Antibodies and immunoassays for soybean antigens
Soybean derivatives are ubiquitous components of foods. Soybean may cause food allergy. Soybean derivatives are also extremely popular as cheap alternative of other foods (meat, milk) and may be used to adulterate different foods.
We have obtained monoclonal antibodies towards two major targets of soybean: SBTI and “soybean globulin”. SBTI is common in all soybean derivatives and relatively heat stable; therefore we recommend to use SBTI assay for ultrasensitive detection of trace amounts of soybean antigens (allergens).
For massive adulteration of foodstuff, the sensitivity of the assay is not the issue; it is more important to obtain a test which is minimally dependent on the matrix effect of tested material (the physical and chemical properties).
Antibodies against soybean trypsin inhibitor (SBTI)
We have obtained mAbs against soybean trypsin inhibitor. The mAbs are divided into three epitope groups by cross-inhibition and matching ability – see the table below
| mAb | Isotype | Epitope group |
| X841 | IgG1 | A |
| X842 | IgG1 | A |
| X832 | IgG1 | B |
| X833 | IgG1 | C |
| X834 | IgG1 | A |
| X837 | IgG1 | B |
| X844 | IgG1 | A |
SBTI EIA (K384T)was designed using a.m. mAbs. This kit is intended for research purposes to determine SBTI content in food. Due to partial heat-sensitivity of the determined epitope and results of SBTI determination in heated products cannot be directly calculated in % (w/w) of soybean in product.
Kit performance data:
Test principle: sandwich, monoclonal antibodies
Total time of analysis: 30/30/15 min
Sensitivity: 1 ng/ml
Material: food, soybean derivatives
Application of K384T SBTI EIA: heterogeneity of SBTI content in different soybean sources
| Tested material | SBTI of dry weight, % |
| Flour 1, Serbia | 0.02 |
| Flour 2, Serbia | 0.02 |
| Soybean concentrate Danpro-H | 0.33 |
| Soybean isolate H-90, China | 2.6 |
| Soybean isolate S-650, China | 0.13 |
| Soybean flour, Aventis | 3.3 |
Soy rapid test -
Soya (X384T) is based on SBTI determination and designed for both allergy and food adulteration control in the kitchen. The pilot lot of this rapid test is under evaluation.
Antibodies against soybean globulin components
A group of monoclonal antibodies was obtained against defatted cooked soybean homogenate. The exact location of target antigens is performed currently, however the existence of at least one immunometric pair shows that there is more than one epitope on the target molecule(s).
Soybean EIA (K384A) for quantitative determination of soybean protein(s) in foods can be made using a single antibody; the food derived soybean antigens are passively adsorbed onto the microplate surface.
Current K384A kit version detects relatively heat stable epitopes of soybean antigens and shows zero level of background in pure meat samples. The performance of current K384A kit version does not allow to designate it as quantitative due to matrix effect, heterogeneity of soybean derivatives used in food industry and lack of external standard of soybean antigen.
Kit performance data
Test principle: adsorption, direct binding of monoclonal antibodies conjugated with HRP
Total time of analysis: 30/30/15 min
Sensitivity: approx. 0.05% w/w soybean in product
Material: food
Soybean (sandwich) EIA (K384) was developed as another alternative– see IFU.
A current version of the K384 shows less sensitivity comparing to adsorption test (K384A), however this test is less dependent on matrix effect (differential adsorption on plastics) and shows better linearity and quantitative performance.
Kit performance data
Test principle: sandwich, monoclonal antibodies
Total time of analysis: 30/30/15 min
Sensitivity: approx. 0.5% w/w soybean in product
Material: food
Applications of XEMA Soybean tests for foods testing
Determination of soybean % (w/w) content in meat products also is a part of quality control procedure for foods. The regulations in some countries imposes to label the foods as “soybean free” if the content of soybean derivatives does not exceed 1% w/w.
