Abstract
An automated, qualitative and semi-quantitative micro-assay for the detection of plant lectins also known as phyto-agglutinins (glycoproteins; have exciting applications in medicines) is described as an alternative to conventional assays. The method developed in this work is based on hemagglutination (HA) assay that can simultaneously detect the presence and concentration (titer) of lectins in as many as 96 different samples without the aid of an expert eye. We used rice (Oryza sativa L.) seeds for making clarified lectin extracts and Arabic gum as positive control in phosphate buffer saline; the method is applicable to all kinds and parts of the plants. Rabbit red blood cells were used in order to carry out the HA assay in a miniaturized experiment using U-welled microplates (MPs). 25 µL of plant extract is sufficient to carry out HA micro-assay at incubation temperature of 38°C for 20 min. The method was standardized with an expert eye and automated by using MP reader. Moreover, a standard curve for the direct interpretation of lectin concentration is also developed by conversion of absorbance values into titer. The method described will save time, material, labor, and simplify the rapid semi-quantification of plant lectins.
1 Introduction
Automation of microplate (MP) reading has been potentially utilized by many researchers in many fields including the study of hemagglutination (HA) [1,2]. Due to the lack of digital record, the titer determinations (the reciprocal of the highest two-fold dilution), the initial interpretation by experts is tiresome and commonly done in replicates. The range of potential variables and differences between expert readers make comparing inter-laboratory results difficult [3]. Therefore, the purpose of this study was to initiate an effort for the automation or digitalization of the process.
HA assays are convenient and quick way of measuring the presence and amount of hemagglutinin molecules primarily viruses in a given sample. Beside viruses, HA studies are used for the detection of macromolecules such as glycoproteins or lectins of any origin including plants that are able to bind the sialic acid on red blood cell’s (RBC’s) surfaces causing blood agglutination. This is not a part of the life cycle of viruses or mode of action of lectins to cause blood agglutination but it is a very convenient method to detect these entities and molecules in laboratory settings [3,4,5].
Lectins are ubiquitous carbohydrates binding proteins specifically binding diverse sugars on cell surfaces and results in cell agglutination or initiating a variety of physiological pathways. The role of plant lectins in abiotic and biotic stress resistance, symbiosis, medicine, glycobiology, and in industry has been highlighted by many researchers. Some of its important roles include effectiveness towards plant and human pathogenic fungi, insects, bacteria, and viruses viz. HIV and SARS-Covid-19. The potential applications of plant lectins in various fields of glycobiology, medicines, and agriculture are approved [6,7,8]. Classically, a method of visual observation is widely employed in laboratories for routine coagulology or HA assays due to their simplicity [6]. HA titer can also be easily determined by visual observations [9]. Since properties of blood samples strongly depend on time lapse after blood collection, and the number of samples, which can be analyzed simultaneously by visual method, is limited to a few, and the problem of proper interpretation of the obtained results arises very often due to human error. It concerns, especially, lectins agglutination studies both in crude extracts and in purified form. In this case, the laboratory instrument, an MP reader, can effectively solve the problem. With this, we can monitor changes in light extinction (absorption or transmission) in as many as 96 samples simultaneously [10]. The convenience of an MP reader in aggregation studies has been reported [11,12] and the recent high-quality version allows us to incubate samples at a chosen temperature with shaking, while the most recent one is portable version for on-site detection based on a smart phone [10].
The current report presents miniaturizing, standardizing, and automating blood aggregation studies when used for plant lectins with rabbit RBC’s – by comparison of HA assay results obtained with a visual screening and with a MP reader. To our knowledge, this is the first study to present the semi-quantification of lectin activities in a micro-assay format using MP reader for the interpretation of the results.
2 Materials and methods
The study was carried out in the Institute of Biotechnology and Genetic Engineering, Agricultural University Peshawar, Khyber Pakhtunkhwa, Pakistan. In our laboratory, the method of MP reader photometry was developed for routine measurements of hemagglutinin activities within small samples. Especially, HA activities of lectins were extracted from plants using rabbit RBC’s. We have used Arabic gum also called acacia gum as positive control, simple phosphate buffer saline (PBS) as negative control, and different varieties of rice seed extracts with unknown lectin activities. Acacia sp. have been found to have potent hemagglutinin activities, while their gum enhances the HA activity of other lectins [13]. We also demonstrated that the assay is suitable for the semi-quantification and characterization of lectins in plant extracts. The results obtained by micro-assay, using MP reader, were comparable to the values determined by visual interpretation, but with improved convenience and cost efficiency, effectively by miniaturization. To our knowledge, this is the first study to present the semi-quantification of lectin activities in a micro-assay format using MP reader for the interpretation of the results.
