2 MATERIALS AND METHODS

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Unless otherwise credited with the manufacturers of reagents and equipment, commonly used buffers including TE, TAE, PBS, TBS, and bacterial culture media like LB, SB, and SOC, as well as LB plates with the appropriate antibiotics were all prepared as recommended by Sambrook et. al., 1989.

2.1  PATIENT

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A girl (SH), aged 8 years and 5 months was transferred to our hospital because of acute myocarditis with clinical signs of cardiac failure. Two days earlier, upon her admission to another hospital, she had presented with ascites, peripheral and pulmonary edema, and a weight gain of 4 kg due to progressive heart failure. Also present were an elevated antistpreptolysin O titer of 800 IU/ml (which increased to maximum of 1,170 IU/ml), erythema marginatum, fever, hematuria, and proteinuria. Since she met 2 of the major Jones criteria (cardiac failure most likely due to preceding carditis and erythema marginatum) and at least 1 of the minor Jones criteria - fever (Jones 1944), and since she had no other specific symptoms, she was treated for rheumatic fever. Specific virologic examinations to rule out viral carditis did not provide significant evidence of an acute infection. The patient was seropositive for several antinuclear antibodies (ANA) including, anti-Ro/SSA, anti-LA/SSB, anti-Sm, antihistone, and anti-Scl 70 (Topoisomerase I), anti-ribosome. Anti-dsDNA antibodies were positive only once by ELISA at a borderline level of 6.3 IU/ml (cutoff 5.97 IU/ml). Repetitive DNA ELISAs and the immunofluorescence test for Crithidia luciliae were always negative. After undergoing IVIG, high-dose penicillin and aspirin therapy, our patient was discharged for further outpatient care. Her antistreptolyson titer had dropped to 507 IU/ml during therapy. Several days prior to a scheduled admission for heart catheterization (since cardiac function was still borderline), the patient had to be readmitted for fever, headache and vomiting. Upon examination, stomatitis, macrohematuria, a sore throat, and cervical lymphadenopathy were present. Heart biopsy upon catheterization did not show signs of viral infection or vasculitis. At this point, however, renal biopsy showed mesangial lupus nephritis (World Health Organization grade II). In the course of clinical follow up, ANA remained positive (Osei et. al., 2000).

2.2 ISOLATION OF LYMPHOCYTES FROM PERIPHERAL BLOOD LYMPHOCYTES

Isolation of lymphocytes from peripheral blood lymphocytes (PBLs) was carried out by using Ficoll-Paque reagent (Pharmacia, Freiburg, Germany). Ficoll-Paque, a sodium diatrizoate solution, is a low viscousity medium which offers rapid isolation and high quality lymphocytes. Diluted anticoagulant-treated blood is carefully layered on the Ficoll-Paque solution and centrifuged for a short period of time. Differential migration of the components of blood during centrifugation results in the formation of layers containing different cell types; erythrocytes and granulocytes sediment to the bottom, and lower density lymphocytes are sandwiched between an upper layer plasma and the Ficoll-Paque reagent. The lymphocytes are then recovered from the interface and washed with PBS to remove platelets, Ficoll-Paque and plasma that might be present. The upper layer plasma which is essentially free of cells may be saved for future use.

20 ml peripheral blood was obtained from the systemic erythematosus and rheumatic fever patient for phage display library construction. 90 ml Ficoll-Paque was pipetted into two 50 ml centrifuge tubes and 10 ml vol/vol PBS:blood was carefully and slowly layered on top of each with the tube held at a slanting position of about 50 degrees. The mixture was centrifuged for 30 minutes at 1,500 rpm in a Beckman GS-6 Centrifuge (Beckman Instruments Inc, Palo Alto, Ca, USA) with the brakes turned off. Using a Pasteur pipette, the upper layer plasma was carefully aspirated, aliquoted into 1.5 ml microfuge tubes and stored at -80 oC. The lymphocytes were aspirated, pooled into new 50 ml tube, topped to 20 ml with PBS and homogenized by gentle swirling, and ca. 30 μl was saved for lymphocyte count on a hemocytometer (HBG, Germany) using Jenamed2 flourescence microscope (Carl Zeiss Jena, Germany). The lymphocytes were centrifuged for 10 minutes at 1,200 rpm with the brakes at 'low' position, and the pellet was used for total RNA isolation.

