Chinese Journal of Natural Medicines 2014, 12(11): 08470852
Chinese Journal of Natural Medicines
Development of a quantitative ELISA kit for human secreted CD306/LAIR-2 and its application TAO Yuan, LIN Jun*, HU You-Jia, ZHOU Bin, ZHU Bao-Quan* State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 200040, China Available online 20 Nov. 2014
[ABSTRACT] AIM: A quantitative ELISA kit for the detection of human secreted CD306/LAIR-2 was developed using monoclonal and polyclonal antibodies which were raised against a highly purified recombinant human secreted CD306/LAIR-2. METHODS: Anti-human secreted CD306/LAIR-2 monoclonal and polyclonal antibodies were raised by immunizing mouse or rabbit with recombinant human secreted CD306/LAIR-2. The monoclonal antibody was purified by protein G affinity, whereas the polyclonal antibody was purified by protein A affinity. The best match pair of antibodies were found and used to develop a double antibody sandwich ELISA kit for the detection of human secreted CD306/LAIR-2 in human samples. RESULTS: A human secreted CD306/LAIR-2 ELISA kit was formulated with highly purified recombinant human secreted CD306/LAIR-2, highly specific monoclonal and polyclonal antibodies. This kit realized the quantitative measurement of recombinant human CD306/LAIR-2 and natural CD306/LAIR-2 in human serum samples. CONCLUSIONS: The developed human secreted CD306/LAIR-2 ELISA kit is a reliable quantitation immunoassay kit. [KEY WORDS] Human Secreted CD306/LAIR-2; Monoclonal antibody; Polyclonal antibody; ELISA kit
[CLC Number] R965
[Document code] A
[Article ID] 2095-6975(2014)11-0847-06
Introduction Human CD306, also known as leukocyte-associated Ig-like receptor-2 (LAIR-2), is a newly found cluster of differentiation (CD) molecules. It belongs to the Ig super family [1]. CD306/LAIR-2 gene maps to a region of 19q13.4. The protein encoded by this gene has 135 amino acids. As a secreted protein, human CD306/LAIR-2 has three subtypes. Until now, researchers have only found the CD306/LAIR-2 molecule in humans. There is no report on mouse CD306/LAIR-2 yet. The CD306/LAIR-2 molecule is secreted mainly by CD4+T [Received on] 16-Dec.-2013 [Research funding] This project was supported by the National Important Special Foundation of the New Drug Development (No. 2010ZX 09401-403) and the Shanghai Industry-University-ResearchMedical Cooperation Project (No. 12DZ1931903). [*Corresponding author] LIN Jun: Dr., Tel/Fax: +86-21- 6247 9808, E-mail: [email protected]. ZHU Bao-Quan: Prof., Tel/Fax: +86-216279 1661, E-mail: [email protected]. These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
cells in circulating blood, but there could also be other cells or tissues that secrete the CD306/LAIR-2 molecule [1]. The human LAIR family has two members: CD305/LAIR-1 and CD306/LAIR-2. These proteins are encoded by two genes and have about 84% sequence homology and similar structures. Both of their genes map to 19q13.4, but are transcribed for different purposes. CD306/LAIR-2 is a secreted protein, whereas CD305/LAIR-1 is a transmembrane protein [1]. The CD305/LAIR-1 molecule is an inhibitory immune receptor in humans. It can provide inhibitory signals to immune cells, keeping the immune system in a balanced condition. Collagen is the ligand of both CD305/LAIR-1 and CD306/LAIR-2 molecules, but CD306/LAIR-2 has a higher affinity for the ligand, which prevents the CD305/LAIR-1 molecule from providing the inhibitory signals to the body [1]. When the human body can no longer keep the balance of the immune system, autoimmune diseases and other diseases may occur [2]. Researchers have also found that in some patients, there are higher levels of the CD306/LAIR-2 molecule in the blood or tissue than those of healthy individuals. CD306/LAIR-2 gene expression and immunohistochemical tests have shown down-regulated levels in the first
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trimester of pre-eclampsia pregnancies [3]. This may indicate that CD306/LAIR-2 is involved in the development of pre-eclampsia pregnancies. Hence, the quantification and dynamic monitoring of CD306/ LAIR-2 levels in the human body are important to research on the mechanisms of inflammation, allergic reaction, pre-eclampsia pregnancies, and leukemia, etc. Due to the importance of this molecule we have developed an ELISA kit that could provide quantitative determination of secreted human CD306/LAIR-2.