In random control of processed meats, dry milk and baby’s formula in local supermarket, we have located adulteration cases – both with inadequate presence and absence of soybean declared on the label.
| Product | SBTI | Soybean antigen | Data on product label |
| Sausage 1 | - | - | Soybean free |
| Sausage 2 | - | - |
| Dry milk 2 | - | - |
| Protein Mix, infant formula | - | - |
| Sausage 4 | + | ++ | Soybean free |
| Sausage 5 | - | - | Contents soybean |
| Dry milk 1 | ++ | ++ | Soybean free |
| Sausage 3 | +++ | +++ | Contents soybean |
| Canned pork 1 | + | ++ |
| Canned beef 1 | ++ | ++ |
| Polony | + | ++ |
| Soybean flour 1 | ++++ | ++++ |
| Soybean flour 2 | ++++ | ++++ |
Comparative study of XEMA Soybean tests with PCR method
Comparative study of soy content in sausages and raw matherials was performed in collaboration with All-Russian Research Institute of Meat Industry to estimate sensitivity and quantitativity of differnet XEMA EIA and IHA test systems
Soy content was determined in 13 samples of sausages and 2 samples of raw matherials (egg powder and wheat flour) using 3 XEMA EIA test kit (Cat N 384, 384A,384T), rapid test (X384T) and PCR as a reference method.
Table 1. Quantitative determination of soy content by different methods
| Sample number | K384T (SBTI), ng/g of product | K384, % soy w/w | K384A, qualitative/ OD 450 nm | X384T (SBTI), rapid test, qualitative |
% of soy content (w/w) by recipe | PCR, % soy (w/w) |
| ¹1 | 1585 | 0,3 | 8+ /4000 | 3+ | 2 | 3,4 |
| ¹2 | 1924 | 0,4 | 6+ /3000 | 2+ | 2 | 1,6 |
| ¹3 | 1922 | 1,5 | 5+ /2500 | 4+ | 2 | 4,7 |
| ¹4 | >2500 | 6,7 | 7+ /3400 | 8+ | 6,9 | 7,2 |
| ¹5 | 266 | 0,9 | 1+ /500 | 1+ | 5 | 2,6 |
| ¹6 | >2500 | 2,2 | 8+ /4000 | 8+ | 8 | 6,4 |
| ¹7 | 1501 | 0,3 | 8+ /4000 | 3+ | 4,2 | 2,4 |
| ¹8 | >2500 | 2,7 | 7+ /3500 | 8+ | 7,1 | 6,5 |
| ¹9 | >2500 | 3,6 | 7+ /3500 | 5+ | 1 | 1,5 |
| ¹10 | 1670 | 0,3 | 7+ /3400 | 4+ | 1 | 2,3 |
| ¹11 | 1664 | 1 | 6+ /2800 | 4+ | 1 | 2,6 |
| ¹12 | 1867 | 0,7 | 6+ /2900 | 4+ | 1 | 4,2 |
| ¹13 | 676 | 0,3 | 7+ /3300 | 2+ | 1 | 2 |
| PBS | 0 | 0 | 0 | 0 | 0 | N/A |
We performed 2 separate runs during 1 month, samples were stored at -20C.
Totally EIA kits shows bad or no correlation in low soy content range but all samples were positive, there were no falsenegative results.
Qualitative sensitivity of K384A seems to be equivalent to PCR.
Photo 1. Qualitative determination of SBTI by X384T in sausages. All samples were positive, PCR showed the same results.
Photo 2. Qualitative determination of SBTI by X384T in egg powder and wheat flour. Egg powder was strong SBTI positive, wheat powder was negative. PCR showed both positive, with “traces” diagnosis in wheat powder. K384T EIA confirmed presence of SBTI traces in wheat powder.
Correlation between SBTI content visually accepted by X384T and % of soy by PCR method
Arrows show mismatching results in both sides
Results that were showed on previous picture divide between different manufacturers. Dotted line and black triangles illustrated assigned level of soy content by manufacturer initial recipe. It is common production rule to put soy greater to compensate losses during production process.
