Production and Characterization of Monoclonal Antibodies Against Primo Vascular System of Rat

Abstract

Background

The primo vascular system (PVS) has been difficult to detect due to its small diameter and translucent features of the threadlike network. Thus, contrast-enhancing dyes including Alcian blue, Trypan blue and Janus green B had to be used for finding and taking out PVS from rat and mouse.

Objective

Generation of monoclonal antibodies (mAbs) against PVS of rat was intended to use as a detector for PVS and a biological tool for functional study of PVS.

Materials and methods

Primo vessel (PV) and Primo node (PN) were isolated from organ surfaces of rat and then their proteins were isolated and injected into mouse as an immunogen. The classical traditional method was applied for production of mAbs against PVS. The various techniques, such as cell fusion, screening of hybridoma, ELISA, Western blotting (WB), immunofluorescence microscopy (IF), and limiting dilution, were used to generate mAbs against PVS.

Results

Among 16 mAbs generated, 4 representative mAbs were characterized with their specificities in ELISA, WB, and IF. α-rPVS-m1-1 and α-rPVS-m4-6 had strong binding affinities to PVS in both ELISA and WB but did not show specificities in IF at all. On the contrary, α-rPVS-m3-2 and α-rPVS-m3-4 almost did not respond in WB but had strong binding affinities in ELISA and specificities in IF. Two mAbs stained predominantly at extra cellular matrix and cell membrane of PVS of rat in IF, and they were able to discriminate PVS from blood vessel (BV) and lymphatic vessel (LV).

Conclusions

4 representative mAbs against PVS of rat were characterized by ELISA, WB, and IF. α-rPVS-m3-2 and α-rPVS-m3-4, which had strong specificities in IF, can be used as a tool in discriminating PVS from other similar tissues and in elucidate biological function of PVS.

1. Introduction

A Bonghan corpuscle (BHC) and a Bonghan duct (BHD) is a novel vascular network system that was first reported in 1963 by Dr. Bonghan Kim. He claimed that there was a new circulatory system by BHC/BHD distributed throughout the body of all vertebrae, and that BHC and BHD corresponded to an Acupuncture point and Acupuncture meridian, respectively [1]. Recently, BHC/BHD has been re-discovered by Dr. Soh [2], and renamed as “primo vascular system (PVS)” at an international conference in Jecheon, South Korea [3].

The unique characteristics of PVS have been revealed with more than 10 years’ studies on PVS. First, the PVS primarily consists of primo vessels (PVs) and primo nodes (PNs), and PNs often branch out into several PVs [4–6]. Second, the PVS is composed of a bundle of small sub-ducts and this bundle structure is morphologically different from that of a lymphatic vessel (LV) [7]. Third, rod-shaped nuclei are linearly aligned along the longitudinal axis of the PVS [8,9]. Fourth, the PVS contains various immune cells and has their unique cellular composition [7,10,11].

Although there are many demonstrations of anatomical differences between PVS and LV [12], some scientists still raise a question on that PV is a totally different from LV. Thus, PVS still needs further research and characterization to distinguish them from blood vessels (BVs) or LVs. As lymphatic vessel endothelial receptor 1 (LYVE-1) and CD31 was developed as a biomarker for lymphatic endothelial cells [13] and BVs endothelial cells [14] respectively, it would be very helpful for detecting PVS or tracing down action of PVS depending on cell status if the specific components from PVS can be unearthed as biomarkers.

In this study, monoclonal antibodies (mAbs) against PV and PN of rat were produced and their characteristics were investigated in terms of ELISA, Western blotting (WB), and immunofluorescence microscopy (IF). The generated mAbs against PVS can be used not only for discriminating it from LV/BV, but also for a powerful tool in analyzing function of PVS in rat, such as a physiological change in the process of disease.