Materials and Methods Production and purification of antigen and standard protein A DNA sequence encoding the mature form of human CD306/LAIR-2 (Q22-P152) with 6 His-tag on the N-terminus was expressed in E. coli, and then the recombinant protein was purified by Ni-NTA column (Aviscera Bioscience, Santa Clara,USA). Dialysis of the purified protein was performed against PBS and exchanged ten times at 4 °C. The purity was determined by SDS-PAGE gel staining. The protein concentration was measured by two different methods: A280 nm and Bradford assay (Aviscera Bioscience, Santa Clara,USA). Immunization and production of anti-human CD306/LAIR-2 monoclonal antibodies Two BALB/c mice aged 6–8 weeks were immunized with intraperitoneal injections of human recombinant CD306/LAIR-2 protein (100 μg) together with Freund’s adjuvant, over a period of six weeks, at one-week intervals. Afterwards, there was a fusion of spleen cells from an immunized mouse with mouse myeloma cells. The positive cells were cloned by the limiting dilution technique and expanded intraperitoneally in BALB/c mice. Four strains of monoclonal antibodies were named 306-1, 306-2, 306-3, and 306-4. The monoclonal antibodies from the cell cultures were purified using protein G columns and dialysis against PBS. The antibody concentration was measured by two different methods: A280 nm and Bradford assay (Aviscera Bioscience, Santa Clara,USA). Immunization and production of anti-human CD306/LAIR-2 polyclonal antibodies One New Zealand rabbit aged twelve weeks was immunized with a muscle injection of human recombinant CD306/LAIR-2 protein (100 μg ) together with Frend’s adjuvant, over a period of seven weeks, at one-week intervals. Blood was collected seven days following a booster injection and centrifuged to obtain the serum. All of the collected serum was mixed and kept at –70 °C. The rabbit anti-human CD306/LAIR-2 IgG was purified by protein A affinity column (Aviscera Bioscience, Santa Clara,USA), and dialysis against PBS. The antibody concentration was measured by two different methods: A280 nm and the Bradford assay (Aviscera Bioscience, Santa Clara,USA). Antibody titration by indirect ELISA A sample (100 ng per well) of human CD306/LAIR-2 protein in PBS (pH 7.4) was coated on a 96-well microplate and incubated at 4 °C overnight. The liquid in the wells was discarded the following day. The microplate was washed with PBST buffer (with 1% BSA) to remove all of the protein that
did not bind. The microplate was blocked with PBS buffer (with 2% BSA) and incubated at room temperature for 2 h. The blocking buffer was discarded and the microplate was allowed to dry. The microplate was now ready for the addition of antibodies. A two-fold serial dilution was made for each of the monoclonal and polyclonal antibodies. The starting concentration of monoclonal antibodies was 5 μg·mL−1, and the starting concentration of the polyclonal antibody was 2 μg·mL−1. For each dilution, 100 µL per well were added to the microplate that was coated with human CD306/LAIR-2 protein. The microplate was incubated for 2 h at room temperature on a microplate shaker at 300 r·min−1. Next, the microplate was washed with PBST buffer (with 1% BSA), followed by aspiration of all the liquid. Then, goat anti-mouse IgG HRP conjugate was added to detect monoclonal antibodies, and goat anti-rabbit IgG HRP conjugate was added to detect polyclonal antibodies. The microplate was incubated at room temperate on the microplate shaker for 1 h. Another washing and aspiration step was done before the addition of TMB substrate solution. The reaction was stopped with 2 mol·L−1 HCl, and the microplate was read in a microplate reader at 450 nm. Formulation of human secreted CD306/LAIR-2 ELISA kit According to the results of the titer from the indirect ELISA, one monoclonal antibody was chosen to coat the plate. A 96-well microplate was coated with the purified mouse anti-human CD306/LAIR-2 monoclonal antibody 306-4 at 5 g·mL−1 in PBS (pH 7.4). The plates were incubated overnight at 4 °C. Then, the plates were washed with PBST buffer with 1% BSA and aspirated. The plates were blocked with PBS buffer with 2% BSA, and were incubated for 2 h at room temperature before discarding the liquid and allowing the plates to dry. The highly purified recombinant human CD306/LAIR-2 was used as a standard. A solution of 20 ng·mL−1 of human CD306/LAIR-2 protein in PBS buffer with 1% BSA was prepared as the highest standard concentration. A seven point standard curve was made using two-fold serial dilution with 20 ng·mL−1 as the highest standard. PBS containing 1% BSA without any standard protein was used as the negative control. The plate was incubated on the microplate shaker for 2 h at room temperature, and then washed and aspirated. Next, purified rabbit anti human CD306/LAIR-2 IgG (100 µL) was added as detection antibody. Again, the plate was incubated on the microplate shaker for 2 h at room temperature with another wash and aspiration step. Finally, goat anti-rabbit IgG HRP conjugate was added to the wells, and the plate was incubated on a microplate shaker at room temperature for 1 h in the absence of light. After the final wash and aspiration step, TMB substrate solution was added to all of the wells for the color to develop. The reaction was stopped with 2 mol·L−1 HCl, and the absorbance of the plate was read on a microplate reader at 450 nm. The purified normal rabbit IgG without immunization of human CD306/LAIR-2 was used as the as
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say antibody negative control to replace the primary antibody.
Results In a previous study from this laboratory, the recombinant human mature form CD306/LAIR-2 His-tag was electrophoresed on 15% SDS-PAGE gel under reducing conditions. The purity of this human CD306/LAIR-2 recombinant was more than 95% by gel staining. The MW band is ~16 KDa on gel staining. Two BALB/c mice were immunized with intraperitoneal injections of 100 μg of human recombinant CD306/LAIR-2 protein together with Freund’s adjuvant. Afterwards, there was a fusion of spleen cells from the immunized mouse with mouse myeloma cells. As a result, four purified anti-human CD306/LAIR-2 monoclonal antibodies (306-1, 306-2, 306-3, and 306-4) on indirect ELISA indicated specific immunobinding. According to the curves (Fig. 1), the binding of monoclonal antibodies 306-1 and 306-2 with human CD306/LAIR-2 protein is saturated at high concentration. Both antibodies also show a slow decrease in binding in the protein’s lower concentration range. The binding of monoclonal antibodies 306-3 and 306-4 has a better linear rate.
