Monoclonal Antibodies to Sea Cucumber Polysaccharide and Their Use in a Sandwich ELISA Assay

Abstract

Antigen was synthesized with L-SCP, a sea cucumber polysaccharide isolated from Pearsonothuria graeffei (Semper) and bovine serum albumin (BSA) as a carrier protein. Spleen cells with high titer antibody producing ability were removed and fused with myeloma cells of SP2/0-Ag14 origin. Three stable murine monoclonal antibodies (MAb ascites) producing cell lines to L-SCP were generated according to a conventional immunization protocol. Their epitope mapping and binding specificity, which was characterized by blocking and inhibition enzyme-linked immunosorbent assay (ELISA) indicated that these specific MAb ascites have similar binding patterns. A sandwich ELISA was developed on the basis of employing L-SCP specific antibodies including MAb 3G6 as capture antibody and HRP-3G6 as detection antibody. The working range for L-SCP in aqueous solution from this method was 100–10,000 ng/mL with good sensitivity, specificity, and precision (relative standard deviation ≤7.9%). Thus the developed ELISA can be used as a convenient tool for the rapid detection of L-SCP in biological examples in the future.

Introduction

Sea cucumbers are economically important remedies that have been widely cultured in China and other Asian countries for thousands of years. They have been used for healing various internal and external wounds and are believed to have strong digestive and autolytic capabilities.^(1,2) In past decades, a number of novel polysaccharides were extracted from the body wall of the sea cucumber, which have presented as potentially useful therapeutics for various biomedical applications. The polysaccharides have been found to exhibit various biological activities, such as anti-thrombotic, anti-tumor,⁽³⁾ angiogenesis, and anticoagulant properties.^(4–9)

L-SCP, a new polysaccharide isolated from Pearsonothuria graeffei (Semper) with a low molecular weight of around 90 kDa, consists of GalNAc, fucose, GlcUA, and ester sulfate in the ratio of 1:1.2:0.9:5.5, respectively. Current characterization of the polysaccharide content in biological examples was estimated from the content of total sugar by colorimetric methods. However, the physicochemical properties of the polysaccharide have not been fully identified and characterized due to their limited specificity and sensitivity. Moreover, using the high performance liquid chromatography method to measure the content of polysaccharides requires more advanced purification processes and time.⁽¹⁰⁾ Therefore, it is necessary to develop a special microanalysis assay for measuring L-SCP during the extraction process and pharmacokinetic study.

In this study, the immunogenicity of L-SCP was augmented by using neoglycoprotein of conjugated bovine serum albumin (BSA). We raised three MAb ascites against L-SCP and developed a sandwich ELISA assay for this polysaccharide.

Materials and Methods

Materials and apparatus

L-SCP was kindly provided by Xi'an Xintong Pharmaceutical Research Co. (Xi'an, China). BALB/c mice (female, 6–8 weeks old) and myeloma cell line of SP 2/0-Ag14 origin were supplied by Yangzhou University (Yangzhou, China). RPMI 1640 and calf serum (CS) were supplied by Gibco-BRL (Grand Island, NY); Sephacryl S200, Sephadex G200 gel, and dextran were provided by Pharmacia (Uppsala, Sweden). Horseradish peroxidase (HRP) conjugated to goat anti-mouse IgG, IgM, rabbit anti-goat IgG, mouse monoclonal antibody isotyping reagents, poly-L-lysine (PLL, 30 kDa), CDAP, pristane (2,6,10,14-tetramethyl-pentadecane), Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), hypoxathine and thymidine (HT), hypoxathine-aminopterin-thymidine (HAT), bovine serum albumin (BSA), polyethylene glycol (PEG) 1400, and o-phenylene-diamine (OPD) were all purchased from Sigma-Aldrich (St. Louis, MO). All other chemical reagents were of analytical grade. Multiskan Spectrum Microplate Spectrophotometer and CO₂ incubator were from Thermo Electron (Waltham, MA). The microtiter plate shaker was the product of Seoulin Bioscience Co. (Seoul, Korea). ELISA microtiter plates were from JET Bio-filtration Products Co. (GuangZhou, China).

