Ma’arif B, Abada I, Mahardiani A, Hakim A, Maulina N, Purwitasari N, Hidayah K, Jing S. L. A Systematic Review: Comparison of Immunocytochemistry, ELISA, and Western Blot Methods in Alkaline phosphatase Measurement at Genistein-induced Osteoblast Cell. Biomed Pharmacol J 2022;15(4).

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Manuscript received on :04-10-2022
Manuscript accepted on :23-11-2022
Published online on: 07-12-2022

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Plagiarism Check: Yes
Reviewed by: Dr. Yogendra Singh
Second Review by: Dr. Kartik Salwe
Final Approval by: Dr. Patorn Promchai

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Burhan Ma’arif¹ , Iffatul Abada¹, Anisah Mahardiani¹, Abdul Hakim^1*, Novia Maulina¹, Neny Purwitasari², Khoirul Hidayah³ and Seow Lay Jing⁴

¹Department of Pharmacy, Faculty of Medical and Health Science, Maulana Malik Ibrahim State Islamic University, Malang, Indonesia

²Department of Pharmaceutical Science, Faculty of Pharmacy, Universitas Airlangga, Surabaya, Indonesia

³Faculty of Sharia, Maulana Malik Ibrahim State Islamic University, Malang, Indonesia

⁴Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Health Sciences, Universiti Kuala Lumpur Royal College of Medicine, Perak, Malaysia

Corresponding Author E-mail: ahakim@farmasi.uin-malang.ac.id

DOI : https://dx.doi.org/10.13005/bpj/2523

Abstract

Osteoporosis is a bone disorder characterized by the decrease of bone mass along with bone micro-architecture damage and has a risk become a fracture. One of the causes of osteoporosis is estrogen deficiency. Genistein is a phytoestrogen compound in the isoflavone group containing a similar structure compared to 17β-estradiol, thus it can bind to estrogen receptors and produce an estrogenic effect. Genistein induction can stimulate bone formation and promote the increase of alkaline phosphate (ALP) activities in osteoblast cells which can be observed by immunocytochemistry or Enzyme-linked Immunosorbent Assay (ELISA) or Western blot method. Using the PRISMA guideline technique, choose and strategize article searches by reading the title, abstract, and then the whole text of the article. Articles with the keywords "genistein or osteoblast cells or alkaline phosphate or immunocytochemistry or immunofluorescence or ELISA or western blot" were retrieved from databases including Google Scholar, PubMed, Researchgate, and Sciencedirect. 24 relevant research articles were uncovered as a result of this systematic review. Comparison of immunocytochemistry and ELISA methods in order to analyze the activities of ALP in osteoblast induced by genistein includes selectivity, sensitivity, processing time, and cost efficiency parameters. The immunocytochemistry method has a higher level of sensitivity and a faster processing time, whereas the ELISA method has a higher level of selectivity and less cost efficiency. The western blot method has selectivity for detecting complex-level protein expression.

Keywords

Cost Efficiency; ELISA; Immunocytochemistry; Selectivity; Sensitivity, Processing Time; Western Blot

Introduction

Osteoporosis prevalence increases with increasing age in women. Over 30% of women aged 60-70 years suffer from osteoporosis, this data can increase to 70% at 80 years.¹ Elderly woman will face the postmenopausal phase due to estrogen deficiency, which is one of the factors causing osteoporosis.^2-5 Osteoporosis is a bone disorder characterized by a decrease in bone mass and bone micro-architecture damage, leading to an increased risk of fractures.⁶

Treating estrogen deficiency in postmenopausal women can be treated using hormone replacement therapy (HRT).⁷ Long-term use of HRT can cause side effects such as endometrium cancer, breast cancer, and stroke.^8,9 Research on the use of natural ingredients to replace estrogen function with minimal side effects has been done and the results have led to the use of phytoestrogen compound.^10-12