Fig. 1 Mouse anti-human CD306/LAIR-2 monoclonal antibody titer
One New Zealand rabbit was immunized with muscle injection of human recombinant CD306/LAIR-2 protein (100 μg) together with Freund’s adjuvant. Blood was collected seven days following a booster injection and centrifuged to obtain the serum. As shown in Fig. 2, the binding of the polyclonal antibody with human CD306/LAIR-2 protein was saturated at higher concentrations (> 0.25 µg·mL−1). However, the specific binding of rabbit anti human CD306/LAIR-2 antibody with human CD306/LAIR-2 protein indicated a linear relationship at lower concentrations (< 0.125 µg·mL−1). The 67 ng·mL−1 of the purified rabbit anti-human CD306/LAIR-2 IgG was located in the linear binding range, and was used as the detection antibody concentration for direct ELISA formulation. Paired antibody screening Each of the purified antibodies of mouse anti-human
Fig. 2 Rabbit anti-human CD306/LAIR-2 polyclonal antibody titer
CD306/LAIR-2 monoclonal was coated onto a 96-well microplate at different concentrations: 10, 5, and 2.5 µg·mL−1. After washing the plate to remove any unbound captured antibody and blocking it with PBS buffer with 2% BSA, a ten-fold serial dilution of human CD306/LAIR-2 recombinant starting from 10 000 to 0.1 ng·mL−1 was applied into each well and incubated for 2 h. After a wash and aspiration step, detection antibody was added at a concentration of 67 ng·mL−1. After another wash and aspiration step, anti-rabbit IgG HRP was added to obtain the standard curves for direct ELISA. According to the curves (Figs. 3A-D), when the standard concentration is lower than 20 ng·mL−1, monoclonal antibody 306-4, as the capture antibody at 5 µg·mL−1, has the best linear curve. The results indicated that monoclonal antibody 306-4 was the best capture antibody that can pair with the polyclonal antibody to formulate an ELISA kit for human CD306/ LAIR-2 immunoassay. The human secreted CD306/LAIR-2 immunoassay is a solid phase ELISA designed to measure human secreted CD306/LAIR-2 in human serum. This ELISA kit contains recombinant human CD306/LAIR-2, and monoclonal and polyclonal antibodies that were raised against this protein. It has been shown to accurately quantify recombinant human CD306/LAIR-2. Results obtained with naturally occurring CD306/LAIR-2 samples showed linear curves. These results indicate that the immunoassay kit can be used to determine relative mass values for natural human CD306/LAIR-2. The human secreted CD306/LAIR-2 ELISA assay employs the quantitative sandwich enzyme immunoassay technique. Monoclonal antibody 306-4 specific for human secreted CD306/LAIR-2 was coated onto a microplate at a concentration of 5 µg·mL−1. Standards and samples are pipetted into the wells, and any CD306/LAIR-2 present is bound by the immobilized antibody. Serum samples from twenty healthy subjects were used in this ELISA kit to determine the concentration of human CD306/LAIR-2. After washing away any unbound substances, the polyclonal antibody specific for
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Fig. 3 Different concentrations of monoclonal antibody 306 as the capture antibody, polyclonal antibody (67 ng/mL) as the detection antibody. A: 306-1, B: 306-2, C: 306-3, and D: 306-4
human secreted CD306/LAIR-2 at 67 ng·mL−1 was added to the wells. Following a wash to remove any unbound antibody, anti-rabbit IgG HRP conjugate was added to the wells. After washing away any unbound enzyme, a substrate solution was added to the wells. The color developed in proportion to the amount of human secreted CD306/LAIR-2 bound in the initial step. The color development is stopped and the intensity of the color is measured. To analyze the data, a standard curve must be created. First, the average duplicate readings were taken for each standard, positive control, and samples, and then the average
zero standard optical density was sutracted. Next, the standard curve was created (Fig. 4) by reducing the data using computer software capable of generating a log-log curve fit. The results indicate that the median serum level of secreted CD306/ LAIR-2 is (1.34 ± 0.17) ng·mL−1 (Fig. 5). The concentration of secreted CD306/LAIR-2 in human plasma samples was under the detectable limit of this ELISA kit. The pooled research human serum samples were diluted with PBS containing 1% BSA solution. The recovery and linearity of the samples were measured by Human Secreted CD306/LAIR-2 ELISA Kit. Diluted specimens demonstrated
Fig. 4 Standard curve of human secreted CD306/LAIR-2 sandwich ELISA assay
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Fig. 5 Tested human serum samples using the Human Secreted CD306/LAIR-2 ELISA Kit
excellent linearity and recovery when compared to the neat
concentration of human secreted CD306/LAIR-2. As shown in Table 1, the results indicate that this CD306/LAIR-2 ELISA can accurately determine the concentration of this molecule in serum samples in this assay matrix. The complete sequence of the human CD306/LAIR-2 protein was compared with sequences in the protein identification resource and the Swiss-Protein data bases. No significant homology with other human proteins was found. The specificity of human secreted CD306/LAIR-2 ELISA kit was tested with other highly purified soluble recombinant: human CD305/LAIR-1, CD209, CD320, and CD36. Each analyte was spiked at 1 000 ng·mL−1 in the assay matrix and run as an unknown in the assay. The results indicate that this ELISA kit does not have any cross-reactivity with these soluble CD proteins (Table 2).
Table 1 Linearity and recovery of human secreted CD306/LAIR-2 ELISA kit Sample Type
Dilution Factor
Assayed Levels (ng·mL−1)
Final Levels (ng·mL−1)
Recovery (%)
Human Serum
1X
1.493
1.493
100
Human Serum
2X
0.727
1.454
Human Serum
4X
0.382
1.528
Table 2 Specificity of the human secreted CD306/LAIR-2 ELISA kit
97.4 102
Table 3 Sensitivity test results of human secreted CD306/ LAIR-2 ELISA kit
Protein
Cross-reactivity
Human Secreted CD306/LAIR-2
Yes
Human Soluble CD305/LAIR-1
No
5 000
1.480
0.004
Human Soluble CD209
No
2 500
1.101
0.007
Human Soluble CD320
No
1 250
0.83
0.011
Human Soluble CD36
No
625
0.543
0.012
312.5
0.308
0.002
156.3
0.168
0.001
78.1
0.092
0.005
39.1
0.055
0.010
19.5
0.031
0.008
9.8
0.029
0.004
0
0
0.005
0.024
0.005
Standard concentrations (pg·mL−1)
The sensitivity of the human secreted CD306/LAIR-2 ELISA was determined by testing different concentrations of CD306/LAIR-2 standard 25 times and the lowest standard concentration that is detectable by this kit is around 10 pg·mL−1 (Table 3). The sensitivity of the human secreted CD306/LAIR-2 ELISA was determined by adding two standard derivations to the mean optical density of twenty five replicates of the zero standard and calculated from the standard curve. The results show that this ELISA kit has a sensitivity of 10 pg·mL−1.
