Summary
This is an ELISA (Enzyme Linked ImmunoSorbent Assay) kit for measurement of human apo B-48 with high sensitivity using Sandwich assay principle.
[Advantage]
(1) Rapid assay (total reaction time: 2 hours 50 min).
(2) A small sample volume.
(3) An ecologically-friendly preservative is used.
(4) Excellent precision and reproducibility.
[Components]
Reagents Amounts
(A) Anti-apo B-48-coated plate 96 wells (8x12) / 1 plate
(B) Standard human apo B-48 (Freeze-dried) 128ng/1 vial
(C) Dilution Buffer solution 60ml/1 vial
(D) Biotin-conjugated anti-apo B-48 antibody 100µl/ 1 vial
(E) HRP-conjugated streptavidin 100 µl / 1 vial
(F) Chromogenic substrate reagent (TMB) 12ml/ 1 vial
(H) Reaction Stop Solution (1M H2SO4) 12ml/ 1 vial
(I) Concentrated washing buffer (10x) 100ml/ 1 bottle
Assay sample
Human serum or plasma (heparin or EDTA as anticoagulant) diluted to 100x with buffer (C).
Purpose of assay
Quantitative measurement of apolipoprotein B-48
Assay range
2.5 – 160 ng/ml
Assay operation
1. Equipment necessary but not included in the kit.
(1) Micropipette (a micropipette able to deliver sample volume with high precision.), and a pipette for repetitive dispensing.
(2) Microplate washing apparatus (a microplate washer or a flashing bottle with nozzle).
(3) A microplate reader (A densitometer for microplate).
2. Preparation of reagents
(1) Washing buffer: Dilute the concentrated washing buffer (I) to 10X with purified water.
(2) Biotin-conjugated anti-apo B-48 (D): Dilute to 100X with the buffer solution(C).
(3) HRP-conjugated streptavidin (E): Dilute to 100X with the buffer solution(C).
(4) Other reagents are ready-to-use.
(5) All the reagent solutions should be used after allowing them to reach room temperature (20-25 º C).
3. Assay sample dilution
Dilute assay samples (human sera or plasma) to 100X with buffer (C).
4. An example of preparing standard solutions
Prepare the original standard solution by adding 400 µl of distilled water to the vial (B) and mix gently and thoroughly to dissolve the content, then prepare a series of standard solutions by a dilution program shown below. After reconstitution, the original standard solution should be used within 24 hours. Only freeze and thaw the original standard solution once.
The use of either polyethylene or polypropylene test tubes is recommended for the dilution process. Test tubes of other materials are not suitable due to high protein absorption.
Concentration (ng/ml) 160 80 40 20 10 5.0 2.5 0
Std. Sol. (µl ) 200**200* 200* 200* 200* 200* 200* 0
Dil.Buffer (µl )-C 200 200 200 200 200 200 200 200
**Original standard solution, *One rank higher standard solution
5. Assay procedure
(1) Remove the cover sheet of the microplate after bringing it up to room temperature.
(2) Rinse the antibody coated wells (A) 4 times by filling the wells with 350µl of Wash Buffer and then discarding the buffer. Remove residual buffer in the wells by striking the plate upside-down onto several sheets of folded paper towel.
(3) Pipette 50 µl of diluted sample into the sample wells.
(4) Pipette 50 µl of the standard solution to the wells designated for standards.
(5) Shake the plate on a plate shaker at 800-1,000rpm for approximately 5 to 10 seconds.
(6) Incubate for 1 hour at room temperature (20-25ºC).
(7) Discard the reaction mixture, and then wash wells 4 times as described in (2).
(8) Pipette 50 µl of biotin-conjugated anti-apo B-48 solution to all wells. Then shake on a plate shaker as (5).
(9) Incubate the plate for 1 hour at room temperature.
(10) Discard the reaction mixture, and then wash the plate 4 times as in (2).
(11) Pipette 50 µl of HRP-conjugated avidin solution to all wells, and shake as (5).
(12) Incubate for 30 minute at room temperature.
(13) Discard the reaction mixture, and wash the plate 4 times as (2).
(14) Pipette 50 µl of chromogenic substrate solution to wells, and shake as (5).
(15) Let the plate stand for 20 minutes at room temperature. Avoid exposing the microtiter plate to direct sunlight. Covering the plate with aluminum foil is recommended.
(16) Add 50 µl of the reaction Stop Solution (H) to all wells and shake as (5).
(17) Measure the absorbance at 450 nm (sub-wave length, 620nm) in a plate reader within 30 minutes.
[Calculation of apo B-48 concentration]
(1) Prepare a standard curve using semi-logarithmic or logarithmic graph paper by plotting absorbance* (Y-axis) against apo B-48 concentration (ng/ml) on X-axis.
*Absorbance at 450nm minus absorbance at 620nm.
(2) Using the standard curve, read the apo B-48 concentration of a sample from its absorbance*, and multiply the assay value by dilution rate. Though the assay range is wide enough, in case the absorbance of some samples are higher than that of the highest standard, please repeat the assay after proper dilution (upper limit is 200X) of samples with the buffer solution.
* We recommend the use of 3rd order regression curve or 4-parameter method in computer calculation.
[Important notice in the treatments]
1. Treatment of assay samples
(1) Use serum or plasma samples obtained by standard collection method. Please, avoid using NaF-containing blood sampling tube, because fluoride ion is a peroxidase inhibitor, and may reduce the coloration even after washing.
(2) Turbid samples or those containing insoluble materials should be centrifuged before assay and remove those materials.
(3) Measure the samples as soon as possible after sampling.
(4) Dilution of assay samples should be made using microtubes before starting assay.
2. Storage of assay samples.
If assay samples have to be stored for a long period, freeze samples and store below –35C. The original standard solution should be prepared immediately before assay. But if you have to store it, store it below –35C. Avoid repeated freezing and thawing for both samples and the standard solution. |