction there being an excellent correlation between peak levels of CRP and creatine phosphokinase.

REAGENT

  1. QUANTIA-CRP Activation Buffer (R1): Ready to use.
  2. QUANTIA -CRP reagent (R2): Ready to use solution of anti-CRP antibody.
  3. QUANTIA-CRP Calibrator: A lyophilized preparation of serum equivalent to the stated amount of CRP on a mg/dl basis, when hydrated appropriately. The QUANTIA-CRP calibrator is traceable to the W.H.O. International Reference Standard (85/506) for Human C-reactive protein

    Each batch of reagents undergoes rigorous quality control at various stages of manufacture for its specificity, sensitivity, and performance.

REAGENT STORAGE AND STABILITY

  1. Store the reagents at 2 8C. DO NOT FREEZE.
  2. The shelf life of the reagent, activation buffer and the calibrator is as per the expiry date mentioned on the respective vial label.
  3. The reconstituted Quantia-CRP calibrator is stable for 7 days at 2 8C and 48 hours at 25C 30C (RT.).

PRINCIPLE

Quantia CRP is a turbidimetric immunoassay for determination of C-reactive protein in human serum and is based on the principle of agglutination reaction. The test specimen is mixed with activation buffer ( R1), Quantia-CRP reagent (R2) is then added and allowed to react. Presence of CRP in the test specimen results in the formation of an insoluble complex producing a turbidity, which is measured at 340 nm wavelength. The increase in turbidity corresponds to the concentration of CRP in the test specimen.

NOTE

  1. In vitro diagnostic reagent for laboratory and professional use only. Not for medicinal use.
  2. All the reagents derived from human source have been tested for HBsAg and HIV antibodies and are found to be non-reactive. However handle the material as if infectious.
  3. Reagents contain 0.1% Sodium Azide as preservative. Avoid contact with skin and mucosa. On disposal flush with large quantities of water
  4. The reagents can be damaged due to microbial contamination or on exposure to extreme temperatures. It is recommended that the performance of the reagents be verified using known controls periodically.
  5. Gently mix the Quantia-CRP reagents well before use to improve test performance.
  6. Do not use vortex mixers for mixing. Gently mix the reagents and samples during test procedures.
  7. As the reagents within lots have been matched, reagents from different lots must not be interchanged.
  8. Calibrators of different manufacturers must not be used with Quantia-CRP reagents.
  9. The calibration curve must be validated periodically with known controls.
  10. The Quantia-CRP assay is recommended only for analyzers with cuvette mode. Though any semi automated analyzer with appropriate programming facility can be used, for best results it is recommended to use Quantiamate analyzer. Fully automated analyzers may be used, provided the reagent has been standardized on the system.
  11. The procedures mentioned in this pack insert are based on a minimum reading volume of 500 ml (0.5 ml). In case of instruments where minimum volume required for reading absorbance is 1.0 ml, use double the quantity of reagents and samples mentioned in the test procedure.

SPECIMEN COLLECTION AND PREPARATION

No special preparation of the patient is required prior to specimen collection by approved techniques.

Only serum should be used for testing. Should a delay in testing occur, store the samples at 2 - 8C. Samples can be stored for upto a week at 2 - 8C, provided they are not contaminated. Do not use hemolysed, icteric, or highly turbid serum. Turbid or particulate serum samples must be clarified by centrifugation at 2000 rpm for 15 minutes. Use the clear supernatant for testing.

ADDITIONAL MATERIAL REQUIRED

Spectrophotometer with 340 nm wavelength filter and cuvette mode, stopwatch, well calibrated micropipettes, disposable tips, isotonic saline, particulate free distilled water, test-tube rack, incubator/ waterbath set at 37C, optically clean disposable cuvettes such as Quantiamate semi micro cuvettes/ glass cuvettes.

TEST PROCEDURE

Bring reagent and specimen to room temperature before use. Parameters for instruments

Wavelength 340 nm
Reaction temperature 37C
Cuvette 1 cm pathlength

Method for preparation of CRP Calibration Curve

The Quantia-CRP calibrator must be reconstituted exactly with 1.0 ml of distilled water, wait for 10 minutes, gently swirl the vial till the solution attains homogeneity. Once reconstituted it is ready to use for preparing the CRP calibration curve.

The Concentration of CRP (S) in the reconstituted calibrator is as mentioned at the end of the package insert.

Dilute the calibrator serially as mentioned below for preparation of calibration curve.

Test Tube No.
1
2
3
4
5
6
Calibrator dilution No.
D1
D2
D3
D4
D5
D6
Saline
-
100 ml
100 ml
100 ml
100 ml
100 ml
Calibrator (S)
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
Conc. of CRP in mg/dl
10
5
25
12
0.6
0.3

Five dilutions of the calibrator including the highest 10 mg/dl (D1) and lowest 0.3/mgdl (D6) concentrations of measuring range must be used for the preparation of the calibration curve. Select any three dilutions from D2 to D4 in addition to D1 and D6 to prepare the calibration curve.