Discussion Immune responses are controlled by the opposing actions of activating and inhibitory immune receptors. The inhibitory signal is important in stopping the immune system from overreacting, which can lead to the development of autoimmune disease. Many times, an inhibitory signal is present in an inhibitory receptor, such as CD305/LAIR-1. Researchers have not discovered how this regulation process works; there are two possible ways. One way is to regulate the expression of the ligand to the inhibitory receptors to control the binding
Background
Average O.D. minus background from 25 Tests
Standard deviation
between these two molecules. Another way is to secrete another receptor to competitively bind to the same ligand as the inhibitory receptor. In vivo, collagens are natural ligands for CD305/LAIR-1. The interaction between collagen and CD305/LAIR-1 can inhibit the activation of immune cells. This interaction reveals a novel mechanism of peripheral immune regulation by inhibitory immune receptors binding to extracellular matrix collagens. This interaction can be blocked by CD306/ LAIR-2, a secreted member of the LAIR family. Thus, CD305/LAIR-1 and CD306/LAIR-2 are both involved in the
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immune regulation of the human body. Another important ligand of CD305/LAIR-1 is C1q. C1q-mediated inhibition of monocyte-DC differentiation and C1q-mediated inhibition of IFN-α production by plasmacytoid DCs were both reversed by CD306/LAIR-2 [4]. In some patients with rheumatoid arthritis (RA), there is evidence of higher levels of CD306/LAIR-2 than those of healthy subjects [5]. When damage of the joint capsule happens, the chemokines secreted around there can attract large amounts of CD4+ T cells to the site. CD4+ T cells can secrete large amounts of the CD306/LAIR-2 molecule, which can block the CD305/LAIR-1 molecule from signaling inhibition [6]. The local immune cells will activate to attack the collagen which causes RA [1]. Research also shows that CD306/LAIR-2 can be a biomarker of inflammation [1]. Checking the CD306/LAIR-2 levels of RA patients may help to monitor the development of this disease. In the human body, type I and type III collagen have many binding sites for CD305/LAIR-1 and CD306/ LAIR-2 molecules. Platelet activation by collagen can be inhibited by CD306/LAIR-2/Fc. Thus, CD306/LAIR-2 may be used to treat thromboembolic disease. CD306/LAIR-2 is a human-derived molecule, and compared with non-human antithrombotic therapy, it may have less of an allergic reaction and patients may benefit from the treatment [6]. The CD306/LAIR-2 molecule is also involved in cancer research. Ep-CAM is a ligand of CD305/LAIR-1 and CD306/ LAIR-2. Ep-CAM interacts with the LAIR molecules through its first epidermal growth factor domain [7]. CD305/ LAIR1-Ep-CAM interaction may contribute to mucosal tolerance of cancer, and CD306/LAIR-2 can possibly modulate this function [8]. In order to correctly monitor the level of CD306/LAIR-2 in patients, an effective quantification tool is needed. Differing levels of CD306/LAIR-2 can reveal some important mechanisms of the disease, or can help in the observation of the development of the disease. Nowadays, sandwich ELISAs are a common tool used to detect and quantify antigens of interest. The antigen to be detected does not need to be purified, and most of the time does not even need to be diluted. It is more sensitive and less costly for large sample quantitation. The human secreted CD306/LAIR-2 ELISA assay employs the quantitative sandwich enzyme immunoassay technique with high sensitivity. This kit offers an effective and fast way to quantify any human secreted CD306/LAIR-2 present in
serum samples.
Conclusions The human secreted CD306/LAIR-2 immunoassay is formulated as a solid phase ELISA designed to measure human secreted CD306/LAIR-2 in human serum. It contains recombinant human CD306/LAIR-2, monoclonal and polyclonal antibodies that were raised against this protein. It has been shown to accurately quantify recombinant human CD306/ LAIR-2. The Results obtained with naturally occurring CD306/LAIR-2 samples show linear curves. These results indicate that a human secreted CD306/LAIR-2 immunoassay kit with high specificity can be used to determine relative mass values for natural human CD306/LAIR-2 in serum samples.
References [1]
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[8]
Lebbink RJ, van den Berg MC, de Ruiter T, et al. The soluble leukocyte-associated Ig-like receptor (LAIR)-2 antagonizes the collagen/LAIR-1 inhibitory immune interaction [J]. J Immunol, 2008, 180 (3): 1662-1669. Simone R, Pesce G, Antola P, et al. Serum LAIR-2 is increased in autoimmune thyroid disease [J]. PLoS One, 2013, 8 (5): e63282. Founds SA, Fallert-Junecko B, Reinhart TA, et al. LAIR2 localizes specifically to sites of extravillous trophoblast invasion [J]. Placenta, 2010, 31 (10): 880-885. Son M, Santiago-Schwarz F, Al-Abed Y, et al. C1q limits dendritic cell differentiation and activation by engaging LAIR-1 [J], Proc Natl Acad Sci USA, 2012, 109 (46): E3160-3167. Olde Nordkamp MJ, van Roon JA, Douwes M, et al. Enhanced secretion of leukocyte-associated immunoglobulin-like receptor 2 (LAIR-2) and soluble LAIR-1 in rheumatoid arthritis: LAIR-2 is a more efficient antagonist of the LAIR-1-collagen inhibitory interaction than is soluble LAIR-1 [J]. Arthritis Rheum, 2011, 63 (12): 3749-3757. Lenting PJ, Westerlaken GH，Denis CV, et al. Efficient inhibition of collagen-induced platelet activation and adhesion by LAIR-2, a soluble Ig-like receptor family member [J]. PLoS One, 2010, 5 (8): e12174. Meyaard L, van der Vuurst de Vries AR, de Ruiter T, et al. The epithelial cellular adhesion molecule (Ep-CAM) is a ligand for the leukocyte-associated immunoglobulin-like receptor (LAIR) [J]. J Exp Med, 2001, 194 (1): 107-112. Xie X, Cao YX, Wang W, et al. Preparation and identification of monoclonal antibodies against leukocyte-associated immunoglobulin-like receptor-2 (LAIR-2) and establishment of sandwich ELISA for detecting LAIR-2 [J]. Curr Immunol, 2004, 24 (1): 32-35.
