Pathological Sera Research Articles

Cathepsin B/cystatin C complex levels in sera from patients with lung and colorectal cancer.

A sandwich-type ELISA has been developed for quantification of the complex between the cysteine proteinase cathepsin B (CB) and its reversible tight-binding inhibitor cystatin C (CC) in normal and pathological sera. The assay is based on a combination of catching Ab (3E1), raised against CB, and a horseradish peroxidase-labelled detection Ab (1A2), raised against CC. Only the CB/CC complex is able to evoke a signal in this assay. The detection limit of the assay was 15.5 nM and the working range between 31.3-200 nM. The within and between-run coefficients of variance (CV) varied from 4.7% to 9.4% and 11% to 12.8%, respectively, demonstrating satisfactory reproducibility of the method. The concentration of the CB/CC complex was determined in sera from 90 healthy controls, 32 patients with non-cancerous lung diseases, 148 patients with lung and 32 patients with colorectal cancer. The CB/CC complex was significantly less abundant in sera of patients bearing malignant lung tumours than in those with non-cancerous lung diseases or healthy controls (p<0.001). In colorectal cancer sera its level was significantly lower in advanced stages C and D than in early Dukes' stages A and B (p=0.02). Our results show that the increased levels of CB in malignant sera are not impaired effectively by CC and support the hypothesis of hindered inhibitory capability during cancer progression.

Antibodies for trypsinized human red blood cells in human sera.

During studies of agglutination of trypsinized human red blood cells (RBC) by anti-Rh sera, it was noticed that some human sera without Rh antibodies agglutinated these erythrocytes. Of 933 normal and pathological sera subsequently screened, 116 produced such agglutination. The agglutinins were of IgM nature and were directed against an antigen exposed or created by tryptic digestion of RBC. They reacted equally well with autologous as with allogeneic trypsinized RBC and were different from T agglutinins and from Paul-Bunnell antibodies. The mode of formation of these antibodies remains unknown.

Unusual Precipitins in Pathological Human Sera

In conducting double diffusion gel precipitation tests, unexpected, strong precipitation reactions were noticed between a serum originating from a renal graft recipient and many pathological human sera. The highest frequency of positive reactions were produced by sera of patients with infectious mononucleosis (83%), chronic Epstein-Barr virus infections (72%), rheumatoid arthritis (57%), lepromatous leprosy (57%), and AIDS (44%). The precipitin in these pathological sera was identified as an antibody of IgM variety and the precipitinogen in the transplantation serum was shown to be a thermostable component with beta-globulin mobility. No explanation of the nature of reactions observed can be given at present.

Enzyme immunoassay for IgG rheumatoid factor combining with homologous IgG.

An enzyme immunoassay (EIA) was developed to detect, in human sera, IgG rheumatoid factor (RF) combining with human IgG. Wells of the EIA plate were coated with the Fc fragment of human IgG. Binding of RF was detected by goat antibodies to F(ab')2 of human IgG followed by rabbit anti-goat IgG conjugated with alkaline phosphatase and finally by the substrate. Using this procedure, RF was detected in 35%-85% of various pathological human sera, but only in 7% of normal human sera. An analogous procedure was also described for detection, in rabbit sera, of an RF combining with rabbit IgG.

Specificity of antibodies to Newcastle disease virus.

Specificity of antibodies to Victoria strain of Newcastle disease virus (NDV) found in infectious mononucleosis (IM) and other pathologic sera was investigated by agglutination of NDV-modified human O red blood cells, as well as by immunodiffusion and enzyme immunoassay with various preparations of the virus. These studies clearly demonstrated that the NDV antibodies are distinct from P-B or H-D antibodies. The unexpected observation that guinea pig kidney (GPK) tissues absorbed NDV antibodies allowed their classification into a group of 'GPK-positive' heterophile antibodies. The simultaneous occurrence of the NDV antibodies and H-D antibodies in IM and other diseases suggests the possibility that multiple new antigenic determinants, especially those of carbohydrate nature, may appear due to the alteration of self-antigens as a result of various pathologic processes.

