Babesia caballi Antibody Test, cELISA Kit
2 plates, maximum 182 samples
Article number:
273-2
Content:
Units
Selling unit:
Kit
This Babesia caballi Antibody Test Kit is a competitive enzyme-linked immunosorbent assay (cELISA). Sample serum B. caballi antibody inhibits binding of primary monoclonal antibody. The binding of primary monoclonal antibody to the antigen-coated plate is detected with HRP-labeled secondary antibody. Finally, the presence of HRP-labeled secondary antibody is quantified by the addition of enzyme substrate and subsequent color product development. Strong color development indicates little or no inhibition of primary monoclonal antibody binding and therefore the absence of B. caballi antibody in sample sera. Weak color development due to inhibition of the primary monoclonal antibody binding to the antigen on the solid phase indicates the presence of B. caballi antibodies in sample sera.
Test Validation
The mean of the Negative Controls must produce an optical density ≥ 0.300 and ≤ 2.000.
The mean of the Positive Controls must have an inhibition of ≥ 40%.
Interpreting the Results
If a test sample produces < 40% inhibition, it is negative.
If a test sample produces ≥ 40% inhibition, it is positive.
Storage at 2–8 °C
PO Box 502
Pullman, WA 99163 USA
P: 509.334.5815
F: 509.332.5356
support@vmrd.com
www.vmrd.com
Test Validation
• The mean of the Negative Controls must produce an optical density ≥ 0.300
and ≤ 2.000.
• The mean of the Positive Controls must have an inhibition of ≥ 40%.
Interpreting the Results
• If a test sample produces ≥ 40% inhibition, it is positive.
• If a test sample produces < 40% inhibition, it is negative.
Precautions
Kit components should be handled and disposed of as potentially hazardous.
Do not eat, drink, or smoke where serum samples and kit reagents are
handled. Do not pipette by mouth. Some reagents may be harmful if ingested.
If ingested, seek medical attention. Do not use expired or contaminated
reagents, or reagents from other kits or serials. Do not mix reagents from
different serials of this same product.
Component B, Positive Control, contains sodium azide as a preservative.
Component C, Negative Control, contains sodium azide as a preservative.
Component D, 100X Primary Antibody, contains ProClin 300,
methylisothiazolone, and bromonitrodioxane as preservatives.
Component E, 100X Secondary Antibody-Peroxidase Conjugate, contains
ProClin 300, methylisothiazolone, bromonitrodioxane, and thimerosal as
preservatives.
Component F, Antibody Diluting Buffer, contains ProClin 300,
methylisothiazolone and bromonitrodioxane as preservatives.
Component G, Serum Diluting Buffer, contains sodium azide as a preservative.
Component J, Stop Solution, contains sodium fl uoride.
Version 200813
FOR VETERINARY USE ONLY
BABESIA CABALLI
ANTIBODY TEST KIT,
cELISA
Assay instructions for catalog number 273-2
General Description
This competitive, enzyme-linked, immunosorbent assay (cELISA) detects
antibodies to
Babesia caballi
in equine sera. Sample serum
B. caballi
antibody
inhibits binding of primary monoclonal antibody. The binding of primary
monoclonal antibody to the antigen-coated plate is detected with horseradish-
peroxidase (HRP)-labeled secondary antibody. Binding of the HRP-labeled
secondary antibody is detected by the addition of enzyme substrate and
quantifi ed by subsequent color product development. Strong color development
indicates little or no inhibition of primary monoclonal antibody binding and
therefore the absence of
B. caballi
antibody in sample sera. Weak color
development due to inhibition of the primary monoclonal antibody binding to
the antigen on the solid phase indicates the presence of
B. caballi
antibodies in
sample sera.
Kit Contents
Component
A Antigen-Coated Plates 2 plates
B Positive Control 2 ml
C Negative Control 2 ml
D 100X Primary Antibody 0.3 ml
E 100X Secondary Antibody-Peroxidase Conjugate 0.3 ml
F Antibody Diluting Buffer 60 ml
G Serum Diluting Buffer 10.5 ml
H 10X Wash Solution Concentrate 120 ml
I Substrate Solution 30 ml
J Stop Solution 30 ml
Assay Instructions
Materials Required But Not Included in the Test Kit
Single and multichannel adjustable-volume pipettors and disposable plastic
tips, non-antigen-coated transfer plate(s), ELISA microplate absorbance
spectrophotometer with 620, 630 or 650 nm fi lter, deionized or distilled water,
paper towels, graduated cylinder, timer, multichannel pipettor reservoirs, wash
bottle, manual multichannel washing device or automatic plate washer
VLN/PCN 332/501A.20
Storage and Stability
Store all reagents at 2-8°C. Do not freeze. Reagents will remain stable until the
expiration date when stored as instructed. Do not use test kit past the expiration
date.
