FR2738576A1 - New monoclonal antibody specific for type I 5 alpha-reductase - Google Patents

Abstract

Monoclonal antibody (MAb) specific for type I 5 alpha -reductase (I) is new. Also claimed are:(a) cell lines that produce MAb, and(b) diagnostic kits contg. the monoclonal antibodies.

Description

 The present invention relates to antibodies specific for human 5α-reductase, and their applications, particularly in the field of diagnosis.

 5α-reductase is a membrane-bound enzyme found in the prostate, seminal vesicles, sebaceous glands, brain and other tissues. It catalyzes the testosterone-dependent NAPH reduction (T) to 5-dihydrotestosterone (DHT).

 This is a decisive step in androgen metabolism, which regulates the normal or pathological cell growth of the prostate. This enzyme plays a role in several endocrine disorders such as prostate hyperplasia, prostate and skin cancer, acne and hirsutism.

 For many years, studies have been undertaken to characterize, locate and inhibit the enzyme.

 Thus two forms of isoenzymes have been demonstrated and characterized in humans (IS.C .: Delos, JBS 94).

 They have maximum activities at neutral-basic pH (type 1) and acid (type 2) and are encoded by two different genes that have been cloned.

 They respond differently to various inhibitors (azasteroids) or modulators such as lipidosterol extract Serenoa Repens (ELSSR).

 After many controversies it is now established that both isoforms are expressed in the human prostate but that their expression depends on the type of cell (stroma or epithelium) or pathological (benign or malignant tumor).

 In order to better study the respective activity of the inhibitors on the two enzymatic types, cloning and gene expression of these two enzymes in a eukaryotic system has been developed.

 It would therefore be desirable to have antibodies specific for this enzyme, in particular to be able to detect or measure it.

 The subject of the invention is therefore monoclonal antibodies specifically directed against type I reductase.

 More particularly, the subject of the invention is the antibodies designated by 3D2, 14C6, 2E2 and 12B12.

 Obtaining such antibodies requires the production of a large amount of the corresponding isoform of the reductase. This is possible in a baculovirus system, in which the genes of the Q reductase have been inserted; SF9 insect cells are then infected as described by Lehle et al (J. Steroid Biochem Molec Biol 46, 1993, 177-182).

 After culturing, cells infected with baculovirus expressing Type I 5α-reductase (SaRi) are harvested, lysed, homogenized and centrifuged.

 According to a particularly advantageous aspect of the invention, the pellet is resuspended in a buffer solution containing testosterone and NADPH. A solution of phospholipids is added under conditions leading to the formation of liposomes, having encapsulated SQ R1. Unexpectedly, the encapsulation of S ctR1 in liposomes allows its purification while maintaining the affinity and activity of the enzyme.

 The liposomal preparation thus obtained can then be directly injected into mice in order to provoke an immune reaction.

 The mouse splenocytes thus immunized are removed and fused to prepare hybridomas. These are then tested on DU145 cells, which produce only isotype 1 of the Q-reductase (positive control). This screening makes it possible to select hybridomas with a higher reactivity. The clones secreting an antibody in high concentration and having a high reactivity with 5aR1 are selected by ELISA and immunoblot techniques.

 An advantageous application of these antibodies is their use in in vitro diagnostic methods for measuring the production of SaR1 in biological samples, in particular prostate tissue.

 The subject of the invention is therefore an immunological diagnostic kit comprising such antibodies.

 The diagnoses in question may be kits of known type, in particular of the Sandwich type, with monoclonal antibodies and in particular of the ELISA or RIA type, for example.

 SaRI specific antibodies are preferably labeled, in particular with chromogenic, fluorescent or radioactive substances.

 Antibodies according to the invention may also be included in imaging products for in situ detection of the antigen.

 The antibodies according to the invention will be particularly useful for diagnosing androgen-dependent diseases leading to hyperconcentration of 5aR1, for example in prostate cancer.

 The examples which follow are intended to illustrate the invention without in any way limiting its scope.

Example 1: Preparation of SQ reductase type 1
Spodoptera frugiperda (SF9) cells (ATCC, CRL1711) are maintained at 27 ° C. in Grace's insect medium, supplemented with 10% fetal calf serum, 3.3 g / l yeastolate, 3.3 g / l. l of lactalbumin hydrolyzate (Gibco / BRL) 50 μg / ml of gentamycin sulfate and 2.5 μg / ml of amphotericin B (Sigma, France). The cells are transfected with Autographa californica polyhedra virus (AcNPV) DNA (Invitrogen Co.,
CA, USA) to obtain wild-type viral stocks.