Antibodies to Newcastle disease virus in various human diseases.

Sera of patients with infectious mononucleosis (IM) and various other diseases were studied for agglutinins against Newcastle disease virus (NDV)-modified human group O red blood cells (NDVO) and antibodies to the NDV preparations. In agreement with previous studies, the NDVO antibodies are found in a wide variety of diseases in addition to IM, including Japanese IM-like syndrome (22%), syphilis (24%), lepromatous leprosy (30%), systemic lupus erythematosus (29%), multiple sclerosis (18%) and cancer (17%); these antibodies were also found in patients with renal allografts (29%). It was also noted that the Victoria (VIC), Roakin and Herts strains, but not B1 strain of NDV are active in the NDVO agglutination, and VIC and Roakin strains, but not B1 strain in the immunodiffusion. Immunodiffusion and enzyme immunoassay with various preparations of the VIC strain revealed that the major antigen(s) of the virus under study is carried by the hemagglutinin-neuraminidase (H-N) glycoproteins. The H-N molecule was also shown to be able to modify human erythrocytes for the agglutination by the pathologic sera.

Immunochemical studies on the nature of the serum component (SAA) related to secondary amyloidosis.

The relative occurrence of the amyloid-related serum protein SAA in various disease states and in healthy subjects has been compared by both solid phase radioimmunoassay ((RIA) and immunodiffusion techniques which employ antibodies to purified amyloid fibril proten AA of hemogeneous size and charge. SAA levels were elevated above normal in certain categories of neoplastic, inflammatory, and infectious diseases as well as in secondary amyloidosis. The lowest median value, 8 ng./ml., was observed for approximately 150 normal sera, with no age-related increase in subjects ranging in age from 16 to 70 years. The results are consistent with several recent observations that SAA is normal acute phase reactant, and hence the RIA for SAA has no prognostic or diagnostic significance for secondary amyloidosis. The sensitivity of RIA for the detection of SAA is lower than would be expected when AA cross-reactivity values for sera are correlated with their reaction with anti-AA antibodies in immunodiffusion. This observation, along with others reported elsewhere suggests that those determinants which cross-react with anti-AA antibodies are relatively hidden in native SAA. Myeloma sera were less reactive than other groups of pathologic sera in immunodiffusion, although they were similar to other patients' sera when analyzed by RIA. Antibodies to highly purified AA were also used to investigate the structue of SAA by a double-antibody immunoprecipitation method. Precipitated SAA was partially dissociated during sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis to a 12,500 molecular weight moiety designated, SAAL. Multiple radiolabeled species of molecular weight intermidiate to SAA and SAAL were also detected and appeared to represent imcompletely dissociated SAA. The results suggest the SAA is an aggregate of several SAAL chains.

Streptolysin O inhibition by serum gamma G-globulin and beta-lipoprotein after blocking of nonesterified cholesterol by digitonin.

Abstract Physicochemical blocking of serum nonesterified cholesterol by digitonin and removal of the uncombined digitonin by transdehydroandrosterone eliminate the cholesterol molecules accessible to streptolysin O. In the presence of digitonin, denatured or pathological β-lipoproteins lose their streptolysin-inhibiting activity while inhibition by pure γ-globulin is not affected. Twenty normal and 198 pathological sera with high titers were studied. After digitonin fixation, they show a drop in the inhibition titer often greater than the one after β-lipoprotein precipitation by dextran sulphate. The difference was statistically significant at P=0.05, and it is assumed that β-lipoprotein precipitation does not totally suppress the non-specific inhibition of streptolysin while the digitonin method does. In normal subjects and in 89 cases with streptococcal diseases, both methods gave average titers corresponding to 30 to 50 per cent of the preliminary titers; "specific" and serum titers were more or less parallel. On the contrary, the specific titers were low in patients with unexplained high titers. In these cases there was no correlation between specific and serum titers. Therefore, the classic Todd procedure for antistreptolysin (ASL) determination can be considered as valuable in streptococcal disease but uncertain in some other conditions. In 3 cases with extremely high serum inhibition capacity related to the gamma globulin (which was monoclonal in 2 cases), the digitonin treatment did not change the titers after removal of the β-lipoprotein. Cholesterol blocking prior to titration of streptolysin inhibition allows a very specific determination of antistreptolysin.

