Screening of expression libraries constructed in λ phage vectors
In typical immunoscreening experiments, libraries of λ phage expression vector constructs should be spread on plates of E. coli strains that do not contain isopropylthio-β-D-galactoside (IPTG). The lack of inducers ensures that no fusion proteins toxic to the host are produced before the successful formation of phage plaques, and after 2~4 h, the plates are moved from 42℃ to 37℃ to stabilize all the fusion proteins of the temperature-sensitive type. This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Screening of expression libraries constructed in λ phage vectors.
Materials and Instruments
Blocking Buffer Chloroform IPTG Antigen-Antibody Complex Detection Reagent SM TNT Buffer Wash Buffer Radioiodine Labeled Secondary Antibody Primary Antibody LB Agar Plate LB Top Agar Plate Cellulose Nitrate Filter Membrane λ Phage Expression Library E.coli Move makings For more product details, please visit Aladdin Scientific website.
Air incubator preset at 42°C Culture tubes Flat-tip tweezers Syringe with 18-gauge needle filled with waterproof black ink (India ink)
Buffers
The components of the storage solution, buffer and reagents are listed in Appendix 1, and the storage solution is diluted to the appropriate concentration.
Closed buffer
10 mmol/L Tris-Cl (pH 8.0)
150 mmol/L NaCl
0.05%(V/V) Tween-20
20%(V/V) Fetal bovine blood淸
Alternatively, 5%(V/V) skimmed milk powder or TNT buffer with 1% gelatin (m/V) and 3%(m/V) bovine serum albumin may be used as a blocking buffer. Different laboratories differ as to which reagent is best. It is recommended that the researcher decide the best choice by pre-testing. The blocking buffer is stored at 4°C and can be reused several times. A final concentration of 0.05% sodium azide is added to the liquid solution to inhibit the growth of microorganisms.
Chloroform
IPTG(10 mmol/L)
Before step 4, dissolve 0.6 g of solid IPTG in 250 ml of water. Sterilize by passing through a 0.25um filter membrane.
Antigen-Antibody Complex Detection Reagent:
Alkaline phosphatase (AP) coupled antibody for colorimetric reaction screening reagents
Horseradish Peroxidase (HRP) colorimetric screening reagent with coupled antibody
Chemiluminescence Screening Reagent
For the reagents used for each screening method, see Step 15, and for common methods of detecting antibodies, see Immunoscreening with Antibodies at the end of this chapter. For details, see Appendix 9.
SM
Store a 50 ml aliquot of SM at room temperature. Discard after use to avoid contamination.
TNT Buffer
10 mmol/L Tris-Cl (pH 8.0)
150 mmol/LNaCl
0.05%(V/V)Tween-20
Approximately 1 liter of TNT buffer is required for every 10 membranes screened. Store at room temperature.
Wash the buffer:
TNT buffer with 0.1% (m/V) bovine blood albumin.
TNT buffer with 0.1% (m/V) bovine albumin and 0.1% (V/V) NP-40
These buffers do not contain sodium azide
Radioactive compounds
125I-labeled protein A or 125I-labeled immunoglobulin (optional)
Radioactive iodine labeled secondary antibody
This antibody may be used if an antigen-antibody complex is to be detected by the radiolabeled secondary antibody in step 15.
Antibody
Primary antibody
For the selection of primary and secondary antibodies, see the information section on immunoscreening using antibodies at the end of this chapter.
Medium
LB agar plates
The 90 mm Petri dishes should contain 30-35 ml of agar medium, and the 150 mm plates about 50 ml. The plates must be dry, otherwise the top layer of agarose will peel off when the nitrocellulose filter membrane is removed. Plates made two days ago are usually well dried by leaving them slightly uncovered at 37°C for 1-2 h. Ampicillin-free LB agar plates should be used for the screening process, as E. coli Y1090 hsdR grows slowly in the presence of antibiotics.
LB top agar plate
Melt the top layer of agar in a microwave oven for a short time, cool to 47°C, divide it into 3 ml aliquots (for 90 mm plates) or 7.5 ml aliquots (for 150 mm plates) in sterile tubes, and place these aliquots in a dry or water bath at 47°C to prevent coagulation. 