Synthesis and purification of L-SCP-BSA conjugate

L-SCP-BSA conjugate was prepared as described by Shafer and colleagues.⁽¹¹⁾ Briefly, 100 μL of 1-cyano dimethylamino pyridinium tetrafluorate (100 mg/mL in acetonitrile) were added to 2 mL of L-SCP (13.9 mg/mL in water). The pH of the reaction mixture was subsequently raised to 9.0 by addition of 300 mM triethylamine. After the addition of 2 mL of BSA (9.9 mg/mL in 150 mM NaCl), and overnight reaction at 4°C, the reaction mixture was quenched with 1 mL of 500 mM ethanolamine. The reaction mixture was dialyzed against phosphate-buffered saline (PBS, pH 7.4), then applied to a Sephacryl S200 column (100 × 1.6 cm) and eluted with PBS. The fractions containing the conjugate were pooled and dialyzed against distilled water. The amount of polysaccharide in the conjugate was determined by the anthrone–sulfuric acid method,⁽¹²⁾ and the content of protein was determined by Lowry's method.⁽¹³⁾ The carrier-hapten ratio was estimated as mole of polysaccharide per mole of protein. In all, 10 mg of the conjugate with a carrier-hapten ratio of ≈7 was obtained from the reaction. The conjugate appeared as a single peak in the void volume and entirely separate from free BSA and/or L-SCP. The conjugate was further sterilized through a 0.22 mm filter and stored at −20°C for immunization.

Immunization of mice

Immunization

Five 7-week-old female BALB/c mice (Yangzhou University, China) were used for immunization. Each injection was intraperitoneally administered in a single volume of 0.2 mL of a 1:1 mixture of Freund's complete adjuvant (initial) or Freund's incomplete adjuvant (subsequent) and antigen (L-SCP-BSA) in PBS. Freund's complete adjuvant was used for the first injection, followed at 14-day intervals with injections of Freund's incomplete adjuvant for a period of 2 months. Mice were bled 10 days after each boost, and the collected serum samples were stored at −20°C before being assayed. A pre-fusion boost was administered 4 days before fusion (double doses in PBS).

Indirect ELISA

Indirect ELISA was used to determine the serum samples and MAb titers. The high binding plates used in the ELISA assay (JET Bio-filtration Products Co., GuangZhou, China) were pre-coated with PLL⁽¹⁴⁾ (5 μg/mL in PBS, 100 μL/well) overnight at 37°C. The plates were then washed three times with phosphate-buffered saline (PBS, pH 7.4) containing 0.05% Tween-20 (PBST). L-SCP (10 μg/mL dissolved in 0.2 mol/L carbonate buffer [pH 9.6] 100 μL/well) was coated for 2 h, and then washed with PBST. The gelatin solution (1% w/v in PBS, 150 μL/well) was added to block nonspecific sites on the plates for 2 h, followed by three washings. Then samples were added, and the plates were incubated for 2 h at 37°C. After washing three times with PBST, the plates were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO) for 1 h at 37°C. The plates were washed (as above), and the immune complexes were located using the horseradish peroxidase substrates, which consisted of 40 μL of 3% hydrogen peroxide and 4 mg/mL OPD in 10 mL of citrate buffer (pH 5.4). The reaction was stopped by adding 2.0 M sulphuric acid 50 μL/well, and the plates were measured at 492 nm with 630 nm reference on a Multiskan Spectrum Microplate Spectrophotometer from Thermo Electron (Waltham, MA).

Production of MAb ascites against L-SCP

After the last immunization, splenocytes were collected and fused with SP 2/0-Ag14 myeloma cells. Produced hybridomas were screened against L-SCP in an indirect ELISA performed as follows. The selected positive cultures were cloned by the limiting dilution method. Antibody-producing hybridomas were thrice sub-cloned and the resulting hybridomas were expanded for antibody production by being injected intraperitoneally into pristine primed BALB/c mice to obtain ascetic fluid. The purified MAb ascites were conjugated to horseradish peroxidase (HRP) by using the periodate method⁽¹⁵⁾ for further study.

Characterization of MAb ascites

Titer, isotype and affinity measurement

The titers of the MAb ascites were measured by the above-mentioned indirect ELISA. The MAb ascites were isotyped with a mouse MAb isotyping test kit (Sigma, St. Louis, MO) according to the manufacturer's recommendations. Affinity measurements were performed as mentioned by Friguet and associates.⁽¹⁶⁾

Epitope localization

The epitope localizations of L-SCP recognized by each MAb ascites were mapped according to the blocking ELISA method. Briefly, the L-SCP-coated microplates were incubated with each MAb ascites with indicated titration at 37°C for 2 h. After washing, the HRP-conjugated MAb ascites was diluted to give an absorbance value of 1.0 in a direct ELISA tested as described above. In this assay, an absorbance value read at 492 nm with 630 nm reference on a Multiskan Spectrum Microplate Spectrophotometer close to zero indicates that the two tested MAb ascites recognize the same or two closely associated epitopes, while a value close to 1.0 shows the MAb ascites binding to distinct epitopes.

