Ml Of Antiserum Research Articles

High volume antisera yields from rabbits using plasmapheresis

A simple procedure for plasmapheresis of rabbits is described, permitting collection of approximately 75 ml of antisera to be obtained at weekly intervals. Evidence for maintenance of antibody titer throughout this interval is presented.

Quantitative determination of titers of anti‐zona serum

The titers of rabbit antiserum against isolated mouse zonae pellucidae were examined by several methods in connection with inhibitory effect on fertilization. The titers determined by zona precipitate, zona dissolution, indirect immunofluorescence and in vitro fertilization tests were 2(4), 2(1) to 2(4), 2(7) and 2(4), respectively. Cytotoxic effect could not be detected from zona antibody. The indirect immunofluorescence was most sensitive for detection of zona antibody but did not represent the extent of inhibitory effect on fertilization. The titer obtained by zona precipitate test was most close to the titer obtained by inhibitory effect on in vitro fertilization. The present study also demonstrated that at least 0.0375 ml of antiserum per female mouse, equivalent to 0.15-0.25% of body weight, was necessary for inhibition of fertilization in vivo by passive immunization with anti-zona serum.

実験的緑膿菌感染症の化学療法に関する基礎的研究

Pseudomonas aeruginosa is an ubiquitous organism in nature and has been thought to be low-virulent. The discovery and practical use of penicillin followed by exploitation of various antibiotics such as streptomycin and chloramphenicol, lead to the effective treatment of infectious diseases that were due to certain bacterial species. Gram-positive pyogenic cocci, representative of such organism, were successfully eradicated by therapeutic usage of these drugs whereas the cases of infection due to Gram-negative rods known as low-virulent or even non-pathogenic became increasingly important. This phenomenon is understood as “microbisme sèlectioné et substitute” or “opportunistic infection”.Although the discovery of gentamicin (GM) and penicillin delivatives such as carbenicillin and sulbenicillin resulted in fairly effective control of human P. aeruginosa infection, certain predisposed patients developed fatal infection due to this organism.In the present study, the author attempted to find an effective way to control human P. aeruginosa infection with a combined administration of GM, one of the most eff ective antibiotics for P. aeruginosa, and several samples of γ globulin (γ-G) in tests on experimental P. aeruginosa infection in mice. The bacterial strain used in this experiment was P. aeruginosa NC-5. Minimum inhibitory concentration (MIC) of GM against P. aeruginosa NC-5, estimated by broth dilution method, was 6.25 mcg/ml.ICR strain male mice,3 to 4 weeks old (about 15 grams), w ere infected with an intraperitoneal injection of 0.5 ml of bacterial suspension. GM combined with or without -G was intramuscularly injected shortly after the inoculation. The mice were o brserved for survival up to 3 days.The median lethal doses (LD50) of strain NC-5 to mice, estimated with and without mucin, were ca 10 3.2 cells/ml and ca 10 6.2 cells/ml, respectively.The results of therapeutic experiments obtained were as follows:1. The median effective doses of GM for the mice challenged with 1,000 LD50 and 100LD50 of NC-5 were 312.5 mcg/0.1 ml and 70.7 mcg/0.1 ml, respectively.2. Neither fractionated γ-G (150 mcg/0.1 ml) nor γ-G in. whole serum (500 mcg/0.1ml) of normal rabbits protected the mice against the challenge of 1,000 LD50 of the organism. But therapeutic effects were observed in the combined administration of 50mcg/0.1 ml (1/6 ED50) of GM and 250 mcg/O.1 ml of serum (or 150 mcg/0.1 ml of γ-G).3. Anti-NC-5 rabbit serum was more effective than normal serum. That is, therapeutic effects were observed in the group treated with 200 mcg/0.1 ml of the antiserum or its γ-G. The combined usage of 25 mcg/0.1 ml (1/8 ED50) of antiserum γ-G and a 50 mcg/0.1 ml (1/6 ED50) of GM was markedly synergized.4. The effect, of γ-G combined with GM was verified as the activity of a specific antibody by its absorption test with corresponding antigen.5. IgG and IgM fractions from rabbit antiserum for NC-5 were compared with each other for their therapeutic effect. IgG, was more effective than IgM despite its lower agglutination titer.6. In the group of mice treated with GM combined with γ-G (or IgG) the viable bacterial number in peritoneal exsudate was decreased and the animals survived. In the use of IgM instead of IgG, however, the decrease of viable bacterial number was less and the mice failed to survive.From the above described results, it was concluded that synergism between the antiserum or its γ-G and GM was observed in the treatment of experimental P. aeruginosa infection in mice. The mechanism of this synergism claimed that the bacteria were easily phagocytized by the opsonization with the antiserum or its γ-G, and GM exert bactericidal activity in cooperation with the host cells.