Specialized equipment
Air incubator preset at 42°C
Culture tubes (sterile 13 mmX100 mm)
Flat-tip tweezers
Syringe with waterproof black ink (India ink) with 18-gauge needle
Nitrocellulose filter membrane
Filter membranes suitable for binding proteins and immunoblotting reactions include Triton-X100-free nitrocellulose filter membranes (Millipore HATF or equivalent) and nitrocellulose derivatives such as Hybond-Cextra (approved by Amersham Pharmacia Biotech), whereas nylon or polarized nylon membranes are not suitable for immunoscreening because of their high background and weak ability to immobilize proteins. Nylon or polarized nylon membranes are not suitable for immunoscreening because of the high background and weak protein fixation.
Mark the membrane with a soft pencil or ballpoint pen, moisten with water, sandwich between dry Whatman 3 mm filter papers, wrap the stack of papers in aluminum foil, and autoclave at 10 psi (0.70 kg/cm2 ).
Additional Reagents
Step 1 of this protocol requires the reagents listed in Chapter 2, Protocol 1.
Step 17 of this protocol requires the reagents listed in Chapter 2, Protocol 1.
Vectors and Bacterial Strains
λ Phage Expression Library
The cDNA libraries can be constructed using suitable expression vectors as described in Chapter 11 or can be purchased from the supplier. Before starting the experiment, the potency of the phage solution should be determined as described in Scheme 1 in Chapter 2.
E.coli
Screening with different host strains requires different media, e.g. E.coli Y1090 hsdR, which is often used as a host for cDNA libraries constructed from λgtll, carries a mutation in the gene encoding an ATP-dependent protease. The fusion protein expressed in this strain is more stable than that expressed in strains that do not have this mutation. This strain also carries the pMC9 plasmid, which encodes a lac-blocking protein that prevents the β-galactosidase promoter from initiating the synthesis of cytotoxic fusion proteins.9 The pMC9 plasmid also carries a selection marker (amp), which ensures that the plasmid will not be lost. E.coli Y1090 hsdR strains should be grown in medium containing 50ug/ml ampicillin (except for screening, see the note on LB agar plates above). LB agar plates). For libraries constructed from λZAP and its derivatives, immunoscreening was performed with E.coli BB4 and XL1-Blue strains. This strain carries the lacl gene and the tetracycline resistance marker (tet) on the F' factor. These strains should be grown in medium containing 12.5ug/ml tetracycline.
Before applying these host bacteria, familiarize yourself with their genotypes and any special growth requirements.
Methods
Plate culture of λ phage
1. Take a monoclonal clone of the appropriate E. coli strain for inoculation, and prepare cultured bacteria for plate spreading as described in Scheme 1 of Chapter 2.
2. Calculate the number of dishes required for library screening, assuming that 2X104 and 5X104 phage plaques can be grown in each 90 mm and 150 mm dish, respectively. Prepare one sterile test tube (13 mmxl00 mm) for each dish and line up the tubes on the test tube rack. Add 0.1 ml of bacteria for plate spreading and 0.1 ml of SM solution containing the required number of λ phage expression libraries to each test tube, and incubate at 37°C for 20 min.
The soft results of immunoscreening were best when the density of phage spots was low. The color produced by the chromogenic reaction in the outer periphery of the phage plaques was the most obvious. Thus, well-separated immunopositive phage plaques have a characteristic ring shape, whereas overly dense plaques have a lamellar morphology and the intensity of the color on the common periphery between neighboring phage plaques is greatly diminished, although positive phage plaques can be identified as long as a portion of the periphery does not join the used edge of the neighboring phage plaques. At densities greater than about 500 phage/cm2 , the periphery of neighboring phage disappears and antigen-positive phage become more difficult to distinguish. Therefore, the number of phages per plate given above should be considered as the upper limit for immunoscreening.
3. Continue to add 2.5 ml (90 mm dish) or 7.5 ml (150 mm dish) of melted top agarose to each tube, mix well, and pour immediately onto an LB agar plate. After solidification, the plates were incubated at 42°C for 3.5 h. The plates were then incubated for 3.5 h at 42°C.