Genistein is a phytoestrogen compound in the isoflavone group, it has estrogenic activities because of the presence of phenolic rings that bind to estrogen receptors to treat target cells.^13,14 In vitro, genistein can suppress osteoclast function and bone resorption, furthermore, induce the apoptosis of mature osteoclast cells. In vivo, genistein can prevent trabecular bone loss in the male rats’ model.^15-19 In silico, a proof that genistein has an active compound identity range like 17β-estradiol. ^20,21

Administration of genistein causes a direct effect on the growth and the differentiation of osteoblast and expresses a protein marker for bone formation, one of them is ALP. ^2,22-24 Immunocytochemistry and enzyme-linked immunosorbent assay (ELISA) methods can do the measurement of ALP expression.^25-28 These methods have their advantages, several parameters to analyze both methods are selectivity, sensitivity, processing time, and cost efficiency parameters.

Important parameters to be used in analyzing an observational method, when doing observation must pay attention to how much the ability of a method to recognize and measure a protein in a sample affects the success of a study. Also, short observational duration and less cost efficiency are parameters that must be considered in research, thus producing effective and efficient research. This systematic review is expected to summarize the decision of the selection of an appropriate method for in vitro observation, especially to observe bone formation activity in vitro.

Materials and Methods

Materials

Criteria for collecting data

The inclusion criteria in this review article were (i) studies related to the effect of genistein on increasing ALP expression in osteoblast with immunocytochemistry or ELISA or western blot method; (ii) studies related to genistein compound; (iii) English articles; (iv) full text and open access articles that published in 2010-2020.

The exclusion criteria in this review article were (i) studies related to other than the effect of genistein on increasing ALP expression in osteoblast cells with immunocytochemistry or ELISA or western blot method; (ii) studies related to other than genistein; (iii) articles in foreign languages other than English; (iv) articles were not full text and open access that published before 2011.

Strategy for article selection and search

The data used in the systematic review was primer data by collecting published articles from Google Scholar, PubMed, Researchgate, and Sciencedirect databases with keywords “genistein or osteoblasts or alkaline phosphatase or immunocytochemistry or immunofluorescence or ELISA or western blot.” Afterward, those articles were screened and selected based on their title and abstract. Then, a feasibility test was conducted by reading the entire article according to inclusion and exclusion criteria.

Methods 

The method used in the systematic review uses the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline.^29,30

Data analysis 

Data analysis according to PRISMA guideline, which describes the results of research articles and provides exposure to relevant results in the form of article summary tables.

Results

Strategy for article selection and search

After selecting a specific and systematic article search strategy using the PRISMA guideline method with four databases, namely Google Scholar, PubMed, Researchgate, and Sciencedirect, 24 research articles were relevant to the topic of discussion, including 8 research articles from GoogleScholar, 7 from PubMed, 4 from ResearchGate, and 5 from ScienceDirect (Figure 1). Then every research article found was published in less than the last 10 years (2010-2020) (Figure 2).

Figure 1: PRISMA Guideline diagram Click here to view figure

Figure 2: The year of publication of the article found Click here to view figure

In Vitro Analysis

The marker factor observed in this literature study was ALP. ALP protein is produced in the early stage of osteoblast differentiation and is one of the specific marker factors, also plays an important role in bone formation and mineralization.^18,29-33 Type of osteoblast cell can be primary cell cultures from cells, tissues, and organs obtained directly from its origin organism, however, cell lines culture is obtained from the first subculture of primary culture. Primary cell culture refers to the culture starting primary cell culture has several weaknesses, for instance, needs an experimental animal as a raw material for culture and it most likely occurs virus or microbe contamination. This contamination can infect experimental animals used as culture stocks.³⁴ The increase of ALP activity as a marker of bone formation can occur through various types of osteoblast cells obtained from primary culture or cell line with the process of bone formation around >10% to >50%.³⁵