Haemoglobin-binding capacity of the seromucoid fraction of human serum

In normal sera, the concentrations of seromucoid, total haptoglobin and sulphosalicylo-soluble Hp (SS-Hp) were determined. The seromucoid derived from serum incubated with Hb, was separated by paper electrophoresis at pH 8.6. A significant decrease was found in the α 2-seromucoid content. The Hb-binding capacity of the concentrated seromucoid was investigated. Seromucoid showed only a small part of the total Hb-binding capacity of serum and the results agreed with the previously performed SS-Hp determination. The lost Hp activity could not be traced among proteins precipitable by sulphosalicylic acid. Determinations of SS-Hp were also made in some pathological sera and significant differences were found. The results of determinations of electrophoretic seromucoid fractions compared with determinations of SS-Hp indicated on more or less marked heterogeneity of α 2-seromucoid, which sometimes can consist entirely of SS-Hp. The importance of SS-Hp and its relationship to the total Hp of serum are discussed.

The estimation of fatty acid esters in serum by the hydroxamate method

Hydroxamate methods for the estimation of fatty acid esters in serum generally involve extractions, nitrations and/or evaporations. It is shown, however, that the hydroxamate reaction may be applied directly to serum. The influence of different factors is investigated; the hydroxylamine and sodium hydroxide solutions, the nature and water content of the solvent, the time and temperature of the hydroxamate reaction. Diluted homogenized, sterilized mixed milk is proposed as a simple and stable standard. The coefficient of variation of the proposed method, applied to pooled serum, was 2.5% of the mean value. A hydroxamate method including previous extraction, etc. had a variation coefficient of 6.5%. The two methods gave similar average results. Interference of non-lipids was negligible for pooled serum. The blank correction was inadequate for a grossly bile-pigmented serum. Otherwise, lipid analysis on 34 suspected sera justifies the expectation that interference in pathological sera does not prevent direct application of the hydroxamate method. The procedure is recommended for use in routine laboratories.

Metabolism of human leukocytes in vitro. V. Inhibition by human serum of formate and glycine incorporations.

SummaryThe presence of one or more chemical substances in normal and pathological human sera capable of decreasing the rate of incorporation of formate and glycine into human leukemic leukocytes in vitro has been shown. Human leukemia sera as a group demonstrated a slightly subnormal inhibitor activity.

Electrophoretic patterns of blood sera in poliomyelitis and Guillain-Barré's disease (encephalomyeloradiculitis)

Summary 1. Electrophoretic study was made of the blood sera of 4 “normal” children,23 cases of poliomyelitis, 5 cases of Guillain-Barre's disease (encephalomyeloradiculitis), 1 case of pseudohypertrophic progressive muscular dystrophy, and 1 case of paralysis of unknown etiology. 2. All of the electrophoretic patterns of the sera of the “normal” childrenagreed well with those in the literature for adults. 3. All of the cases of poliomyelitis, with a single exception, showed definite abnormalities in the beta disturbance, although the time from onset of illness ranged from 6 days to 22 years. 4. In one case of poliomyelitis no abnormality in the beta disturbance was noted at the eighth day of illness but there was a definite abnormalitiy at the nineteenth day. 5. In 2 cases of poliomyelitis there was a definite decrease in the abnormalityof the beta disturbance after periods of ninety-seven days and two hundred and twenty-four days had elapsed following the first determination. 6. The electrophoretic patterns of the sera of patients with Guillain-Barre'sdisease (encephalomyeloradiculitis) did not differ appreciably from those of patients with poliomyelitis. 7. Electrophoretic patterns of the serum of the one patient with pseudohypertrophicprogressive muscular dystrophy and of the one patient with paralysis of unknown etiology were within normal limits. 8. Calculations of the proportions of the various electrophoretic components present did not reveal any consistent differences between the normal and the pathologic sera studied.