rev:   February 18, 2004

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  ANTIBODIES  

(anti-Human and others as indicated)

RDI Division of Fitzgerald Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (neutralization, blotting, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below.


Bacillus Thurigiensis  CRY1 (antibody, antigen and kit see below)

-Cry 1Ac, protein and rabbit polyclonal

-Cry 1F, protein, rabbit and chicken polyclonals

-Cry 2A, protein, rabbit and chicken polyclonals

-Cry 2A, protein, rabbit and chicken polyclonals

-Cry 9C, protein, rabbit and chicken polyclonals


anti-Bt Cry 1Ab Monoclonal

CATALOG # RDI-BTCRY1abm      $625.00/200ug

                       RDI-BTCRY1abmX   $1250.00/500ug


IMMUNOGEN Purified Bt Cry 1Ab protein

HOST Mouse (monoclonal)

PURIFICATION METHOD Protein A column

PROTEIN CONCENTRATION 1mg/ml (after reconstitution) in Phosphate, pH 7.3. 0.5 mg/vial

STORAGE Lyophilized: store at 4 °C. After reconstitution, if Not used within a week, aliquot and store at -20 °C

STABILITY/SHELF LIFE Two years lyophilized

RECONSTITUTION Reconstitute with 0.5 ml of Phosphate, pH 7.3. Mix gently, Wash the sides of the vial and wait 5-10 minutes before use.

For ELISA use as a capture antibody, dilute to 1-2 ug/mL in phosphate pH 7.3 and coat plates with 125 uL per well. Incubate overnight, wash plates 3X with 250 uL of appropriate wash solution and post coat for 2 hours with 250 ul of 0.1% BSA in phosphate post coating buffer

SPECIFICITY

Reacts with Bt Cry 1Ab and Cry 1Ac toxins. Very low cross-reactivity has been observed with Cry 1F, Cry 2A, Cry 3B, Cry 9C.

APPLICATIONS ELISA, Western blot, Immunoprecipitation,.

SENSITIVITY When used in conjunction with cat#RDI-BTCRY1abR  polyclonal anti-Cry 1Ab and a goat anti-rabbit HRP a sensitivity of < 0.125 ng/mL has been observed

FOR RESEARCH USE ONLY


Rabbit anti-Bt Cry 1Ab Polyclonal

CATALOG # RDI-BTCRY1abR $625.00/200ug


CATALOG # RDI-BTCRY1abrX $1250.00/0.5mg

IMMUNOGEN Purified Bt Cry 1Ab protein

HOST Rabbit (polyclonal)

PURIFICATION METHOD Protein A column

PROTEIN CONCENTRATION 1mg/ml (after reconstitution) in Phosphate, pH 7.3. 0.2 mg/vial

STORAGE Lyophilized: store at 4 °C. After reconstitution, if Not used within a week, aliquot and store at -20 °C

STABILITY/SHELF LIFE Two years lyophilized

RECONSTITUTION Reconstitute with 0.2 ml of Phosphate, pH 7.3. Mix gently, Wash the sides of the vial and wait 5-10 minutes before use. For ELISA use as a reporter antibody, dilute to (1:500-1:800) in appropriate diluent

SPECIFICITY Reacts with Bt Cry 1Ab and Cry 1Ac toxins. Very low cross-reactivity has been observed with Cry 1F, Cry 2A, Cry 3B, Cry 9C.

APPLICATIONS ELISA, Western blot, Immunoprecipitation.

SENSITIVITY When using a sample size of 100 uL, and used in conjunction with monoclonal anti-Cry 1Ab (cat#RDI-BTCRY1abm) and a goat anti-rabbit HRP a sensitivity of < 0.125 ng/mL has been observed.

FOR RESEARCH USE ONLY


Bt Cry 1Ab Protein

CATALOG # RDI-BTCR1-AG   $500.00 /10ug


SOURCE Bacillus Thurigiensis (Bt)

PURIFICATION METHOD Isolated from Bt spores

PROTEIN CONCENTRATION 10 ug/mlTris, 0.1% BSA pH7.4. 1 mL/vial

STORAGE If not used within a week, aliquot and store at < -20 °C

STABILITY/SHELF LIFE Two years if stored at < -20 °C

DILUTION FOR ELISA STDS. For ELISA standards, dilute in appropriate buffer. Suggested standard concentration are: 0.25, 0.50, 1.0, 2.0, and 4.0 ng/mL.