2.3 RNA ISOLATION FROM LYMPHOCYTES

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Isolation of total RNA from lymphocytes was carried out by using TRI reagent (MOLECULAR RESEARCH CENTER, INC, Cincinnati, OH, USA) according to the manufacturer´s protocol. TRI reagent contains phenol and guanidine thiocynate in a monophase solution. The RNA isolation steps basically include homogenizing or lysing the sample in TRI reagent, phase seperation from the aqueous phase by adding chloroform and centrifuging, RNA precipitation from the aqueous phase by additon of isopropanol, RNA wash using ethanol, and RNA pellet resuspended in appropriate buffer.

The lymphocytes (ca. 106-107 cells/ml) were homogenized in 1 ml TRI reagent and incubated at room temperature for 5 minutes. 200 μl chloroform (Merck GmbH, Darmstadt, Germany) was added, vigorously shaken for 15 seconds and incubated at room temperature for a further15 minutes, and centrifuged at 12,000 xg (ca. 11,500 rpm) for 15 minutes at 4 oC. The aqueous phase was carefully pipetted into a new microfuge tube, mixed with 500 μl isopropanol (Merck, Germany), incubated at room temperature for 10 minutes, and centrifuged at 12,000 xg for 10 minutes at 4 oC. The pellet was washed with 1 ml of 75% ethanol (Merck, Germany), centrifuged as above, air-dried briefly for about 5 minutes and resuspended in 50 μl TE buffer. 10 μl of the RNA was analysed on 1% agarose (Gibco BRL, USA) gel electrophoresed at 200 V for 15 minutes. The rest of the RNA was stored at -80 oC for synthesis of cDNA.

2.4 SYNTHESIS OF Fd, λ AND κ cDNA BY REVERSE TRANSCRIPTION

Total RNA was used for the synthesis of first strand cDNA using 3' primers that were subsequently used in the PCR. The 3' primers specifically associate to the 3' end of the light chain (CL) constant region or the CH1/hinge (Heavy chain) allowing for incorporation of the interchain cystein codon of each chain in the product (Persson et. al. 1991; Kang et. al., 1991b).

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10 μl of total RNA was added to 2 μl of either heavy (Fd; VH + CL1), or light chain (κ and λ) 3' constant region primer in a 1.5 ml microfuge tube, 8 ml of sterile water was added, and incubated at 70 oC for 10 minutes, chilled for 5 minutes on ice and centrifuged for 5 seconds. 7 μl sterilized water, 10 μl 5x reverse transcriptase buffer (Gibco BRL, Life Technologies, USA), 5 μl DTT (Gibco BRL, USA), 2 μl RNAase Inhibitor (Promega, Madison, WI, USA), 3 μl dNTP-mix (Promega, USA), were added, mixed and incubated at 42 oC for 2 minutes, and 3 μl Superscript reverse transcriptase (Gibco BRL, USA), was added and incubated at 42 oC for 50 minutes. The reaction was centrifuged for about 10 seconds, incubated at 70 oC for 15 minutes, briefly centrifuged for about 10 seconds and incubated for 10 minutes on ice. 1 μl RNAase H (Promega, USA) was added, and incubated at 37 oC for 20 minutes. The resulting cDNA was shortly centrifuged and stored at -20 oC for PCR.

2.5 RT-PCR AMPLIFICATION OF Fd, λ, AND κ CHAINS

Each PCR was run in a 0.5 ml microfuge tube in a total volume of 100 μl and contained 75.2 μl sterile water, 10 μl of 10x PCR buffer (without Mg2+-ions; Promega, USA), 5 μl (1.25 mM) MgCl2 (Promega, USA), 0.8 μl dNTP-mix (final concentration 200 μM), 3 μl of either 3' constant region primer namely, cG1z, cK1d, cL2, 3 μl of the appropriate 5' variable region primer, 1 μl (5 Units) Taq DNA polymerase (Promega, USA), a drop of mineral oil (Sigma Chemical Co., St. Louis, MO, USA), and 2 μl of the corresponding 3' constant region primer cDNA (template). For either positive or negative control reactions, the following 5' variable region primers were used: conGa (VH chains), conKa (kappa chains) and conL1 (lambda chains), [Table 1,1]. In negative control reactions however, no cDNA were added, rather water was added. Finally, the reaction was centrifuged for about 30 seconds at 12,000 rpm and thermocycling was carried out in a HYBAID Omnigene DNA Thermocycler (MWG-BioTech GmbH, Germany), using the following reaction conditions; initial denaturation: x1 (94 oC) for 3 minutes, cycling: x40 with primer annealing - 52 oC for 1 minute, extension - 74 oC for 1 minute, denaturation - 93 oC for 30 seconds, and final extention - 74 oC for 10 minutes. At the end of the thermocycling, 10 μl of the PCR reaction was run on a 2% agarose gel using ΦX 174/Hae III marker (Gibco BRL, Life Technologies, USA). A band of ca. 680 bp for VH and LH fragments, 330 bp for the positive controls, and no amplication product(s) in the negative controls were indicative of a successful PCR.