The reproducibility and linearity of the absorbance signals were determined by a U-welled MP using TC-96 ELISA Microplate Reader (TECO DIAGNOSTICS USA) with a wavelength of 595 nm for HA studies [14].
A standard curve was prepared using the extract of Arabic gum (0.2 g/mL) as positive control at one end point (maximum lectins) and 1× PBS (zero lectins) as negative control at another end point ranging in concentration from 0 HU/mL to a maximum of 4,096 or 5,120 HU/mL (i.e., titer, a reciprocal of two-fold serial dilution).
2.1 Extraction of lectins from plants
Lectin assay buffer i.e., PBS (1×; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4) was used to extract total lectins from the plant material available in our lab i.e., rice seeds and Arabic gum powder (Sigma Aldrich) by the method used by Bhagyawant et al. [15]. Ground rice seeds and pure Arabic gum powder was taken and dissolved in 1× PBS for centrifugation at 10,000 rpm for 15 min. After which the supernatant was transferred into another tube and stored at −20°C. The same method is applicable for the extraction of all types of plants and plant parts, except that seeds require extra step of fat removal to ease the process of lectin analysis [16].
2.2 Preparation of rabbit RBCs preparation
About 2 mL of rabbit blood was obtained in ethylenediaminetetraacetic acid tubes from the animal husbandry of Agriculture University, Peshawar, Khyber Pakhtunkhwa, Pakistan. It was immediately centrifuged at 500×g for 10 min and the serum discarded. After that PBS was added to the pellet, gently mixed, and again centrifuged at 500×g for 10 min. The washing process with PBS was repeated twice. After that the pellet (packed rabbit RBCs) was used to make 5% suspension of rabbit RBCs in PBS and stored at 4°C until use. Five percent formulation can be reduced depending on the consistency of the source blood and clearance of our HA assay results [6,15].
2.3 Serial dilution, HA assay, and visual reading of MP
The micro-assay requires 25–100 µL of extract and 5% Californian bred rabbit [6,15]. First a microtiter plate with U-bottomed wells was taken and equal volume (50 µL) of clarified rice seeds’ lectin extract was added in the first well of each row which was then serially diluted with 1× PBS through well 10. Last two wells were reserved for positive (Arabic gum) and negative (PBS) controls, respectively. Then, half volume (25 µL) of 5% rabbit RBC suspension was added into each well and incubated for 20 min at 38–39°C. If incubator is not available, time of incubation can be extended up to 1 h at room temperature. The results are recorded as strong, intermediate, and weak or no agglutination with an expert eye and pictures were taken with eight Megapixel camera and saved for comparison with other observation techniques [6,16].
2.4 HA assay with MP reader
The microtiter plate was prepared by the same method as above for visual analysis and subjected to MP reader (Figure 1). All the 96 samples were read at 595 nm concurrently without shaking and the absorbance recorded [14,15,16]. More than two observations were taken with a time interval of 10–15 min for all the samples. The positive control (Arabic gum) and negative control PBS were taken as standard and their reproducibility was checked. The visual readings and the absorbance of all the samples were compared and interpreted accordingly. Measurements were taken every 10–20 min for 1–2 h. It is especially important to adjust the volume and always use the same sample volume to get the same optical path. In MP reader, the meniscus of the sample in each well works as an optical lens and refracts the optical path. We have used 75 µL, i.e., 50 µL sample and 25 µL RBCs suspension throughout the experiment [17,18]. Endpoint measurements with positive control and negative control were done exactly after predetermined periods, for e.g., every 10–20 min for 1–2 h at 595 nm. The mean absorbance values of positive control were taken as one end and that of negative control as another end obtaining two end points of 0 and 1.5 at 595 nm (Figures 1–4).