Determination of plasma oxytocin by radioimmunoassay (author's transl)

A simple radioimmunoassay was applied to the measurement of oxytocin in human plasma.Ten mg of synthetic oxytocin and 10 mg of porcine gamma globulin were dissolved in 4 ml of distilled water, and then added 150 mg of carbodiimide, after adjusted pH to 6-7. The solution was incubated 25°C for 4 hours and dialyzed overnight at 4°C against distilled water. An antibody to oxytocin was produced by intramuscular administration of the conjugate of oxytocin and globulin, emulsified with an equal volume of Freund's complete adjuvant, to rabbits at 2 weeks intervals. Oxytocin was radioiodinated by chloramine-T method, and then purified by gel filtration using Sephadex G-25. Plasma oxytocin was adsorbed by Florisil and extracted with acetone containing 1% acetic acid. Radioimmunoassay of oxytocin was performed by adding 0.1 ml of 125I-oxytocin and 0.1 ml of antisera to plasma extracts diluted with 0.05 M phosphate buffer containing 0.1% lysozyme (pH 7.4). After incubation at 4°C for 72 hours, antibody-bound antigen was separated using dextran-coated charcoal.A high specificity of immunoassay was demonstrated by the fact that large excess of angiotensin I and II, and ACTH did not displace labelled oxytocin from the antibody. Lysine-8-vasopressin and arginine-8-vasopressin showed very little cross-reaction in the assay, possessing only 0.002% of the immunological potency of oxytocin. The specific activity of 125 I-oxytocin was 166 μCi/μg. Adsorption and extraction capacities of Florisil were 96.6±2.1% and 85.7±2.5%, respectively. Intra-and inter-assay variability were 7.2±4.9% and 4.3±2.2%, respectively. The sensitivity of the assay was below 1pg/tube.Normal levels of plasma oxytocin were 0-2.2 pg/ml (n=13) in males and 0-10.4 pg/ml (n=10) in females. Plasma oxytocin levels in the 39th and 40th weeks of pregnancy were 27.9±4.14 pg/ml (n=4) and 29.8±17.1 pg/ml (n=13), respectively. The levels in creased to 33.1±12.1 pg/ml (n=7) and 37.1±17.5 pg/ml (n=7) in the first and third stages of labor, and decreased to 13.6±5.25 pg/ml (n=6) on the 2nd to 8th day after labor.The radioimmunoassay for oxytocin in plasma is considered to be sufficiently applicable for clinical use.

Immunological studies with the gonadotropins and their subunits from the green sea turtle Chelonia mydas