Preparation
a. Warm reagents: Bring the serum samples, reagents and plate(s) to room
temperature (23 ± 2°C) prior to starting the test.
b. Prepare controls and samples: The Positive and Negative Controls (B &
C) and test serum samples must be diluted 1/2 in Serum Diluting Buffer
(G) for use in this test. It is recommended that these dilutions be made in
a non-antigen-coated transfer plate. Add Serum Diluting Buffer (G) to the
appropriate wells of a transfer plate according to your setup record. Add
an equal volume of Positive Control (B), Negative Control (C), or sample
to each well and cycle the pipettor at least three time to mix. Change tips
between samples. The volume in the transfer plate must be in excess of 50
l in order to transfer 50 l from it. Load Positive Control (B) in duplicate and
Negative Control (C) in triplicate, regardless of the number of serum samples
to be tested. When whole plates are used, it is best to put the controls in wells
on different areas of the plate. The controls must be loaded every time the
assay is performed and on each plate if multiple plates are used.
c. Prepare plates: Remove the plate(s) from the foil pouch(es) (A). If applicable:
Return any unused strips to the pouch and securely seal it. Place strips
to be used in the frame and number the top of each strip to maintain
orientation. Always mark the strips in case they dislodge from the frame
during washing.
d. Prepare primary antibody: Prepare 1X Primary Antibody by diluting 1 part of
the 100X Primary Antibody (D) with 99 parts of Antibody Diluting buffer (F).
Example: For 96 wells, mix 60 l of 100X Primary Antibody (D) with 5.940
ml of Antibody Diluting Buffer (F) to yield 6 ml of 1X Primary Antibody. Fifty
microliters (50 l) are needed per well. Allow extra quantity for reservoirs,
tubing, pipetting, etc.
e. Prepare secondary antibody-peroxidase conjugate: Prepare 1X Secondary
Antibody-Peroxidase Conjugate by diluting 1 part of the 100X Secondary
Antibody-Peroxidase Conjugate (E) with 99 parts of Antibody Diluting buffer
(F). Example: For 96 wells, mix 60 l of 100X Secondary Antibody-Peroxidase
Conjugate (E) with 5.940 ml of Antibody Diluting Buffer (F) to yield 6 ml of 1X
Secondary Antibody-Peroxidase Conjugate. Fifty microliters (50 l) are needed
per well. Allow extra quantity for reservoirs, tubing, pipetting, etc.
f. Prepare wash solution: Prepare 1X Wash Solution by diluting 1 part of the
10X Wash Solution Concentrate (H) with 9 parts of deionized or distilled
water. Approximately 1.8 ml are needed per well. Allow extra quantity for
reservoirs, tubing, pipetting, etc.
Test Procedure
1. Load controls and serum samples: Using a pipettor set at 50 µl, transfer
PO Box 502
Pullman, WA 99163 USA
P: 509.334.5815
F: 509.332.5356
support@vmrd.com
www.vmrd.com
diluted controls and serum samples into the Antigen-Coated Plate (A). Serum
samples and controls should be loaded into the Antigen-Coated Plate (A)
as quickly as possible. Change tips between samples. Tap the side of the
Antigen-Coated Plate (A) several times to make sure the samples coat the
bottom of the wells. Use care not to spill samples from well to well. Incubate
the plate 30 minutes at room temperature (23 ± 2°C).
2. Wash wells: After the 30-minute incubation, wash the plate 3 times:
If an automatic washer is used, place the plate on the washing apparatus
and wash plate 3 times, fi lling the wells each time with 1X Wash Solution.
If manual washing is used, dump well contents and remove remaining sera
and controls by sharply striking the inverted plate 4 times on a clean paper
towel, striking a clean area each time. Immediately fi ll each well with 1X
Wash Solution using a multichannel fi lling device or a wash bottle. Empty
the wash solution from the plate and strike the inverted plate sharply on a
clean paper towel as above. Fill and empty the plate by the same method 2
additional times for a total of 3 washes.
3. Add primary antibody: Add 50 l of diluted (1X) Primary Antibody to each
well. Tap the side of the loaded assay plate several times to make sure the
primary antibody coats the bottom of the wells. Incubate for 30 minutes at
room temperature (23 ± 2°C).
4. Wash wells: After the 30-minute incubation, wash the plate 3 times as in Step
2.
5. Add secondary antibody-peroxidase conjugate: Add 50 l of diluted (1X)
Secondary Antibody-Peroxidase Conjugate to each well. Tap the side of
the loaded assay plate several times to make sure the conjugate coats the
bottom of the wells. Incubate for 30 minutes at room temperature (23 ± 2°C).
6. Wash wells: After the 30-minute incubation, wash the plate 3 times as in Step
2.
7. Add substrate solution: Add 50 µl of Substrate Solution (I) to each well. Tap
the side of the loaded assay plate several times to make sure the substrate
coats the bottom of the wells. Incubate 15 minutes at room temperature (23
± 2°C). Avoid leaving the plate in direct sunlight. Do not empty wells.
8. Add stop solution: Add 50 µl of Stop Solution (J) to each well. Tap the side of
the loaded assay plate several times to mix the Substrate Solution and the
Stop Solution. Do not empty wells.
9. Read and record the test results: Immediately after adding the Stop Solution,
the plate should be read on a microplate absorbance spectrophotometer.
Set the optical density (OD) reading wavelength to 620, 630 or 650 nm and
read plate(s). Some readers require an empty well on the plate for blanking.
In this case, no reagents should be added to this well.
10. Return all remaining kit reagents to 2-8°C for storage.
Calculation of % Inhibition (%I):
% Inhibition (%I) = 100 [1-(Sample OD ÷ NC OD)]
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