Cite this article as: TAO Yuan, LIN Jun, HU You-Jia, ZHOU Bin, ZHU Bao-Quan. Development of a quantitative ELISA kit for human secreted CD306/LAIR-2 and its application [J]. Chinese Journal of Natural Medicines, 2014, 12 (11): 847-852
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Chinese Journal of Natural Medicines
Development of a quantitative ELISA kit for human secreted CD306/LAIR-2 and its application TAO Yuan, LIN Jun*, HU You-Jia, ZHOU Bin, ZHU Bao-Quan* State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 200040, China Available online 20 Nov. 2014
[ABSTRACT] AIM: A quantitative ELISA kit for the detection of human secreted CD306/LAIR-2 was developed using monoclonal and polyclonal antibodies which were raised against a highly purified recombinant human secreted CD306/LAIR-2. METHODS: Anti-human secreted CD306/LAIR-2 monoclonal and polyclonal antibodies were raised by immunizing mouse or rabbit with recombinant human secreted CD306/LAIR-2. The monoclonal antibody was purified by protein G affinity, whereas the polyclonal antibody was purified by protein A affinity. The best match pair of antibodies were found and used to develop a double antibody sandwich ELISA kit for the detection of human secreted CD306/LAIR-2 in human samples. RESULTS: A human secreted CD306/LAIR-2 ELISA kit was formulated with highly purified recombinant human secreted CD306/LAIR-2, highly specific monoclonal and polyclonal antibodies. This kit realized the quantitative measurement of recombinant human CD306/LAIR-2 and natural CD306/LAIR-2 in human serum samples. CONCLUSIONS: The developed human secreted CD306/LAIR-2 ELISA kit is a reliable quantitation immunoassay kit. [KEY WORDS] Human Secreted CD306/LAIR-2; Monoclonal antibody; Polyclonal antibody; ELISA kit
[CLC Number] R965
[Document code] A
[Article ID] 2095-6975(2014)11-0847-06
Introduction Human CD306, also known as leukocyte-associated Ig-like receptor-2 (LAIR-2), is a newly found cluster of differentiation (CD) molecules. It belongs to the Ig super family [1]. CD306/LAIR-2 gene maps to a region of 19q13.4. The protein encoded by this gene has 135 amino acids. As a secreted protein, human CD306/LAIR-2 has three subtypes. Until now, researchers have only found the CD306/LAIR-2 molecule in humans. There is no report on mouse CD306/LAIR-2 yet. The CD306/LAIR-2 molecule is secreted mainly by CD4+T [Received on] 16-Dec.-2013 [Research funding] This project was supported by the National Important Special Foundation of the New Drug Development (No. 2010ZX 09401-403) and the Shanghai Industry-University-ResearchMedical Cooperation Project (No. 12DZ1931903). [*Corresponding author] LIN Jun: Dr., Tel/Fax: +86-21- 6247 9808, E-mail: [email protected]. ZHU Bao-Quan: Prof., Tel/Fax: +86-216279 1661, E-mail: [email protected]. These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
cells in circulating blood, but there could also be other cells or tissues that secrete the CD306/LAIR-2 molecule [1]. The human LAIR family has two members: CD305/LAIR-1 and CD306/LAIR-2. These proteins are encoded by two genes and have about 84% sequence homology and similar structures. Both of their genes map to 19q13.4, but are transcribed for different purposes. CD306/LAIR-2 is a secreted protein, whereas CD305/LAIR-1 is a transmembrane protein [1]. The CD305/LAIR-1 molecule is an inhibitory immune receptor in humans. It can provide inhibitory signals to immune cells, keeping the immune system in a balanced condition. Collagen is the ligand of both CD305/LAIR-1 and CD306/LAIR-2 molecules, but CD306/LAIR-2 has a higher affinity for the ligand, which prevents the CD305/LAIR-1 molecule from providing the inhibitory signals to the body [1]. When the human body can no longer keep the balance of the immune system, autoimmune diseases and other diseases may occur [2]. Researchers have also found that in some patients, there are higher levels of the CD306/LAIR-2 molecule in the blood or tissue than those of healthy individuals. CD306/LAIR-2 gene expression and immunohistochemical tests have shown down-regulated levels in the first
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TAO Yuan, et al. / Chin J Nat Med, 2014, 12(11): 847852
trimester of pre-eclampsia pregnancies [3]. This may indicate that CD306/LAIR-2 is involved in the development of pre-eclampsia pregnancies. Hence, the quantification and dynamic monitoring of CD306/ LAIR-2 levels in the human body are important to research on the mechanisms of inflammation, allergic reaction, pre-eclampsia pregnancies, and leukemia, etc. Due to the importance of this molecule we have developed an ELISA kit that could provide quantitative determination of secreted human CD306/LAIR-2.