Antisera generated in rabbits against highly purified follicle-stimulating hormone (FSH), luteinizing hormone (LH), and the β subunit of LH from the green sea turtle, Chelonia mydas, were used to obtain additional biochemical information on the chemical and biological relatedness between the two turtle gonadotropins. Evidence that the antisera were directed against the biologically active gonadotropins was obtained from neutralization studies. At concentrations of 1 ml of antiserum per 100 μg of hormone, FSH and LH could each be selectively neutralized with homologous antisera. These results provide additional evidence that both gonadotropins have intrinsic biological activities in assays related to testis growth and androgen production in lizards and in vitro androgen production by snake testes. Each antiserum ( a s ) showed some binding to the heterologous radioiodinated hormone, but binding was clearly maximal with homologous tracer, and some of this cross-reaction was shown to be due to heterogeneity in antibody populations. Highly specific radioimmunoassays (RIAs) were developed for both FSH and LH with these antisera. When FSH a s was used in conjunction with 125I-labeled FSH, Chelonia LH and thyroid-stimulating hormone (TSH) showing virtually no cross-reaction (e.g., contamination of LH with FSH was < 1%). Alternatively, RIA using either LHβ a s or LH a s combined with 125I-labeled LHβ as tracer was highly specific for LH and especially the β subunit; there was no cross-reaction with either FSH, TSH, or the α subunit of LH. When LH a s was used with 125I-labeled α as tracer, the specificity of the RIA was reduced; in this case, LHβ alone showed no cross-reaction but LHα was highly potent. Thus, these systems provide a means for studying the importance of the two separate subunits in immunorelatedness among pituitary glycoprotein hormones and for measurement of circulating gonadotropin levels.

Rapid Two-Dimensional Immunoelectrophoresis of Human Serum Proteins

Abstract We describe two versions of a two-dimensional immunoelectrophoretic system and illustrate their use for separating and estimating human serum proteins. Each is sensitive, rapid, and simple to perform, and requires no special apparatus and only 0.1-0.6 ml of antiserum. Both procedures appear to be potentially useful in the clinical laboratory. Time required to obtain a stained pattern varies from 4.5 to 7 h, depending on the intended purpose and sensitivity (number of possible precipitin peaks) desired. With the faster method ("rapid screening") 25 to 35 precipitin peaks are detected. With the slower but more sensitive method 40 to &amp;gt; 50 peaks are detected. The sensitivity of either version is a function of the titer of the polyvalent antiserum to whole human serum. Of the precipitin peaks thus far detected, 35 have been identified.

A micromethod for membrane immunofluorescence

A micromethod of membrane immunofluorescence is described which permits satisfactory staining with 10 5 cells and 0.005 ml of antiserum. Results by this technique in titrating anti-thymocyte serum were comparable to those obtained by a routinely used membrane immunofluorescence procedure.

Autoregulation of renal blood flow in rabbits immunized with angiotensin II.

The role of renin in the autoregulation of renal blood flow was examined in rabbits actively immunized with angiotensin. Autoregulation was found to be unaffected by high serum concentrations of antiangiotensin antibody. On average, 1 ml of antiserum inactivated the pressor effect of 5.23 ± 4.95 μg of angiotensin (mean ± S.D.), range 0.64–16.0, n=17. Similar antibody concentrations were found in serum and in renal lymph, excluding major differences in antibody litres of renal interstitial fluid and serum. To evaluate the amount of antibody present in arteriolar walls, isolated hind quarters from immunized rabbits were artificially perfused. When adding angiotensin to the perfusion fluid, about 17 times higher doses were needed in immunized than in control rabbit hind quarters to give similar pressor responses. Accordingly, in the immunized rabbits antiangiotensin antibody was present both in arteriolar walls and in renal interstitial fluid, thus providing strong evidence that angiotensin is not essential in the autoregulation of renal blood flow.

Possible Application of the IHI Test in the Species Identification of Hair

Immunological species differentiation of keratein from hair has previously been demonstrated by the precipitin ring test in tubes (Pillemer et al. 1939) and by the indirect hemagglutination test (Simonsen 1970). In the present study the possibility of species identification of s-carboxymethylkeratein (SGMK) from single hairs was examined. SCMK and rabbit anti-SCMK sera from man, horse, dog and ox were prepared according to methods described by Gillespie (1962) and Simonsen (1970, 1971). Suitable antisera were used for the indirect hemagglutination-inhibition (IHI) test (Stavitsky 1954). The antisera were absorbed with heterologous SCMK and the inhibition test performed using SCMK extracted from 5 cm stretches of hairs by reduction and alkylation in 1 ml fluid volumes. To each vial containing 0.5 ml of antiserum in a serial 2-fold dilution row of the respective antisera was added 0.05 ml of a homologous or a heterologous single-hair SCMK. After incubation at 37 °C for 30 min. SCMK-coated goat erythrocytes were added and the test read after incubation at 20 °C for 18 hrs.