Production and isolation of recombinant baculovirus
This step was detailed for SaR1 by lehlé et al (J. Steroid
Biochem. Nlolec. Biol., 1993, 177-182). The expression of the SaR1 protein by the selected recombinant baculovirus designated by CS and pure polyhedron was confirmed by assays of S a reductase activity in the cell pellets, in the presence of testosterone and NADPH.

Partial purification of recombinant 5a reductase 1
SF9 cells infected with baculoviruses expressing ScxR1 were harvested 4 days after infection and homogenized in a high ionic strength buffer (100 mM Tris, pH 7.2, 100 mM sodium citrate, 100 mM KCl, D1T mM, 1 mM EDTA, 20% glycerol) (buffer A) containing 10 μg / ml aprotinin, 10 μg / ml leupeptin and 10 μg / ml of
TAME, as described in Sargent et al (J. Steroid Biochem Molec Biol., 1991, 38, 73-77) .The homogenate is subjected to centrifugation for one hour at 105,000 g, and 4 C, then the pellet. The cell is resuspended in buffer A containing 1 μM testosterone and 5 mM NADPH to obtain a microsomal preparation.

A methanol-chloroform solution (2: 1) of phospholipids from beef brain (Sigma, France) is evaporated to dryness under vacuum, resuspended under sonication in a solution of n-octyl -
D-glucopyranoside, then added directly to the microsomal preparation to give a final lipid concentration of 5 mg / ml and octyl glucoside of 0.8% w / v. The mixture is kept on ice for 90 minutes, stirred at regular intervals, and then dialyzed against 3.5 I of buffer A at 4 ° C for 3 days (Molecular Cutoff Threshold of Spectrapore 12,000 - 14,000). (5aR1 encapsulated in liposomes) was used as a source for enzymatic kinetics studies.The activity of 5aR1 was measured in the initial cell pellet and after liposome encapsulation.The protein concentration was measured using the BCA technique (Pierce, IL, USA).

 Liposome encapsulation of the type 1 isoenzyme preserves activity and affinity and allows purification. The encapsulated isoenzyme has an apparent Km of 1.8 FM and a specific activity of 6.5 nmol / mg protein / min, whereas they are respectively 2.9 FM and 2.4 nmol / mg protein / min before encapsulation; we observe a slight but not significant decrease in Km and a decrease of nearly 3 times in the specific activity.

Example 2: Immunizations
Female BALB / c mice of 16-18 g (~ 6 weeks) were immunized with the liposomes, the dates of injection, the concentration of antigen and the adjuvants used are reported hereinafter.

