Hemolysis In Gel Research Articles

Inhibition of complement-mediated hemolysis in gel by rheumatoid factors.

When subjected to a hemolysis in gel (HIG) assay for the detection of complement deficiency, 9 of 37 sera from patients with classical rheumatoid arthritis produced impaired lysis of sensitized sheep erythrocytes. All sera were normal in a test for the alternative pathway and no major abnormalities were found within the complement system. Using a two-step HIG technique, with guinea-pig serum as the complement source, all sera were shown to inhibit lysis of sheep erythrocytes sensitized with rabbit IgG. Lysis of IgM-coated erythrocytes was not inhibited. The agglutination titers in a Waaler-Rose test, and the areas of inhibition in the two-step HIG assay with IgG-sensitized erythrocytes, were correlated (r = 0.80, p less than 0.001). Absorption of serum with rabbit IgG coupled to Sepharose 4B, reduced the capacity to inhibit immune hemolysis. The eluate from IgG-Sepharose contained rheumatoid factors and also inhibited immune hemolysis. The findings suggested that rheumatoid factors in serum were responsible for inhibition in the HIG assays used.

Duration of rubella immunity induced by two-dose measles, mumps and rubella (MMR) vaccination. A 15-year follow-up in Finland

A national two-dose vaccination program with a combined measles, mumps and rubella (MMR-II) vaccine was introduced in Finland, in 1982, immunizing children at the ages of 14–18 months and 6 years. Antibody levels were determined from serial samples from a group of originally 350 children during 15 years. The latest samples were taken 15.5 years after the first vaccination and 70% of the children could still be reached. The aim of this study was to determine the kinetics of rubella antibodies induced by the MMR-II vaccine in these individuals. Rubella antibodies were analyzed from three different cohorts: Group I seronegative children ( n=166) vaccinated at 14–18 months and 6 years, Group II seronegative children ( n=139) and Group III seropositive children ( n=16) vaccinated at 6 and 11–13 years. Samples collected 0–9 years after vaccination were analyzed by hemolysis-in-gel (HIG) and later samples by enzyme immunoassay (EIA) techniques. The primary vaccination induced 100% seropositivity in vaccinees with a mean zone diameter of 10 (±1.3), 10.2 (±1.1) and 11.5 (±0.9) mm, in Groups I, II and III, respectively. The seropositivity rate was still high at 15 years, 99%, 100% and 100% with the geometric mean titer 23, 46 and 105 IU/ml, respectively. At 15 years, antibody levels <15 IU/ml which is the suggested protective level, were found in 31, 9 and 0% of children in Groups I, II and III, respectively. Because almost a third of the individuals in Group I now, at the age of 17 years, had low levels of rubella antibodies, it is possible that rubella infections may re-emerge during pregnancy. A careful surveillance including serological follow-up is therefore very important.

Effects of collagenolytic protease preparations from invertebrates on the immune system.

We studied the effects of an active ingredient of new enzyme preparations containing collagenolytic proteases from various hydrobionts (Collagenase from hydrobionts, Polycollagenase-K, and Fermenkol). These substances in therapeutic doses did not affect the humoral immune response estimated by hemagglutination and local hemolysis in gel and caused no local irritation and allergic reactions after long-term application to the skin.

Changes of the immunological patterns against measles, mumps and rubella. A vaccination programme studied 3 to 7 years after the introduction of a two-dose schedule

A two-dose vaccination programme using a combined measles, mumps and rubella vaccine (MMR) and administration at the ages of 18 months and 12 years was introduced in 1982. The 12-year-old schoolchildren were tested yearly from 1985 to 1989 on serum samples obtained prior to and after vaccination. Each year between 420 and 756 children were tested. The method used for antibody testing was the haemolysis-in-gel (HIG) assay. For measles also the enzyme-linked immunosorbent assay (ELISA) and the neutralization titre (NT) were applied. Only minor variations of the prevaccination immunity to measles were seen during the period 3–7 years after introduction of the programme. The age groups studied had partly been vaccinated against measles earlier. Between 12 and 16% lacked prevaccination immunity. In contrast the immunity to mumps and rubella of the 12-year-old children decreased considerably during the study period. No general vaccination against these diseases had been performed. Thus the susceptibility to mumps increased from 14% in 1985 to 39% in 1989 and to rubella from 41 to 57%. The seroconversion rate of children seronegative for measles was high, i.e. 100% in 1985 and later varied between 96 and 97%. For mumps, the seroconversion rate was lower and varied between 72 and 88%. All sera converted to rubella. During the follow-up period there was a declining incidence of measles, mumps and rubella. The relationship between the vaccination and reduction of disease and natural immunity strongly suggests that the association is causal and that this vaccination policy reduced the transmission of infection.

