Porcine heavy chain Immunoglobulin ELISA KIT 猪免疫球蛋白重链 ELISA KIT 试剂盒

Porcine heavy chain Immunoglobulin     ELISA Kit

96 Tests

Catalogue Number: E07I0023

Store all reagents at 2-8°C

Collect sample:

Serum or blood plasma

FOR LABORATORY RESEARCH USE ONLY. NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!

INTENDED USE

This B.G Ig-Hc ELISA kit is intended Laboratory for research use only and is not for use in diagnostic or therapeutic procedures. The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of Ig-Hc in the sample, this Ig-Hc ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versus Ig-Hc concentration. The concentration of Ig-Hc in the samples is then determined by comparing the O.D. of the samples to the standard curve.

PRINCIPLE OF THE ASSAY

This Ig-Hc enzyme linked immunosorbent assay 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 Ig-Hc. Standards or samples are then added to the microtiter plate wells and Ig-Hc if present, will bind to the antibody pre-coated wells. In order to quantitatively determine the amount of Ig-Hc present in the sample, a standardized preparation of horseradish peroxidase （HRP）-conjugated polyclonal antibody, specific for Ig-Hc are added to each well to “sandwich” the Ig-Hc immobilized on the plate. The microtiter plate undergoes incubation, and then the wells are thoroughly washed to remove all unbound components. Next, A and B 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 Ig-Hc and enzyme-conjugated antibody will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wave length of 450 nm.

REAGENTS PROVIDED

All reagents provided are stored at 2-8° C. Refer to the expiration date on the label.

1.          MICROTITER PLATE  96 wells

2.          ENZYME CONJUGATE  10.0 mL  1 vial

3.          STANDARD.1   0 mg/dl  1 vial

4.          STANDARD.2   2.5 mg/dl  1 vial

5.          STANDARD.3   5.0 mg/dl  1 vial

6.          STANDARD.4   10 mg/dl  1 vial

7.          STANDARD.5   25 mg/dl  1 vial

8.          STANDARD.6   50 mg/dl   1 vial

9.          SUBSTRATE A  6.0 mL  1 vial

10.       SUBSTRATE B  6.0 mL  1 vial

11.       STOP SOLUTION  6.0 mL  1 vial

12.       WASH SOLUTION x100  10 mL  1 vial

13.       INSTRUCTION   1

SAMPLE COLLECTION AND STORAGE

Serum-Use a serum separator tube（SST） and allow samples to clot for 30minutes before centrifugation for 15minutes at approximay 1000 x g.Remove serum and assay immediay or aliquot and store samples at -20 °C or -80°C.

Plasma-Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2-8°C within 30minutes of collection. Store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles.

Cell culture fluid and other biological fluids-Remove particulates by centrifugation and assay immediay or aliquot and store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles.

NOTE: Serum, plasma, and cell culture fluid samples to be used within 7 days may be stored at 2-8°C, otherwise samples must stored at -20°C（≤2months） or -80°C（≤6months） to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles .When performing the assay slowly bring samples to room temperature.

DO NOT USE HEAT-TREATED SPECIMENS.

MATERIALS REQUIRED BUT NOT SUPPLIED

1.          Microplate reader capable of measuring absorbance at 450 nm.

2.          Precision pipettes to deliver 2 ml to 1 ml volumes.

3.          Adjustable 10ml -100ml pipettes for reagent preparation.

4.          Adjustable 10ml -100ml pipettes for reagent preparation.

5.          100 ml and 1 liter graduated cylinders.

6.          Calibrated adjustable precision pipettes, preferably with disposable plastic tips. （A manifold multi-channel pipette is desirable for large assays.）

7.          Absorbent paper.

8.          37°C incubator.

9.          Distilled or deionized water.

10.       Data analysis and graphing software. Graph paper: linear （Cartesian）, log-log or semi-log, or log-logit as desired.

11.       Tubes to prepare standard or sample dilutions.

PRECAUTIONS

1.          Do not substitute reagents from one kit lot to another. Standard, conjugate and microtiter plates are matched for optimal performance. Use only the reagents supplied by manufacturer.

2.          Allow kit reagents and materials to reach room temperature （20-25°C） before use. Do not use water baths to thaw samples or reagents.

3.          Do not use kit components beyond their expiration date.

4.          Use only deionized or distilled water to dilute reagents.

5.          Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.

6.          Use fresh disposable pipette tips for each transfer to avoid contamination.

7.          Do not mix acid and sodium hypochlorite solutions.

8.          Serum and plasma should be handled as potentially hazardous and capable of transmitting disease. Disposable gloves must be worn during the assay procedure, since no known test method can offer complete assurance that products derived from human blood will not transmit infectious agents. Therefore, all blood derivatives should be considered potentially infectious and good laboratory practices should be followed.

9.          All samples should be disposed of in a manner that will inactivate viruses.

10.       Solid Waste: Autoclave 60 min. at 121°C.

11.       Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste should be allowed to stand for a minimum of 30 minutes to inactivate the viruses before disposal.

12.       Substrate Solution is easily contaminated. If bluish prior to use, do not use.

13.       Substrate B contains 20% acetone, keep this reagent away from sources of heat or flame.

14.       Remove all kit reagents from refrigerator and allow them to reach room temperature （ 20-25°C）.

ASSAY PROCEDURE

Prepare all Standards before starting assay procedure （see Preparation Reagents）. It is recommended that all Standards and Samples be added

1.          in duplicate to the Microtiter Plate.

2.          First, secure the desired number of coated wells in the holder, then add 50 μL of Standards or Samples to the appropriate well of the antibody pre-coated Microtiter Plate.

3.          Add 100 μL of Conjugate to each well. Mix well. Complete mixing in this step is important. Cover and incubate for 1 hours at 37°C.

4.          Prepare Substrate Solution no more than 15 minutes before end of incubation （see Preparation of Reagents）.

5.          Wash the Microtiter Plate using one of the specified methods indicated below:

6.          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 compley with distilled or de-ionized water, then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure four more times for a total of FIVE 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 frame firmly hen washing the plate to assure that all strips remain securely in frame.

7.          Automated Washing: Aspirate all wells, then wash plate FIVE times using distilled or de-ionized water. Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350 μL/well/wash （range: 350-400 μL）. 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.

8.          Add 50 μL Substrate A&B to each well. Cover and incubate for 15 minutes at 20-25°C.

9.          Add 50 μL of Stop Solution to each well. Mix well.

10.       Read the Optical Density （O.D.） at 450 nm using a microtiter plate reader within 30 minutes.

B.G CALCULATION OF RESULTS

1.        This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. （450 nm） obtained for each of the six standard concentrations on the vertical （Y） axis versus the corresponding concentration on the horizontal （X） axis.

2.        First, calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software.

3.        To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration.

4.        Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.

5.        The sensitivity by this assay is 0.01 mg/dl

6.        Standard curve

Disposal Note Safety

1.          This kit contains materials with small quantities of sodium azide. Sodium azide reacts with lead and copper plumbing to form explosive metal azides. Upon disposal, flush drains with a large volume of water to prevent azide accumulation. Avoid ingestion and contact with eyes, skin and mucous membranes. In case of contact, rinse affected area with plenty of water. Observe all federal, state and local regulations for disposal.