Nucleoside-coated bacteriophage T4: a method for the detection and characterization of anti-nucleoside antibodies

A general method for coupling nucleosides to bacteriophage T4 is described. The method is based on opening the C2′–C3′ bond in the ribose moiety of the nucleoside with sodium periodate, reacting the product with bacteriophage T4, and stabilizing the bond formed by reduction with sodium borohydride. The phage conjugates that survive the coupling process are specifically inactivated by the corresponding anti-nucleoside serum, and are thus used for the detection of minute amounts of anti-nucleoside or anti-nucleotide antibodies. As little as 2 ng of antibodies per ml of antiserum can be detected. Conjugates of adenosine, guanosine, inosine, uridine adn cytidine to bacteriophage T4 were prepared by the above-described method. The ability of different specific antisera to inactive these various nucleoside-T4 conjugates was studied. It was found that anti-purine antibodies inactivated the homologous or the heterologous purine-T4 conjugate and, likewise, anti-pyrimidine antibodies inactivated the homologous or the heterologous pyrimidine-T4 conjugates. Moreover, uridine antibodies were able to inactivate both adenosine- and guanosine-T4 conjugates. Antibodies against denatured DNA were able to inactivate all the nucleoside-modified bacteriophage conjugates. Antibodies cross-reacting with the various nucleosides were also detected in the sera of rabbits immunized with yeast tRNA in complete Freund's adjuvant. The inactivation of the nucleoside-T4 conjugate can be inhibited by the homologous nucleoside or by immunologically cross-reacting materials. Such inhibition may serve as an assay for the sensitive detection and quantitation of various nucleosides and other inhibitors.

Preparation of protein-bacteriophage conjugates and their use in detection of anti-protein antibodies

Protein bacteriophage T 4 conjugates can be prepared, making use of bifunctional reagents such as tolylene-2,4-diisocyanate, glutaraldehyde and bis-diazobenzidine. The conjugates surviving the coupling process are inactivated by the specific anti-protein sera. This inactivation can serve as an assay for detection and quantitation of anti-protein antibodies. Using the assay described, as little as 0.2–2 ng of antibody per 1 ml of antiserum could be detected. Insulin bacteriophage conjugate was inactivated to a significant extent by sera of diabetic patients, who had received injections of insulin, diluted 100–10 000-fold.

Antigen-Antibody Reactions in Agar

Summary A micromodification of the agar gel precipitin reaction of Oudin is presented; the method uses the optical densities of antigen-antibody precipitate bands formed in reaction tubes for determining the antibody concentration of rabbit antiprotein antisera in absolute weight units, mg N/ml. When microreaction tubes were used, it was possible to obtain standard O.D. vs. antiserum concentration curves (1.56% to 12.5% antiserum) which required only 0.4 ml of antiserum and less than 0.2 ml of 1% antigen in agar. Optical density values could be obtained at a single antiserum concentration (6.25%) through the use of 0.05 ml of antisera and less than 0.02 ml of 1% protein antigen in agar. These values were used to obtain total precipitate nitrogen values from a standard curve relating O.D. to total precipitate nitrogen at equivalence in liquid media. Total nitrogen values were converted to antibody nitrogen values by substituting them in an equation which expresses antibody nitrogen as a function of total nitrogen and the ratio of antibody to antigen at equivalence. This method was used to monitor weekly the production of antisera to human serum albumin in 12 rabbits. Optical density measurements made at single antiserum concentrations were used to predict the final antibody content of two pools of antisera. The mieromodification of the agar gel precipitin reaction offers considerable savings in time and materials, since a complete analysis can be obtained from O.D. measurements at a single antiserum concentration, using a minimum of antigen, antiserum and other reagents after only 2 days of incubation at 25°C.

