The methods described in this section are only for the preparation of antisera containing relatively low-titer anti-E. coli antibodies. If the treated antiserum still cross-reacts at a high level with host components, it must be further purified by affinity chromatography on a chromatography column containing immobilized E. coli components. This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Antibody assay for removal of cross-reactivity in antisera
Materials and Instruments
Closure Buffer Closure Reagent Antibody LB Top Agarose LB Plate Agar Medium Move makings For more product details, please visit Aladdin Scientific website.
Nitrocellulose filter membrane λ phage vector cDNA library
Buffers and solutions
See Appendix 12 for components of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
Confinement buffer
10 mmol/L Tris-Cl (pH 8.0)
150 mmol/L NaCl
0.05%(V/V) Tween-20
Sealing reagent [1% (m/V) gelatin, 3% (m/V) bovine serum albumin, or 5% (m/V) skimmed milk powder]
Different laboratories disagree on the actual merits of these closures. We recommend that the user perform a pre-test to determine which of these containment solutions is optimal for encapsulation with the primary and secondary antibodies of choice. The blocking solution can be stored at 4°C and reused several times. Sodium azide is added to a final concentration of 0.05% (m/V) to inhibit microbial growth.
Antibodies
Preparation of antibodies for library screening
Media
LB plate agar medium
30-35 ml of agar medium per 90 mm Petri dish. About 50 ml of agar medium per 150 mm plate. Plates must be dry or the top layer of agarose will be removed when the nitrocellulose filter membrane is removed. Usually, plates prepared two days before are good if they are slightly uncovered and incubated at 37°C for 1-2 h before use.
LB Top Layer Agarose
Melt the top layer of agarose in a microwave oven for a short time before use, then cool to 47°C. Divide the melted top layer of agar into 3 ml aliquots (for 90 mm plates) or 7.5 ml aliquots (for 150 mm plates) in sterile tubes. Place the aliquots in a dryer or water bath at 47°C to prevent the top layer of agar from solidifying.
Specialized Equipment
Nitrocellulose filter membrane
Suitable for protein binding and immunoblotting include Triton X-100 nitrocellulose filter membranes (Millipore HATF or equivalent) and nitrocellulose-derived supports such as Hjrbond-C extra [Amersham Pharmarcia Biotech]. Nylon membranes and polarized nylon membranes are not suitable for immunoscreening due to poor protein immobilization and high background.
Additional reagents
Step 1 of this section requires the reagents listed in Protocol 1 of Chapter 2.
Step 2 of this section requires the reagents listed in Scheme 1 of this chapter.
Vectors and Strains
λ phage vector
Use expression vectors and strains to construct the desired cDNA library
Methods
1. Spread non-recombinant λ phage on 10 agar plates to obtain semi-fusion lysates (see Scheme 1 in Chapter 2).
2. Prepare lysate blots on nitrocellulose filter membranes as described in Steps 5 to 12 of Scheme 1, but omitting the IPTG treatment.
It is not necessary to label the membranes as described in Step 8 of Scheme 1.
3. dilute the antibody preparation for screening 1:10 with the blocking solution.
4. incubate the membrane with the diluted antibody for 6 h. Add 0.05% sodium azide to the treated antibody and store at 4°C for use in immunoscreening.
With minor modifications, this method can be used for candidate screening of bacterial colonies for the removal of anti-E. coli antibodies by immobilizing a number of bacterial colonies on nitrocellulose filter membranes that do not contain the inserted cDNA expression vectors (see Scheme 2). Cell lysis and membrane washing are then performed according to steps 10-16 of Scheme 2. Finally, the antiserum is processed according to steps 3 and 4 above. 