2. Materials and Methods

2.1. Animals

Male Sprague-Dawley rats aged 4–8 weeks were obtained from Dhamul Science (Daejeon, Korea) and kept in 20–26°C temperature-controlled environment with 50–70% humidity, and provided with water and commercial rat chow ad libitum. The procedures involving the animals and their care were in full compliance with current international laws and policies [Guide for the Care and Use of Laboratory Animals. National Academy Press 1996].

2.2. Preparation of primo vessels (PVs) and primo nodes (PNs) from organ surface and abdominal wall of rat

The rats were anesthetized by an intramuscular injection of a mixed solution of 0.5 mL of Alfaxalone and 0.1 mL of Rompun. PVs and PNs were harvested, according to previously described methods [15]. Briefly, the PVs/PNs were sampled using small surgical instruments from the organ surface in thoracic and abdominal cavities, and abdominal wall after skin incision at the linea alba of the abdomen. All procedures of observations were performed under a stereomicroscope (SZX16, Olympus, Japan). In order to use as a negative control for selection of hybridoma cells, BV and LV were also sampled.

2.3. Production of monoclonal antibodies (mAbs) against PVs and PNs

2.3.1. Preparation of immunogen

PVs and PNs obtained from 10–15 rats were washed with 150 mM phosphate-buffered saline (PBS) by centrifugation at 12,000 rpm at 4°C and stored in PBS containing protease inhibitor cocktail (Pierce, USA) at −70°C. After thawing them, total proteins from PVs and PNs were isolated by sonicate processor and centrifugation, and then stored at −70°C until to use.

2.3.2. Production of mAbs against PVs and PNs

The isolated proteins were used as an immunogen for the production of mAbs. The immunization of a Balb/c mouse with the isolated proteins of PVS, fusion between immunized mouse spleen cell and SP2/0-Ag14 myeloma cell, screening of hybridomas by ELISA, and cloning by limiting dilution were conducted according to previously described methods [16]. In brief, the 0.5 mL of protein suspension was mixed with an equal volume of complete Freund’s adjuvant and injected into the peritoneal cavity of female Balb/c mice (6–8 weeks old). The first injection was followed by four boost injections with mixture of protein suspension and incomplete adjuvant at 2–3 week intervals prior to sacrificing mouse for cell fusion.

2.3.3. ELISA

For the screening of mAbs, 96-well microtiter plates were incubated with 50 mL of the isolated proteins overnight at 4°C. After washing with PBS-Tween 20 (PBST) extensively, the plate was treated with 50 µL of 3% BSA blocking solution for 30 min and then incubated in 100 µL of the supernatant of hybridoma cell for 1 hour at 37°C, and then in HRP-conjugated 2^nd Ab in PBS (1:1000 dilution) for additional 1 hour. Following a final rinse with PBST, the color reaction for the detection of immunogen-Ab complex was initiated with the addition of 50 µL of substrate solution (ABTS + 0.03% H₂O₂) for 15 minutes and then stopped by adding 50 µL of 100 mM HCl. The absorbance was measured at 405 nm in an automatic ELISA reader (Model 550, Bio-Rad, Hercules, CA, USA).

2.3.4. SDS-PAGE and Western blotting (WB)

The isolated proteins was denatured by boiling for 3 minutes after adding 2X sodium dodecyl sulfate (SDS) treatment buffer and then separated on a 12% SDS-polyacrylamide gel. Proteins on the gels was either stained with Coomassie blue (CB) or electro-transferred to a polyvinylidene membrane for WB. The blot was blocked with blocking buffer (5% sucrose and 5% nonfat milk in PBS) and probed with supernatant of hybridoma overnight at room temperature (RT). After the membrane was thoroughly rinsed with PBST, the blot was visualized using HRP-conjugated 2^nd Ab in PBS and substrate solution (4-chloro-1-naphtole).

