rev:    January 31, 2005

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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.

See also new PHAGE Titration Elisa cat#RDI-PRPHAGE   $500.00/1kt  

See also New HYPERPHAGE  cat#RDI-PRHYPE    $940.00/1set (5vials X 2ml each)  

Mouse anti-M13,df,F1 Filamentous Phages Monoclonal

       RDI-PRO61097         $250.00/vial 100ug

cat#RDI-PRO61397   $750.00/vial  500ug

-also available Biotin conjugated cat#RDI-PRO61597 $344.00/0.25ml

-also available FITC conjugated: cat#RDI-PRO61497 $500.00/0.25ml

-also available HRP conjugated   cat#RDI-PRO61197 $345.00/750ul

Clone               B62-FE2

Category          Mouse monoclonal

Purification:     Protein A affinity chromatogrpahy

Ig Class            IgG2b

Immunogen       fd phages from E. Coli F+ strain (JM109)

Specificity/Clinical Application  clone B62-FE2 abs binds to an internal epitope (AEGDDPAKA) on the N-terminus of pVIII (Phage coat protein)

Application    -Phage display (immunoassays for the identification of recombinant antigen- or antibody phages)

                        Detection limit:10 E07 phage particles

                       -immunoelectron microscopy

                      - immunoblotting (western)

Working Dilution 1:10,000 for ELISA

Storage At 2-8 DEG C stable for one year after reconstitution when stored at 2-8 DEG C. STRONGLY recommend adding 0.5% BSA to diluents and a suitable preservative (as 0.05-0.09% sodium azide or equivalent) Aliquot and store at -20 DEG C. For prolonged storage aliquots should be kept at -20 DEG C. Avoid repeated thawing and freezing. IMPORTANT NOTE: Antibody shows a tendency to precipitate at neutral pH, recommend keeping at >pH7.4)

Reconstitution Reconstitute in 1 ml dist. water (final solution contains 0.09% % NaN3, 0.5% BSA in PBS buffer, pH 7.4)

Quantity: 100ug or 500ug (lyophilized) BULK without BSA available on request

ref:

Micheel B, Heymann S, Scharte G, Böttger V, Vogel V, Dübel S, Breitling F, Little M, Behrsing O: Production of monoclonal antibodies against epitopes of the main coat protein of filamentous fd phages. J. Immunol. Methods 171, 103-109 (1994).

Kneissel S, Queitsch I, Petersen G, Behrsing O, Micheel B, Dübel S. Epitope structures recognised by antibodies against the major coat protein (g8p) of filamentous bacteriophage fd (Inoviridae). J. Mol. Biol. 288, 21-28 (1999)

Rondot S, Koch J, Breitling F, Dübel S: A helper phage to improve single-chain antibody presentation in phage display. Nature Biotechnology 19, 75-78 (2001).

Bulk quotes upon request (available without BSA in bulk) 1 miiligram (no BSA but with 0.09% NaN3) is cat#RDI-PRO61097-1X $2188.00/

For In Vitro Research Use Only-Not For Use In Or On Humans or Animals-Not For Diagnostics

cat#RDI-PRHYPE   $925.00/set    

Clone determination:        409-1(M13K07deltapIII)

Category                            Hyperphage for packaging of display plasmid

Purification:                      PEG precipitation

Specificity:                       Infection of bacteria via pIII

Application:                     Provides helper phage function in packaging a common phage

                                        display phagemid (for example pSEX, pHen2)

Working dilution:             The concentration of hyperphage particles per ml after

                                         reconstitution is indictaed on the label. For infection use a Moi of 20 (ref2)

Stability/Storage:             1 year at 2-8 DEG C(lyophilized) after reconstitution use immediately

Reconstitution:                 Reconstitute each glass vial in 2ml H20 dist.

Quantity:                          5 vials (2ml lyophilized hyperphage each)

                                        sample data: titer particles 1 X 10 E12/ml

ref:  1) Nature Biotechnology 19, 75-78, 2001

       2) Koch J et al, Generation of antibody libraries fromhuman donors.In:Antibody engineering (eds Kontermann R and Dubel S) Springer Verlag, Heidelberg/New York, 2001

General comments: The pSEX vector is generally more useful for single chain antibodies, the pHEN2 system is preferred for Fab ones. The advantage of pHEN2 is also the greater stability and as a consequence of this feature, better screening results are obtained.

The hyperphage can be titrated directly without additional pretreatment. Titration can be performed using e.g. our phage titration ELISA (cat.no. RDI-PRPHAGE   $500.00/1kit  $438.00/3+). For titration we would like to recommend the use of P8 rather than P3. We would not expect interference with infectivity; but for each subsequent panning round a new hyperphage preparation is required.