The incubation at 42°C is to ensure inactivation of the λ-deterrent protein (encoded by the clts857 allele) and activation of the lysis program during λ-phage development.
Induction of protein expression
4. Use a soft pencil or ballpoint pen to number the nitrocellulose filter membranes. Gloves should be worn when holding the filter membrane so that oils on the skin do not interfere with the transfer of proteins. Soak the membrane in isopropylthio-β-D-galactopyranoside solution (IPTG,10 mmol/L) for a few minutes, then remove it with flat-tipped tweezers (e.g., Millipore tweezers) and place it on a stack of Kimwipes paper, and allow it to dry at room temperature.
5. Remove the plate from the incubator and quickly cover the surface with an IPTG-impregnated nitrocellulose filter membrane without moving the membrane after contact with the plate. Place the plates at 37°C and repeat several times until all plates are covered with nitrocellulose filters.
The easiest way to place the membrane on the plate without harboring air bubbles is to hold the edge of the membrane, bend it slightly so that the center is first in contact with the center of the plate, and use the wicking action to draw the membrane onto the plate.
It is important to place the membrane on the plate quickly so that the temperature of the plate does not fall below 37°C. Below 30°C, the growth of the λ phage is severely stunted.
6. Allow the plates to warm at 37°C for at least 4 h.
It is difficult to distinguish between immunoreactive phage and false positive results when initially screening the library. Careful handling of the filter membrane, the nature of the particular antiserum, the method of detection of the antigen-antibody complex, and a variety of other factors can affect the frequency of false positives. In general, color-borne reactions are associated with fewer false positives than either immunochemical or chemiluminescent reactions. However, even under optimal conditions, false positives will occur at a frequency of approximately 1 per plate. For this reason, we recommend screening on photocopy filter membranes to avoid the labor-intensive process of screening large numbers of phage spots a second time.
If you do not use a replica membrane for library screening, the original membrane can be left on a flat trigger for 10-12 h. If a replica membrane is required. Please see Step 9 for details.
7. Lift the lid off the dish and continue to incubate at 37°C for 20 min. This step strengthens the connection between the soft agar and the plate.
8. Bring the plates to room temperature in batches and, using a syringe with waterproof black ink and an 18-gauge needle, poke through the membrane to the agar underneath, marking at least 3 asymmetric locations.
9. Peel the membrane from the plate with flat-tipped forceps and quickly immerse it in a large amount of TNT buffer, gently swirl the membrane in the buffer to wash away any remaining agarose, and shake the TNT to prevent the membranes from sticking to each other.
If large pieces of agarose stick to the nitrocellulose membrane, pre-cool the plate at 4°C for 30 min or -20°C for 5 min before peeling off the membrane.
10. When all membranes have been transferred to TNT buffer, wrap the plate in plastic wrap and store at 4°C until results of the immunoscreening are available. 
Detection of phage expression of target fusion proteins
IMPORTANT: Do not allow the membranes to dry completely in the following steps. Antibodies that would otherwise bind non-specifically and reversibly to the moist membranes will remain permanently on the membranes as soon as the membranes are dried. In addition, the membranes should be immersed in various buffers and antibody solutions in such a way as to prevent them from binding to each other. For this purpose, the membranes can be divided into batches (5 membranes per batch) and each batch can be placed in a single large flat dish or crystallization dish, thus minimizing the problem of interconnection. The dishes can be stacked on top of each other on a low-speed platform shaker.
11. After all membranes have been stripped and rinsed, place them one by one in fresh TNT. After all membranes have been transferred, continue to gently agitate the buffer for 30 minutes at room temperature.
If necessary, the membranes can be removed from the buffer at this point, wrapped in Satan wrap and stored at 4°C for 24 hours.
12. Using flat-tipped forceps, transfer the membranes one by one to a glass dish containing containment buffer (7.5 ml per 82 mm membrane, 15 ml per 138 mm membrane), submerge all the membranes and slowly shake the solution on a rotating platform for 30 min at room temperature.
