This Human CRP ELISA kit is to be used for the in vitro quantitative determination of human C-Reactive Protein (CRP) concentrations in serum. It is intended for research use only and not for use in diagnostic procedures.
Principle:This CRP enzyme-linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific for CRP. Standards or samples are then added to the appropriate microtiter plate wells and incubated. CRP, if present, will bind and become immobilized by the antibody pre-coated on the wells. The microtiter plate wells are thoroughly washed to remove unbound CRP and other components of sample.
In order to quantitate the amount of CRP present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated antibody specific for CRP is added to each well to "sandwich" the CRP immobilized during the second incubation. The wells are thoroughly washed to remove all unbound HRP-conjugated antibodies and a TMB (3,3'5,5' tetramethyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain CRP and enzyme-conjugated antibody will exhibit a change in colour. The enzyme-substrate reaction is terminated by the addition of an acid solution and the colour change is measured spectrophotometrically at a wavelength of 450nm±2nm.
In order to measure the concentration of CRP in the samples, this kit standard (ready-to use) is assayed at the same time as the samples (diluted if necessary with Sample Diluent). This allows the operator to produce a standard curve of Optical Density (O.D.) versus CRP concentration (mg/L). The concentration of CRP in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Sensitivity:0.03mg/L
Detection Range:0.005 -0.100mg/L
Kit Components:351744A: Microtiter Plate, 1x96wells, Pre-coated with a monoclonal anti-human CRP351744B: CRP Ab (HRP), 1x12ml. Anti-human CRP labeled with HRP351744C: Standard 1, 0mg/L, 1x1ml. Buffered protein base with preservative
351744D: Standard 2, 0.005mg/L, 1x1ml. CRP in buffered protein base with preservative
351744E: Standard 3, 0.010mg/L, 1x1ml. CRP in buffered protein base with preservative
351744F: Standard 4 , 0.025mg/L, 1x1ml. CRP in buffered protein base with preservative
351744G: Standard 5, 0.050mg/L, 1x1ml. CRP in buffered protein base with preservative
351744H: Standard 6, 0.100mg/L, 1x1ml. CRP in buffered protein base with preservative
351744J : TMB Substrate, 1x11ml. Ready to use
351744K: Stop Solution, 1x11ml. 2N HCl. Caution: Caustic Material!351744L: CRP Sample Diluent, 1x50ml. PBS buffer with BSA as preservative.
Storage and Stability:Store components at 4°C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Materials Required but not Supplied:1. Single or multi-channel precision pipettes with disposable tips: 5-10ul, 100ul and 500ul 2. Multi-channel pipette reservoir or equivalent reagent container3. Distilled or deionized water4. Test tubes and racks5. Polypropylene tubes or containers (25ml)6. Erlenmeyer flasks: 100ml, 400ml, 1L and 2L7. Microtiter plate reader (450nm±2nm)8. Automatic microtiter plate washer or squirt bottle9. Sodium hypochlorite solution, 5.25% (household liquid bleach)10. Deionized or distilled water11. Plastic plate cover12. Disposable gloves13. Absorbent paper
Protocol:1. Add 10ul of Standard or Sample to the appropriate well of the antibody pre-coated Microtiter Plate.
2. Dispense 100ul of conjugate to each well. Thoroughly mix for 30-60 seconds.
3. Cover plate and incubate for 45 minutes at room T￥temperature (18-25°C).
4. Wash the Microtiter Plate five times using 1X Wash Buffer, using one of the specified methods below:Manual Washing: Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well completely with distilled or deionized water, then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure four more times for a total of 5 washes. After final wash, invert plate and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate firmly when washing the plate to assure that all strips remain securely in frame
Automated Washing: Aspirate all wells, then wash plates 5 times using distilled or deionized water. Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350ul/well/wash (range: 350-400ul). After final wash, invert plate and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. It is recommended that the washer be set for a soaking time of 10 seconds or shaking time of 5 seconds between washes.
5. Add 100ul TMB Substrate to each well. Cover andiIncubate for 10-15 minutes at room temperature.
6. Add 100ul Stop Solution to each well. Mix well.
7. Read the Optical Density (O.D.) at 450nm using a microtiter plate reader within 30 minutes.