APPLICATIONS ELISA

For Research Use Only


anti-CRY1Ac (rabbit polyclonal)

cat#RDI-BTCRY1ACabr  $625.00/200ug    $1250.00/0.5mg

Bt Cry 1AC Protein

CATALOG # RDI-BTCR1AC-AG   $500.00 /10ug


anti-CRY1F (rabbit polyclonal)

cat#RDI-BTCRY1Fabr  $625.00/200ug  $1250.00/0.5mg

Bt Cry 1F Protein

CATALOG # RDI-BTCR1F-AG   $500.00 /10ug


anti-CRY1F (chicken polyclonal)

cat#RDI-BTCRY1Fabck  $625.00/200ug  $1250.00/0.5mg

Bt Cry 1F Protein

CATALOG # RDI-BTCR1F-AG   $500.00 /10ug


anti-CRY2A (rabbit polyclonal)

cat#RDI-BTCRY2Aabr  $625.00/200ug  $1250.00/0.5mg

Bt Cry 2A Protein

CATALOG # RDI-BTCR2A-AG   $500.00 /10ug


anti-CRY2A (chicken polyclonal)

cat#RDI-BTCRY2Aabck  $625.00/200ug  $1250.00/0.5mg

Bt Cry 2A Protein

CATALOG # RDI-BTCR2A-AG   $500.00 /10ug


anti-CRY3B (rabbit polyclonal)

cat#RDI-BTCRY3Babr  $625.00/200ug  $1250.00/0.5mg

Bt Cry 3B Protein

CATALOG # RDI-BTCR3B-AG   $500.00 /10ug


anti-CRY3B (Chicken polyclonal)

cat#RDI-BTCRY3Babck  $625.00/200ug  $1250.00/0.5mg

Bt Cry 3B Protein

CATALOG # RDI-BTCR3B-AG   $500.00 /10ug


anti-CRY9C (rabbit polyclonal)

cat#RDI-BTCRY9Cabr  $625.00/200ug  $1250.00/0.5mg

Bt Cry 9C Protein

CATALOG # RDI-BTCR9C-AG   $500.00 /10ug


anti-CRY9C (chicken polyclonal)

cat#RDI-BTCRY9Cabr  $625.00/200ug  $1250.00/0.5mg

Bt Cry 9C Protein

CATALOG # RDI-BTCR9C-AG   $500.00 /10ug




CRY1 ELisa Plate Kit cat#RDI-BTCRY1-KT  $625.00  $562.00/kit 5+

* Intended Use (For In Vitro research Use Only)

For the detection and quantitation of Bt Cry1Ab and Cry1Ac (MON810, Bt11, Bt176) endotoxin residues in corn tissues and cotton leaf tissues. For use with other sample matrixes, contact the company for application bulletins and/or specific matrix validation guidelines

The Bt Cry1Ab/Cry1Ac Microtiter Plate Kit is a "sandwich" enzyme linked immunosorbent assay (ELISA). In the assay system, standards, controls, or sample extracts are added to wells coated with monoclonal antibodies raised against Cry1Ab/Cry1Ac endotoxin. Any endotoxin residues found in the standard or sample extracts bind to the antibodies on the wells. The "sandwich" is completed by the addition of polyclonal antibodies raised against the same endotoxin. An enzyme labeled conjugate is then added and the enzyme activity bound to the wells is measured using a substrate to develop a colored product. Since the formation of a "sandwich complex" occurs only in the presence of a Cry1Ac/Cr1Ab molecule, the enzyme activity of the bound sandwich complex is directly proportional to the amount of endotoxin in the sample.

A dose response curve of absorbance of the colored product formed vs. concentration is generated using results obtained from the standards. Concentration of Cry1Ab/Cry1Ac present in the control and sample extracts are determined directly from this curve.

Lighter color = Lower concentration Darker color = Higher concentration

* Reagents

The Bt Cry1Ab/Cry1Ac Kit contains the following items:

1. Cry1Ab/Cry1Ac Antibody coated wells

Monoclonal antibody specific for Cry1Ab/Cry1Ac endotoxin adsorbed to plastic wells.

8 strips of 12 antibody coated wells and strip holder (1).

2. Cry1Ab/Cry1Ac Antiserum solution

Polyclonal antibody (rabbit) specific for Cry1Ab/Cry1Ac endotoxin.  One vial containing 11 mL

3.Goat anti-rabbit Enzyme Conjugate (100x)

Horseradish peroxidase (HRP) labeled goat anti-rabbit. Supplied as a liquid concentrate 100x with preservative and stabilizers.  One vial containing 0.25 mL

4. Conjugate Diluting Buffer

Bufferd solution with preservative and stabilizers used to dilute the conjugate  One bottle containing 12 mL

5. Bt Standards (Cry1Ab)

Five concentrations (0, 0.25, 0.5, 1.0, 2.0, 4.0 ng/mL) of Bt calibrators in a buffered solution with preservative and stabilizers. Each vial contains 2.0 mL.

