Bence-Jones proteins detection

On examination of the urine and serum of numerous patients with suspected paraproteinemia in both Jamaicans and Africans between 1962 and 1966, it was concluded that whenever a low total serum y-globulin level with a normal serum electrophoretic pattern were encountered in a suspected case of multiple myelomatosis, it was then essential to obtain also a specimen of urine from such a patient for further electrophoretic examination. Invariably simultaneous electrophoresis of such sera and urines proved to be diagnostic, even when the classical heat test for Bence Jones protein was negative. Consequently it was found that concurrent electrophoresis of scrum and urine was the first means of detecting multiple myelomatosis in no less than 20% of the patients, which were subsequently confirmed either by bone marrow biopsy or X-ray examination or both (M3). 

Ultracentrijugation. In those laboratories where an ultracentrifuge is available, the pattern obtained of the serum of a patient with Waldenstrom macroglobulinemia is very characteristic. Frequently more than 30% of the total serum proteins could be accounted for by the 19 S peak. Bence Jones protein is not an uncommon finding in patients with Waldenstrom macroglobulinemia and was detected in patients both in Nigeria and Jamacia with the disease. 

Monoclonal protein can be detected in serum, urine, or both in greater than 95% of patients with multiple myeloma (D16). Bone marrow plasma cells exceed 10%. Patients with advanced disease may excrete Bence-Jones proteins in urine. Both hypercalcemia and Bence-Jones proteinuria can contribute to renal failure (A6). 

The dipstick test for total protein includes a cellulose test pad impregnated with tetrabromphenol blue and a citrate pH 3 buffer. The reaction is based on the protein error of indicators phenomenon in which certain chemical indicators demonstrate one color in the presence of protein and another in its absence. Thus tetrabromphenol blue is green in the presence of protem at pH 3 but yellow in its absence. The color is read after exactly 60s and the test has a lower detection limit of 150 to 300mg/L, depending on the type and proportions of protein present. The reagent is most sensitive to albumin and less sensitive to globulins, Bence Jones protein, mucoproteins, and hemoglobin. 

Beetham R. Detection of Bence Jones protein in practice. Ann Clin Biochem 2000 37 563-70. 

Our best evidence of this is a 3-year follow-up of 402 patients in whom Bence Jones protein had been detected in our laboratory. Dr. Corbett obtained biopsy evidence of malignant immunocytoma in 400. The various forms such malignant immunocytomata can take are listed in Table 7, which also includes the benign varieties in which I personally have not yet found immunoglobulin fragments. 

Six published and personal examples have been detected of Bence Jones protein precipitating in the cold, but all under 20°C, and causing no symptoms in the patients. They were mostly type L, and this class shows an excess among monoclonal cryoimmunoglobulins. Rarely, true crystals may form in the cold, and any such protein would be welcome to X-ray crystallographers. 

Bundles, Coonrad, and Arends (R13) surveyed 35 patients with leukemia and detected paraproteins in at least 4, and possibly in 2 others. Since then there have been numerous reports of paraproteinemia with all kinds of lymphoma or leukemia, although almost no further surveys until 1969 when 19 of 76 patients were found to have excess light-chain excretion (L6). Personal surveys have revealed serum paraproteins in 3 atypical cases out of 124 consecutive patients with Hodgkin s disease, 26 of 207 with lymphosarcoma (see Section 7.7.2), 1 of 45 with reticulo-sarcoma, 0 of 31 with giant follicular lymphoma, 3 of 84 with chronic lymphatic leukemia, 0 of 43 with chronic myeloid leukemia, 0 of 57 with acute leukemia, and 1 of 5 with chronic monocytic leukemia as established by high lysozyme levels. Urine has revealed Bence Jones proteins in many of those with serum paraproteins and in addition only Bence Jones in 1 atypical Hodgkin s, 2 lymphosarcoma, 1 giant follicular lymphoma, yet 8 with chronic lymphatic leukemia. 

W7. Wetter, 0., Fragments of Bence Jones proteins their detection and biological significance. Protides Biol. Fluids, Proc. Colloq. 17, 137-139 (1969). 