Methods for screening the naturally acquired and vaccine-induced immunity to the measles virus

Since the measles, mumps and rubella (MMR) vaccine was introduced into Sweden in 1982, a yearly evaluation of the immunity patterns and sero-conversion rates in 12-year-old children has been carried out. Since 1977, about half of the pre-school children have been vaccinated against measles. This study includes two study groups. (1) 145 selected pre- and post-vaccination samples tested by the haemolysis-in-gel (HIG) technique and the neutralization test (NT). The selection was made from 1298 12-year-old schoolchildren in 1986 and 1987, whose pre-vaccination sera had shown negative or borderline reactions to the HIG technique. (2) Consecutive pre- and post-vaccination samples obtained from 190 vaccinees in 1988 and 1989. These samples were studied by an enzyme-linked, immunosorbent assay (ELISA) and compared to the NT. The NT and the HIG tests yielded congruent results in early post-vaccination sera from children susceptible to measles prior to vaccination. In late post-vaccination samples, the NT and the HIG tests were discordant, up to 25% of the NT-positive samples being negative by the HIG technique. In no instance did the ELISA produce discrepant results, compared with those of the NT. With both this assays significantly lower antibody levels were detected in late post-vaccination sera (8–11 years) compared to early post-vaccination samples ( P < 0·001) or to sera obtained after natural infection.

Methods for screening the naturally acquired and vaccine-induced immunity to the mumps virus

Since the introduction of the measles, mumps and rubella (MMR) vaccine in Sweden in 1982, a yearly evaluation of the immunity pattern and seroconversion rate of 12-year-old children has been carried out. To be able to realize large-scale, follow-up studies, techniques have to be used which are not too labour-intensive and time-consuming but are sensitive enough to detect past immunity and post-vaccination titres. We report tests with haemolysis-in-gel (HIG) technique and an enzyme-linked, immunosorbent assay (ELISA) compared with neutralization (NT) for the detection of mumps antibodies. This study comprises 226 paired serum samples obtained between 1985 and 1989. One hundred and forty-one of the paired samples had been selected because they had given negative or borderline readings, using HIG technique. The remaining samples were consecutive pre- and post-vaccination sera obtained in 1989 from 85 vaccinees from one area in Sweden. HIG technique gave both false positive and false negative readings, compared with NT as also the false positive sera were detected. Non-inactivated sera could not be used in the NT test against mumps virus, owing to unspecific NT reactions. No differences between non-inactivated and inactivated sera by NT were seen, as against other paramyxoviruses. Cross-reactivity between mumps and parainfluenzae in NT tests was not demonstrated. The ELISA test proved more reliable and specific than HIG and was more sensitive than NT. Some post-vaccination sera from vaccinees who failed to seroconvert by NT contained high levels of mumps antibody detectable by the ELISA. Late post-vaccination sera which were negative by the NT also showed high levels of mumps-specific antibody detectable by the ELISA. Using ELISA test it was demonstrated that sera from children naturally immunized with mumps virus had significantly higher ELISA values than post-vaccination sera from children earlier vaccinated ( P < 0.001). A significantly higher booster effect after vaccination was also seen by the ELISA compared with the NT test.

Effect of low level immunity on response to live rubella virus vaccine

Serum specimens of 1075 young adults representing nursing and medical staff were collected for determination of rubella immunity using the radial haemolysis (RH) technique, also known as the haemolysis in gel (HIG) test. Of the sera 84 (7.8%) were negative (RH titre <4 mm) and an additional 93 (8.7%) gave an RH titre of 4–5 mm, which was regarded as the limit of immunity. Initially, 64 persons of these 177 were vaccinated with the RA27/3 strain of live attenuated rubella virus. Their serum samples were collected at the time of vaccination and at three weeks and three months after vaccination. Altogether 54 vaccinees could be followed for their immune response throughout the study. It became obvious that vaccination generally caused seroconversion only after three months rather than within three weeks. Only one person remained seronegative - even after a booster dose of vaccine. The mean final antibody titres in individuals with pre-existing low level immunity was 6 mm whereas in initially seronegative persons the antibody titre after vaccination was 8.5 mm on average. Thus a pre-existing low level immunity will effectively block the immune response to live rubella virus vaccine and this phenomenon may explain some apparent vaccination failures.