A microtiter plate prepared for the HA assay of plant lectins; in each row (a–h), well #1 is undiluted plant extract, wells #2–9 are serial dilution of the extract, well #10 is glucose + extract for inhibition assay, well #11 is positive control, and well #12 is negative control.
Absorbance of HA assay (for the presence of lectins in plant extracts with known strength) via MP reader is plotted on x-axis against titer or lectin’s concentration of the known samples on y-axis to develop a standard curve that will help in finding the titer of samples with unknown lectin concentration.
Absorbance of HA assay for the presence of lectins in plant extracts in microplate is plotted against time (before, during, and after incubation period), showing settling of RBCs for strong positive (below curve) having the lowest absorbance values, as compared to the negative (below curve) and moderate positive (the middle curve).
Illustration of HA in a U-bottomed MP: Complete and strong agglutination will appear as a complete lattice (a) with lowest absorbance value of 0–0.4, partial/incomplete agglutination will appear as a diffused button (b) with absorbance value of 0.4–0.8, and non-agglutinated blood appears as sharp button with absorbance value of 1–1.3 obtained at 595 nm (c).
2.5 Standard curve
Standard curve of known samples (Arabic gum and PBS) using the average of their absorbance were plotted on y-axis and the respective concentrations or titer (HU/mL) on x-axis, which was further employed to find the concentration (x-axis) of unknown rice seed lectins by comparing their absorbance (y-axis) with the standard curve.
3 Results
3.1 Visual observation of HA
In this assay (HA), we used U-bottomed MPs, which relay on a convenient factor of sinking blood cells at bottom under gravity that can be seen with our naked eye. The RBCs naturally sink, sliding to the very bottom of the well forming a button at the very tip as shown in Figures 1 and 4. Strong positive results were recorded as lattice, moderate as diffused button, and negative as sharp button [11,14,16].
3.2 HA reading by MP reader
MP reader has been recommended by many scientists as a convenient tool in coagulology studies (1997). The MP was prepared by the method described in Section 2.3 and subjected to analysis via MP reader at 595 nm. We have analyzed 96 samples at a time in a MP using MP reader by taking multiple observations of absorbance for each sample at time interval of 10–15 min up to 60 min. This enabled us to trace, compare, and interpret visually the kind of changes in absorbance taking place in the samples with time due to agglutination. The range of absorbance at 595 nm recorded for the analysis of lectins in the samples was 0–1.4 depicting strong positive to negative HA (Tables 1 and 2).
Comparison of the HA assay results obtained from trained technician and pictures taken with 8 Megapixel camera after incubation
| Unaided eye vs camera eye | Unaided eye vs auto/absorbance | |||||
|---|---|---|---|---|---|---|
| Samples tested | Undiluted sample | Positive control | Negative control | Undiluted sample | Strong positive control | Negative control |
| Agreement | 98% | 98% | 98% | 98% | 98% | 98% |
| Absorbance range | 0.4–0.9 | 0.3–0.6 | 0.98–1.4 | |||
Results taken (unaided eye vs camera eye) and trained technician vs absorbance taken by microplate reader (Unaided eye vs auto/absorbance) for the presence of plant lectins in undiluted sample/plant extract, positive control (gum Arabic) and negative control/blank (PBS).
Comparison of the HA assay results obtained between trained technician and pictures taken with 8 Megapixel camera after incubation and results recorded (unaided eye vs camera eye) and trained technician vs absorbance taken by MP reader after incubation (unaided eye vs auto/absorbance) for the presence of plant lectins in serial dilution of plant extract
| Unaided eye vs camera eye | Unaided eye vs auto (absorbance) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Serial dilution | 2−1 | 2−2 | 2−3 | 2−4 | 2−5 | 2−1 | 2−2 | 2−3 | 2−4 | 2−5 |
| agreement | 99% | 90% | 70% | 50% | 30% | 99% | 99% | 99% | 99% | 99% |
| Absorbance range | Depends on the concentration of phytoagglutinins, strong positive (0.25–0.65), moderate to weak positive (0.65–0.95), and negative (0.98–1.4) | |||||||||
3.3 Standard curve
For the ease of comparison, standardization, and interpretation of the visual observation and results taken by MP reader, Arabic gum was taken as strong positive control and simple PBS solution was taken as negative control.