Radioimmunoassay of Human Chorionic Gonadotropin (HCG) and Luteinizing Hormone (LH) in the Urine

In 1965, Wilde et al tried to use radioimmunoassay for estimating human chorionic gonadotropin (HCG) and Bagshawe et al, Odell and Midgley et al. also used this method for assaying HCG and luteinizing hormone (LH). One of our aims in this paper was to determine whether even partially purified HCG (5,000 I.U./mg) could be used for radioimmunoassay or not, and the other was to determine the proper experimental condition for each step of the reactions, in order to get the best results.Anti-HCG sera were prepared by giving two injections to four rabbits at 2-week intervals of 1,000 I.U. of crude preparation of HCG (1,000 I.U./mg) in 1 ml of phosphate saline buffer, freshly emulsified with 1 ml of complete Freiund's adjuvant (Difco) each time. The injections were made in the footpads and i.d. After 3 weeks of rest, a series of booster injections with 1,000 I.U. of HCG without adjuvant were given i.p. 5 times every other day. Rabbist were bled 7 days after the last injection and the sera were obtained. The antibody titers were from 1 : 6,400 to 1 : 25,600 by hemagglutination.For preparing absorbed anti-HCG serum, one ml of antiserum was incubated with 30 mg of an alcohol precipitate of urine from a child and 30 mg of human serum protein at 37°C for 60 min., and was kept at 4°C over-night. After centrifuging the mixture, the supernatant, the absorbed anti-HCG serum, showed only one precipitin band to HCG antigen on immuno-electrophoresis. Radioiodination of HCG by 131I was carried out by the modified chloramine T method of Greenwood.All materials were diluted with 0.5% bovine serum albumin (BSA) in borate buffered saline (BBS) -diluent-, and the 2nd IRP-HMG and Pergonal 500TM were used as standard LH.In radioiodination, the ratio of chloramine T to HCG was 0.4-1.3 : 1. The high specific activity of 131I-HCG which was more than 200 mc/mg, was obtained, and around 60% of the 131I-HCG preparation was precipitable with absorbed anti-HCG serum.The procedure of radioimmunoassay was as follows : The diluted anti-HCG serum was mixed with sample (HCG) and 131I-HCG. After incubation, the inactivated diluted rabbit serum (as carrier protein) and horse anti (rabbit serum r-globulin) serum globulin were added and then incubated. Th resulting precipitate was washed once with diluent and hydrolyzed with 1 ml of 2N NaOH and then counted with Scintillation Counter.Th proper experimental conditions for each step of radioimmunoassay were determined. 1. The combination of 131I-HCG and anti-HCG needed 48 hrs. for the maximum reaction (1 st reaction). 2. Cold HCG (sample) should be added to anti-HCG serum at least 4 hrs. before the addition of 131I-HCG in the 1st reaction. 3. The proper temperature during incubation in the 1st and 2nd reaction was 4°C. 4. As diluent, 0.5% bovine serum albumin in BBS was necessary and enough. 5. In the 2nd reaction, 0.1 ml of diluted (1 : 2,000) rabbit serum as carrier protein, gave the maximum precipitate with 0.2 mg of our horse anti (rabbit serum r-globulin) serum globulin. 6. The 2nd reaction needed more than 6 hrs. at 4°C.The standard curve of HCG shows that the amount of HCG from 0.0025 I.U./ml to 0.3125 I.U./ml was sufficient to be estimated quantitatively and the amount as much as 0.001 I.U./ml of HCG was also qualitatively detectable by this method. The amount of HCG in the urine was measured simultaneously by radioimmunoassay, hemagglutination inhibition rection and bioassay. The results showed that the radioimniunoassay could be clinically used. This method was also very useful to detect the low titer of HCG in the urine which could not be detected by other methods.The amount of LH in the urine of adult women having normal ovarian cycle, was radioimmunologically assayed, and it was shown that the peak of LH appeared one to three days before ovulation assumed by B.B.T.