2.5.1 Purification and Quantification of PCR products

PCR DNA was eluted and purified from agarose gel using S & S Biotrap (Schleicher & Schuell GmbH, Dassel, Germany). The Biotrap is an electro-seperation system for elution and purification of charged macromolecules e.g. proteins as well as nucleic acid fragments from 14 to 150,000 base pairs without losses due to denaturation.

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Except the PCR product controls using conGa (Fd), and conKa/conL1 (light chain) that were discarded, the rest of the PCR products were pooled as follows; all Fd products were combined together and, all light chain products (κ and λ) also combined together. Each was extracted with 1:1 vol./vol. of Phenol:Chloroform (Roti-Phenol, Carl Roth, Germany). The PCR DNA was run on 2% agarose gel at 150 V for 1-1/2 hours. The heavy and light chain PCR products were excised out of the agarose gel and electroeluted from the gel slice at 200 V for 1-1/2 hours using the S & S Biotrap, extracted with 1:1 vol./vol. Phenol:chloroform, and each resuspended in 50 μl TE buffer. 1 μl of the DNA was analysed on a 2% agarose gel and the concentration of the light and heavy chain PCR products were determined to be 0.491 μg/ μl and 0.598 μg/ μl respectively.

2.5.2 Restriction of PCR products

Preparation of antibody light and heavy chain inserts for cloning was carried out by enzymatic double restriction using Sac I (40 U/ μl) and Xba I (40 U/ μl) which utilize buffer A for light chain DNA and, Xho I (40 U/ μl) and Spe I (40 U/ μl) which utilize buffer H for heavy chain DNA (Bohringer Mannheim GmbH, Germany).

10 μg light chain PCR DNA (0.491 μg/ μl) was restricted using 1.5x excess units of the light chain restriction enzymes i.e. 57 U Sac I/μg DNA and 112.5 U Xba I/ μg DNA. The reaction was incubated at 37 oC for 3h, ethanol precipitated, electroeluted as above, resuspended in TE buffer, and quantitated. Similarly, 10 μg heavy chain PCR DNA (0.598 μg/ μl) was restricted using 1.5x excess units of the heavy chain restriction enzymes i.e.109.5 U Xho I/ μg DNA and 25.8 U Spe I/ μg DNA, at 37 oC for 3h, ethanol precipitated, electroeluted, resuspended in TE buffer, and the amount measured.

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PCR PRIMERS

Table 1.1 Human variable and constant region PCR primers used for phage antibody library construction (Kang et. al., 1991b).

Human Heavy Chain Variable Domain 5' Primers

VH1a

5' - CAG GTG CAG CTC GAG CAG TCT GGG - 3'

(24 mer)

VH1f

5' - CAG GTG CAG CTG CTC GAG TCT GGG - 3'

(24 mer)

VH2f

5' - CAG GTG CAG CTA CTC GAG TCG GG - 3'

(23 mer)

VH3a

5' - GAG GTG CAG CTC GAG GAG TCT GGG - 3'

(24 mer)

VH3f

5' - GAG GTG CAG CTG CTC GAG TCT GGG - 3'

(24 mer)

VH4f

5' - CAG GTG CAG CTG CTC GAG TCG GG - 3'

(23 mer)

VH4g

5' - CAG GTG CAG CTA CTC GAG TGG GG - 3'

(23 mer)

VH6a

5' - CAG GTA CAG CTC GAG CAG TCA GG - 3'

(23 mer)

Human Heavy Chain constant domain 3' Primer

IgG1

CG1z

5'- GCA TGT ACT AGT TTT GTC ACA AGA TTT GGG - 3'

(30 mer)

Human Heavy Chain constant domain control 5' Primers

IgG1

ConGa

5' - TCC ACC AAG GGC CCA TCG - 3'

(18 mer)

Human Light Chain Primers

Kappa chain variable domain 5' primers

VK1a

5' - GAC ATC GAG CTC ACC CAG TCT CCA -3'

(24 mer)