2.3.5. Immunofluorescence microscopy (IF)

To test the specificity of mAbs, IF was conducted with sections of paraffin-embedded PV and PN slides. Briefly, sections were deparaffinized by treatment with xylenes and rehydrated in a graded alcohol series. Antigen retrieval was carried out by boiling sections in sodium citrate buffer (10 mM sodium citrate; and 0.05% Tween 20, pH 6.0) for 10 min in a microwave oven. After being allowed to cool to RT, the sections were rinsed with water. Sections were incubated with 5% BSA in PBS for 30 min at RT in order to block nonspecific binding. They were then incubated with mAbs against PVS overnight at 4°C. The complexes of immune reaction were detected using 2^nd Ab (goat anti-mouse IgG) Dylight-488 (1:200) in 1% BSA for 30 min in dark condition. After thoroughly washed with PBST, 4′,6-diamidino-2-phenylindole (DAPI) was added to identify the nuclei and then mounted on cover glass with Mowiol solution.

3. Results and discussion

3.1. Preparation of samples

The PVS has been identified in various sites, such as subcutaneous tissue [15,17], internal organs surface [18], brain ventricles [19,20], and blood and lymphatic vessels [21] in several animal species. About 10 pieces of PVS sample from one rat were randomly collected from various tissues/organs for immunization of mouse and test of mAbs. Among various tissues and organs, subcutaneous tissue, small intestine and bladder were main spots that the PVS pieces were easily found and taken out with a fine pincette. PV and PNs on small intestine in Fig. 1A represented a typical PVS interconnecting between tissues/organs. PV sample residing in LV was also prepared for IF image to test mAbs. Fig. 1B showed Alcian blue stained-PV inside LV 15 min after injection of 0.1% Alcian blue solution into lumbar lymph node. LV containing Alcian blue stained-PV was taken out, fixed, paraffin embedded, and processed for IF.

1

Light microscopic images of primo-vascular system. (A) Primo vessel (PV) and primo nodes (PN) on surface of small intestine. Free movable PVS is found as part of larger network on/between internal organs. Note transparent PV and PN compared with scarlet blood vessels (BV). (B) PV inside lymphatic vessel (LV) stained with Alcian blue. PV inside LV was clearly visible as a blue line 15 min after injection of 0.1% Alcian blue into lumbar lymph node. Scale bars indicate 200 mm.

3.2. Characterization of the produced α-rhPVS-mAb

From several fusion experiments, 8 hybridoma cells were primarily screened with the proteins from PV/PN by ELISA. They were selected twice more as hybridoma cell culture were expanded from 24-well to 6-well to T-25 flask. And then 16 α-rPVS-mAbs were produced by limiting dilution method at final stage. Names and numbers of 16 mAbs were originated from three mother hybridoma cells; #1, #3, and #4. Thus, names and numbers of all the 16 mAbs were labeled as α-rPVS-m1-1~4, α-rPVS-m3-1~6, and α-rPVS-m4-1~6. ELISA, WB, and IF were processed for further characterization of mAbs, and their results were summarized in Table 1. The colonies from same mother cell showed very similar results from the conducted tests (data not shown). Thus, among the test results from 16 mAbs, those of only 4 representative mAbs were shown in Table 1.

Table 1 Characteristics of mAbs against primo-vascular system of rat.

The affinity of each mAb to immunogen on ELISA was measured with value of OD_405. When values of OD₄₀₅ were higher than 0.8, between 0.7 and 0.8, and between 0.7 and 0.6, they were registered as +++, ++, +, respectively in Table 1. As shown in a graph profile of OD₄₀₅ vs clone name (Fig. 2A), α-rPVS-m3-4 exhibited the strongest affinity (0.814) to the proteins of PVS among the registered mAbs. The results of WB analysis were also designated as same way according to degrees of positive signal strength (Table 1). α-rPVS-m3-2 recognized a single protein band of 25-kDa of molecular weight (Fig. 2C lane 1) but α-rPVS-m3-4, which originated from same mother hybridoma cell of #3 with α-rPVS-m3-2, did not bind to any of the proteins of PVS (data not shown). The WBs of α-rPVS-m1-1 and α-rPVS-m4-6 displayed quite different patterns. While α-rPVS-m1-1 was bound to many proteins between 100 to 15-kDa with strong signal (Fig. 2C lane 2), α-rPVS-m4-6 recognized two bands, 70-kDa protein with very strong signal and 50-kDa protein with strong signal (Fig. 2C lane 3).