immunisalions <SEP> lev. <SEP> of the <SEP> immunisalion <SEP> pediatric, <SEP> of the <SEP> immunization <SEP> sampled. <SEP><SEP> immunization <SEP> lev. <SEP>
<tb> 0B / 06/1993 <SEP> 23/06/06/1993 <SEP> sera <SEP> 0B / 07/1993 <SEP> sera <SEP> 27/07/1993 <SEP> sera <SEP> 26 / 11/1993 <SEP> serums
<tb> Mouse <SEP> 1 <SEP> (S1) <SEP> OJ. <SEP> lipos # ACF <SEP>J15;<SEP> lipos # AIF <SEP> J28: <SEP> 05/07 <SEP> J70 <SEP> lipos # AIF <SEP> J42: <SEP> 20/07
<tb> Mouse <SEP> 2 <SEP> (S2) <SEP> OJ. <SEP> lipos # ACF <SEP>J15;<SEP> lipos # AIF <SEP> J28: <SEP> 05/07 <SEP> J30 <SEP> lipos # AIF <SEP> J42: <SEP> 20/07
<tb> Mouse <SEP> 3 <SEP> (S3) <SEP> JO <SEP> Papl # ACF <SEP> J15: <SEP> lipos # AIF <SEP> J27: <SEP> 20/07 <SEP> J34 <SEP> pepl # AIF <SEP> J44: <SEP> 06/08 <SEP> J156: <SEP> pepl # AIF <SEP> J166: <SEP> 06/12
<tb> Mouse <SEP> 4 <SEP> (S4) <SEP> OJ. <SEP> Pepl # ACF <SEP> J15. <SEP> lipos # AIF <SEP> J27: <SE> 20/07 <SEP> J34: <SEP> pepl # AIF <SEP> J44: <SE> 06/08 <SEP> J156: <SEP> pepl # AIF <SEP> J166: <SEP> 06/12
<tb> Mouse <SEP> 5 <SEP> (S5) <SEP> OJ. <SEP> pepl # ACF <SEP> J15. <SEP> pepl # AIF <SEP> J27: <SEP> 20/07 <SEP> J34: <SEP> pepl # AIF <SEP> J44: <SEP> 06/08 <SEP> J156: <SEP> Pepl # AIF <SEP> J166: <SEP> 06/12
<tb> Mouse <SEP> 6 <SEP> (S6) <SEP> OJ. <SEP> pepl # ACF <SEP> J15. <SEP> pept # AIF <SEP> J27: <SEP> 20/07 <SEP> J34. <SEP> pepl # AIF <SEP> J44: <SE> 06/08 <SEP> J156: <SEP> pepl # AIF <SEP> J166: <SEP> 06/12
<tb> Sepia <SEP> pie-immune <SEP> S1 # S2 <SEP> J.3. <SEP> 05/06 <SEP> J.3: <SEP> 05/06
<tb> Sepia <SEP> pié-immune <SEP> S1 # S5 <SEP> J.3. <SEP> 20/06 <SEP> J.3: <SEP> 20/06
<Tb>

ACF: adjuvanl complel of Freund
AIF: adjuvanlincomplel de Freund lipos: liposomes (100 μl of liposomes per mouse and by injection) pepl: peptide (25 g of peptide per mouse and by injection)
Control of immunizations
The sera of the mice were taken every 10 days after each booster and tested on cytospins (wild-type and transfected SF9 cells, DU145 cells) by an immunolabeling technique.

MUNICULAR TECHNIQUE OF CELLS - about 20,000 cells are projected on a microscopy slide.
using a Shandon cytocentrifuge, - the cells are air-dried, - fixing with a 3.7% formaldehyde solution, - rinsing with PBS, - fixing in methanol and acetone at -20 C, air drying and rinsing in PBS, the endogenous peroxidases are blocked in PBS with 0.3% H2O2 (2).
ml of 30% H2O2 in 198 ml of PBS), the non-specific sites are saturated with normal goat serum, the cells are incubated with the supernatants used pure, after rinsing, the antibodies specifically bound are revealed with the
biotin avidin-peroxidase complex using a binding antibody
biotinylated goat anti-mouse, - visualization of complexes through a chromogenic reaction that
uses as substrate DAB (diaminobenzidine), - before reading the slides are counterstained with hematoxylin.

 The results clearly show an increase in the serum titer of the sera of the S1 and S2 mice on the DU145 cells. The reactivity observed on SF9 + (wild-type) cells, on the other hand, remained low, indicating a low background noise during this enzymatic technique.

 Cell fusion from S1 mouse splenocytes was therefore performed.

Example 3 Preparation of hybridomas 1) S1 cell fusion (mouse immunized with 5 &alpha; R1 liposomes):
Three days before the fusion, 40 μl of liposomes under 150 μl of sterile PBS were injected intravenously into the mouse S1.

80 x 106 splenocytes were placed in the presence of 20 million syngeneic mouse myeloma cells (X63Ag8 / 653 myeloma) and incubated according to the Koehler lymphocyte hybridization technique and
Milstein (Nature 256. 445-447, 1975) with polyethylene glycol (PEG 1500
Sigma) at 40%.

number of plates prepared 16 96-well plates in the presence of 10,000 peritoneal macrophages of Balb / c mice per well 384 wells (4 plates) at 105 splenocytes per well 384 wells (4 plates at 5 × 104 splenocytes per well 768 wells (8 plates) plates) at 2.5 x 104 splenocytes per well
Medium used: RPMI 1640 (BioWhittaker) + 20% fetal calf serum origin SVF MultiSer Cytosystem Australia n lot 203 271 13 + HAT concentration H100cim, at 0.4 m, T 16pm, N Glutamine concentration 2mN1 + antibiotics concentration Peni 200 U / mL, Strepto 200> g / mL (penicillinestreptomycin).