VK1s

5' - GAC ATC GAG CTC ACC CAG TCT CC -3'

(23 mer)

VK2a

5' - GAT ATT GAG CTC ACT CAG TCT CCA -3'

(24 mer)

VK3a

5' - GAA ATT GAG CTC ACG CAG TCT CCA -3'

(24 mer)

VK3b

5' - GAA ATT GAG CTC AC(G/A) CAG TCT CCA -3'

(24 mer)

Kappa chain constant domain 3' primers

CK1d

5' - GCG CCG TCT AGA ATT AAC ACT CTC CCC TGT TGA AGC TCT TTG TGA CGG GCG AAC TCA G -3'

(57 mer)

Kappa chain constant domain control 5' primers

CONKa

5' - act gtg gct gca cca tct g -3'

(19 mer)

Lambda chain variable domain 5' primers

VL1

5' - AAT TTT GAG CTC ACT CAG CCC CAC -3'

(24 mer)

VL2

5' - TCT GCC GAG CTC CAG CCT GCC TCC GTG -3'

(27 mer)

VL3

5' - TCT GTG GAG CTC CAG CCG CCC TCA GTG -3'

(27 mer)

VL4

5' - TCT GAA GAG CTC CAG GAC CCT GTT GTG TCT GTG -3'

(30 mer)

VL5

5' - CAG TCT GAG CTC ACG CAG CCG CCC -3'

(24 mer)

VL6

5' - CAG ACT GAG CTC ACT CAG GAG CCC -3'

(24 mer)

VL7

5' - CAG GTT GAG CTC ACT CAA CCG CCC -3'

(24 mer)

VL8

5' - CAG GCT GAG CTC ACT CAG CCG TCT TCC -3'

(27 mer)

Lambda chain constant domain 3' primers

CL2

5' - CG CCG TCT AGA ATT ATG AAC ATT CTG TAG G -3'

(30 mer)

Lambda chain constant domain control 5' primers

CONL1

5' - AAG GCT GCC CCC ACG GTC ACT CTG -3'

(24 mer)

Underlined nucleotides represent primer-encoded restriction sites

2.6 PREPARATION OF E. coli STRAIN XL1-Blue FOR CLONING

The preparation of bacterial cells for high efficiency transformation involves growing the bacteria to early-to-midlog phase. This offers a period of greatest competence where the cells are still growing rapidly at densities (and volumes) high enough to recover very large number of cells. The cells are harvested by chilling and centrifuging, and for electroporation, washed several times with water and resuspended in 10% Glycerol in deionised water to reduce the ionic strength of the final cell suspension (Gene Pulser Controller, Operating Instructions and Applications Guide, BIO RAD, USA). Increasing cell concentration increases yield of transformants, therefore cells are resuspended to very high concentration, aliqouted and stored at -80 oC. The E. coli strain XL1-Blue (Stratagene, U.S.A) has the following genotype:

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recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac -

F' (proAB + , lacI q , ZdelM15, Tn10).

XL1-Blue allows high efficiency transformation, up to 109 colonies per μg of pUC DNA when using electroporation (Cold Spring Harbor Laboratory Course Manual, 1993). XL1-Blue is recombination-deficient (rec A1) which is necessary to maintain stability of plasmids propagated in this strain. In addition, transposon 10 (Tn 10) which harbors the gene coding for resistance towards the antibiotic tetracycline (tetr) has been intergrated into the F' factor. This ensures the expression of the F' factor in media containing tetracycline and maintains the ability of the bacteria to act as a host for male-specific phages such as M13.