2

Binding affinity of α-rPVS-mAb to the proteins of PVS, and images of Coomassie blue (CB)-stained SDS gel and WB. (A) Binding affinity of each α-rPVS-mAb to the protein of PVS by ELISA. The isolated proteins of rat PVS was coated onto a microplate and processed with supernatant of each hybridoma cell. Binding affinity was shown as OD₄₀₅ value after color reaction of immunogen-antibody. α-rPVS-m3-4 showed the strongest affinity to proteins of PVS. (B) CB stained SDS polyacrylamide gel. Lane M and P lane were electrophoresed with a size marker of molecular weight and isolated total proteins of PVS. (C) Corresponding WB of lane P in (B). Lane 1, 2, and 3 was probed with α-rPVS-m3-2, α-rPVS-m1-1, and α-rPVS-m4-6, respectively. Note WB signal strength of each mAb to proteins of PVS from Table 1.

3.3. Immunofluorescence microscopic (IF) image of PVS with α-rPVS-mAb

To check the specificity of α-rPVS-mAb to PVS of rat, IF was conducted with produced α-rPVS-mAbs on paraffin sections of PVS sample. Sections of BV and LV sample were used for negative controls for selection of α-rPVS-mAbs responding positively in IF. When α-rPVS-m3-2 expressing very strong specificity as shown in Table 1 was probed to sections of BV and LV sample, no did a positive signal come out with both samples (Fig. 3B and E). It indicated that α-rPVS-m3-2 did not have specificity to BV and LV in rat with IF. And those were true of α-rPVS-m3-4 (data not shown) that had strong specificity to PV/PN in IF. Although the α-rPVS-m1-1 presented fairly strong specificity in ELISA and WB, it did not respond at all to section of PV in IF (Fig. 3H) as registered in Table 1. The result of IF with α-rPVS-m4-6 appeared almost same as that of IF with α-rPVS-m1-1 (data not shown).

3

Immunofluorescence microscopic (IF) images of blood vessel (BV), lymphatic vessel (LV), and primo vessel (PV) as negative controls. Longitudinally sectioned BV (A–C) and LV (D–F) were probed with α-rPVS-m3-2. DNA and signal of immune reaction on sections were detected by DAPI (A, D) and 2^nd Ab of goat anti-mouse IgG-Dylight 488 (B, E), respectively. Images of IF in B and E represent those probed with all the α-rPVS-mAb, indicating that the generated mAbs did not bind to BV and LV in rat. Section of PV (G–I) was probed with α-rPVS-m1-1 and then treated with DAPI (G) and 2^nd Ab (H) to detect DNA and signal of immune reaction. α-rPVS-m1-1 showed no signal in IF of PVS although it exhibited positive signals both ELISA and WB as shown in Table 1. Scale bars indicate 100 mm.

The specificity of α-rPVS-m3-2 in IF was examined on paraffin-embedded sections of PV and PN. The sections were stained with α-rPVS-m3-2 and 2^nd Ab (goat anti-mouse IgG) Dylight-488. Enhanced α-rPVS-m3-2 signal was predominantly localized at peripheries in cross and longitudinal sections of PV and PN (arrow in Fig. 4B, E and H). Since the signal sizes of α-rPVS-m3-2 in PV and PN were beyond the general thickness of plasma membrane of cell, it suggested that α-rPVS-m3-2 would recognize extra cellular matrix and cell membrane of PVS. But some components in cell cytoplasm wouldn’t be rule out as immunogens against α-rPVS-m3-2 (arrow in Fig. 4C, F and I).