2) Screening
Wells in which only one clone grew were identified and the supernatants removed.

320 supernatants were tested on fixed cell cytospins
DU145 (positive control) and SF9 + (negative control).

 27 hybridomas were thus selected and frozen (Table 1).

Table 1
LIST OF ANTI-SIR HYBRIDOMES 1
selected for their responsiveness
on DU145 1: 1D11 10 7G10 19 llD7 2: 1E3 11 8C5 20 11F5 3 2D2 12 8D2 21: 12A5 4: 2D5 13 8H1 22 12B12 5 2E2 14 8H8 23 12G10 6 3C10 15 9C8 24 13D11 7: 3D2 16 9H10 25 14C1 8 5B6 17 10C4 26 14C6 9 6H11 18 lIdI 27: 15B6
Example 4
A fine specificity analysis was performed with three complementary techniques.

a) reactivity on SF9-fixed cells transfected with the cDNA of 5α-R1 by the immunolabeling technique.

 11 hybridomas showed greater reactivity on transfected DU145 and SF9 cells than on the negative control, native SF9.

 The supernatants of these hybridomas were then analyzed by immunoblotting.

Table 2
REACTIVITY OF CELL CULTURING SURNAGEANTS
Hybridomas DU145 SF9 / 5 R1 SF9 [+] 1 D 11 +++ +++ + 1 E 3 ++ + + 2D2 + + 2D5 +++ + + 2E2 + 3C10 + 3D2 + 5 B 6 + 6H11 + 7G10 + 8 D 2 ++ - 8 H 1 +++ +++ 8 H 8 ++ + 9 C 8 +++ + 9 H 10 ++ +++ + 10 C 4 ++ + 11D4 + 11 D 7 + + + 11 F 5 ++ ++ + 12 A 5 - - 12 B 12 +++ ++ + 12 G 10 ++ + 14C 1 +/- ++ + 14 C 6 + 15 B 6 ++ ++ + b) Immunoblot Reactivity on Protein Extracts of SF9 + and SF9 5 &alpha; R1:
IMMUNOBLOT TECHNIQUE ON PROTEIN EXTRACTS :: - electrophoretic protein migration, - nitrocellulose protein transfer, - antibody incubation, - mouse Ig revelation by biotin-coupled anti-mouse Ig antibody, - addition of streptavidin coupled with alkaline phosphatase, - addition of the chromogen.

Results (Table 3):
Several hybridoma supernatants showed reactivity against different proteins of different molecular weight than 5α-R1. At the expected level of SaRi (29Kd) four antibodies showed reactivity:
Table 3
IMMUNOBLOT ON PROTEIN EXTRACTS
Hybridomas SF9 / SaR1 SF9 [+] 3D2 29kD BF to 25kD 11 F 5 29kD 28-30kD 12G10 29kD 13 D11 29kD
BF = background noise c) ELISA reactivity on the peptide (amino acids 234 to 258 of the coding sequence of 5 &alpha; R1):
TECHNIQUE ENLISA ON PEPTIDE: PASSIVE COUPLING 1) passive coating of the peptide on a rigid plastic ELISA plate - "Coating": 10 g of peptide "5 &alpha;R1" per well on a rigid plastic ELISA plate, round bottom (IMMULON 2 Dynatech) in carbonatebicarbonate buffer (SIGMA capsule ref: C 3441) dissolve a capsule in 100 ml of distilled water (final solution: 0.5 M PH 9.6); distribution of 50 l per well; overnight at 4 ° C., 2 washes in PBS-Tween 20 0.05%, saturation with 3% skim milk in PBS 200 l / well; ; 1 hour to
Ambient temperature (RT), 2 washes in PBS-Tween 20 0.05%, incubation with 50 μl / well of samples (pure supernatants) overnight at 4 ° C., 3 washes in PBS-Tween 20 0.05 Incubation with peroxidase-conjugated mouse IgG (HL) (Jackson) 115-036-003: 1: 5000 dilution in 0.1% PBS-BSA.

50 sipucts; lh at RT, - 3 washes in PBS-Tween 20 0.05%, - substrate
Substrate Dilution Buffer: phosphate / citrate pHS with sodium perborate (H202 substitute) ref Sigma P4922. Dissolve one capsule in 100 ml of distilled water.