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300 μl glycerol stockof E. coli strain XL1-blue (Stratagene Cloning Systems, La Jolla, Ca., USA) was resuspended in 5 ml SB medium supplemented with 20 μl of 10 mg/ml tetracycline (Merck, Germany) and incubated overnight with shaking (300 rpm) at 37 oC. 3 ml of the overnight culture was transferred into a 37 oC pre-warmed 300 ml SB medium containing 600 μl tetracycline (10 mg/ml) and incubated as above until mid-log phase (for ca. 3 h), when the OD 600 = 0.8 - 1.0. The culture was incubated on ice for 20 minutes and divided into 6 pre-chilled 50 ml tubes, and centrifuged at 4,800 rpm (3,838 xg) for 10 minutes at 4 oC. The supernatant was discarded and the pellet resuspended in 10 ml ice-cold water, topped to 50 ml and re-centrifuged as above. The pellet was resuspended in 25 ml ice-cold water and centrifuged again, and was resuspended in 1 ml 10% glycerol (Carl Roth, Germany). The cells were pooled into two tubes and centrifuged as above, pooled and resuspended in 3 ml of 10% glycerol and aliqouted at 200 μl into 1.5 ml microfuge tubes directly onto dry ice and stored at -80 oC. To test whether the cells were viable and devoid of contamination, 50 μl were plated on carbenicillin (Merck, Germany) [100 μg/ml LB medium], kanamycin (Sigma, USA) [70 μg/ml LB medium], and tetracycline (10 μg/ml LB medium) plates, and incubated overnight at 37 oC. Inspection of the plates next morning showed that there were no growth on the carbenicillin and kanamycin plates indicating that the XL1-blue strain had not acquired resistance to these two markers whilst there were clones on the tetracycline plates showing that the cells were viable. 1 μg wildtype phagemid, pComb3HSS (Dr. Barbas, Scripps Research Institute, La Jolla, USA) was electroporated into 200 μl of the cells to determine transformation efficiency of the cells. This was determined to be 3.2 x 107 colonies/ μg DNA.

2.7 PREPARTION OF pComb3H VECTOR

2.7.1 Transformation of E. coli strain XL1-blue by High Voltage-Electroporation (Electrotransformation).

Transformation of bacteria by high voltage electroporation is a convenient and reliable way to achieve success in cloning. Frozen glycerol stock of bacteria are thawed on ice and used for transformation. Electroporation is carried out in a special high voltage mini-electrode and chilled cuvettes. An optimal balance between the strength of the electrical field, the length of the electrical pulse and the concentration of DNA results in higher transformation efficiencies (Sambrook et. al., 1989).

1 μg of pComb3HSS DNA resuspended in 10 μl sterile deionised water was mixed with 200 μl thawed glycerol stock of E. coli strain XL1-blue cells and transferred into a chilled 0.2 cm gap cuvette (Bio-Rad, USA), and pulsed at 2.5 kV, 25 μFD and 200 ohms for 4.5 seconds at a field strength of 12.5 kV/cm in a Gene Pulser (Bio-Rad, USA). The electrotransformed cells were rapidly flushed with 3 ml SOC medium into a 10 ml tube and incubated at 37 oC for 1h, shaking (200 rpm), in a G 24 Incubator shaker (New Brunswick Scientific Co. Inc., Edison, NJ, USA). The culture was transferred into a 50 ml tube containing pre-warmed (37 oC) 10 ml SB medium supplemented with 2.6 μl carbenicillin,100 μl of 1M MgCl2 (Merck, Germany), and incubated as above. Prior to the incubation, about 80 μl was saved for determining the efficiency of the transformation by plating 50 μl, 1.0 μl, and 0.1 μl on LB plates containing 100 μg/ml carbenicillin. The culture was transferred into a 1l conical flask containing 100 ml SB medium supplemented with 56.0 μl of 100 μg/ml carbenicillin, 1 ml of 1M MgCl2 and incubated overnight as above. The plasmid DNA was extracted with the QIAGEN MIDI Plasmid kit the next day.

2.7.2 pComb3HSS: QIAGEN MIDI plasmid purification

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pComb3HSS DNA was purified using the QIAGEN MIDI plasmid purification kit according to the manufacturers instructions. The QIAGEN plasmid kit is an alkaline lysis based purification protocoll. It allows the purification of ultrapure plasmid DNA (yield ca. 100 μg DNA) without the use of phenol, chloroform, or cesium chloride (QIAGEN Plasmid Purification Handbook). Essentially, the protocol involves lysis of bacterial cells under alkaline conditions and after centrifugation, the lysate is applied under defined salt conditions to a pre-equilibrated QIAGEN resin tip. The resin ensures that only plasmid DNA is bound while degraded RNA, cellular proteins, and metabolites are washed down the column under gravity. A high salt buffer efficiently elutes plasmid DNA from the resin and the DNA is desalted and precipitated by isopropanol. Finally, it is washed in 70% ethanol, dried briefly and resuspended in an appropriate buffer.

The overnight pComb3HSS electrotransformed culture was divided 50 ml each into two 50 ml centrifuge tubes and centrifuged at 3,000 rpm (1,500 xg), for 20 minutes at 4 oC, Beckmann GS-15R centrifuge. One pellet was used for purification of the wildtype plasmid using the Qiagen Plasmid purification kit. 1 μg of the plasmid DNA was analysed on 1% agarose gel with 1 kb DNA ladder (Gibco BRL, USA) as a marker.