13. Using flat-tipped forceps, transfer the membranes to fresh glass dishes containing diluted primary antibody in blocking buffer (7.5 ml per 82 mm membrane, 15 ml per 138 mm membrane), using the highest antibody dilution that produces a background that is not too high but still detects 50-100 pg of denatured antigen. All membranes are submerged and the solution is slowly shaken on a rotating platform for 30 min at room temperature.
Antibodies can be stored at 4°C and reused several times. Microbial growth is inhibited by the addition of sodium azide to the solution at a final concentration of 0.05%.
14. Wash the membranes for 10 min in each of the following buffers, transferring the membranes sheet by sheet between buffers, 7.5 ml of buffer per 82 mm sheet; 15 ml per 138 mm sheet.
TNT Buffer with 0.1% Bovine Serum Albumin
TNT Buffer with 0.1% bovine serum albumin and 0.1% NP-40 (NondidetP-40)
TNT Buffer with 0.1% Bovine Serum Albumin
15. Detection of antigen-antibody complexes by radiochemical, colorimetric or chemiluminescent reagents of choice.
Radiochemical Screening
Approximately 1uCi of 125I-labeled protein A or immunoglobulin is required per membrane, and radiolabeled protein A is commercially available (specific activity 2-100uCi/ug) Radioiodinated secondary antibodies can be purchased from vendors or prepared as per the radioactive iodine labeling of IgG in the pre-materials information section.
a. Dilute the radiolabeled ligand with containment buffer (7.5 ml per 8 mm filter membrane, 15 ml per 138 mm filter membrane).
b. Allow to incubate for lh at room temperature, rinse several times with TNT, and perform autoradiography as described in Appendix 9.
Continue to step 16.
Color Reaction Screening
Antibodies coupled to horseradish peroxidase (HRP) or alkaline phosphatase (AP) that recognize the species-specific determinant of the first antibody can be purchased from the manufacturer and used at the dilutions and requirements recommended in the product insert. Typically 5ul of coupled antiserum can be added to 7.5ul of blocking buffer (no sodium azide) per 82 mm membrane, see Appendix 9 for more information on HRP or AP.
Localization of Antigen-Antibody-Antibody-AP Complexes by Combined Application of 5-Bromo-4-Chloro-3-Indole Phosphate (BCIP) Substrate and Nitroblue Tetrazolium (NBT) 
a. Place the membrane in a solution containing AP-coupled antibody and slowly moisten it for 1.5 to 2 h at room temperature.
b. Wash the membrane according to step 14.
c. Prepare BCIP (50 mg/ml in 100% dimethylformamide) and NBT (50 mg/ml in 70% dimethylformamide) storage solution and store away from light.
d. Prepare BCIP/NET developer solution before use:
i. Add 33ul of NBT solution to AP buffer and mix well. ii.
i. Add 33ul of NBT solution to AP buffer, mix well. ii. Add 16.5ul of BCIP storage solution, mix well, store away from light and use within 1h. iii.
e. Blot filter membrane dry with paper towel.
f. Submerge the membrane in BCIP/NBT Developer (7.5 ml per 82 mm membrane, 15 ml per 138 mm membrane) and incubate for several hours at room temperature.
g. Wash twice with distilled water to achieve a dark purple coloration at the antigen-antibody complex.
Continue to step 16.
Localization of antigen-antibody-antibody-HRP complexes using 4-chloro-1-naphthol 
a. Place the membrane in a solution containing the HRP-coupled antibody and shake gently for 1.5 to 2 h at room temperature.
b. Wash the membrane according to step 14.
c. Dissolve 60 mg of 4-chloro-1-naphthol in 20 ml of ice-cooled methanol to make developer solution. Before use, mix with 100 m [10 mmol/L Tris-Cl (pH 7.5), 150 mmol/L NaCl solution containing 60ul of 30% H2O2.
d. Blot the water from the membrane with a paper towel and rinse it with 10 mmol/LTris-Cl (pH 7.5), 150 mmol/LNaCl solution.
e. Immerse the membrane in 4-chloro-1-naphthol developer (10 ml for each 82 mm membrane, 25 ml for each 138 mm membrane) and incubate at room temperature for 15-20 min.
f. Wash twice with distilled water to reveal a dark purple color at the antigen-antibody complex.