Materials and Methods Production and purification of antigen and standard protein A DNA sequence encoding the mature form of human CD306/LAIR-2 (Q22-P152) with 6 His-tag on the N-terminus was expressed in E. coli, and then the recombinant protein was purified by Ni-NTA column (Aviscera Bioscience, Santa Clara,USA). Dialysis of the purified protein was performed against PBS and exchanged ten times at 4 °C. The purity was determined by SDS-PAGE gel staining. The protein concentration was measured by two different methods: A280 nm and Bradford assay (Aviscera Bioscience, Santa Clara,USA). Immunization and production of anti-human CD306/LAIR-2 monoclonal antibodies Two BALB/c mice aged 6–8 weeks were immunized with intraperitoneal injections of human recombinant CD306/LAIR-2 protein (100 μg) together with Freund’s adjuvant, over a period of six weeks, at one-week intervals. Afterwards, there was a fusion of spleen cells from an immunized mouse with mouse myeloma cells. The positive cells were cloned by the limiting dilution technique and expanded intraperitoneally in BALB/c mice. Four strains of monoclonal antibodies were named 306-1, 306-2, 306-3, and 306-4. The monoclonal antibodies from the cell cultures were purified using protein G columns and dialysis against PBS. The antibody concentration was measured by two different methods: A280 nm and Bradford assay (Aviscera Bioscience, Santa Clara,USA). Immunization and production of anti-human CD306/LAIR-2 polyclonal antibodies One New Zealand rabbit aged twelve weeks was immunized with a muscle injection of human recombinant CD306/LAIR-2 protein (100 μg ) together with Frend’s adjuvant, over a period of seven weeks, at one-week intervals. Blood was collected seven days following a booster injection and centrifuged to obtain the serum. All of the collected serum was mixed and kept at –70 °C. The rabbit anti-human CD306/LAIR-2 IgG was purified by protein A affinity column (Aviscera Bioscience, Santa Clara,USA), and dialysis against PBS. The antibody concentration was measured by two different methods: A280 nm and the Bradford assay (Aviscera Bioscience, Santa Clara,USA). Antibody titration by indirect ELISA A sample (100 ng per well) of human CD306/LAIR-2 protein in PBS (pH 7.4) was coated on a 96-well microplate and incubated at 4 °C overnight. The liquid in the wells was discarded the following day. The microplate was washed with PBST buffer (with 1% BSA) to remove all of the protein that
did not bind. The microplate was blocked with PBS buffer (with 2% BSA) and incubated at room temperature for 2 h. The blocking buffer was discarded and the microplate was allowed to dry. The microplate was now ready for the addition of antibodies. A two-fold serial dilution was made for each of the monoclonal and polyclonal antibodies. The starting concentration of monoclonal antibodies was 5 μg·mL−1, and the starting concentration of the polyclonal antibody was 2 μg·mL−1. For each dilution, 100 µL per well were added to the microplate that was coated with human CD306/LAIR-2 protein. The microplate was incubated for 2 h at room temperature on a microplate shaker at 300 r·min−1. Next, the microplate was washed with PBST buffer (with 1% BSA), followed by aspiration of all the liquid. Then, goat anti-mouse IgG HRP conjugate was added to detect monoclonal antibodies, and goat anti-rabbit IgG HRP conjugate was added to detect polyclonal antibodies. The microplate was incubated at room temperate on the microplate shaker for 1 h. Another washing and aspiration step was done before the addition of TMB substrate solution. The reaction was stopped with 2 mol·L−1 HCl, and the microplate was read in a microplate reader at 450 nm. Formulation of human secreted CD306/LAIR-2 ELISA kit According to the results of the titer from the indirect ELISA, one monoclonal antibody was chosen to coat the plate. A 96-well microplate was coated with the purified mouse anti-human CD306/LAIR-2 monoclonal antibody 306-4 at 5 g·mL−1 in PBS (pH 7.4). The plates were incubated overnight at 4 °C. Then, the plates were washed with PBST buffer with 1% BSA and aspirated. The plates were blocked with PBS buffer with 2% BSA, and were incubated for 2 h at room temperature before discarding the liquid and allowing the plates to dry. The highly purified recombinant human CD306/LAIR-2 was used as a standard. A solution of 20 ng·mL−1 of human CD306/LAIR-2 protein in PBS buffer with 1% BSA was prepared as the highest standard concentration. A seven point standard curve was made using two-fold serial dilution with 20 ng·mL−1 as the highest standard. PBS containing 1% BSA without any standard protein was used as the negative control. The plate was incubated on the microplate shaker for 2 h at room temperature, and then washed and aspirated. Next, purified rabbit anti human CD306/LAIR-2 IgG (100 µL) was added as detection antibody. Again, the plate was incubated on the microplate shaker for 2 h at room temperature with another wash and aspiration step. Finally, goat anti-rabbit IgG HRP conjugate was added to the wells, and the plate was incubated on a microplate shaker at room temperature for 1 h in the absence of light. After the final wash and aspiration step, TMB substrate solution was added to all of the wells for the color to develop. The reaction was stopped with 2 mol·L−1 HCl, and the absorbance of the plate was read on a microplate reader at 450 nm. The purified normal rabbit IgG without immunization of human CD306/LAIR-2 was used as the as
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say antibody negative control to replace the primary antibody.
Results In a previous study from this laboratory, the recombinant human mature form CD306/LAIR-2 His-tag was electrophoresed on 15% SDS-PAGE gel under reducing conditions. The purity of this human CD306/LAIR-2 recombinant was more than 95% by gel staining. The MW band is ~16 KDa on gel staining. Two BALB/c mice were immunized with intraperitoneal injections of 100 μg of human recombinant CD306/LAIR-2 protein together with Freund’s adjuvant. Afterwards, there was a fusion of spleen cells from the immunized mouse with mouse myeloma cells. As a result, four purified anti-human CD306/LAIR-2 monoclonal antibodies (306-1, 306-2, 306-3, and 306-4) on indirect ELISA indicated specific immunobinding. According to the curves (Fig. 1), the binding of monoclonal antibodies 306-1 and 306-2 with human CD306/LAIR-2 protein is saturated at high concentration. Both antibodies also show a slow decrease in binding in the protein’s lower concentration range. The binding of monoclonal antibodies 306-3 and 306-4 has a better linear rate.