 Substrate: O.P.D. ref Sigma P8287 at 0.8 mg / ml in the dilution buffer (1 pellet of 10 mg in 12.5 ml) 50 l / well; 10 min at TA and in the dark.

stopping the reaction with 50 μl / well of 1M H2SO4.

- reading at 492 nm.

Test of the 27 supernatants used pure on peptide coated at 4 C or 37
C (results table 4).

Table 4
TEST OF SURNAGEANTS ON PEPTIDE PASSIVELY COUPLED BY
AN ELISA TECHNIQUE
Results expressed in multiple of background noise (BF). Optical Density (OD).

<Tb>

Fus. <SEP> 5aR1 <SEP> ooet @ n @ <SEP> и <SEP><SEP> 37 c <SEP> coeting <SEP> A <SEP> 4 c
<tb> Hybridomas <SEP> petide <SEP> peptide
<tb><SEP> 1 <SEP> D <SEP> 11 <SEP> 1.5 <SEP> 1.4
<tb><SEP> 1 <SEP> E <SEP> 3 <SEP> 2 <SEP> L <SEP> 1.7
<tb><SEP> 2 <SEP> D <SEP> 2 <SEP> 1.2 <SEP> 1.5
<tb><SEP> 2 <SEP> D <SEP> 5 <SEP> 1.3 <SEP> 1.6
<tb><SEP> 2 <SEP> E <SEP> 1 <SEP> 1.8 <SEP> 1.4
<tb><SEP> 3 <SEP> C <SEP> 10 <SEP> 1.7 <SEP> 2,2
<tb><SEP> 3 <SEP> D <SEP> 2 <SEP> 1,3 <SEP> 1,3
<tb><SEP> 5 <SEP> B <SEP> 6 <SEP> 1.6 <SEP> 1.4
<tb><SEP> 6 <SEP> H <SEP> 11 <SEP> 1.5 <SEP> 1.7
<tb><SEP> 7 <SEP> G <SEP> 10 <SEP> 1.3 <SEP> 1.4
<tb><SEP> 8 <SEP> C <SEP> s <SEP> 1.7 <SEP> 2
<tb><SEP> 8 <SEP> 8 <SEP> D <SEP> 2 <SEP> 1.5
<tb><SEP> 8 <SEP> H <SEP> i <SEP> 1.5 <SEP> 1,2
<tb><SEP> 8 <SEP> H <SEP> 8 <SEP> 2
<tb><SEP> 9 <SEP> C <SEP> 8 <SEP> 1.6 <SEP> 1.9
<tb><SEP> 9 <SEP> H <SEP> 10 <SEP> 1,9 <SEP> 1,4
<tb><SEP> 10 <SEP> C <SEP> 4 <SEP> 1.5 <SEP> 1.6
<tb><SEP> 11 <SEP> D <SEP> 4 <SEP> 1.3 <SEP> 1.5
<tb><SEP> 11 <SEP> D <SEP> 7 <SEP> 1.8 <SEP> 1.7
<tb><SEP> 11 <SEP> F <SEP> 5 <SEP> 1.7 <SEP> 1.8
<tb><SEP> 12 <SEP> A <SEP> 6 <SEP> 1.8 <SEP> 1.4
<tb><SEP> 12 <SEP> B <SEP> 12 <SEP> 6 <SEP> 2
<tb><SEP> 12 <SEP> G <SEP> 10 <SEP> 1.3 <SEP> 1.4
<tb><SEP> 13 <SEP> D <SEP> 11 <SEP> 1.3 <SEP> 1.7
<tb><SEP> 14 <SEP> C <SEP> 1 <SEP> 1.3 <SEP> 1.3
<tb><SEP> 14 <SEP> C <SEP> 6 <SEP> 2 <SEP> 1,3
<tb><SEP> 15 <SEP> B <SEP> 6 <SEP> 1.3 <SEP> 1.3
<tb> noise <SEP> d2 <SEP> background <SEP> 0.117 <SEP> 0.141
<tb><SEP> DO
<Tb>
2nd peptide ELISA protocol: covalent coupling of the peptide on the plastic:
TECHNIQUE ELISA ON PEPTIDE: COVALENT COUPLING - "Coating":
Hybridomas tested: 1E3,2E2,3D2,8H1,9C8,14C6.

 CovaLink plates (Nunc, Cat No. 478042).