2.8 PREPARATION OF LIGHT CHAIN VECTOR FOR CLONING

The pComb3H-SS1 vector contains a light chain stuffer sequence of about 1,200 bp and a heavy chain stuffer of 300 bp

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10 μg pComb3H-SS DNA was restricted with Sac I and Xba I (6.25 U/ μg and 12.5 U/ μg respectively) at 37 oC, ethanol precipitated, run on 0.7% low melting agarose (Sigma, USA) at 90 V, for 1 h and both vector DNA and light chain stuffer fragments excised out, and electroeluted (seperately) in an S & S Biotrap. The DNA was ethanol precipitated, resuspended in TE buffer, the amount measured and stored at -20 oC.

2.9 TEST LIGATION-LIGHT CHAIN INSERT

Important parameters to consider during ligation of an insert (foreign DNA) to a vector include an optimal ratio of insert to vector and the concentration of each DNA in the reaction mixture (Sambrook et. al., 1989). To monitor the efficiency of ligation, it is invaluable to carry out test ligations which should include the optimal ratio of cut vector DNA to insert, the cut vector with ligase but without insert, and the cut vector DNA only without neither insert nor ligase. These serve as a useful guide for the subsequent ligation.

The light chain insert test ligation was as follows:

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  1. 43.2 ng pComb3H-S vector + 12.1 ng light chain insert + ligase
  2. 86.4 ng pComb3H-S vector + 12.1 ng light chain insert + ligase
  3. 86.4 ng pComb3H-S vector + ligase (no insert)
  4. 86.4 ng pComb3H-S vector (no insert, no ligase)

Each reaction was carried out in a total volume of 5 μl, incubated at 16 oC overnight, electroporated into 200 μl of E. coli strain XL1-blue, and titrated overnight as above. Phenol/chloroform prepartion of (total) DNA and restriction enzyme analysis using the light chain enzymes Sac I and Xba I for each test ligation was carried out to determine which of the test ligations was (more) successful.

2.9.1 Light Chain Library construction: ligation of Light Chain insert to Light Chain vector

2.2 μg pComb3H-S DNA was ligated to 0.605 μg light chain DNA overnight at 16 oC i.e. 50x the concentrations of both light chain vector and insert from test ligation (a) above, was used in the construction of the light chain library. 50 μl, 1.0 μl, and 0.1 μl of the library were plated on carbenicillin plates (100 μg/ml LB medium). The light chain library total DNA was extracted by using the QIAGEN Midi Plasmid preparation kit.

2.9.2 Light Chain Library: Single clones minipreps culture

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Single clones from the carbenicillin plates were cultured overnight in SB medium supplemented with 100 μg/ml carbenicillin. Minipreps were prepared by phenol/chloroform extraction and the DNA analysed on agarose gel by light chain restriction enzymes to determine the percentage of clones that had the light chain fragment (insert).

2.10 THE COMPLETE FAB LIBRARY CONSTRUCTION

A series of background test ligations, and restriction enzyme analyses of the ligations, revealed that a 3:1 vector to insert molar ratio gave a higher percentage of desired clones with about 70% having both the light and heavy chain inserts and this was used during the actual ligation of the heavy chain insert to the heavy chain vector.

4.95 μg heavy chain vector DNA and 1.65 μg heavy chain insert were ligated overnight, ethanol precipitated the next day, resuspended in 10 μl water and electroporated into XL1-blue cells. The cells were grown at 37 oC for 1h with shaking and, transferred into a prewarmed (37 oC) 10 ml SB medium containing 2.6 μl carbenicillin (low concentration), and 100 μl 1M MgCl2. 50 μl, 1.0 μl, and 0.1 μl were titrated on carbenicillin LB plates, and the rest incubated for 1h as above. The culture was transferred into a 37 oC prewarmed 100 ml SB medium supplemented with 56 μl carbenicillin (high concentration) and 1 ml 1M MgCl2 and incubated for 1 h as above. 200 μl (7.0 x 1011 t.u/ml) of VCSM13 helper phage (Stratagene, Ca., USA) was added to the culture and incubated for 2 h as above. 160 μl kanamycin was added to the culture and incubated under the same conditions as above, overnight.