Development of electrophoretic protein separation techniques have been paralleled by improvements in protein detection methods. Protein detection in early electrophoretic applications, utilizing electrophoretic separations of solutions or colloidal suspensions from about 1816 to 1937, was limited to direct visualization of proteins coated onto microspheres, or studies of naturally colored proteins such as hemoglobin, myoglobin, or ferritin <1-4). An increase in sensitivity and the ability to detect non-colored proteins was achieved by the use of the specific absorption, by proteins, of ultraviolet light. This detection technique permitted Tiselius,in 1937, to demonstrate the quantitative electrophoretic separation of ovalbumin, serum globulin fractions and Bence Jones proteins (S). Tiselius also employed the shadows, or schlieren, created by the boundaries, due to the different concentrations of proteins in the electrophoretic system to detect protein position and concentration ( ). These detection methods served as the main methods for protein detection in the liquid electrophoresis systems. However,... 

Proteins with lysine at position 190 are designated Oz(-l-) those with arginine are Oz(—). This substitution can be detected with an antiserum which gives a positive reaction only with Oz(-f) Bence Jones proteins (and with normal sera) (20). A rabbit antiserum which recognizes the presence of glycine at position 152 has also been developed reactive proteins, designated Kem(-I-), comprised 8 of 38 monoclonal X chains studied (21a). 

As in the human, a large proportion of mice with multiple myeloma excrete a urinary Bence Jones protein which, in an individual animal, is either k or. The concentration is highly variable, ranging from a barely detectable level to a concentration as high as 50 mg/nJ. 

A test for the detection of Bence Jones proteinuria which consists of adding acetic acid and sodium chloride solution to the urine. Precipitation occurs if Bence-Jones protein is present. 

The sensitivity and specificity of the quantitative precipitin method make it applicable to the identification and estimation of abnormal plasma proteins in certain diseases, particularly when present in concentrations too low to be detected by other methods. Goettsch and her associates (103, 104) and Kendall (181) employed immunochemical techniques in the study of nephrotic sera Kendall (181) also investigated the distribution of his immunologically distinct globulin subfractions in hyperglobulinemia due to lymphogranuloma venereum, cirrhosis of the liver, and rheumatoid arthritis Kabat (261) was able to demonstrate the presence of as little as 0.15 gram per cent Bence-Jones protein in the serum of a patient with multiple myeloma. Many other applications of immunochemical techniques are noted by Kabat (167) and Treffers (358) in recent reviews. 

With regard to those sera with apparently normal electrophoretic patterns obtained in patients excreting large amounts of Bence-Jones protdn in the mine, it should be borne in mind that free urinary excretion of Benc Jones proteins is apt to keep the blood Bence-Jones protein levels low, too low to be detectable by salt or electrophoretic fractionation. It may be presumed that a small amount of Bence-Jones proton is present in the blood, as can be demonstmted by immunochemical methods. 

Kabat (251) was able to show by immunochemical methods that the main component of the serum of case 11 (Table VII) was, in fact, a Bence-Jones protein. Rabbit antiserum to this patient s urinary Bence-Jones protein gave a strong precipitin reaction with a 1 625 dilution of the patient s serum. This reaction appeared to be due entirely to Bence-Jones protein since after absorption of the rabbit antiserum with case 11 myeloma serum no additional precipitin reaction could be obtained with the patient s urine or purified Bence-Jones protein nor did the (absorbed) antiserum react with normal serum protein constituents. Qualitative dilution tests indicated a concentration of Bence-Jones protein in the serum of the same order of magnitude as was obtained for the main abnormal component by other methods. In case 1 (Table VII) Kabat was also able to demonstrate by immunochemical methods that while the main component was not a Bence-Jones protein, 0.15 to 0.2 gram per cent of a Bence-Jones protein nevertheless was present and could be identified and estimated by emplojdng the quantitative precipitin method. This concentration, approximately 1.5% of the total protein content of this serum, obviously could not be detected by salting-out, electrophoretic, or ultracentrifugal methods. 

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