The process of osteoblast cell differentiation can be divided into several stages, namely proliferation, matrix maturation (early differentiation), and mineralization (late differentiation). The main marker protein in osteoblast cells differentiation is ALP, collagen type 1, osteopontin (OPN), and osteocalcin (OSC), with runt-related transcription factor 2 (Runx2) and osterix (OSX) as transcription factors.^18,36-39 Besides, differentiated osteoblasts will express receptor activators of nuclear factor kβ-ligand (RANKL) and osteoprotegerin (OPG). RANKL and OPG can be used as markers to observe osteoclast cell function.³⁷

Measurement of ALP marker factor shows osteoblastic activity from genistein compound administration. Genistein is an isoflavone group and has structural similarities with 17β-estradiol (Figure 3). Genistein can bind to ER, thus it shows as a phytoestrogen that produces the estrogenic effect.^18,37-43 Administration of genistein causes a direct effect on the growth and differentiation of osteoblast cells and the release a protein.^18,31,41,43 Therefore, increasing the production of mRNA from several markers of osteoblast cell differentiation, namely ALP.^36,44

Figure 3: Structure of (A) Genistein; (B) 17β-estradiol (pubchem.ncbi.nlm.nih.gov). Click here to view figure

Genistein can affect several molecular pathways that stimulate bone formation and inhibit bone resorption. Administration of genistein can increase the production of ALP, OPN, and OSC and activate the Runx2 transcription factor, hence causing osteogenesis. Genistein can stimulate the occurrence of proliferation and differentiation through nitric oxide (NO) or cyclic guanosine monophosphate (cGMP) pathway and can inhibit osteoclastic activity. Also, genistein can affect the nuclear factor kappa–light–chain-enhancer of activated B cells (NF-ĸB) pathway, it stimulates the occurrence of osteoclastogenesis through RANKL and RANK bonds. RANKL is expressed by osteoblast cell surface and RANK is expressed by osteoclast cell surface.⁴⁵ Osteoclastogenesis can be inhibited by OPG, as a result, RANKL binds to OPG and it will maintain the balance of bone formation and bone absorption in the process of bone remodeling. RANKL is a cytokine and plays a role in recruitment, differentiation, and osteoclast cell activation, meanwhile, OPG is a cytokine and plays a role in the inhibition of osteoclastogenesis through its bonds to RANKL.^{15,31,36,37,46,47}

Genistein compound induced in osteoblast cells will enter the cell membrane and then translocate into the nucleus and binds to ER or activated estrogen receptor (ER*). The bond between genistein with ER will cause transcription by transcription factor Runx2 and encourages osteogenesis through osteoblast cell differentiation and produce ALP.^{2,3,38,44,48,49} Measurement of ALP by induction of genistein compound in osteoblast cells was performed by immunocytochemistry or ELISA method.

Characteristics regarding the potential genistein increasing or decreasing ALP activities in various types of osteoblasts by measuring immunocytochemistry or ELISA methods and the mechanism of osteogenesis and osteoclastogenesis was summarized in (Table 1 and Figure 4).

Table 1: Result of Genistein-induced.

Figure 4: Mechanism osteogenesis and osteoclastogenesis after genistein induced osteoblast cell. Click here to view figure

Comparative Analysis Method

The most used method in ALP analysis as a bone formation biomarker is immunocytochemistry and ELISA methods. The immunocytochemistry method is a common method used in laboratories (in vitro) to visualize the location or place of a specific protein or antigen in the cells using primary antibodies which specifically bind to these proteins or antigens. Primary antigens allow visualization of protein under fluorescence microscopy after extending binding with secondary antibodies that have conjugated with fluorophore groups.²⁴ The ELISA method is a serological method based on the specific reaction between antigen and antibody. The method has high selectivity and sensitivity owing to the change of substrate color (colorimetric) as an indicator for enzyme measurement.^52-54