2.11 PREPARATION OF THE NAIVE SURFACE DISPLAYED FAB PHAGES

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The overnight culture was divided into two 50 ml tubes and centrifuged at 3,000 rpm (ca. 1,500 xg) for 20 minutes at 20 oC in a Beckmann GS-15R centrifuge. The supernatant was transferred into a GSA rotor flask and 4 g (4% w/v) PEG-8000 (Sigma, USA) and 3 g (3% w/v) NaCl (Merck, Germany) was added and incubated on ice for 20 minutes. The bacterial pellet containing the unselected (total) library DNA was saved for DNA isolation using the QIAGEN DNA Preparation kit. The PEG precipitated phage suspension was centrifuged at 8,600 rpm for 20 minutes at 4 oC (in a Sorvall RC 5B Plus centrifuge using a GSA rotor). The supernatant was discarded and the pellet was resuspended in 2 ml of 1% Casein (Sigma)/TBS (supplemented with 0.02% NaN3 [Merck, Germany]), and divided 1 ml each into two 1.5 ml microfuge tubes and centrifuged for 5 minutes at 12,500. The two Fab phage suspensions (supernatants) were stored at 4 oC (for biopanning), and -20 oC (for long term storage) whilst the pellets were discarded.

2.12 BIOPANNING

2.12.1 Coating of antigens

Maxisorp immunotubes (Nunc,Wiesbaden, Germany) were coated with 9 μg (300 μl) IVIG (30 mg/ml) preparation (Sandoglobin, Sandoz, Basel, Switzerland) and stored at 4 oC.

2.12.2 Day 1

2.12.2.1 Bacterial culture

200 μl glycerol stock of E. coli strain XL1-blue was thawed on ice and resuspended in 10 ml SB medium supplemented with 20 μl tetracyclin (10 μg/ml, and incubated at 37 oC for about 1 h (until mid-log phase, Absorbance at OD600nm = 0.8 - 1.2) with shaking.

2.12.2.2 Panning methodology

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Antigen-coated immunotube number 1 was washed 3x with wash buffer (0.2% PBS/Casein-0.05% Tween 20 (Fluka, Switzerland) and once with PBS, blocked completely with blocking buffer (1% Casein/PBS) and incubated at 37 oC for 1 h. The blocked tube was washed 2x with wash buffer and once with PBS, and 300 μl of the Fab phage suspension was added and incubated at 37 oC for 2 h inside a humidified chamber.

Using a Pasteur pipette, the Fab phage suspension were pipetted from the tube and unbound phages washed off as follows: Panning 1; 1 ml wash buffer was pipetted into the tube, and allowed to stand for 10 minutes at room temperature and aspirated. This was repeated again and then rinsed with PBS. During subsequent panning rounds however, the wash buffer was allowed to stand for 2 minutes (repeated 10x) and finally rinsed once with PBS. 300 μl elution buffer [0.1M HCl (Merck)/glycine (Sigma), pH 2.2] was added to the tube, vigorously pipetted up and down, and incubated 10 minutes at room temperature. The eluted phages were collected from the tube, and neutralized with 60 μl neutralization buffer containing 2M Tris (Carl Roth, Germany)/HCl pH 9.0, and used to infect 3 ml of E. coli strain XL1-blue culture (from above) and incubated at 37 oC for 20 minutes in a shaking incubator.

50 μl of the bacterial culture was plated on carbenicillin and tetracyclin plates and incubated at 37 oC overnight as a control to ascertain that no phagemid and helper phage contaminations had taken place in the bacterial hosts. The transformed 3 ml E. coli was added to a 37 oC pre-warmed 10 ml SB medium supplemented with 2.6 μl carbenicillin and 100 μl of 1M MgCl2, and briefly vortexed. 60 μl was saved for output titration of the Fab phage (by plating 50 μl, 1.0 μl, and 0.1 μl on carbenicillin plates) with the rest of the culture incubated further at 37 oC for 1 h, with shaking. Similarly for input Fab phage titration, 10 μl of 10-7, 10-8, and 10-9 c.f.u/ml serial dilutions of the infecting (PEG-precipitated) phage suspension were incubated with 50 μl of the bacterial culture at 37 oC for 20 minutes and were plated on carbenicillin plates for Fab phageinput titration. The transformed 13 ml culture was added to a 37 oC-prewarmed 100 ml SB medium supplemented with 56 μl carbenicillin and 1 ml of 1M MgCl2, and incubated at 37oC for 1 h, with shaking, and 200 μl (7.0 x 1011 t.u/ml) VCSM13 helper phage was added, and incubated at for 2h as above. 160 μl kanamycin (70 μg/ml) was added to the culture and grown overnight as above.