Four concentrations of peptide are tested - 1.2 g of peptide + 300 ng of NHS (Pierce, Cat No. 24510, lot 931021159), - 0.6 g of peptide + 150 ng of NHS, - 0.3 g of peptide + 75 μg of NHS, 0.15 μg of peptide + 37.5 μg of NHS,
Final volume: 50 sl, in distilled water.

 Add 50 μl per well of EDAC (Sigma Cat E-7750, Lot 23H0063).

 Incubate for 2 hours at room temperature (RT).

3 washes in PBS, 0.05% Tween 20, 2M NaCl, 50 mM MgCl 2, incubation with 50 μl / well of the samples (pure supernatants)
TA, 3 washes in PBS, Tween 20, 0.05%, 2 M NaCl, 50 mM MgCl 2, incubation with peroxidase-conjugated mouse anti-IgG (H + L) (Jackson), reference 115-036 -003: 1 / 5000th dilution in 0.1% PBS-BSA.

50 μl / well; 1 hour at RT, 3 washes in PBS-Tween 20 0.05%, substrate
Substrate Dilution Buffer: phosphate / citrate pH5 with sodium perborate (H202 substitute) ref Sigma P4922. Dissolve one capsule in 100 ml of distilled water.

 Substrate: O.PD ref Sigma P8287 at 0.8 mg / ml in the dilution buffer (1 pellet of 10 mg in 12.5 ml) 50 Ill / well; 10 min at TA and in the dark.

- Stopping the reaction with 50 l / well of 1M H2SO4, - Reading at 492 nm,
Results (Table 5)
Table 5
ENLISA TEST ON PEPTIDE COUPLE BY COVALENT METHOD
Supernatants tested Nb of times BF Nb of times BF
on non plaque peptide
plate coated
5aR11E3 2.8 1 5aR1 2E2 19 1 5 &alpha; R1 3D2 1 1
SaR1 8H1 2 1 5 &alpha; R1 9C8 2,3 1 5 &alpha; R1 14C6 14 1 * Results a) 5aR1-3D2 immunoblot reagent on a 29kD protein.

 This hybridoma was cloned followed by screening from the immunoglobulin assay values in the clone supernatants. The reactivity of the supernatants with the highest concentration of immunoglobulin was then monitored. The most reactive clone: 3D2 / 1F7 was produced in sufficient quantity to be injected into mice in order to obtain ten ml of ascites. Specific antibody produced by 3D2 / 1F7 was isotyped as IgM.

b) 5aR1-14C6 and 5aR1-2E2
These hybridomas were selected because their supernatants are ELISA-reactive on the peptide covalently coupled to the plastic.

These antibodies do not react on the immunoblot protein.

 These hybridomas were cloned. The reactivity of the clone supernatants with the highest concentration of immunoglobulin was then monitored.

c) 5 &alpha; R1-12B12
The supernatant exhibits strong cell reactivity and a strong peptide ELISA signal.

 This hybridoma has been cloned. The reactivity of the clone supernatants with the highest concentration of immunoglobulin was then monitored.

Claims (7)

 1. Specific monoclonal antibody against Type I 5-reductase.  2. monoclonal antibody according to claim 1, characterized in that it is selected from the group consisting of antibodies secreted by one of the hybridomas: 3D2, 14C6, 2E2, and 12B12, which exhibit a reactivity in ELISA on a peptide comprising the sequence between amino acids 234 to 258 of the sequence of Q1 R1, and / or immunoblot on a 29 Kd protein.  3. A cell line characterized in that it produces an antibody according to one of claims 1 or 2.  4. Diagnostic kit characterized in that it contains an antibody according to one of claims 1 or 2.  5. Method for in vitro diagnosis of androgen-dependent diseases, characterized in that a biological sample is brought into contact with an antibody according to one of Claims 1 or 2, labeled and the 5-reductase is dosed into the sample.  6. Method according to claim 5, characterized in that the biological sample consists of prostate tissue for the diagnosis of cancer.  7. Process for the production of an antibody according to one of claims 1 or 2, characterized in that a) eukaryotic cells are transfected in such a way as to make them  produce recombinant type 1 Q reductase, b) said cells are harvested, lysed and resuspended, c) a phospholipid solution is added to the suspension, so that  liposomes, d) the liposomes, having encapsulated the reductase, are recovered, and then  injected into mice, to make them produce specific antibodies  of this type 1 reductase.