Fig. 1 Mouse anti-human CD306/LAIR-2 monoclonal antibody titer
One New Zealand rabbit was immunized with muscle injection of human recombinant CD306/LAIR-2 protein (100 μg) together with Freund’s adjuvant. Blood was collected seven days following a booster injection and centrifuged to obtain the serum. As shown in Fig. 2, the binding of the polyclonal antibody with human CD306/LAIR-2 protein was saturated at higher concentrations (> 0.25 µg·mL−1). However, the specific binding of rabbit anti human CD306/LAIR-2 antibody with human CD306/LAIR-2 protein indicated a linear relationship at lower concentrations (< 0.125 µg·mL−1). The 67 ng·mL−1 of the purified rabbit anti-human CD306/LAIR-2 IgG was located in the linear binding range, and was used as the detection antibody concentration for direct ELISA formulation. Paired antibody screening Each of the purified antibodies of mouse anti-human
Fig. 2 Rabbit anti-human CD306/LAIR-2 polyclonal antibody titer
CD306/LAIR-2 monoclonal was coated onto a 96-well microplate at different concentrations: 10, 5, and 2.5 µg·mL−1. After washing the plate to remove any unbound captured antibody and blocking it with PBS buffer with 2% BSA, a ten-fold serial dilution of human CD306/LAIR-2 recombinant starting from 10 000 to 0.1 ng·mL−1 was applied into each well and incubated for 2 h. After a wash and aspiration step, detection antibody was added at a concentration of 67 ng·mL−1. After another wash and aspiration step, anti-rabbit IgG HRP was added to obtain the standard curves for direct ELISA. According to the curves (Figs. 3A-D), when the standard concentration is lower than 20 ng·mL−1, monoclonal antibody 306-4, as the capture antibody at 5 µg·mL−1, has the best linear curve. The results indicated that monoclonal antibody 306-4 was the best capture antibody that can pair with the polyclonal antibody to formulate an ELISA kit for human CD306/ LAIR-2 immunoassay. The human secreted CD306/LAIR-2 immunoassay is a solid phase ELISA designed to measure human secreted CD306/LAIR-2 in human serum. This ELISA kit contains recombinant human CD306/LAIR-2, and monoclonal and polyclonal antibodies that were raised against this protein. It has been shown to accurately quantify recombinant human CD306/LAIR-2. Results obtained with naturally occurring CD306/LAIR-2 samples showed linear curves. These results indicate that the immunoassay kit can be used to determine relative mass values for natural human CD306/LAIR-2. The human secreted CD306/LAIR-2 ELISA assay employs the quantitative sandwich enzyme immunoassay technique. Monoclonal antibody 306-4 specific for human secreted CD306/LAIR-2 was coated onto a microplate at a concentration of 5 µg·mL−1. Standards and samples are pipetted into the wells, and any CD306/LAIR-2 present is bound by the immobilized antibody. Serum samples from twenty healthy subjects were used in this ELISA kit to determine the concentration of human CD306/LAIR-2. After washing away any unbound substances, the polyclonal antibody specific for
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Fig. 3 Different concentrations of monoclonal antibody 306 as the capture antibody, polyclonal antibody (67 ng/mL) as the detection antibody. A: 306-1, B: 306-2, C: 306-3, and D: 306-4
human secreted CD306/LAIR-2 at 67 ng·mL−1 was added to the wells. Following a wash to remove any unbound antibody, anti-rabbit IgG HRP conjugate was added to the wells. After washing away any unbound enzyme, a substrate solution was added to the wells. The color developed in proportion to the amount of human secreted CD306/LAIR-2 bound in the initial step. The color development is stopped and the intensity of the color is measured. To analyze the data, a standard curve must be created. First, the average duplicate readings were taken for each standard, positive control, and samples, and then the average
zero standard optical density was sutracted. Next, the standard curve was created (Fig. 4) by reducing the data using computer software capable of generating a log-log curve fit. The results indicate that the median serum level of secreted CD306/ LAIR-2 is (1.34 ± 0.17) ng·mL−1 (Fig. 5). The concentration of secreted CD306/LAIR-2 in human plasma samples was under the detectable limit of this ELISA kit. The pooled research human serum samples were diluted with PBS containing 1% BSA solution. The recovery and linearity of the samples were measured by Human Secreted CD306/LAIR-2 ELISA Kit. Diluted specimens demonstrated
Fig. 4 Standard curve of human secreted CD306/LAIR-2 sandwich ELISA assay
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Fig. 5 Tested human serum samples using the Human Secreted CD306/LAIR-2 ELISA Kit
excellent linearity and recovery when compared to the neat
concentration of human secreted CD306/LAIR-2. As shown in Table 1, the results indicate that this CD306/LAIR-2 ELISA can accurately determine the concentration of this molecule in serum samples in this assay matrix. The complete sequence of the human CD306/LAIR-2 protein was compared with sequences in the protein identification resource and the Swiss-Protein data bases. No significant homology with other human proteins was found. The specificity of human secreted CD306/LAIR-2 ELISA kit was tested with other highly purified soluble recombinant: human CD305/LAIR-1, CD209, CD320, and CD36. Each analyte was spiked at 1 000 ng·mL−1 in the assay matrix and run as an unknown in the assay. The results indicate that this ELISA kit does not have any cross-reactivity with these soluble CD proteins (Table 2).