Cross-reactivity of MAb ascites

The cross-reactivity of the MAb ascites was determined by inhibition ELISA using a set of mono-(GalNAc, fucose), oligo-(sucrose), or polysaccharides (dextran, amidulin, and heparin) structurally related to L-SCP as potential inhibitors. The amount of each MAb ascites used in the inhibition ELISA was determined by testing against L-SCP as antigen to yield an absorbance value of approximately 1.0.⁽¹⁷⁾ The MAb ascites were pre-incubated overnight at 4°C with serially diluted inhibitors at concentrations between 0.048 and 200 μg/mL. After incubation, 100 μL of the mixture were added to L-SCP-coated ELISA plates. Further steps followed the indirect-ELISA as described above.

Determination of L-SCP based on sandwich ELISA

A sandwich ELISA was developed using MAb 3G6 (1:5000) against L-SCP as the capture antibody, HRP-conjugated MAb 3G6 (1:400) as the detection antibody, and free L-SCP in aqueous with a detection concentration range of 100 and 10,000 ng/mL as the standards. Results were reported as the mean of the two separate light absorbance values subtracted from the negative control value. Furthermore, the sandwich ELISA was validated by following experiments of specificity and precision.

Results

Characterization of anti-L-SCP MAb ascites

Five BALB/c mice immunized with the conjugate preparation showed reactivity to immunogen in indirect ELISA (data not shown). From three single fusions of spleen cells, we obtained hybridomas secreting antibodies that reacted with L-SCP. We selected three anti-L-SCP MAb ascites (3G6, 9H2, and 5H4) from these hybridomas for further development. The characteristics of the MAb ascites are summarized in Table 1. The MAb 3G6 belongs to the immunoglobulin IgG3 isotype, while MAb ascites 9H2 and 5H4 belong to the immunoglobulin IgM. The affinity constants determined by competitive ELISA were 1.40 × 10⁹, 3.74 × 10⁷, and 3.64 × 10⁸ (L/M), respectively.

Table 1.

Characterization of Anti-L-SCP MAb Ascites

MAb ascites	3G6	9H2	5H4	Isotype	Titer	Affinity constant (L/M) ^b
3G6	±^a	±	±	IgG3	2 × 10⁵	1.40 × 10⁹
9H2	±	±	±	IgM	1 × 10⁵	3.74 × 10⁷
5H4	±	±	±	IgM	1 × 10⁵	3.64 × 10⁸

The two MAb ascites can mutually and partly inhibit each other from binding to L-SCP.

Affinities of MAb ascites were estimated by incubating MAb ascites with L-SCP in solution. The unsaturated MAb ascites at equilibrium is measured by classical indirect ELISA with L-SCP captured onto the solid phase.

The epitope specificity of the MAb ascites was further investigated by inhibition ELISA. Results are shown in Table 2. In comparison with the IC₅₀ value, which was <0.048 μg/mL for L-SCP itself, heparin showed the IC₅₀ values of 50 μg/mL for all three MAb ascites, whereas mono-(GalNAc, fucose), oligo-(sucrose), dextran, and amidulin failed to inhibit the specific binding of the MAb ascites to L-SCP, even at a high concentration of 200 μg/mL. To our knowledge, there is structural similarity between heparin and L-SCP, where both compounds are branched glycosaminoglycans that largely contain GlcUAs and ester sulfates. But, L-SCP still consists of GalNAc, fucose, and GlcUA, which gave a different result from heparin. These results demonstrated the high specificity of the MAb ascites for L-SCP.

Table 2.

Concentrations of Inhibitors Needed to Obtain 50% Inhibition of Binding of HRP-conjugated MAb Ascites to L-SCP as Coating Antigen

	IC₅₀ (μg/mL) ^a
Inhibitors	GalNAc	Fucose	Sucrose	Dextran	Amidulin	Heparin	L-SCP (10 μg/mL)
3G6	>200	>200	>200	>200	>200	50	<0.048
9H2	>200	>200	>200	>200	>200	50	<0.048
5H4	>200	>200	>200	>200	>200	50	<0.048

Means from at least two experiments.

ELISA assay for L-SCP

Choosing the MAb pair for sandwich ELISA

The three antibodies obtained above could form nine different combinations with L-SCP. In order to identify the specific combination, a crisscross ELISA was carried out. The concentration of L-SCP used in this assay was 10 μg/mL, which supplied a condition of saturated L-SCP. All three MAb ascites were tested individually as capture antibodies and detection antibodies (Table 3). Most of the MAb combinations, even for the same MAb ascites, provided a similar sensitivity for L-SCP. It may be expected that there is a great amount of epitopes identified by these MAb ascites. L-SCP contains largely ester sulfates and possesses a linear structure with a lot of repeating oligosaccharide units, which may contribute to this result. Among the combinations, the MAb 3G6 as the capture antibody and HRP-3G6 as the detection antibody gave the highest signal-noise ratio. Therefore, the 3G6/3G6 pair was chosen for further characterization.