FOR IN VITRO RESEARCH USE ONLY

SAMPLE INSERT see insert with each kit for batch specific information

Cat. No.:               RDI-PRPHAGE   $500.00   $469.00/kit 5+

Size of the Kit: 12 x 8 Determinations

Storage:             2 - 8° C

                           In vitro Test FOR RESEARCH USE ONLY

1. Introduction

The phage display technology is a powerful and well-established tool for the investigation of protein-protein interactions and for the generation of human antibodies for in vitro diagnostics and in vivo therapy (Koch et al. 2000). Generally several rounds of selection on an immobilized antigen are required to enrich and isolate the binding phage. The need to determine phage titers before and after each round to monitor the panning process still represents a major time and material consuming factor within this procedure. Usually, the phages are titered by infecting E. coli with serial dilutions of phage, plating the bacteria on agar plates and counting the colony (cfu) or plaque forming units (pfu) after over-night incubation (Koch et al., 2000). To determine therelative number of phage particles, however, immunotitration by PROGEN's Phage Titration ELISA offers a fast, sensitive and reproducible alternative for titration of M13 bacteriophage preparations.

2. Test Principle

The assay is based on a sandwich ELISA technique. A monoclonal antibody specific for the pVIII protein on the surface of the bacteriophage M13 is coated onto microtiter strips and is used to capture M13 particles from the specimen. Captured M13 particles are detected by a peroxidase conjugated monoclonal antibody to pVIII, which is bound to the immune complex. Addition of substrate solution results in a color reaction which is proportional to the amount of specifically bound phage particles. The absorbance is measured photometrically at 450 nm. The kit control provided contains a lyophilized M13 particle preparation. It shows a typical titration curve when used in a serial dilution (Fig. 1) and allows the quantitation of samples of an unknown particle titer.

3. Material Required

· Precision pipettes

· Sterile pipette tips

· Distilled water

· Vials for specimen dilutions

· 37°C Incubator

· Microtiter plate spectrophotometer (450 nm)

4. Contents of Test Kit

12 x 8-well microtiter strips, coated with mouse monoclonal antibodies to M13

KC Kit Control (purified M13 particles), lyoph., 2 vials

WB Wash Buffer (20x), 20 ml

C Anti-M13, Peroxidase Conjugate (20x), lyoph.

S TMB Substrate Concentrate (20x), 750 µl

SS Stop Solution (ready-to-use), 13 ml

Adhesion foil

Resealable plastic bag

All components contain Thimerosal as preservative!

5. Preparation of Reagents

Allow kit to reach RT. Buffer concentrates may contain salt cristals which dissolve quickly at 37°C. Store unused strips in resealable plastic bag with desiccant at 2-8°C. Dilute required volumes of reagents immediately before use!

Preparation of Reagents

WB Dilute 1:20 with distilled water for ready-to-use wash buffer.

KC Reconstitute with 500 µl distilled water; contains approx. 1 x 10 8 particles/ml (see label for exact concentration).

C* Reconstitute with 750 µl distilled water. Dilute 1:20 with ready-to-use wash buffer for ready-to-use antibody peroxidase conjugate

S* Dilute 1:20 with distilled water for ready-to-use substrate.

CAUTION: dilute substrate in glass or polypropylene tubes only!

*Dilute immediately before use.

6. Example for Kit Control and Specimen

Dilution

The linear range of the ELISA covers 5x 10 6 – 1x 10 8 particles. Dilute specimen containing M13 particles to reach a concentration within the linear range of the ELISA using ready-to-use wash buffer. Dilute the reconstituted Kit Control also in ready-to-use wash buffer. For measurement dilute further in steps of 1:2. A minimum of 2-3 different dilutions should be tested.

7. Test Procedure

1. Pipette 100 µl of ready-to-use wash buffer (Blank), serial dilutions of Kit Control, and specimen (diluted in ready-to-use wash buffer) into the wells of the microtiter strips. Seal strips with adhesion foil provided and incubate for 1h at 37°C.

2. Empty contents of microtiter strips. Fill wells with 200 µl each of ready-to-use wash buffer, incubate approx. 5 sec, empty and tap inverted plate. Repeat washing step 2x.

3. Pipette 100 µl per well of ready-to-use peroxidase conjugate. Seal strips with adhesion foil and incubate for 1h at 37°C.

4. Repeat washing step as described in 2.

5. Pipette 100 µl per well of ready-to-use substrate. Incubate for 10 - 15 min at RT.

6. Stop color reaction by adding 100 µl of stop solution into each well.

7. Measure intensity of color reaction with a photometer at 450 nm within 30 min.

Fig. 1 - Example of a Titration Curve

8. Calculation of Results

Create a titration curve by using semilogarithmic paper and plotting the OD readings (y-axis) of the serial dilution of the Kit Control (x-axis) analogue to Fig. 1.

Use this standard curve for the calculation of the particle titer of unknown specimens.

9. Quality Control

Kit Control (undiluted) OD > 1.5

Blank OD < 0.15

10. References

1. Koch J, Breitling F, and Dübel S (2000): Rapid titration of filamentous bacteriophage (M13) on nitrocellulose membranes. BioTechniques, 29, 1196-1202.

2. Rondot S, Koch J, Breitling F, and Dübel S (2001): A helper phage to improve single-chain antibody presentation in phage display. Nature Biotechnology 19, 75-78.

34 Junction Square Drive

Concord MA 01742-3049

USA

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

fax     (978) 371-2266

EMAIL:antibodies@fitzgerald-fii.com

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