Immunocytochemistry Method

The immunocytochemistry method is divided into 2 types, direct immunocytochemistry, and indirect immunocytochemistry⁵⁵ (Figure 5).  Direct immunocytochemistry using primary antibody only, this antibody acts as specific antibodies that have conjugated with the fluorophore group and localize the antigen precisely and specifically, therefore it can be observed on fluorescence microscopy. While indirect immunocytochemistry uses primary and secondary antibodies, the secondary antibody acts as specific antibodies that have conjugated with fluorophore groups and localize antigens precisely and specifically, as a result, it can be observed under fluorescence microscopy.^56,57

Figure 5: Immunocytochemistry Method. Click here to view figure

ELISA Method

There are four types of ELISA methods, for instance, direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA (Figure 6). Direct ELISA using a conjugated primary antibody, can bind directly to the antigen. Indirect ELISA uses a primary antibody that binds to the antigen and then ca conjugated secondary antibody is added. Sandwich ELISA using a suitable pair of antibodies, each specific antibody will bind to a different epitope of the antigen. The first antibody is known as the trapping antibody and the second antibody is known as the detection antibody. Meanwhile, competitive ELISA occurs through a competitive process among antigens in the microplate. After the antibodies are incubated in the sample, next, it is inserted into a micro-pcontainingtains the antigens and then rinsed to remove specific antibodies that do not bind to the antigen. The more antigens in the sample, the more antigens, and antibodies bound are formed, thus fewer antibodies that do not bound will cause those antibodies to bind to the antigens in the micro-plate.^58,59

Figure 6: ELISA Method. Click here to view figure

Western Blot Method

Western blot or immunoblotting is a qualitative technique used to identify specific antibodies from complex protein samples with a certain molecular weight that have been separated. This method can show proteins by reacting antibodies and antigens that are used to detect cross-reactions (Figure 7).⁶⁰ Western blot is very effective in detecting antigens that have small sizes in solutions that contain a lot of protein. The stages begin with sample preparation (protein purification), measurement of the concentration and amount of protein used, separation of proteins by polyacrylamide gel electrophoresis, transfer of proteins from gel to a nitrocellulose membrane, membrane blocking, incubation of primary and secondary antibodies, detection of target proteins, and calculation of protein bands. Even though the working technique is simple, specific, and precise in implementation, sometimes there are difficulties with unclear, non-specific, and bubble protein bands.

Discussion

Parameter Analysis

Parameters used in the analysis of Immunocytochemistry, ELISA, and Western blot methods are selectivity, sensitivity, processing time, and cost efficiency parameters. The following is an explanation of each parameter and a summary can be seen in (Table 2).

Table 2: Parameter Analysis Results

Description: sign “✓” is a method that has advantages according to each parameter

Selectivity

The selectivity parameter is used to describe the ability of a method to observe protein in the complex sample accurately.⁵⁸ ELISA method a has higher selectivity than immunocytochemistry because the antigen can be detected by using antibodies marked with an enzyme.^59,66 In addition, the western blot method has selectivity in detecting targets at endogenous levels in complex samples and can optimize antibody and assay performance.⁶⁴  The results in detecting antigens and antibodies bound to detect autoimmune disorders in humans like a pemphigus foliaceous, bullous pemphigoid, dermatitis herpetiformis, myositis, autoimmune hepatitis, etc, when most studies have used Hep2 cells from Ali et al., 2016, Karumanchi and Oommen, 2018, and Menezes et al., 2020 were performed by immunocytochemistry/immunofluorescence and ELISA methods.^67,73-74 Result of selectivity analysis in immunocytochemistry/immunofluorescence and ELISA methods used kappa analysis and cohen’s kappa coefficient. Showed ELISA method had a higher selectivity around 84-99%, whereas the immunocytochemistry/immunofluorescence method about 40-95%.