2.12.3 DAY 2 (and other subsequent rounds of panning)

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Preparation of surface-displayed Fabs for the next round of panning was done exactly as under section 2.10, above. For subsequent panning rounds, 300 μl of the PEG precipitated Fab Phage suspension (from the previous panning) was incubated with the coated antigen in the immunotubes, washed, eluted, neutralized and used to re-infect freshly grown bacteria (as in Panning 1), and the panning steps continued as above. Total library DNA was isolated from the bacterial pellet from the last round of panning for further analysis, unlike the earlier panning rounds when the bacterial pellet was discarded (during PEG/NaCl precipitation of the phages).

At the end of the last round of panning, single clones growing on carbenicillin plates were cultured in 48-well plates containing 300 μl SB supplemented with carbenicillin and 10 mM MgCl2 for 5 - 6 h at 37 oC with shaking. VCSM13 helper phages were added and grown further for 1 h. Thereafter, kanamycin was added and grown overnight. The overnight culture was centrifuged at 2000 rpm in a Beckman GS-15R centrifuge using a plate rotor (Beckman Instruments, Palo Alto, Ca., USA). The supernatant was transferred into a blocked 96-well microtitre plate and used in Fab phage ELISA. DNA were prepared from the pellets from clones that were positive in the ELISA and analyzed by restriction enzyme on agarose to identify clones that had both heavy and light chain inserts.

2.13 ELISA PROCEDURES

Microtitre plates (Coastar, Corning, Wiesbaden, Germany) were coated with equal concentrations of antigens (600 ng/well) and kept in a humidified chamber at 4oC overnight. Wells were blocked with 1% Casein-PBS for 1 h at 37 oC and incubated with Fab phage supernatants for 1 h. Bound Fab phages were detected by HRP-conjugated mouse anti-M13 phage antibodies (Pharmacia, Freiburg, Germany), and read in a DYNATECH MR 5000 ELISA reader.

2.14 DETERMINATION AND ANALYSIS OF NUCLEOTIDE AND AMINO ACID SEQUENCES

↓26

Fab phage clones that were positive in the ELISA and had light and heavy chain DNA inserts as determined by restriction enzyme analysis were cultured overnight in SB medium containing carbenicillin. DNA preparations were done either by the Promega Wizard Miniprep kit (Promega, Madison, WI) or Qiagen miniprep kit (Qiagen, Hilden, Germany) according to the manufacturers instructions. Cycle sequencing reaction was performed using Amersham cycle sequencing kit (Amersham, Braumscheig, Germany) with infrared flourophore (IRD41)-labelled primers (MWG Biotech, Eberserg, Germany) (Fischer et. al., 1999). The sequencing primers were PelB and SeqGb for the heavy chains, and OmpA, SeqLb or SeqKb for the light chains , (Fischer et. al., 1999; Jendreyko et. al., 1998; Graus et. al., 1997).

Table 1.2: Sequencing primers.

A. HEAVY CHAIN

(+) strand:

SEQGb

5'- GTC GTT GAC CAG GCA GCC CAG -3 ' (21-mer)

(-) strand:

Pelb

5'- ACC TAT TGC CTA CGG CAG CCG - 3' (21-mer)

B. LIGHT CHAIN

(+) strand:

SEQkb

5'- ATA GAA GTT GTT CAG CAG GCA - 3' (21-mer)

(+) strand:

SEQlb

5'- GAA GTC ACT TAT GAG ACA ACA C - 3' (22-mer)

(-) strand:

OmpA

5'- AAG ACA GCT ATC GCG ATT GCA G - 3' (22-mer)

Nucleotide sequences were analyzed with the MacDNAsis program, and manually controlled by scf-files. Sequencing was repeated for ambiguous or unclear sequences. Clones expressing functionally rearranged heavy and light chain sequences were further evaluated by VBASE via the Internet (http://www.mrc-cpecam.ac.uk/imt-doc/publicINTRO.html) using the program DNA-Plot developed by W. Muller and H.-H. Althaus, University of Cologne, Germany for determination of germline segments and mutations. To enable easy comparison of our findings with those of others in this field, we applied the widely used Kabat definition of complementarity-detemining regions (CDRs) and framework regions (FRs) as well as the V-BASE nomenclature. Thus we used the full length of the germline V-genes in these calculations.


Footnotes and Endnotes

1 

pComb3H-SS: wild type plasmid

pComb3H-S: light chain vector (with the light chain stuffer fragment excised out)



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