As the results of positive control and negative control were 100% reproducible both visually and via MP reader, thus these were taken as standard and the standard curve was established to find out the titer of unknown samples. The mean absorbance values (0.3–0.6) of positive control (Arabic gum) were taken and plotted against the highest possible titer value (2,048). Similarly, the mean absorbance values (0.9–1.4) of the negative control (PBS) were taken and plotted against the lowest possible titer value (0) as shown in Figures 3 and 4. This standard curve can be used to easily interpret the presence as well as concentration (titer) of unknown lectins in the sample by simply comparing its absorbance value (Figures 1–4). Closer the absorbance values to the positive control, greater is the possibility that the sample contains high concentration of Phyto-agglutinins and vice versa. Similarly, its concentration can be interpreted accordingly if the experts for visual observation are not available.
4 Discussion
In this work, we have demonstrated that the HA assay is suitable for the semi-quantification and characterization of lectins in plant extracts and the procedure can be miniaturized and subjected to automation via MP reader for rapid and accurate observations. The results obtained by micro-assay using MP reader were comparable to the values determined by visual interpretation, but with improved convenience, time, and cost efficiency. These results validated the applicability of MP reader photometry for lectin assays in large number of samples in a short time. A small amount of extract (25–100 µL) is more than sufficient to run all the lectin assays and to quantify proteins as well. The method enables photometric HA assays to be conducted on samples other than plants containing hemagglutinins. Even though the described method has advantages, it also has some disadvantages. The volume of the sample extracts must be consistent, uniform, and adjusted in every well because the MP reader gives different results with different volumes in the same plate especially in case of estimation of protein concentration using miniaturizing. For uniform results, bubbles should also be removed. A skillful laboratory worker may be needed to validate the results visually at the initial stage. While analyzing many samples, a multichannel pipette is required as using a simple micropipette may take longer time while filling all the 96 wells. This method is very well-suited for the explorative screening of samples on a laboratory scale [19].
The miniaturization did not affect the precision of the 96-well MPs method and sugar concentrations [20], during analysis of non-structural sugar analysis in ligneous plants. They also found that the reproducibility and accuracy of MP method in comparison with high-performance liquid chromatography (HPLC) were excellent, more specific, and improved. Gomez et al. [20] validated the micro well method to be reliable, rapid, and simple to perform and less expensive than HPLC or other classic and expensive enzymatic methods while examining different sugars in ligneous plants. Wolberg et al. [21] studied fibrin clot structure emphasizing that plate-reading spectrophotometer can provide a qualitative measure of fiber structure for both purified fibrinogen and plasma and suggested that plate-reading spectrophotometers can be a convenient, inexpensive, and rapid means of analyzing fibrin clot structure.
Visual observation of HA always requires negative and positive control even by expert’s eye to compare with an unknown sample. In a U-bottom MP, strong positive samples will show a lattice or network spread throughout the bottom. Diffused button at the bottom shows partial agglutination and sharp button shows negative agglutination, as shown in Figures 1 and 4.
In an MP, one of the rules of selection is that the agglutination should settle fast and non-agglutination should settle slow. First well of the microtiter plate will definitely form a lattice when both the lectins and RBCs are equivalent. While the next wells with lectins and RBCs will agglutinate faster than non-agglutinating control well. If these criteria of interpretation are used, then we should take the results within 5–10 min while visualizing constantly. By this method, in rice seeds, agglutination up to well no. 7 (10−6) was observed. This method of interpretation is good for few samples to be analyzed but not for many samples at the same time, e.g., in a microtiter plate with 96 wells.
Visually, HA can be monitored in many different ways. If the samples are few in number it can be carried out on a glass slide like that of monitoring ABO blood group system [6]. But if the samples are large in number, less in quantity, and titer has to be find out through serial dilution, then microtiter plate is a best option to save time and samples [11,14].
When the right agglutinins with right quantity are present in the test sample added to the RBCs suspension, instead of getting a button at the bottom of the tube a shield or lattice is formed (Figures 1 and 4). This happens because the carbohydrate part of the lectins is able to bind to the molecules of sialic acid present on RBC’s surfaces by cross-linking. Thus, when RBCs cross-linked with lectins reach the bottom of the tube, they form a lattice which spreads around the bottom and does not sink to form a button [6,22].