6. Control (Cry1Ab)

A concentration (approximately 1.5 ng/mL) of Bt Cry1Ab in a bufferd solution with preservative and stabilizers. One vial containing 2.0 mL.

7. Extaction Solution/Sample Diluent (5x)

Buffered solution 10x concentrate with preservative and stabilizers without any detectable Bt endotoxin.  One bottle containing 30 mL.

8. Color Solution

A solution of hydrogen peroxide and 3,3',5,5'-tetramethyl benzidine in an organic base.   One bottle containing 12 mL

9. Stopping Solution

A solution of diluted acid.   One bottle containing 6 mL  Avoid contact of Stopping Solution (diluted sulfuric acid) with skin and mucous membranes. If this reagent comes in contact with skin, wash with water

10. Washing Buffer (5x) Concentrate

Preserved buffered 5x concentrate.  One bottle containing 110 mL

* Reagent Storage and Stability

Store all reagents at 2-8*C. Do not freeze. Reagents may be used until the expiration date on the box.

Consult state, local and federal regulations for proper disposal of all reagents.

* Materials Required but Not Provided

In addition to the reagents provided, the following items are essential for the performance of the test:

Precision pipets capable of delivering 20, 100, 500, and 1000 uL , and tips

Disposable Tissue Extractors,  cat#RDI-PN510010

Test tubes for dilution of sample extracts

Marking pen (indelible)

Tape or Parafilmâ

Timer

Vortex mixer

Distilled or deionized water for diluting Wash buffer and the 10x Cry1Ab/Cry1Ac Extraction/Dilution Buffer

Storage bottles with 300 mL capacity for the storage of 1x Extraction/Dilution buffer and 1000 mL capacity for storage of 1x Wash buffer

Microplate or strip reader capable of reading absorbance at 450 nm

Wash bottle (Nalgene cat # 03-409-10E or equivalent) if performing manual plate washing

Test tube rack

* Materials Recommended but Not Required

Multi-channel pipette (100 uL)

Reagent reservoirs for multi-channel dispensing

Automated plate washer

* Sample Information

This procedure is recommended fo use with corn tissues and cotton leaf tissues. For testing of Cry1Ab and Cry1Ac in corn and cotton seeds, and in bulk corn grain, refer to application bulletin. Other samples may require modifications to the procedure and should be thoroughly validated.

Samples containing gross particulate matter should be filtered using a low protein binding filter such as Pall Gelman Sciences cat # 4184 or equivalent. Alternatively the samples can be centrifuged at 5000 x g for 5 minutes.

Sample Extraction

1. Take 2 leaf punch samples (approximately 10 mg each) by snapping the tube cap of the Sample extraction Device down on the leaf. Insert the pestle into the tube and grind the tissue by rotating the pestle against the sides of the tube with a twisting motion. Continue the process for about 30 seconds, or until the leaf tissue is well ground. To prevent sample contamination, a new extraction device and pestle must be used with each plant tissue sample

* Limitations

This Assay will detect Cry1Ab/ Cry1Ac and other related endotoxins to different degrees. Refer to specificity table for data on several of the Cry endotoxins.

* Quality Control

A control solution at approximately 1.5 ng/mL of Cry1Ab is provided with this Assay kit. It is recommended that it be included in every run and treated in the same manner as unknown samples. Acceptable limits should be established by each laboratory.

* Assay Procedure

Read Reagent Preparation, Procedural Notes and Precautions before proceeding.

1. Add 100 uL of standard zero, 100 uL of each Calibrator or Control, and 100 uL of each diluted sample extract to their respective wells. Follow the same order of addition for all reagents. Cover plate to prevent contamination and evaporation.

2. Throroughly mix the contents of the wells by moving the strip holder in a rapid circular motion on the benchtop for a full 20-30 seconds. Be careful not to spill contents.

3. Incubate at ambient temperature for 30 minutes.

4. After incubation, carefully removed the covering and vigorously shake the contents of the wells into a sink or other suitable container. Flood the wells completely with Wash Buffer then shake to empty. Repeat this wash step two times. Tap the strips on to a stack of paper towels to remove residual wash buffer. Alternative, perform these three washes with a microtiter plate or strip washer.