Sensitivity

The sensitivity parameter is used to describe the ability of a method to measure a protein in the cells that can be affected by changing situations.⁶⁵ The immunocytochemistry method has a higher sensitivity than the ELISA method because the observation is performed by using fluorescence microscopy which can detect an extremely sensitive antigen.^59,66 The results in the detection of antigen and antibody bound were formed to detect autoimmune disorders in humans like a pemphigus foliaceous, bullous pemphigoid, dermatitis herpetiformis, myositis, autoimmune hepatitis, etc when most studies have used Hep2 cells from Ali et al., 2016, Karumanchi and Oommen, 2018, and Menezes et al., 2020 were performed by immunocytochemistry/immunofluorescence and ELISA methods.^67,73-74 Result of sensitivity analysis in immunocytochemistry/immunofluorescence and ELISA methods used kappa analysis and cohen’s kappa coefficient. It was shown that the immunocytochemistry/immunofluorescence method had a higher sensitivity of about 50-100%, while the ELISA method was only about 30-97%.

Then, from several studies, the western blot method is ideal to be combined with the ELISA or ICC methods. In the study of Marycz et al. (2021), Western blot was used in observing to detect protein level expression (increased expression of RUNX-2) protein and ICC identified osteopontin (OPN) and fluorescence imaging (reduced OPN expression) with p<0.05.⁶⁸ The same thing was also done in the study of Donoso et al. (2014), using mesenchymal stem cells (MSCs). The western blot method was used to detect protein content such as transcription factors PPARγ, p‐PPARγ, Runx2, bone morphogenetic receptor IA, and the ICC method for cell localization. derived from pSmad1/5/8.⁶⁹ Then in the research of Wang et al. (2017), the ELISA method was used in the analysis of alkaline phosphatase (ALP) and osteocalcin (OC) as well as the western blot method to see protein expressions such as Sirtuin 1 (SIRT1), nuclear factor (NF)-κB and NF-B inhibitor (IkB) with p< 0.01 70.

Processing Time

The processing time parameter describes how long it takes to process the ALP observation in each method. The results in the detection of antigen-antibody bound were formed to detect autoimmune disorders in humans like a pemphigus foliaceous, bullous pemphigoid, dermatitis herpetiformis from Ali et al., 2016 and Tayde et al., 2018.^67,76 The research was performed by using immunocytochemistry/immunofluorescence and ELISA methods, explained that the immunocytochemistry/immunofluorescence method could be run in faster processing time than the ELISA method. The western blot method has a long time starting from preparation to protein calculation, which takes approximately 45 minutes for pretreatment and 1 hour for incubation time.^62,72

Cost Efficiency

The cost-efficiency parameter describes the amount of cost used in the observation of each method. Observation using the immunocytochemistry method or fluorescence is more expensive than the ELISA method. It is because in immunocytochemistry method requires fluorescence microscopy that can observe up to the molecular level.⁷⁷ In the western blot method, using high-quality antibodies to detect the desired protein such as polyacrylamide SDS, and polyvinylidene difluoride (PVDF).⁶⁰ Observation using the ELISA method costs less than immunocytochemistry and western blot method because observation with ELISA methods without fluorescence microscopy and is also easier to apply in the environment, especially in environments that are limited by resources like an operator and equipment.^76,78

Conclusion

The immunocytochemistry and ELISA methods can measure the ALP after genistein induction, which is characterized by an increase or decrease in ALP activities in osteoblast cells. While western blot is used to detect protein expression levels. Both methods have their respective advantages, the immunocytochemistry method has higher sensitivity with faster processing time, meanwhile, the ELISA method has a higher selectivity with less cost efficiency dan the metode western blot has selectivity in detecting targets at the endogenous level of complex samples. This article is expected to be used as a reference by researchers to select suitable methods for the measurement of bone formation markers in vitro.

Acknowledgment

This research is supported and funded by the International Collaborative Development Research Maulana Malik Ibrahim State Islamic University, Malang, Indonesia, in 2022-2023.

Conflict of Interest

There is no conflict of interest.

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