Arabic gum was taken as a positive control, the absorbance of which was seen to be reduced with time when compared to its original (time = 0) reading by MP reader, e.g., from 1.332 to 1.271 at 30 s interval (Tables 1 and 2). It means that if agglutination is occurring, the absorbance of the sample will reduce with time by a factor of 0.07. However, this result is for strong positive samples. This factor may vary for the samples with medium or low strength of the agglutinin present in the sample (Figure 4). Arabic gum used as positive control obtained from Acacia sp. have been found to have potent hemagglutinin activities and its gum also enhances the HA activity of other lectins [23,24].
In HA assay, usually sample and blood are incubated at room temperature for 20–30 min to see if any agglutination has taken place. As we have used rabbit RBCs, the body temperature of which is 38–39°C (100.5–103.5°F), therefore, samples at this temperature were incubated for 20 min inside an incubator.
The literature about the interpretation of HA results is available. However, these observations and interpretations can vary from lab to lab, expert to expert, type and concentration of RBCs, incubation time, nature of the sample agglutinin, and method of HA assay. Also, there is no digital record of the MP or titer determinations so the initial interpretation by experts is tiresome and commonly done in replicates. The range of potential variables and differences between expert readers make comparing inter-laboratory results difficult [25].
The standard curve was linear (r 2 = 0.841) for hemagglutinins concentration in the samples. The measurements showed progressively decreasing absorbance at higher lectin concentrations. Nevertheless, the correlation between the average values obtained by the method gave the coefficient r 2 also equal to 0.841. The reproducibility of the measurements was very good, with an overall average variation of less than 7% (Tables 1 and 2).
As compared to in-between results, the end-point titers were fairly reliable in U-bottomed MPs; however, all values can be taken into consideration. Expression of titer vs absorbance of the positive and negative control is of value in this micro system (Figure 2).
The uniformity in titers was satisfactory since both test-to-test and within-test variations were small. In addition, the test is easy to perform, economical, and fast. Based on these properties, micro-assays were developed and shown to be highly specific and quantitatively accurate for measuring the activity of either the crude extract or purified fractions of lectins in microgram quantities of tissue (i.e., 100–200 µg). The assays have been successfully applied to clarified extracts of a wide variety of tissues, leaves, seeds, stems, etc. [26,27,28].
MP reading has made many labor-some, expensive, and time-consuming lab procedures very expedient. MP method has been successfully used by Zogda and Porter for the first time for screening plant products against different strains of fungi and bacteria as a convenient microdilution method [22]. Zgoda and Porter (2001) presented a microdilution approach for screening natural products in search of new antimicrobial drugs that would be active against these germs because multi-drug resistance of these microorganisms is a significant problem. Cr. albidus and M. smegmatis were utilized as model organisms to enable the screening process under typical laboratory circumstances. The minimum inhibitory concentration (MIC) values of the antibiotics employed as positive controls were within the MIC ranges advised by the National Committee for Clinical Laboratory Standards. Lemna minor and Ilex cornuta organic plant extracts were used to create the microdilution test. The technique can be utilized as a trustworthy tool for finding novel chemical antibacterial agents. At the moment, it is being utilized for screening of more than thousand extracts from the organic extracts at a microgram scale. The same method can be employed for the characterization of plant lectins with potent biological activities [29,30].
5 Conclusion
In conclusion, MP reader photometry is a reliable and reproducible method for the measurement of HA activities within very small amounts of samples and reagents particularly for the presence of lectins. The reduced sample volume allows measurements on small tissue samples, and cultured cells. The method allows analyzing simultaneously a larger number of individual samples, thereby strengthening the statistical validity of any study.
Acknowledgments
The Authors wish to thanks Researchers Supporting Project Number (RSP2023R45) at King Saud University Riyadh Saudi Arabia for financial support.
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Funding information: This research work was supported by Researchers Supporting Project Number (RSP2023R45) at King Saud University Riyadh Saudi Arabia.
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Author contributions: All authors have contributed equally in writing this article.
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Conflict of interest: The authors declare no conflict of interest.
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Ethical approval: The conducted research is not related to either human or animal use.
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Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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