Table 3.

Combinations Between Three MAb Ascites and Their Horseradish Enzyme Conjugates Tested in a Crisscross ELISA with Saturated L-SCP (10 μg/mL) Using MAb Ascites as Capture or Detection Antibody (n = 3)

	Detection antibodies (P/N^a)
Capture antibodies	HRP-3G6	HRP-9H2	HRP-5H4
3G6	3.572 ± 0.026^b	1.184 ± 0.083	2.545 ± 0.021
9H2	3.193 ± 0.083	1.201 ± 0.015	2.705 ± 0.044
5H4	2.885 ± 0.04	1.039 ± 0.056	2.977 ± 0.041

P, absorbance of the positive; N, absorbance of the blank.

Mean ± standard deviation.

Validation of sandwich ELISA

The sandwich ELISA was characterized by evaluating its sensitivity, specificity, and precision. The standard curve for L-SCP was prepared under optimized conditions. As shown in Figure 1, the linear correlation between the light absorbance and the L-SCP concentration was obtained in the range of 100 to 10,000 ng/mL. The detection concentration limit of L-SCP was 100 ng/mL.

FIG. 1.

Standard curve for L-SCP in PBS was determined by sandwich ELISA. Vertical bars represent standard errors for three independent assays. The 100– 10,000 ng of L-SCP per mL linear range is inserted.

The specificity of the immunoassay was evaluated using related polysaccharides at the concentration of 10 μg/mL. The tested polysaccharides including heparin and chondroitin sulfate, whose structures are similarly to L-SCP, did not exhibit measurable cross-reactivity with the 3G6/3G6 pair. These results demonstrate that the ELISA system using the MAb 3G6/3G6 pair was confirmed to be highly specific.

Intra-assay and inter-assay precision were studied continuously with three standard solutions of L-SCP at the concentration of 200, 1000, and 5000 ng/mL, respectively. Intra-assay precision was evaluated by the RSD of determination of L-SCP from well to well (n = 5) in the same plate, and inter-assay precision was obtained from different plates (n = 5). As the results in Table 4 show, the maximum RSD intra-assay was 7.9%, while inter-assay was 4.5%.

Table 4.

Intra and Inter-assay Precision of L-SCP in Aqueous Solution (n = 5)

	Intra-assay			Inter-assay
Concentrations (ng/mL)	Mean	Accuracy (%)	RSD (%)	Mean	Accuracy (%)	RSD (%)
200	191.8	95.9	7.9	218	109.1	4.5
1000	1017.8	101.8	2.3	969	96.9	4.2
5000	5099.5	102.0	1.5	4968	99.4	2.8

RSD (%), relative standard deviation.

Discussion

L-SCP belongs to the glycosaminoglycan family, which is a negatively charged heterogeneous polysaccharide. In previous studies, L-SCP showed various biological functions, especially its anticoagulant and antithrombotic activity.^(18,19) It is well-known that it is hard to establish a fast and convenient method that could be used to determine polysaccharides in complex mixtures such as extracts, blood plasma, or urine. However there is always a need to quantify polysaccharides such as L-SCP in the extraction process and pharmacokinetic studies. Therefore we developed an immunoassay as the preferred method for microanalysis of L-SCP.

In order to establish an immunoassay for sea cucumber polysaccharide, anti-L-SCP MAb ascites were prepared and identified in the present work. Since polysaccharides belong to the T-cell-independent antigens without immunologic memory effects, they cannot generate highly sensitive and specific MAbs directly. In this study, L-SCP was chemically coupled to the carrier protein, BSA, to prepare the immunogen for immunizing BALB/c mice.^(20–23) Three MAb ascites against L-SCP were acquired from successful fusions using spleen cells from three immunized BALB/c mice. Among these MAb ascites, the combination of MAb ascites 3G6 and HRP-3G6 was employed in developing a specific sandwich ELISA, with the advantages of high specificity, good sensitivity (detection concentration limit = 100 ng/mL), and precision (RSD ≤7.9%) (Table 4; Fig.1).

In conclusion, a sandwich ELISA based on the MAb ascites of 3G6/3G6 routinely used not only for optimization of the extraction but also for the purification process of L-SCP was established in this study. Furthermore, it was found that L-SCP mixed with mice plasma could be specifically detected by this method with no endogenous bimolecular interference in preliminary research (data not shown). L-SCP could become a new anticoagulant drug with high performance and low toxicity. Thus the ELISA developed herein can also be used as a convenient tool for monitoring L-SCP levels in future pharmacokinetic studies.

Footnotes

Author Disclosure Statement

The authors have no financial interests to disclose.

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