5. Add 100 uL of Cry1Ab/Cry1Ac antibody solution.

6. Throroughly mix the contents of the wells by moving the strip holder in a rapid circular motion on the benchtop for a full 20-30 seconds. Be careful not to spill contents.

7. Incubate at ambient temperature for 30 minutes.

8. Repeat step 4.

9. Add 100 uL of enzyme conjugate.

10. Throroughly mix the contents of the wells by moving the strip holder in a rapid circular motion on the benchtop for a full 20-30 seconds. Be careful not to spill contents.

11. Incubate at ambient temperature for 30 minutes.

12. Repeat step 4.

13. Add 100 uL of color solution.

14. Incubate at ambient temperature for 20 minutes.

15. Throroughly mix the contents of the wells by moving the strip holder ina rapid circular motion on the benchtop for a full 20-30 seconds. Be careful not to spill contents

16. Add 50 uL of stopping solution. This will turn the well contents to yellow.

17. Read results at 450 nm within 15 minutes after adding the Stopping Solution. Set the plate reader to blank on the zero standard wells.

* Results

Data Interpretation

Average the absorbance readings for the duplicate calibrators, control, and samples.

Semi-Quantitative Results

Compare the OD's of the diluted sample extracts to those of the calibrators to obtain an estimate of the amount of Cry1Ab/ Cry1Ac endotoxin in the sample extract.

Quantitative Results

For a quantitative Cry1Ab/Cry1Ac assay, a linear curve fit for the standard curve should be used if the plate reader used has data reduction capabilities. Otherwise, calculate the results as described in the manual calculations.

Manual Calculations

1. Average the absorbance value for each of the calibrators, control, and samples.

2. Construct a standard curve by plotting the mean OD of each calibrator on the vertical linear (Y) axis against its corresponding Cry1Ab concentration on the horizontal linear (X) axis on the graph paper provided.

3. Determine the endotoxin concentration for controls and samples by finding its OD value and its corresponding concentration level on the graph. Multiply the results by

dilution factor incurred during extraction (500 uL ¸ x mg leaf tissue)/1000 to report as micrograms (ug) of endotoxin per gram of tissue.

* Performance Data

Precision

Cry1Ab fortified samples were analyzed both within a single assay, and in differentassays. The following results were obtained using buffer, leaf and kernel extracts:

Buffer

Control 1 2 3
Mean (ppb) 1.04 2.12 3.94
% CV (within assay) 6.3 5.6 3.4
% CV (between assay) 3.9 4.0 1.9

Leaf Extract
Control 1 2 3
Mean (ppb) 1.30 2.08 3.96
% CV (within assay) 5.4 7.9 7.7
% CV (between assay) 3.2 5.1 4.7

Corn Kernel Extract
Control 1 2 3
Mean (ppb) 1.24 2.16 4.04
% CV (within assay) 6.7 3.5 8.1
% CV (between assay) 4.6 2.6 7.9

Limit of Deteaction

The Bt Cr1Ab/Cry1Ac Assay limit of detection is 0.125 ng/ml (ppb) Cry1Ab in corn leaf extract. The Limit of Detection (LOD) was determined by calculating 3 standard deviations (OD units) from a negative corn leaf sample population and by interpolating from a Cry1Ab standard curve.

Recovery

Corn leaf extract samples were fortified with various levels of Cry1Ab endotoxin and then assayed using the Cry1Ab/Cry1Ac Assay. The following results were obtained:

                                          -- Recovery --------------
Amount of Cry1Ab Added (ppb) Mean .(ppb) S.D(ppb) %
0.750 0.718 0.026 96
1.50 1.49 0.092 99
3.0 2.95 0.090 98
Average 98

Specificity

The Cry1Ab/Cry1Ac detects the presence of various Bt endotoxin to differing degrees. The following table shows the concentration of various Bt endotoxin equivalent to the given concentration of Cry1Ab.

Bt Endotoxin (ng/ml)
Cry1Ab     0.50       1.0  2.0 4.0
Cry1Ac 0.28 0.49 0.95 2.0
Cry1F 18 24 57 460
Cry9C 72 317 1429 >2700
Cry2A >2500 >2500 >2500 >2500

For Research Use Only


-copyright by owner
RDI Division of Fitzgerald Industries Intl

34 Junction Square Drive

Concord MA 01742-3049

USA

phone (800) 370-2222

     or  (978) 371-6446 or (800) 370-2222

fax     (978) 371-2266

EMAIL:antibodies@fitzgerald-fii.com

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