Biotinylated species-specific antibodies and anti-biotin protein-coupled HRP are also commercially available and should be diluted according to the instructions of the different manufacturers and immunoscreened as described above for HRP-coupled antibodies.
After screening by colorimetric reaction, λ phage clones can be picked directly on the blotted filter membranes (Alter and Patriel990). The number of infected particles obtained by scraping the membranes with a dissecting blade is very inconsistent, and can range from 100 to 104pfu per blotted print. The scraped prints can be mixed with 20ul of SM, left at 4°C overnight, and re-laid in a suitable E. coli strain according to a certain dilution gradient. The plates can be re-laid in a suitable E.coli strain at a certain dilution gradient. Of course the traditional method of utilizing the blotted filter membrane aligned with the original phage spot yields a larger number of phages. However, too large a number is not beneficial as the next step will be phage spot purification of the recombinant phage.
If the knife-scraping method is used, it is recommended that the traditional method of blotting the filter membranes and phage spots be used again, especially when screening large cDNA libraries, which can be foolproof.
Chemiluminescent Screening
The chemiluminescence reaction is the most sensitive method for detecting immunopositive phage spots. Secondary antibodies are usually coupled to AP or HRP, and the use of AP-coupled antibodies requires a substrate such as 1,2-dioxetane phosphates. This substrate is phosphorylated to emit light at 466nm, and the HRP-coupled antibody oxidizes the substrate luminal, which emits a strong light at 428nm in the presence of hydrogen peroxide and phenol. In both systems, the light emitted can be captured by autoradiography. Chemiluminescent detection is rapid, sensitive (1-10 pg of antigen can be detected), and results in a permanent record of the screened membrane (x-ray film). Two potential disadvantages are the high cost of the reagents and the need to compare the radioautography film with a master plate to obtain a positive clone, as shown in the typical protocol below. 
a. Place the filter membrane in a solution containing an AP or HRP coupled antibody and shake gently for 1.5 to 2 h at room temperature. b. Place the membrane in a solution containing an AP or HRP coupled antibody and shake gently for 1.5 to 2 h at room temperature.
b. Wash the membrane according to step 14.
c. Prepare the chemiluminescent reaction substrate according to the manufacturer's instructions.
d. Incubate the washed membrane with the chemiluminescent reaction substrate for 1-5 min (determine the optimal exposure time according to the manufacturer's recommendations).
e. Remove excess liquid from the filter membrane and wrap immediately in Saran wrap; do not allow the membrane to dry out.
f. Radiographic autoradiography (see Appendix 9). Generally, an initial exposure of 1 min is used, and the appropriate exposure time is determined at this interval.
Continue to step 16.
16. Localize the positive spot or compare it to a duplicate filter film to determine the matching signal. If screening with a radiolabeled or chemiluminescent probe, compare the results of the radiolabeled autoradiography with an agar plate on the surface of the light box, or if screening with a colorimetric reaction reagent, a visible positive residue may be left on the filter membrane and continue with the following steps:
a. Cover the filter membrane with a Saran or Mylar wrap.
b. Mark the location of the wells on the membrane and the location of the antigen-positive clone on the surface of the Saran Packing Membrane with a waterproof marker of a different color, and mark the Saran Packing Membrane to locate the plate that corresponds to the membrane.
c. Place the Saran packaging membrane on the surface of the reading light box, and place the plate containing the original phage plaque on the membrane for verification.
d. Identify the area of positive phage plaques and use the wide mouth of a Pasteur pipette to pick up a block of agar from this area and place it in 1 ml of SM with 2 drops of chloroform.
e. Save the Saran packaging membrane as a permanent record of positive phage spot localization, as the colored spots on the original membrane will soon fade.
17. After several hours at 4°C, elute the phage from the agar block. Determine the titer of phage in the eluate and re-plate the culture at a density of approximately 3000 phage spots per 90 mm dish. Re-screen the phage spots from step 4 onwards as described above and repeat the screening and plate spreading. Repeat the screening and plate laying until consistent immunopositive recombinant phages are obtained. 