Blocking of antibody complement-dependent effector functions by streptococcal IgG Fc-receptor and staphylococcal protein A.

Using haemolysis in gel, two bacterial IgG-binding substances, an Fc-receptor isolated from group A streptococci type M15, and protein A from Staphylococcus aureus, were shown to inhibit complement-mediated lysis of sheep erythrocytes sensitized with rabbit IgG. When the crude alkaline extracts of ten types of group A streptococci were tested to see whether streptococcal components other than Fc-binding material might affect lysis, the degree of inhibition was found to be correlated with Fc-binding activity. In no case was the lysis of IgM-coated cells inhibited. Opsonophagocytosis experiments showed that both purified streptococcal Fc-receptor and protein A impaired antibody complement-dependent killing by human polymorphonuclear leukocytes of each of two strains of group B streptococci (lacking IgG Fc-receptors). Furthermore, the impairment was ascribable to interference with the fixation of complement to the antibodies, as demonstrated in pre-opsonization experiments with one of the strains. Our results suggest that blocking of the binding of complement to IgG is an important virulence mechanism in Fc-receptor-bearing streptococci and staphylococci.

Comparison between a commercial elisa, rubazyme, and hemolysis-in-gel test for determination of rubella antibodies

A commercial enzyme-linked immunosorbent assay, Rubazyme, was compared with the hemolysis-in-gel (HIG) test for antibodies to rubella in 826 sera. The results were in agreement for 99.4% of the 725 sera tested for immunity. However, the Rubazyme assay was no more efficient that either the hemagglutination-inhibition or HIG test in discriminating between sera with low levels of antibody and negative sera. Thus, it was concluded that the HIG test is the method of choice for immunity testing because of the low cost and simplicity. Rubazyme may be of value to confirm equivocal HIG results.

Vaccination against measles, mumps and rubella (MMR): A comparison between the antibody responses at the ages of 18 months and 12 years and between different methods of antibody titration

In connection with the introduction of the trivalent vaccine against measles, mumps and rubella at 18 months and 12 years of age, an evaluation of the seroconversion and booster effects in the two age-groups was carried out. This also comprised different laboratory-test methods appropriate for follow-up studies after large-scale, vaccination studies. The measles, mumps and rubella antibodies were measured by the haemolysis-in-gel (HIG) method. Measles antibodies were also measured by the haemagglutination-inhibition (HI) test. Borderline values or samples negative to measles or mumps were also tested by the serum-neutralization (SN) test. All but four of 150 18-month-old children lacked antibodies against measles by the HI test and one of these by the HIG method. Against mumps, 99% were seronegative in the HIG test and 97% in the SN test and two against rubella prior to vaccination. Among 247 schoolchildren, 60 (24%) lacked antibodies in the HI test and 28 (11%) of these also in the HIG test. Sixty-six schoolchildren (25%) were negative to mumps and 45% to rubella prior to vaccination. The seroconversion rate for the 18-month-old children was 96% against measles, 93% against mumps and 99% against rubella. The figure for the schoolchildren was 82% against measles, 80% against mumps and 100% against rubella. On comparing the titre levels in seroconverting children, the measles-antibody levels were found to be lower among older children, compared with younger, while the opposite was true for rubella.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of killed and live attenuated influenza vaccine on symptoms and specific airway conductance in asthmatics and healthy subjects.

Killed and live influenza virus vaccines were given to asthmatics and healthy subjects to investigate symptoms and alterations in their respiratory performance after vaccination. Polyvalent killed influenza virus vaccine was given to 16 asthmatics and live attenuated influenza virus vaccine to 23 asthmatics and 21 healthy subjects. Fourteen of the 16 asthmatics vaccinated with the killed vaccine displayed a significant rise in serum antibody level as measured by a single radial haemolysis in gel (SRH test). 11 of the 23 asthmatics and 14 of the 21 healthy subjects vaccinated with the live attenuated vaccine displayed a significant rise in the SRH test. Among the subjects with no measurable initial antibodies and with a significant rise in the SRH test, one asthmatic vaccinated with the killed vaccine experienced symptoms of common cold with fever and dyspnoea 1 week after vaccination. Three asthmatics and four healthy subjects vaccinated with live attenuated vaccine experienced mild symptoms, mainly rhinorrhoea, cough and sore throat 2 to 3 days after vaccination. No alterations in specific airway conductance in asthmatics or in healthy subjects were observed. We conclude that both killed and live attenuated influenza virus vaccines are tolerated well by asthmatics and appear to be safe for asthma patients.