4

Immunofluorescence microscopic (IF) images of PV and PN probed with α-PVS-m3-2. Cross (A–C) and longitudinal section (D–F) of PV and PN (G–H) were probed with α-rPVS-m3-2 and localization of immunogen was detected by 2^nd Ab (goat anti-mouse IgG)-Dylight 488 (B, E, H). α-rPVS-m3-2 was predominantly localized on cell membrane and extra-cellular matrix of PV (arrow in B, E) and PN (arrow in H). Note that there were two different types of sinus in PN; insets by open and white triangle. Sinus by open triangle was not stained but sinus by white triangle was stained at its outer surface. Scale bars indicate 100 mm.

PN was fully occupied with various cells and other components [22]. And many sinuses or cavities were found inside PN as seen in Fig. 4H (open and white triangle). When PN was stained with α-rPVS-m3-2 and 2^nd Ab-Dylight-488, two different types of sinus (open and white triangle) were discernible; sinus by open triangle was not stained but sinus by white triangle was stained at its outer surface. Thus, considering staining pattern of α-rPVS-m3-2 to PV in Fig. 4B and E, it postulated that the sinuses stained with α-rPVS-m3-2 at periphery were the lumens of sub-ducts of PV. The specificity of α-rPVS-m3-4 in IF showed almost same as that of α-rPVS-m3-2 to paraffin-embedded sections of PV and PN (Fig. 5A ~ 5I); α-rPVS-m3-4 was strongly bound to cell peripheries of PV and PN (arrow in Fig. 4B, E and H), suggesting that α-rPVS-m3-4 would recognize extra cellular matrix and cell membrane of PVS.

5

Immunofluorescence microscopic (IF) images of PV and PN probed with α-PVS-m3-4. Cross (A–C) and longitudinal section (D–F) of PV and PN (G–H) were probed with α-rPVS-m3-4 and localization of immunogen was detected by 2^nd Ab-Dylight 488 (B, E, H). α-rPVS-m3-4 was bound to on cell membrane and extra-cellular matrix of PV and PN (arrow in B, E, H). It was also bound to surface of sub-ducts of PV and PN (arrowhead in B, H). Scale bars indicate 100 µm.

Although α-rPVS-m3-2 showed specificity to PVS in IF but not to BV and LV as in Figs. 3 and 4, more direct method to prove that α-rPVS-m3-2 binds to PVS would be staining of α-rPVS-m3-2 with section of PV inside LV. When cross (Fig. 6A, B and C) and longitudinal section (Fig. 6D, E and F) of LV containing PV inside were probed with α-rPVS-m3-2 and then processed to detect complex of immunogen-mAbs by 2^nd Ab-Dylight 488, the signal appeared only on PV inside but not on LV outside (Fig. 6B and E), indicating that α-rPVS-mAb has specificity to PV in rat.

6

Immunofluorescence microscopic (IF) images of PV inside lymphatic vessel (LV). Cross (A–C) and longitudinal section (D–F) of LV were probed with α-rPVS-m3-2, and then processed to visualize reaction of immunogen-mAb by 2^nd Ab-Dylight 488 (B, E). The signal appeared only on PV but not on LV, indicating that α-rPVS-mAb has specificity to PV of rat in IF. Scale bars indicate 100 µm.

IFs were also conducted with sections of PVS of rabbit or mouse to check the cross-reactivity of α-rPVS-mAbs. However, none of α-rPVS-mAbs did not work out in IF.

4. Conclusions

In this study, the total proteins of isolated PV and PN of rat were used as an immunogen for injection of mouse and production of mAbs. 16 α-rPVS-mAbs against PV and PN of rat were generated by a classical fusion method for hybridoma cell. The specificities of representative 4 mAbs (α-rPVS-m1-1 α-rPVS-m3-2, α-rPVS-m3-4, and α-rPVS-m4-6) were characterized with ELISA, WB, and IF. While α-rPVS-m1-1 and α-rPVS-m4-6 did not show specificity to PV and PN in IF, α-rPVS-m3-2 and α-rPVS-m3-4 stained predominantly at extra cellular matrix and cell membrane of PVS in rat. Thus, α-rPVS-m3-2 and α-rPVS-m3-4 can be used as a tool in discriminating PVS from BV/LV and other similar tissues in rat.