Test procedure for preparation of calibration curve

  1. Zero the instrument with distilled water.
  2. Pipette 500 ml of activation buffer (R1) and 50 ml of standard D1 from tube no. 1 in the measuring cuvette. Mix well and incubate for 5 minutes at 37C.
  3. Read absorbance (A1).
  4. Add 50 ml of Quantia-CRP reagent (R2) (preincubated at 370C), mix gently and start the stopwatch simultaneously.
  5. Read absorbance (A2) at the end of exactly five minutes.
  6. Repeat steps No. 2 5 for each diluted calibrator selected for preparing the calibration curve.
  7. Calculate DA (A2 A1) for each diluted calibrator for preparing the calibration curve. Plot a graph of DA versus concentration of CRP on the graph paper provided with the kit.
The calibration curve so obtained is valid only for the same lot of QUANTIA-CRP reagent.

Test procedure for specimen

  1. For determination of CRP concentration in the test specimen please follow steps 2 5 using the test specimen in place of the calibrator.
  2. Calculate DA (A2 A1) for the test specimen.

VALIDATION CRITERIA

If the DA of the test specimen is less than DA obtained for the calibrator of highest concentration (D1) then the concentration of CRP in the test specimen can be determined directly by interpolating DA of the test specimen on the obtained calibration curve.

If the DA of the test specimen is higher than DA of the standard with highest concentration (D1) then the test has to be rerun by carrying out serial dilutions of test specimen till the DA of the diluted test specimen is less than DA of D1. The CRP concentration for such samples can be determined as mentioned below in calculations.

CALCULATIONS

  1. Interpolate DA of the diluted test specimen on the calibration curve and obtain the CRP concentration C of the diluted test specimen.
  2. Multiply the CRP concentration C with the dilution factor (F) of the test specimen for obtaining the concentration of CRP in the neat test specimen.

    Concentration of CRP in the neat test specimen in mg/dl = C x F
    (Where F is the dilution factor of the test specimen, for e.g. 2 for 1:2 dilution of test specimen and so on)

SPECIFIC PERFORMANCE CHARACTERISTICS

Measuring range

The Quantia-CRP reagent has been designed to measure CRP concentrations in the range of 0.3-10 mg/dl.

Detection limit / Analytical Sensitivity

Detection limit: 0.3 mg/dl
The detection limit represents the lowest measurable CRP concentrations that can be distinguished from zero.


Prozone limit

No prozone effect was observed with CRP concentration upto 100 mg/dl.

Precision

Intra-assay precision
n
Mean mg/dl
SD
CV (%)
Sample 1
15
0.55
0.01
2.08
Sample 2
15
1.64
0.03
1.77
Sample 3
15
9.82
0.12
1.23

Inter-assay precision
n
Mean mg/dl
SD
CV (%)
Sample 1
15
0.55
0.01
2.08
Sample 2
15
1.64
0.03
1.77
Sample 3
15
9.82
0.12
1.23

Interference

No interference was observed with:

Interference factor
No interference upto
Glucose
400 mg/dl
Bilirubin
50 mg/dl
Haemoglobin
500 mg/dl
Creatinine
6 gms/L
Urea
2 gms/L

REFERENCE VALUES

The reference values of CRP in normal population are 0.6 mg/dl.

REMARKS

  1. Usage of well-calibrated equipment and accessories and procedures is critical for achieving correct results.
  2. When DA obtained for the test specimen is greater than the DA of the standard with highest concentration then, it indicates that the concentration of CRP in the test specimen is beyond the measuring range of the Quantia-CRP assay. Such specimens should be rerun with further dilutions.
  3. Markedly lipemic, hemolysed, and contaminated serum samples could produce non-specific CRP values.
  4. Use of plasma rather than serum can lead to erroneous CRP values.
  5. Elevated levels of CRP are found to be present after the 1st trimester of pregnancy and persists until delivery.
  6. CRP levels are elevated in women who are on oral contraceptives.
  7. The commonly used anti-inflammatory drugs or immunosuppressive drugs, including steroids do not affect CRP response, unless the disease activity is affected and it covers an exceptionally broad incremental range upto 3000 times.
  8. Do not read results beyond five minutes.
  9. Since CRP production is a non-specific response to tissue injury, it is recommended that results of the test should be correlated with clinical findings to arrive at the final diagnosis.

WARRANTY

This product is designed to perform as described on the label and package insert. The manufacturer disclaims any implied warranty of use and sale for any other purpose.

BIBILIOGRAPHY

  1. Andersen H.C., Mc Carthy M., Am. J. Med., 8 : 445 (1950).
  2. Ward A.N., Cooper E.M., Clin. Chem. Acta, 81, 75 (1977)
  3. Fisher C.L., Nakamura R., Am. J. Clin. Path., 66, 840 (1976).
  4. Connell E. B., Connell J., Am J. Obs. Gynaec., 110, 633 (1971).
  5. Clinical Laboratory Diagnostics, Edited by Lothar Thomas, M.D., 1st Ed., 1998, TH-Books Verlagsgesellschaft mbH, Frankfurt, Germany.
  6. Data on file : Tulip Diagnostics.

TULIP DIAGNOSTICS (P) LTD.
Gitanjali - Tulip Block, Dr. A. A. Rego Bagh, Bambolim P.O., Goa - 403 202, INDIA.
Website: www.tulipgroup.com