Evidence for flavivirus(es) outside of the distribution area for Ixodes ricinus in Norway.

Small rodent (vole) sera were collected from three different locations in Norway. One of these was within the distribution area for Ixodes ricinus, and a tick-borne encephalitis (TBE) virus strain had been isolated from ticks collected there (Traavik & Mehl, 1977). The two other locations were outside the I. ricinus area, one in southern Norway, the other at nearly 70 degrees N. The sera were tested for TBE antibodies by hemagglutination inhibition (HI), hemolysis-in-gel (HIG) and complement fixation (CFT). All sera were also tested for HI antibodies to Uukuniemi (UUK) virus, and some positive TBE HI reactions were verified by separation of immunoglobulins and serum lipoproteins in NaBr gradients. Animals containing TBE virus antibodies reacting in all three serological tests and animals with UUK HI antibodies were detected only from the location within the I. ricinus area. From the two locations outside the I. ricinus area we found animals which had antibodies reacting with TBE virus in HI and HIG, but not in CFT. Antibodies to UUK were not detected. The results indicate that flavivirus(es) related to, but not identical with TBE viruses are transmitted by other vectors than I. ricinus in parts of Norway.

Rubella immune state and rubella vaccination of female university students.

At the Finnish Student Health Service, we have measured rubella antibodies in 3 consecutive academic years--1979-80, 1980-81 and 1981-82--in 2 483 first year female students. In 1979-80, 16.4% were seronegative, the following year the figures were 10.4% and in the third year 7.4%. Among students less than or equal to 19 yr of age the seronegatives decreased from 19 to 2% in 3 yr. This decrease shows that the national rubella vaccination programme which began in 1975 has been more successful than expected. A hemolysis-in-gel (HIG) test has proved a suitable screening method and the health examination of first year students a suitable occasion to test the immunity against rubella. Seroconversion occurred in 81.4% of those vaccinated.

Use of the direct haemolysis-in-gel test for rubella antibodies in the Icelandic prevention programme for congenital rubella.

Untreated earlobe capillary blood samples from 5958 girls were screened for rubella antibodies by placing them directly onto haemolysis-in-gel (HIG) test plates. The method is in our experience quick, reliable and also acceptable to young girls. The method is convenient for testing for natural immunity before vaccination and in most but not all cases satisfactory as a test for seroconversion after vaccination. A comparison was made between naturally infected girls and a group of vaccinated girls. Vaccinated girls had lower values and most of the low or doubtful positives were in this group. Serum samples from 51 low positive girls were tested by the haemagglutination inhibition (HI) test and the serum HIG test and their results compared with their respective direct HIG values.

Chemical linkage of erythrocytes and viral antigen in the hemolysis-in-gel (HIG) test for viral antibodies

The sensitivity of the hemolysis-in-gel (HIG) test with rubella antigen is not improved by chemical linkage of the virus to the erythrocytes, and after such modification, IgM specific antibodies are not detectable. In the influenza HIG test with tetraazotized o-dianisidine (TOD), chromic chloride and potassium periodate as coupling reagents, increased sensitivity was observed with allantoic fluid of infected eggs as antigen. If Tween-ether treated hemagglutinin is used in the HIG test, zones of hemolysis are detectable only after treatment of the erythrocytes with TOD, chromic chloride and potassium periodate.

The immunizing effect and reactogenicity of two live attenuated mumps virus vaccines in Swedish schoolchildren

An evaluation of the seroconversion and booster effects after vaccination with two different mumps vaccines, the Urabe Am 9 strain and the Jeryl Lynn strain, was carried out in schoolchildren. Four hundred and fifty-four schoolchildren aged 11 to 12 years with no previous history of mumps or mumps vaccination were enrolled for the study. The antibody responses were measured by serum neutralization (SN) and haemolysis-in-gel (HIG) tests. Of the 454 subjects, 130 were found to be initially seronegative. Two lots of different strengths of each vaccine were used to evaluate the relationships. The Urabe Am 9 vaccine lots had infectivity titres of 100 000 and 19 000 TCID50 per dose and the Jeryl Lynn vaccine titres of 59 000 and 28 000 TCID50 per dose. Only slight differences in seroconversion rates were seen between the lots. The overall seroconversion rate, measured by SN, was 94% for the Urabe Am 9 vaccine and 91% for the Jeryl Lynn vaccine, whereas the geometric mean titre for virus-neutralizing antibody in seroconverting children was 7.4 with the Urabe Am 9 vaccine and 10.7 with the Jeryl Lynn vaccine. In children who were seropositive prior to vaccination, a marked rise in antibody titre was found 8 weeks after vaccine injection indicating a booster effect. The miscellaneous post-vaccination side-effects were mild and inconsequential.