Table 1 Linearity and recovery of human secreted CD306/LAIR-2 ELISA kit Sample Type
Dilution Factor
Assayed Levels (ng·mL−1)
Final Levels (ng·mL−1)
Recovery (%)
Human Serum
1X
1.493
1.493
100
Human Serum
2X
0.727
1.454
Human Serum
4X
0.382
1.528
Table 2 Specificity of the human secreted CD306/LAIR-2 ELISA kit
97.4 102
Table 3 Sensitivity test results of human secreted CD306/ LAIR-2 ELISA kit
Protein
Cross-reactivity
Human Secreted CD306/LAIR-2
Yes
Human Soluble CD305/LAIR-1
No
5 000
1.480
0.004
Human Soluble CD209
No
2 500
1.101
0.007
Human Soluble CD320
No
1 250
0.83
0.011
Human Soluble CD36
No
625
0.543
0.012
312.5
0.308
0.002
156.3
0.168
0.001
78.1
0.092
0.005
39.1
0.055
0.010
19.5
0.031
0.008
9.8
0.029
0.004
0
0
0.005
0.024
0.005
Standard concentrations (pg·mL−1)
The sensitivity of the human secreted CD306/LAIR-2 ELISA was determined by testing different concentrations of CD306/LAIR-2 standard 25 times and the lowest standard concentration that is detectable by this kit is around 10 pg·mL−1 (Table 3). The sensitivity of the human secreted CD306/LAIR-2 ELISA was determined by adding two standard derivations to the mean optical density of twenty five replicates of the zero standard and calculated from the standard curve. The results show that this ELISA kit has a sensitivity of 10 pg·mL−1.
Discussion Immune responses are controlled by the opposing actions of activating and inhibitory immune receptors. The inhibitory signal is important in stopping the immune system from overreacting, which can lead to the development of autoimmune disease. Many times, an inhibitory signal is present in an inhibitory receptor, such as CD305/LAIR-1. Researchers have not discovered how this regulation process works; there are two possible ways. One way is to regulate the expression of the ligand to the inhibitory receptors to control the binding
Background
Average O.D. minus background from 25 Tests
Standard deviation
between these two molecules. Another way is to secrete another receptor to competitively bind to the same ligand as the inhibitory receptor. In vivo, collagens are natural ligands for CD305/LAIR-1. The interaction between collagen and CD305/LAIR-1 can inhibit the activation of immune cells. This interaction reveals a novel mechanism of peripheral immune regulation by inhibitory immune receptors binding to extracellular matrix collagens. This interaction can be blocked by CD306/ LAIR-2, a secreted member of the LAIR family. Thus, CD305/LAIR-1 and CD306/LAIR-2 are both involved in the
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immune regulation of the human body. Another important ligand of CD305/LAIR-1 is C1q. C1q-mediated inhibition of monocyte-DC differentiation and C1q-mediated inhibition of IFN-α production by plasmacytoid DCs were both reversed by CD306/LAIR-2 [4]. In some patients with rheumatoid arthritis (RA), there is evidence of higher levels of CD306/LAIR-2 than those of healthy subjects [5]. When damage of the joint capsule happens, the chemokines secreted around there can attract large amounts of CD4+ T cells to the site. CD4+ T cells can secrete large amounts of the CD306/LAIR-2 molecule, which can block the CD305/LAIR-1 molecule from signaling inhibition [6]. The local immune cells will activate to attack the collagen which causes RA [1]. Research also shows that CD306/LAIR-2 can be a biomarker of inflammation [1]. Checking the CD306/LAIR-2 levels of RA patients may help to monitor the development of this disease. In the human body, type I and type III collagen have many binding sites for CD305/LAIR-1 and CD306/ LAIR-2 molecules. Platelet activation by collagen can be inhibited by CD306/LAIR-2/Fc. Thus, CD306/LAIR-2 may be used to treat thromboembolic disease. CD306/LAIR-2 is a human-derived molecule, and compared with non-human antithrombotic therapy, it may have less of an allergic reaction and patients may benefit from the treatment [6]. The CD306/LAIR-2 molecule is also involved in cancer research. Ep-CAM is a ligand of CD305/LAIR-1 and CD306/ LAIR-2. Ep-CAM interacts with the LAIR molecules through its first epidermal growth factor domain [7]. CD305/ LAIR1-Ep-CAM interaction may contribute to mucosal tolerance of cancer, and CD306/LAIR-2 can possibly modulate this function [8]. In order to correctly monitor the level of CD306/LAIR-2 in patients, an effective quantification tool is needed. Differing levels of CD306/LAIR-2 can reveal some important mechanisms of the disease, or can help in the observation of the development of the disease. Nowadays, sandwich ELISAs are a common tool used to detect and quantify antigens of interest. The antigen to be detected does not need to be purified, and most of the time does not even need to be diluted. It is more sensitive and less costly for large sample quantitation. The human secreted CD306/LAIR-2 ELISA assay employs the quantitative sandwich enzyme immunoassay technique with high sensitivity. This kit offers an effective and fast way to quantify any human secreted CD306/LAIR-2 present in
serum samples.
Conclusions The human secreted CD306/LAIR-2 immunoassay is formulated as a solid phase ELISA designed to measure human secreted CD306/LAIR-2 in human serum. It contains recombinant human CD306/LAIR-2, monoclonal and polyclonal antibodies that were raised against this protein. It has been shown to accurately quantify recombinant human CD306/ LAIR-2. The Results obtained with naturally occurring CD306/LAIR-2 samples show linear curves. These results indicate that a human secreted CD306/LAIR-2 immunoassay kit with high specificity can be used to determine relative mass values for natural human CD306/LAIR-2 in serum samples.
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Cite this article as: TAO Yuan, LIN Jun, HU You-Jia, ZHOU Bin, ZHU Bao-Quan. Development of a quantitative ELISA kit for human secreted CD306/LAIR-2 and its application [J]. Chinese Journal of Natural Medicines, 2014, 12 (11): 847-852
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