Eine methode zur quantitativen bestimmung von immunpräzipitaten bis zu 1 μg protein

A method of quantitative determination of immune precipitates is described. The precipitation is effected in small tubes. Of each sample, 0.1 ml is transferred with graduated pipettes onto pretreated Chromatographie paper. The nonprecipitated proteins are removed by chromatography. Amido black is used for staining. The intensity of stain attachment is directly proportional to the quantity of precipitate. The removal of excess stain is followed by elution using N/10 NaOH. The extinction of the eluates is determined in a spectrophotometer. Because of its sensitivity, the present technique is particularly suited to the determination of precipitins in low-titer sera. For individual measurements, at the most 0.06 ml of antiserum is required, and in many cases it is possible to use diluted antiserum. With an amount of precipitate of the order of 1 μg, the error of individual measurement is still less than 10%.

A Chromatographic Technique for the Quantitative Study of the Precipitin Reaction

Summary A chromatographic technique for quantitative estimation of precipitin reactions is presented. The main advantageous features of the technique are the small amount of serum required (0.01 ml), the high sensitivity (1 γ of nitrogen), and simplicity of the procedure. The ratio of antigen to antibody in the precipitate can be determined by using the fluorescent antibody. The abbreviated version of the technique can be used for qualitative precipitin reactions or for testing the reactivity of fluorescent antibodies. The minimal concentration of precipitated nitrogen required for quantitative determinations is 100 γ/1 ml of antiserum.

Factors Influencing Leukoagglutination by Antileukocytic Sera

Summary A rapid procedure for leukoagglutination by specific antisera and conditions which influence it are presented. The procedure consists in suspending leukocytes in a small volume of saline, counting the cells with pipette and chamber and measuring out 27,000 cells per mm3 in 0.05 ml of normal saline. To the cells is added 0.5 ml of antiserum diluted with normal saline. The cell-antiserum mixture is centrifuged for one minute and the results are read. The entire procedure is carried out at 22 to 26 C. Agglutination of leukocytes is inhibited at 8 C and the velocity is reduced at 37 C. The optimal temperature is 22 to 26 C. Centrifuging results in the greatest speed of antigen-antibody reaction. Immature leukocytes and especially those outside the body for 2 to 3 days are destroyed more readily than mature cells by hypotonic salt solution. Agglutination requires the presence of intact cells. Electrolytes are necessary for the visible phase of agglutination and the optimal range is 0.85 to 2.5 per cent. Mature leukocytes are agglutinated in lower concentrations. Ageing of cells outside the body interferes with agglutination and mature leukocytes are agglutinated up to the fourteenth day. Immature cells show little or no clumping after the fourth day. Repeated washing of leukocytes does not interfere with agglutination until after the tenth time when most of the cells are destroyed. Hemoglobin up to 50 per cent does not interfere with agglutination. A definite number of leukocytes is necessary for a satisfactory agglutination. Excess or decrease in the number of cells modifies the degree of clumping.

Comparative Quantitative Studies of Guinea-Pig and Rabbit Anti-Hemocyanin Precipitates

Summary and Conclusions Quantitative chemical analyses of precipitates of guinea-pig and rabbit-antibody and hemocyanin from the whelk, Busycon canaliculata, have been made at various points in the precipitative reaction-range. Although the number of determinations with any one serum was limited by the amount of serum available from a single guinea-pig, it appeared that the curve of the composition of the precipitate, as indicated by the ratio of Ab N:Ag N, of guinea-pig antibody and hemocyanin, follows the same general pattern as that of rabbit-antibody, as determined by other investigators. Rabbit- and guinea-pig antisera for hemocyanin had approximately the same Ab N:Ag N ratios at corresponding points in the respective reaction-ranges. The amounts of specifically precipitable protein per ml of antiserum were similar in rabbit- and guinea-pig antisera, showing that when a suitable antigen is used, serum precipitins are produced as readily in guinea-pigs as in rabbits. Guinea-pig antisera failed to produce zones of complete precipitation of antigen and antibody, in contrast to rabbit antisera which produced well-defined neutral zones with neither antigen nor antibody in excess. The Dean and Webb optimal ratio, contrary to findings with other antigens, did not fall in the equivalence-zone with either guinea-pig or rabbit antisera for hemocyanin, but far in the region of excess-antigen. This ratio bore no relation to the antibody-content of the various sera. Ratios by volume at the midpoints of equivalence-zones, however, paralleled the respective antibody-content of the antisera.