Frequent occurrence of anti-rabbit IgM in IgA deficiency.

Analysis of IgA deficient sera revealed impaired hemolysis of sensitized sheep erythrocytes when tested by a hemolysis in gel (HIG) assay developed for detection of complement deficiencies. All sera were normal in a test for the alternative pathway. The impaired hemolysis was not related to complement aberrations but was caused by antibodies to rabbit IgM, demonstrated in 14 of 21 IgA deficient sera, by use of HIG technique and by agglutination. The presence of these antibodies was not related to age, sex or disease. One serum was further examined and the antibodies were shown to be of IgG class.

Alpha-fetoprotein synthesis by clonogenic cells of rat hepatoma McA-RH 7777

Production of alpha-fetoprotein (AFP) was determined in single cells of hepatoma McA-RH7777 and in the clones of their progeny. To elucidate the heritability of this trait in a series of cell generations, a variety of local hemolysis in gel was devised. According to the method the cells and red cells conjugated with protein A were placed on the polylysine covered surface and layered with agarose gel containing antibodies. AFP production by single cells was determined from the formation of plaques--areas of red cell hemolysis. The cells forming the plaques (+AFP) and not forming them (-AFP) were distinguished and their reproduction was followed up. After 7-14 days the cells were fixed and stained by the immunoperoxidase technique with antibodies to AFP. High efficacy of the cloning has been demonstrated for both +AFP- and -AFP-cells (69 and 71%). Negative cells preserved their phenotype more frequently, producing homogenous negative clones, whereas +AFP cells gave "negative" clones in 1/3 of the cases. Both cells gave mixed clones in a small percentage of the cases. At present the AFP trait in these cells is being studied by recloning.

The use of red cells with fused Semliki Forest virus envelope proteins in antibody determinations by hemolysis in gel

Chicken red blood cells with fused Semliki Forest virus (SFV) proteins on the cell membrane were used in the hemolysis-in-gel (HIG) plates. Optimally the plate contained a 1.5 mm thick gel of 1% agarose with 1% red cells and 1 unit/ml gel of complement. 400 ng of SFV was fused to the red cells in 1 ml of the gel (about 20 virions fused to one red cell). Five μl of inactivated (56°C, 30 min) serum samples were pipetted into wells in the gel. After 20 h of incubation at 37°C the diameters of the hemolytic zones were directly proportional to the logarithm of the serum dilution. This made it possible to calculate the antibody titers for the samples using an experimental formula T = 10 φ k ( T is the titer, φ the diameter in mm and k a variable coefficient, which had to be determined for each batch of the plates using a standard serum). Using regression and residual analyses, the formula was shown to fit the experimental results. The fusion-based HIG could be read after as early as 2 h of incubation. The specificity of the test was studied using antisera against Western equine encephalomyelitis, Eastern equine encephalomyelitis, Chikungunya and Uruma viruses, which all gave negative results in the SFV HIG test. Antisera against SFV E1 and E2 proteins were positive, but anti-E3 serum was negative when measured in the SFV HIG test.

Antibody response after application of a new live attenuated mumps vaccine (Pariorix) measured by enzyme-linked immunosorbent assay (ELISA).

Mumps virus specific antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA), hemolysis-in-gel (HIG) test, and hemagglutination-inhibition (HI) test in 17 adult volunteers before and after vaccination with a new live mumps vaccine, Pariorix. The results of the present study indicate that the strain Urabe-Am 9 of the mumps virus (Pariorix) induces antibodies in 100% of seronegative adults when measured by ELISA and in 70.6% and 64.7% in HIG and HI, respectively. ELISA is therefore a very sensitive and reliable method for the demonstration of sero-conversion after vaccination. The vaccine was well tolerated and clinically acceptable in the group of adult volunteers.